1052 Citations
Background Neutrophils the most abundant white blood cells in humans play pivotal roles in innate immunity rapidly migrating to sites of infection and inflammation to phagocytose neutralize and eliminate invading pathogens Neutrophil extracellular trap NET formation is increasingly recognized as an essential rapid innate immune response but when dysregulated it contributes to pathogenesis of sepsis and immunothrombotic disease Objectives Current NETosis models are limited routinely employing nonphysiological triggers that can bypass natural NET regulatory pathways Models utilizing isolated neutrophils and immortalized cell lines do not reflect the complex biology underlying neutrophil activation and NETosis that occurs in whole blood To ... More
Background
Neutrophils, the most abundant white blood cells in humans, play pivotal roles in innate immunity, rapidly migrating to sites of infection and inflammation to phagocytose, neutralize, and eliminate invading pathogens. Neutrophil extracellular trap (NET) formation is increasingly recognized as an essential rapid innate immune response, but when dysregulated, it contributes to pathogenesis of sepsis and immunothrombotic disease.
Objectives
Current NETosis models are limited, routinely employing nonphysiological triggers that can bypass natural NET regulatory pathways. Models utilizing isolated neutrophils and immortalized cell lines do not reflect the complex biology underlying neutrophil activation and NETosis that occurs in whole blood. To our knowledge, we report the first human ex vivo model utilizing naturally occurring molecules to induce NETosis in whole blood. This approach could be used for drug screening and, importantly, inadvertent activators of NETosis.
Methods
Here we describe a novel, high-throughput ex vivo whole blood–induced NETosis model using combinatorial pooling of native NETosis-inducing factors in a more biologically relevant Synthetic-Sepsis model.
Results
We found different combinations of factors evoked distinct neutrophil responses in the rate of NET generation and/or magnitude of NETosis. Despite interdonor variability, similar sets of proinflammatory molecules induced consistent responses across donors. We found that at least 3 biological triggers were necessary to induce NETosis in our system including either tumor necrosis factor-α or lymphotoxin-α.
Conclusion
These findings emphasize the importance of investigating neutrophil physiology in a biologically relevant context to enable a better understanding of disease pathology, risk factors, and therapeutic targets, potentially providing novel strategies for disease intervention and treatment. Less
Neutrophils, the most abundant white blood cells in humans, play pivotal roles in innate immunity, rapidly migrating to sites of infection and inflammation to phagocytose, neutralize, and eliminate invading pathogens. Neutrophil extracellular trap (NET) formation is increasingly recognized as an essential rapid innate immune response, but when dysregulated, it contributes to pathogenesis of sepsis and immunothrombotic disease.
Objectives
Current NETosis models are limited, routinely employing nonphysiological triggers that can bypass natural NET regulatory pathways. Models utilizing isolated neutrophils and immortalized cell lines do not reflect the complex biology underlying neutrophil activation and NETosis that occurs in whole blood. To our knowledge, we report the first human ex vivo model utilizing naturally occurring molecules to induce NETosis in whole blood. This approach could be used for drug screening and, importantly, inadvertent activators of NETosis.
Methods
Here we describe a novel, high-throughput ex vivo whole blood–induced NETosis model using combinatorial pooling of native NETosis-inducing factors in a more biologically relevant Synthetic-Sepsis model.
Results
We found different combinations of factors evoked distinct neutrophil responses in the rate of NET generation and/or magnitude of NETosis. Despite interdonor variability, similar sets of proinflammatory molecules induced consistent responses across donors. We found that at least 3 biological triggers were necessary to induce NETosis in our system including either tumor necrosis factor-α or lymphotoxin-α.
Conclusion
These findings emphasize the importance of investigating neutrophil physiology in a biologically relevant context to enable a better understanding of disease pathology, risk factors, and therapeutic targets, potentially providing novel strategies for disease intervention and treatment. Less
Drug sensitivity testing of patient-derived tumor organoids PDTOs is a promising tool for personalizing cancer treatment Here we present a protocol for generation of and high-throughput drug testing with PDTOs We describe detailed steps for PDTO establishment from colorectal cancer tissues preparation of PDTOs for high-throughput drug testing and quantification of drug testing results using image analysis This protocol provides a standardized workflow for PDTO testing of standard-of-care therapies along with exploring the activity of new agents for translational research
The Maternal-to-Zygotic transition MZT is a reprograming process encompassing zygotic genome activation ZGA and the clearance of maternally-provided mRNAs While some factors regulating MZT have been identified there are thousands of maternal RNAs whose function has not been ascribed yet Here we have performed a proof-of-principle CRISPR-RfxCas d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT Bckdk mRNA knockdown caused epiboly defects ZGA deregulation H K ac reduction and a partial impairment of miR- processing Phospho-proteomic analysis revealed that Phf Baf a a chromatin remodeling factor is ... More
The Maternal-to-Zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT. Bckdk mRNA knockdown caused epiboly defects, ZGA deregulation, H3K27ac reduction and a partial impairment of miR-430 processing. Phospho-proteomic analysis revealed that Phf10/Baf45a, a chromatin remodeling factor, is less phosphorylated upon Bckdk depletion. Further, phf10 mRNA knockdown also altered ZGA and Phf10 constitutively phosphorylated rescued the developmental defects observed after bckdk mRNA depletion. Altogether, our results demonstrate the competence of CRISPR-RfxCas13d screenings to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation. Less
We report the biochemical structural and functional characterization of the protein coded by gene PA in the P aeruginosa PAO genome The PA gene had been annotated as coding a probable bacterioferritin Our structural work shows that the product of gene PA is a protein that adopts the Dps subunit fold which oligomerizes into a -mer quaternary structure Unlike Dps however the ferroxidase di-iron centers and iron coordinating ligands are buried within each subunit in a manner identical to that observed in the ferroxidase center of P aeruginosa bacterioferritin Since these structural characteristics correspond to Dps-like proteins we term the ... More
We report the biochemical, structural, and functional characterization of the protein coded by gene PA4880 in the P. aeruginosa PAO1 genome. The PA4880 gene had been annotated as coding a probable bacterioferritin. Our structural work shows that the product of gene PA4880 is a protein that adopts the Dps subunit fold, which oligomerizes into a 12-mer quaternary structure. Unlike Dps, however, the ferroxidase di-iron centers and iron coordinating ligands are buried within each subunit, in a manner identical to that observed in the ferroxidase center of P. aeruginosa bacterioferritin. Since these structural characteristics correspond to Dps-like proteins, we term the protein as P. aeruginosa Dps-like, or Pa DpsL. The ferroxidase centers in Pa DpsL catalyze the oxidation of Fe2+ utilizing O2 or H2O2 as oxidant, and the resultant Fe3+ is compartmentalized in the interior cavity. Interestingly, incubating Pa DpsL with plasmid DNA results in efficient nicking of the DNA and at higher concentrations of Pa DpsL the DNA is linearized and eventually degraded. The nickase and endonuclease activities suggest that Pa DpsL, in addition to participating in the defense of P. aeruginosa cells against iron-induced toxicity, may also participate in the innate immune mechanisms consisting of restriction endonucleases and cognate methyl transferases. Less
Talin herein referring collectively to talin and couples the actomyosin cytoskeleton to integrins and transmits tension to the extracellular matrix Talin also interacts with numerous additional proteins capable of modulating the actin-integrin linkage and thus downstream mechanosignaling cascades Here we demonstrate that the scaffold protein Caskin interacts directly with the R domain of talin through its C-terminal LD motif Caskin also associates with the WAVE regulatory complex to promote cell migration in an Abi -dependent manner Furthermore we demonstrate that the Caskin Abi interaction is regulated by growth factor-induced phosphorylation of Caskin on serine In MCF and UACC cells which ... More
Talin (herein referring collectively to talin 1 and 2) couples the actomyosin cytoskeleton to integrins and transmits tension to the extracellular matrix. Talin also interacts with numerous additional proteins capable of modulating the actin-integrin linkage and thus downstream mechanosignaling cascades. Here, we demonstrate that the scaffold protein Caskin2 interacts directly with the R8 domain of talin through its C-terminal LD motif. Caskin2 also associates with the WAVE regulatory complex to promote cell migration in an Abi1-dependent manner. Furthermore, we demonstrate that the Caskin2–Abi1 interaction is regulated by growth factor-induced phosphorylation of Caskin2 on serine 878. In MCF7 and UACC893 cells, which contain an amplification of CASKIN2, Caskin2 localizes in plasma membrane-associated plaques and around focal adhesions in cortical microtubule stabilization complexes. Taken together, our results identify Caskin2 as a novel talin-binding protein that might not only connect integrin-mediated adhesion to actin polymerization but could also play a role in crosstalk between integrins and microtubules. Less
Given the crucial role of the main protease Mpro in the replication cycle of SARS-CoV- this viral cysteine protease constitutes a high-profile drug target We investigated peptidomimetic azapeptide nitriles as auspicious irreversibly acting inhibitors of Mpro Our systematic approach combined an Mpro active-site scanning by combinatorially assembled azanitriles with structure-based design Encouraged by the bioactive conformation of open-chain inhibitors we conceptualized the novel chemotype of macrocyclic azanitriles whose binding mode was elucidated by cocrystallization This strategy provided a favorable entropic contribution to target binding and resulted in the development of the extraordinarily potent Mpro inhibitor with an IC value of ... More
Given the crucial role of the main protease (Mpro) in the replication cycle of SARS-CoV-2, this viral cysteine protease constitutes a high-profile drug target. We investigated peptidomimetic azapeptide nitriles as auspicious, irreversibly acting inhibitors of Mpro. Our systematic approach combined an Mpro active-site scanning by combinatorially assembled azanitriles with structure-based design. Encouraged by the bioactive conformation of open-chain inhibitors, we conceptualized the novel chemotype of macrocyclic azanitriles whose binding mode was elucidated by cocrystallization. This strategy provided a favorable entropic contribution to target binding and resulted in the development of the extraordinarily potent Mpro inhibitor 84 with an IC50 value of 3.23 nM and a second-order rate constant of inactivation, kinac/Ki, of 448,000 M–1s–1. The open-chain Mpro inhibitor 58, along with the macrocyclic compounds 83 and 84, a broad-spectrum anticoronaviral agent, demonstrated the highest antiviral activity with EC50 values in the single-digit micromolar range. Our findings are expected to promote the future development of peptidomimetic Mpro inhibitors as anti-SARS-CoV-2 agents. Less
Protein crystallization as opposed to well-established chromatography processes has the benefits to reduce production costs while reaching a comparable high purity However monitoring crystallization processes remains a challenge as the produced crystals may interfere with analytical measurements Especially for capturing proteins from complex feedstock containing various impurities establishing reliable process analytical technology PAT to monitor protein crystallization processes can be complicated In heterogeneous mixtures important product characteristics can be found by multivariate analysis and chemometrics thus contributing to the development of a thorough process understanding In this project an analytical set-up is established combining offline analytics on-line ultraviolet visible light ... More
Protein crystallization as opposed to well-established chromatography processes has the benefits to reduce production costs while reaching a comparable high purity. However, monitoring crystallization processes remains a challenge as the produced crystals may interfere with analytical measurements. Especially for capturing proteins from complex feedstock containing various impurities, establishing reliable process analytical technology (PAT) to monitor protein crystallization processes can be complicated. In heterogeneous mixtures, important product characteristics can be found by multivariate analysis and chemometrics, thus contributing to the development of a thorough process understanding. In this project, an analytical set-up is established combining offline analytics, on-line ultraviolet visible light (UV/Vis) spectroscopy, and in-line Raman spectroscopy to monitor a stirred-batch crystallization process with multiple phases and species being present. As an example process, the enzyme Lactobacillus kefir alcohol dehydrogenase (LkADH) was crystallized from clarified Escherichia coli (E. coli) lysate on a 300 mL scale in five distinct experiments, with the experimental conditions changing in terms of the initial lysate solution preparation method and precipitant concentration. Since UV/Vis spectroscopy is sensitive to particles, a cross-flow filtration (cross-flow filtration)-based bypass enabled the on-line analysis of the liquid phase providing information on the lysate composition regarding the nucleic acid to protein ratio. A principal component analysis (PCA) of in situ Raman spectra supported the identification of spectra and wavenumber ranges associated with productspecific information and revealed that the experiments followed a comparable, spectral trend when crystals were present. Based on preprocessed Raman spectra, a partial least squares (PLS) regression model was optimized to monitor the target molecule concentration in real-time. The off-line sample analysis provided information on the crystal number and crystal geometry by automated image analysis as well as the concentration of LkADH and host cell proteins (HCPs) In spite of a complex lysate suspension containing scattering crystals and various impurities, it was possible to monitor the target molecule concentration in a heterogeneous, multi-phase process using spectroscopic methods. With the presented analytical set-up of off-line, particle-sensitive on-line, and in-line analyzers, a crystallization capture process can be characterized better in terms of the geometry, yield, and purity of the crystals. Less
Eukaryotic innate immune systems use pattern recognition receptors PRRs to sense infection by detecting pathogen-associated molecular patterns which then triggers an immune response Bacteria have similarly evolved immunity proteins that sense certain components of their viral predators known as bacteriophages Although different immunity proteins can recognize different phage-encoded triggers individual bacterial immunity proteins have only been found to sense a single trigger during infection suggesting a one-to-one relationship between bacterial PRRs and their ligands Here we demonstrate that the anti-phage defense protein CapRelSJ in Escherichia coli can directly bind and sense two completely unrelated and structurally different proteins using the ... More
Eukaryotic innate immune systems use pattern recognition receptors (PRRs) to sense infection by detecting pathogen-associated molecular patterns, which then triggers an immune response. Bacteria have similarly evolved immunity proteins that sense certain components of their viral predators known as bacteriophages1–6. Although different immunity proteins can recognize different phage-encoded triggers, individual bacterial immunity proteins have only been found to sense a single trigger during infection, suggesting a one-to-one relationship between bacterial PRRs and their ligands7–11. Here, we demonstrate that the anti-phage defense protein CapRelSJ46 in Escherichia coli can directly bind and sense two completely unrelated and structurally different proteins using the same sensory domain, with overlapping but distinct interfaces. Our results highlight the remarkable versatility of an immune sensory domain, which may be a common property of anti-phage defense systems and enable them to keep pace with their rapidly evolving viral predators. We found that Bas11 phages harbor both trigger proteins that are sensed by CapRelSJ46 during infection, and we demonstrate that such phage can only fully evade CapRelSJ46 defense when both triggers are mutated. Our work reveals how a bacterial immune system that senses more than one trigger can help prevent phages from easily escaping detection, and it may allow detection of a broader range of phages. More generally, our findings illustrate unexpected multifactorial sensing by bacterial defense systems and complex coevolutionary relationships between them and their phage-encoded triggers. Less
Cyanobacterial CO concentrating mechanisms CCMs sequester a globally consequential proportion of carbon into the biosphere Proteinaceous microcompartments called carboxysomes play a critical role in CCM function housing two enzymes to enhance CO fixation carbonic anhydrase CA and Rubisco Despite its importance our current understanding of the carboxysomal CAs found in -cyanobacteria CsoSCA remains limited particularly regarding the regulation of its activity Here we present a structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium sp PCC Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose- -bisphosphate and forms a hexameric trimer of dimers ... More
Cyanobacterial CO2 concentrating mechanisms (CCMs) sequester a globally consequential proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO2 fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in α-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity. Here, we present a structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium sp. PCC7001. Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose-1,5-bisphosphate and forms a hexameric trimer of dimers. Comprehensive phylogenetic and mutational analyses are consistent with this regulation appearing exclusively in cyanobacterial α-carboxysome CAs. These findings clarify the biologically relevant oligomeric state of α-carboxysomal CAs and advance our understanding of the regulation of photosynthesis in this globally dominant lineage. Less
Effector proteins are central to the success of plant pathogens while immunity in hostplants is driven by receptor-mediated recognition of these effectors Understanding the mole-cular details of effector receptor interactions is key for the engineering of novel immunereceptors Here we experimentally determined the crystal structure of the Puccinia graminis f sp tri-tici Pgt effector AvrSr which was not accurately predicted using AlphaFold We charac-terised the role of the conserved cysteine residues in AvrSr using in vitro biochemical assaysand examined Sr -mediated recognition using transient expression in Nicotiana spp andwheat protoplasts The AvrSr structure contains a novel b-strand rich modular fold ... More
Effector proteins are central to the success of plant pathogens, while immunity in hostplants is driven by receptor-mediated recognition of these effectors. Understanding the mole-cular details of effector–receptor interactions is key for the engineering of novel immunereceptors. Here, we experimentally determined the crystal structure of the Puccinia graminis f. sp. tri-tici (Pgt) effector AvrSr27, which was not accurately predicted using AlphaFold2. We charac-terised the role of the conserved cysteine residues in AvrSr27 using in vitro biochemical assaysand examined Sr27-mediated recognition using transient expression in Nicotiana spp. andwheat protoplasts. The AvrSr27 structure contains a novel b-strand rich modular fold consisting of two structu-rally similar domains that bind to Zn2+ ions. The N-terminal domain of AvrSr27 is sufficient forinteraction with Sr27 and triggering cell death. We identified two Pgt proteins structurallyrelated to AvrSr27 but with low sequence identity that can also associate with Sr27, albeitmore weakly. Though only the full-length proteins, trigger Sr27-dependent cell death in tran-sient expression systems. Collectively, our findings have important implications for utilising protein prediction plat-forms for effector proteins, and those embarking on bespoke engineering of immunity recep-tors as solutions to plant disease Less
Pyruvate kinase PK deficiency is a rare genetic disorder that affects this critical enzyme within the glycolysis pathway In recent years Mitapivat MTPV AG- has emerged as a notable allosteric activator for treating PK deficiency However the allosteric regulatory effects exerted on PK by MTPV are yet to be comprehensively elucidated To shed light on the molecular mechanisms of the allosteric effects we employed crystallography and biophysical methods Our efforts yielded a high-resolution crystal structure of the PK tetramer complexed with MTPV at resolution Isothermal titration calorimetry measurements revealed that MTPV binds to human PK with an affinity of M ... More
Pyruvate kinase (PK) deficiency is a rare genetic disorder that affects this critical enzyme within the glycolysis pathway. In recent years, Mitapivat (MTPV, AG-348) has emerged as a notable allosteric activator for treating PK deficiency. However, the allosteric regulatory effects exerted on PK by MTPV are yet to be comprehensively elucidated. To shed light on the molecular mechanisms of the allosteric effects, we employed crystallography and biophysical methods. Our efforts yielded a high-resolution crystal structure of the PK tetramer complexed with MTPV at 2.1 Å resolution. Isothermal titration calorimetry measurements revealed that MTPV binds to human PK with an affinity of 1 μM. The enhanced structural details now allow for unambiguous analysis of the MTPV-filled cavity intricately embedded within the enzyme. Finally, the structure suggests that MTPV binding induces an allosteric effect on the B-domain situated proximal to the active site. In summary, our study provides valuable insights into the allosteric regulation of PK by MTPV and paves the way for further structure-based drug optimization for therapeutic interventions in PK deficiency. Less
Disclosed herein are methods and systems comprising obtaining nucleic acid from a sample that was obtained from a subject capturing and amplifying a target molecule in the nucleic acid using a molecular inversion probe under hybridization conditions ligating an adapter to create a circular molecule sequencing the circular molecule to obtain sequence reads generating a sequencing file comprising the sequence reads of each molecule and a position of each sequence read in a reference genome of a virus and generating a reporting file for the subject comprising a predicted lineage of the virus in the sample
Enteroviruses are the causative agents of paediatric hand-foot-and-mouth disease and a target for pandemic preparedness due to the risk of higher order complications in a large-scale outbreak The A protease of these viruses is responsible for the self-cleavage of the poly protein allowing for correct folding and assembly of capsid proteins in the final stages of viral replication These A proteases are highly conserved between Enterovirus species such as Enterovirus A and Coxsackievirus A Inhibition of the A protease deranges capsid folding and assembly preventing formation of mature virions in host cells and making the protease a valuable target for ... More
Enteroviruses are the causative agents of paediatric hand-foot-and-mouth disease, and a target for pandemic preparedness due to the risk of higher order complications in a large-scale outbreak. The 2A protease of these viruses is responsible for the self-cleavage of the poly protein, allowing for correct folding and assembly of capsid proteins in the final stages of viral replication. These 2A proteases are highly conserved between Enterovirus species, such as Enterovirus A71 and Coxsackievirus A16. Inhibition of the 2A protease deranges capsid folding and assembly, preventing formation of mature virions in host cells and making the protease a valuable target for antiviral activity. Herein, we describe a crystallographic fragment screening campaign that identified 75 fragments which bind to the 2A protease including 38 unique compounds shown to bind within the active site. These fragments reveal a path for the development of non-peptidomimetic inhibitors of the 2A protease with broad-spectrum anti-enteroviral activity. Less
The adenosine A A receptor A AAR belongs to the rhodopsin-like G protein-coupled receptor GPCR family which constitutes the largest class of GPCRs Partial agonists show reduced efficacy as compared to physiological agonists and can even act as antagonists in the presence of a full agonist Here we determined an X-ray crystal structure of the partial A AAR agonist -amino- - H-imidazol- -ylmethyl sulfanyl - -p-hydroxyphenyl- -pyridinedicarbonitrile LUF in complex with the A AAR construct A A-PSB -bRIL stabilized in its inactive conformation and being devoid of any mutations in the ligand binding pocket The determined high-resolution structure resolved water ... More
The adenosine A2A receptor (A2AAR) belongs to the rhodopsin-like G protein-coupled receptor (GPCR) family, which constitutes the largest class of GPCRs. Partial agonists show reduced efficacy as compared to physiological agonists and can even act as antagonists in the presence of a full agonist. Here, we determined an X-ray crystal structure of the partial A2AAR agonist 2-amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-p-hydroxyphenyl-3,5-pyridinedicarbonitrile (LUF5834) in complex with the A2AAR construct A2A-PSB2-bRIL, stabilized in its inactive conformation and being devoid of any mutations in the ligand binding pocket. The determined high-resolution structure (2.43 Å) resolved water networks and crucial binding pocket interactions. A direct hydrogen bond of the p-hydroxy group of LUF5834 with T883.36 was observed, an amino acid that was mutated to alanine in the most frequently used A2AAR crystallization constructs thus preventing the discovery of its interactions in most of the previous A2AAR co-crystal structures. G protein dissociation studies confirmed partial agonistic activity of LUF5834 as compared to that of the full agonist N-ethylcarboxamidoadenosine (NECA). In contrast to NECA, the partial agonist was still able to bind to the receptor construct locked in its inactive conformation by an S913.39K mutation, although with an affinity lower than that at the native receptor. This could explain the compound’s partial agonistic activity: while full A2AAR agonists bind exclusively to the active conformation, likely following conformational selection, partial agonists bind to active as well as inactive conformations, showing higher affinity for the active conformation. This might be a general mechanism of partial agonism also applicable to other GPCRs. Less
The development of effective broad-spectrum antivirals forms an important part of preparing for future pandemics A cause for concern is the currently emerging pathogen Enterovirus D EV-D which primarily spreads through respiratory routes causing mostly mild to severe respiratory illness but in severe cases acute flaccid myelitis The C protease of EV-D is a potential target for the development of antiviral drugs due to its essential role in the viral life cycle and high sequence conservation This protocol was used to grow D C ProB crystals that were applied high-throughput crystallographic follow up compound screening on D C
The crystallization protocol and buffer conditions used to obtain reproducible SARS C V- Nucelocapsid crystals suitable for XChem fragment screening
The crystallization protocol and buffer conditions used to obtain Zika NS helicase crystals suitable for XChem fragment screening The Zika virus ZIKV discovered in Africa in swiftly spread across continents causing significant concern due to its recent association with microcephaly in newborns and Guillain-Barr syndrome in adults Despite a decrease in prevalence the potential for a resurgence remains necessitating urgent therapeutic interventions Like other flaviviruses ZIKV presents promising drug targets within its replication machinery notably the NS helicase NS Hel protein which plays critical roles in viral replication However a lack of structural information impedes the development of specific inhibitors ... More
The crystallization protocol and buffer conditions used to obtain Zika NS3 helicase crystals suitable for XChem fragment screening. The Zika virus (ZIKV), discovered in Africa in 1947, swiftly spread across continents, causing significant concern due to its recent association with microcephaly in newborns and Guillain-Barré syndrome in adults. Despite a decrease in prevalence, the potential for a resurgence remains, necessitating urgent therapeutic interventions. Like other flaviviruses, ZIKV presents promising drug targets within its replication machinery, notably the NS3 helicase (NS3Hel) protein, which plays critical roles in viral replication. However, a lack of structural information impedes the development of specific inhibitors targeting NS3Hel. This protocol was used to grow Zika NS3 crystals that were applied high-throughput crystallographic fragment screening on ZIKV NS3 Helicase. Less
Non-alcoholic fatty liver disease NAFLD - characterized by excess accumulation of fat in the liver - now affects one third of the world s population As NAFLD progresses extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis a major determinant of disease severity and mortality To identify transcriptional regulators of fibrosis we computationally inferred the activity of transcription factors TFs relevant to fibrosis by profiling the matched transcriptomes and epigenomes of human liver biopsies from a deeply-characterized cohort of patients spanning the full histopathologic spectrum of NAFLD CRISPR-based genetic knockout of the top TFs identified ZNF as ... More
Non-alcoholic fatty liver disease (NAFLD) - characterized by excess accumulation of fat in the liver - now affects one third of the world’s population. As NAFLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To identify transcriptional regulators of fibrosis, we computationally inferred the activity of transcription factors (TFs) relevant to fibrosis by profiling the matched transcriptomes and epigenomes of 108 human liver biopsies from a deeply-characterized cohort of patients spanning the full histopathologic spectrum of NAFLD. CRISPR-based genetic knockout of the top 100 TFs identified ZNF469 as a regulator of collagen expression in primary human hepatic stellate cells (HSCs). Gain- and loss-of-function studies established that ZNF469 regulates collagen genes and genes involved in matrix homeostasis through direct binding to gene bodies and regulatory elements. By integrating multiomic large-scale profiling of human biopsies with extensive experimental validation we demonstrate that ZNF469 is a transcriptional regulator of collagen in HSCs. Overall, these data nominate ZNF469 as a previously unrecognized determinant of NAFLD-associated liver fibrosis. Less
This research delves into the early nucleation stages of phycocyanin a protein pivotal for its fluorescent properties and crystalline stability and holding considerable potential for biotechnological applications The paper contrasts traditional crystallization methods with the innovative Langmuir Blodgett nanotemplate approach aiming to enhance molecular assembly and nucleation processes The study employs Langmuir Blodgett nanotemplates alongside second-order nonlinear imaging of chiral crystal SONICC spectroscopy This combination is designed to orderly organize phycocyanin molecules and provide a sensitive visualization of early-stage crystal formation capturing the intricate dynamics of protein crystallization The experiments were conducted under controlled conditions where surface pressure was maintained ... More
This research delves into the early nucleation stages of phycocyanin, a protein pivotal for its fluorescent properties and crystalline stability and holding considerable potential for biotechnological applications. The paper contrasts traditional crystallization methods with the innovative Langmuir–Blodgett nanotemplate approach, aiming to enhance molecular assembly and nucleation processes. The study employs Langmuir–Blodgett nanotemplates alongside second-order nonlinear imaging of chiral crystal (SONICC) spectroscopy. This combination is designed to orderly organize phycocyanin molecules and provide a sensitive visualization of early-stage crystal formation, capturing the intricate dynamics of protein crystallization. The experiments were conducted under controlled conditions, where surface pressure was maintained at 26 mN/m and barrier speed at 70 cm/min to optimize the monolayer formation at the air–water interface. The Langmuir–Blodgett method, compared to traditional vapor diffusion techniques, shows improvements in the uniformity and efficiency of nucleation. The sensitivity of SONICC spectroscopy significantly enhances the visualization of the nucleation process, revealing a more structured and uniform crystalline assembly in the early stages of formation. This method demonstrates a substantial improvement in nucleation dynamics, leading to a more orderly growth process and potentially larger, well-ordered crystals. Integrating Langmuir–Blodgett nanotemplates with SONICC spectroscopy offers a significant step in understanding protein crystallization processes with insights into the nucleation and growth of protein crystals and broad implications for refining crystallography methodologies of protein-based biomaterials, contributing to the advancement of structural biology and materials science. Less
Crimean Congo hemorrhagic fever virus CCHFV is a tick-borne virus that can cause severe disease in humans with case fatality rates of Although structures of CCHFV glycoproteins GP and Gc have provided insights into viral entry and defined epitopes of neutralizing and protective antibodies the structure of glycoprotein Gn and its interactions with GP and Gc have remained elusive Here we used structure-guided protein engineering to produce a stabilized GP -Gn-Gc heterotrimeric glycoprotein complex GP -GnH-DS-Gc A cryo-EM structure of this complex provides the molecular basis for GP s association on the viral surface reveals the structure of Gn and ... More
Crimean–Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that can cause severe disease in humans with case fatality rates of 10–40%. Although structures of CCHFV glycoproteins GP38 and Gc have provided insights into viral entry and defined epitopes of neutralizing and protective antibodies, the structure of glycoprotein Gn and its interactions with GP38 and Gc have remained elusive. Here, we used structure-guided protein engineering to produce a stabilized GP38-Gn-Gc heterotrimeric glycoprotein complex (GP38-GnH-DS-Gc). A cryo-EM structure of this complex provides the molecular basis for GP38’s association on the viral surface, reveals the structure of Gn, and demonstrates that GP38-Gn restrains the Gc fusion loops in the prefusion conformation, facilitated by an N-linked glycan attached to Gn. Immunization with GP38-GnH-DS-Gc conferred 40% protection against lethal IbAr10200 challenge in mice. These data define the architecture of a GP38-Gn-Gc protomer and provide a template for structure-guided vaccine antigen development. Less
Rhomboid intramembrane serine proteases have been implicated in several pathologies and emerge as attractive pharmacological target candidates The most potent and selective rhomboid inhibitors available to date are peptidyl -ketoamides but their selectivity for diverse rhomboid proteases and strategies to modulate it in relevant contexts are poorly understood This gap together with the lack of suitable in vitro models hinders ketoamide development for relevant eukaryotic rhomboid enzymes Here we explore the structure-activity relationship principles of rhomboid inhibiting ketoamides by medicinal chemistry and enzymatic in vitro and in-cell assays with recombinant rhomboid proteases GlpG human mitochondrial rhomboid PARL and human RHBDL ... More
Rhomboid intramembrane serine proteases have been implicated in several pathologies, and emerge as attractive pharmacological target candidates. The most potent and selective rhomboid inhibitors available to date are peptidyl α-ketoamides, but their selectivity for diverse rhomboid proteases and strategies to modulate it in relevant contexts are poorly understood. This gap, together with the lack of suitable in vitro models, hinders ketoamide development for relevant eukaryotic rhomboid enzymes. Here we explore the structure-activity relationship principles of rhomboid inhibiting ketoamides by medicinal chemistry and enzymatic in vitro and in-cell assays with recombinant rhomboid proteases GlpG, human mitochondrial rhomboid PARL and human RHBDL2. We use X-ray crystallography in lipid cubic phase to understand the binding mode of one of the best ketoamide inhibitors synthesised here containing branched terminal substituent bound to GlpG. In addition, to extend the interpretation of the co-crystal structure, we use quantum mechanical calculations and quantify the relative importance of interactions along the inhibitor molecule. These combined experimental analyses implicates that more extensive exploration of chemical space at the prime side is unexpectedly powerful for the selectivity of rhomboid inhibiting ketoamides. Together with variations in the peptide sequence at the non-prime side, or its non-peptidic alternatives, this strategy enables targeted tailoring of potent and selective ketoamides towards diverse rhomboid proteases including disease-relevant ones such as PARL and RHBDL2. Less
Methods of studying eukaryotic cell responses to a perturbation or of stratifying eukaryotic cells or cell lines into one or more subgroups are described The methods involve perturbing a library of cells or cell lines in the same manner and observing how the cells respond to the same perturbation The observation may be via a high throughput screening method for example cell painting and the perturbation may be for example exposure to a therapeutic agent The methods may be used for grouping cells or cell lines that respond similarly to a given therapeutic agent which may be useful for identifying ... More
Methods of studying eukaryotic cell responses to a perturbation, or of stratifying eukaryotic cells or cell lines into one or more subgroups are described. The methods involve perturbing a library of cells or cell lines in the same manner, and observing how the cells respond to the same perturbation. The observation may be via a high throughput screening method, for example, cell painting; and the perturbation may be, for example, exposure to a therapeutic agent.The methods may be used for grouping cells or cell lines that respond similarly to a given therapeutic agent, which may be useful for identifying patient groups and selecting appropriate treatments. Less
Vitamin B cobalamin or Cbl functions as a cofactor in two important enzymatic processes in human cells and life is not sustainable without it B is obtained from food and travels from the stomach through the intestine and into the bloodstream by three B -transporting proteins salivary haptocorrin HC gastric intrinsic factor IF and transcobalamin TC which all bind B with high affinity and require proteolytic degradation to liberate Cbl After intracellular delivery of dietary B Cbl in the aquo hydroxo-Cbl HOCbl form can coordinate various nucleophiles e g glutathione GSH giving rise to glutathionylcobalamin GSCbl a naturally-occurring form of ... More
Vitamin B12 (cobalamin or Cbl) functions as a cofactor in two important enzymatic processes in human cells, and life is not sustainable without it. B12 is obtained from food and travels from the stomach, through the intestine and into the bloodstream by three B12-transporting proteins: salivary haptocorrin (HC), gastric intrinsic factor (IF) and transcobalamin (TC), which all bind B12 with high affinity and require proteolytic degradation to liberate Cbl. After intracellular delivery of dietary B12, Cbl in the aquo/hydroxo-Cbl (HOCbl) form can coordinate various nucleophiles, e.g., glutathione (GSH), giving rise to glutathionylcobalamin (GSCbl), a naturally-occurring form of vitamin B12. Currently there is no data showing whether GSCbl is recognized and transported in the human body. Our crystallographic data shows for the first time the complex between a vitamin B12-transporter and GSCbl, which compared to HOCbl, binds TC equally well. Furthermore, sequence analysis and structural comparisons show that TC recognizes and transports GSCbl and that the residues involved are conserved among TCs from different organisms. Interestingly, HC and IF are not structurally tailored to bind GSCbl. This study provides new insights into the interactions between TC and Cbl. Less
The Swiss Light Source facilitates fragment-based drug-discovery campaigns for academic and industrial users through the Fast Fragment and Compound Screening FFCS software suite This framework is further enriched by the option to utilize the Smart Digital User SDU software for automated data collection across the PXI PXII and PXIII beamlines In this work the newly developed HEIDI webpage https heidi psi ch is introduced a platform crafted using state-of-the-art software architecture and web technologies for sample management of rotational data experiments The HEIDI webpage features a data-review tab for enhanced result visualization and provides programmatic access through a representational state ... More
The Swiss Light Source facilitates fragment-based drug-discovery campaigns for academic and industrial users through the Fast Fragment and Compound Screening (FFCS) software suite. This framework is further enriched by the option to utilize the Smart Digital User (SDU) software for automated data collection across the PXI, PXII and PXIII beamlines. In this work, the newly developed HEIDI webpage (https://heidi.psi.ch) is introduced: a platform crafted using state-of-the-art software architecture and web technologies for sample management of rotational data experiments. The HEIDI webpage features a data-review tab for enhanced result visualization and provides programmatic access through a representational state transfer application programming interface (REST API). The migration of the local FFCS MongoDB instance to the cloud is highlighted and detailed. This transition ensures secure, encrypted and consistently accessible data through a robust and reliable REST API tailored for the FFCS software suite. Collectively, these advancements not only significantly elevate the user experience, but also pave the way for future expansions and improvements in the capabilities of the system. Less
The mammalian Voltage-gated sodium Nav channel is composed of a single subunit kDa a multi-pass membrane protein that renders ion selectivity and two or more Nav subunits kDa that are Type I single-pass membrane proteins and regulate Nav subunit function These subunits are assembled on the plasma membrane of electrically-excitable cells as an intrinsic membrane protein complex and help to initiate and propagate the action potential The four major mammalian Nav -subunit isoforms Nav proteins possess an N-terminal extracellular Immunoglobulin Ig domain ECD a single transmembrane -helix and an intracellular C-terminal region ICD This thesis is mainly focused on the ... More
The mammalian Voltage-gated sodium (Nav) channel is composed of a single α subunit (~ 260
kDa), a multi-pass membrane protein that renders ion selectivity and two or more Navβ subunits
(25‒40 kDa), that are Type I single-pass membrane proteins and regulate Navα subunit function.
These subunits are assembled on the plasma membrane of electrically-excitable cells as an
intrinsic membrane protein complex and help to initiate and propagate the action potential. The
four major mammalian Navβ-subunit isoforms, Navβ1‒4 proteins possess an N-terminal
extracellular Immunoglobulin (Ig) domain (ECD), a single transmembrane α-helix, and an
intracellular C-terminal region (ICD).
This thesis is mainly focused on the structural biology aspects of the human Navβ3 subunit. It
reports the atomic structure of the Navβ3-Ig domain as determined by X-ray crystallography.
Interestingly, the Navβ3-Ig domain is observed as a trimer in the crystal structure. The homotrimer
assembly interface lies at the N-terminus and is constrained by a disulphide bond not
normally present in Ig domains. The Navβ3 subunit Ig domain is known to be glycosylated and
contains four potential N-linked glycosylation sites. However, the X-ray crystallography was
conducted on deglycosylated protein. Using computational modelling, it is shown that glycan
addition would not interfere with Navβ3-Ig domain trimerization. Independent evidence gathered
using Analytical Ultracentrifugation (crosslinked, glycosylated Navβ3-Ig domain, in vitro),
Proximity Ligation Assay (full-length Navβ3, in vivo), Atomic Force Microscopy (isolated fulllength
Navβ3, in vitro) and Photo-activated Localisation Microscopic experiments (full-length
Navβ3, in situ) support the view that the Navβ3 subunit can form trimers when expressed in cells.
The biological significance of Navβ3 subunit trimerization is discussed.
Strategies to express and purify the Navβ1/β2/β4-Ig domains were made. Wild type Navβ2- and
Navβ4-Ig domains exist as monomers and dimers, simultaneously in solution, although crystals
that diffracted to the necessary resolution were not produced. Less
kDa), a multi-pass membrane protein that renders ion selectivity and two or more Navβ subunits
(25‒40 kDa), that are Type I single-pass membrane proteins and regulate Navα subunit function.
These subunits are assembled on the plasma membrane of electrically-excitable cells as an
intrinsic membrane protein complex and help to initiate and propagate the action potential. The
four major mammalian Navβ-subunit isoforms, Navβ1‒4 proteins possess an N-terminal
extracellular Immunoglobulin (Ig) domain (ECD), a single transmembrane α-helix, and an
intracellular C-terminal region (ICD).
This thesis is mainly focused on the structural biology aspects of the human Navβ3 subunit. It
reports the atomic structure of the Navβ3-Ig domain as determined by X-ray crystallography.
Interestingly, the Navβ3-Ig domain is observed as a trimer in the crystal structure. The homotrimer
assembly interface lies at the N-terminus and is constrained by a disulphide bond not
normally present in Ig domains. The Navβ3 subunit Ig domain is known to be glycosylated and
contains four potential N-linked glycosylation sites. However, the X-ray crystallography was
conducted on deglycosylated protein. Using computational modelling, it is shown that glycan
addition would not interfere with Navβ3-Ig domain trimerization. Independent evidence gathered
using Analytical Ultracentrifugation (crosslinked, glycosylated Navβ3-Ig domain, in vitro),
Proximity Ligation Assay (full-length Navβ3, in vivo), Atomic Force Microscopy (isolated fulllength
Navβ3, in vitro) and Photo-activated Localisation Microscopic experiments (full-length
Navβ3, in situ) support the view that the Navβ3 subunit can form trimers when expressed in cells.
The biological significance of Navβ3 subunit trimerization is discussed.
Strategies to express and purify the Navβ1/β2/β4-Ig domains were made. Wild type Navβ2- and
Navβ4-Ig domains exist as monomers and dimers, simultaneously in solution, although crystals
that diffracted to the necessary resolution were not produced. Less
Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy This includes valorization of hemicellulosic fraction of plant biomass the second most abundant biopolymer from plant cell walls aiming to produce prebiotic oligosaccharides widely explored in food and feed industries In this work we conducted biochemical and biophysical characterization of a prokaryotic two-domain R champanellensis xylanase from glycoside hydrolase GH family RcXyn A and evaluated its applicability for XOS production from glucuronoxylan in combination with two endo-xylanases from GH and GH families and a GH xylobiohydrolase RcXyn A liberates ... More
Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy. This includes valorization of hemicellulosic fraction of plant biomass, the second most abundant biopolymer from plant cell walls, aiming to produce prebiotic oligosaccharides, widely explored in food and feed industries. In this work, we conducted biochemical and biophysical characterization of a prokaryotic two-domain R. champanellensis xylanase from glycoside hydrolase (GH) family 30 (RcXyn30A), and evaluated its applicability for XOS production from glucuronoxylan in combination with two endo-xylanases from GH10 and GH11 families and a GH11 xylobiohydrolase. RcXyn30A liberates mainly long monoglucuronylated xylooligosaccharides and is inefficient in cleaving unbranched oligosaccharides. Crystallographic structure of RcXyn30A catalytic domain was solved and refined to 1.37 Å resolution. Structural analysis of the catalytic domain releveled that its high affinity for glucuronic acid substituted xylan is due to the coordination of the substrate decoration by several hydrogen bonds and ionic interactions in the subsite −2. Furthermore, the protein has a larger β5-α5 loop as compared to other GH30 xylanases, which might be crucial for creating an additional aglycone subsite (+3) of the catalytic site. Finally, RcXyn30A activity is synergic to that of GH11 xylobiohydrolase. Less
With the rapid advancements in sequencing technologies the identification of single nucleotide mutations has surged surpassing our capacity for functional characterization Remarkably approximately of these disease-linked point mutations are situated within protein regions devoid of a well-defined D structure known as intrinsically disordered regions IDRs These IDRs are recognized for their pivotal roles in the regulation signaling and control of biological processes They can harbor short linear motifs SLiMs that act as mediators in protein-protein interactions PPIs often subject to regulation through post-translational modifications such as phosphorylation Investigating the impact of these IDR mutations on protein-protein interactions is essential for ... More
With the rapid advancements in sequencing technologies, the identification of
single nucleotide mutations has surged, surpassing our capacity for functional
characterization. Remarkably, approximately 20% of these disease-linked point
mutations are situated within protein regions devoid of a well-defined 3D structure,
known as intrinsically disordered regions (IDRs). These IDRs are recognized for
their pivotal roles in the regulation, signaling, and control of biological processes.
They can harbor short linear motifs (SLiMs) that act as mediators in protein-protein
interactions (PPIs), often subject to regulation through post-translational
modifications such as phosphorylation. Investigating the impact of these IDR
mutations on protein-protein interactions is essential for comprehending the
molecular mechanisms underlying human diseases.
In this doctoral thesis, I present a comprehensive exploration of a peptide-based
proteomics screen, employed to scrutinize 36 disease-associated mutations that
impair phosphorylation sites within IDRs. This approach entailed the immobilization
of synthetic peptides, corresponding to the mutated regions, onto a cellulose
membrane. These peptides were then utilized to capture interacting proteins from
cellular extracts. This method facilitated the simultaneous comparison of
interaction partners among wild-type, phosphorylated, and mutated peptide forms,
enabling the functional assessment of individual mutations. Our analysis
uncovered significant disparities
between the interactomes of phosphorylated and non-phosphorylated peptides,
changes frequently attributed to the disruption of phosphorylation-dependent
SLiMs.
Building on our findings, we placed particular emphasis on the S102P mutation
within the transcription factor GATAD1, a mutation associated with dilated
cardiomyopathy. Our screening demonstrated that this mutation disrupts a crucial
phosphorylation site responsible for 14-3-3 protein binding. To delve deeper into
this interaction, we conducted a thorough investigation, employing techniques such
as isothermal titration calorimetry, X-ray crystallography, and alanine scanning
coupled with mass spectrometry. Our meticulous analyses hinted at the regulatory
role of 14-3-3 binding in GATAD1's nucleocytoplasmic transport, achieved by
masking its nuclear localization signal.
In conclusion, this doctoral thesis focuses on the profound impact of pathogenic
mutations within human phosphorylation sites on protein-protein interactions. The
insights from our research shed fresh light on potential molecular mechanisms
underpinning the development of various human diseases, offering a promising
avenue for further investigation and therapeutic exploration. Less
single nucleotide mutations has surged, surpassing our capacity for functional
characterization. Remarkably, approximately 20% of these disease-linked point
mutations are situated within protein regions devoid of a well-defined 3D structure,
known as intrinsically disordered regions (IDRs). These IDRs are recognized for
their pivotal roles in the regulation, signaling, and control of biological processes.
They can harbor short linear motifs (SLiMs) that act as mediators in protein-protein
interactions (PPIs), often subject to regulation through post-translational
modifications such as phosphorylation. Investigating the impact of these IDR
mutations on protein-protein interactions is essential for comprehending the
molecular mechanisms underlying human diseases.
In this doctoral thesis, I present a comprehensive exploration of a peptide-based
proteomics screen, employed to scrutinize 36 disease-associated mutations that
impair phosphorylation sites within IDRs. This approach entailed the immobilization
of synthetic peptides, corresponding to the mutated regions, onto a cellulose
membrane. These peptides were then utilized to capture interacting proteins from
cellular extracts. This method facilitated the simultaneous comparison of
interaction partners among wild-type, phosphorylated, and mutated peptide forms,
enabling the functional assessment of individual mutations. Our analysis
uncovered significant disparities
between the interactomes of phosphorylated and non-phosphorylated peptides,
changes frequently attributed to the disruption of phosphorylation-dependent
SLiMs.
Building on our findings, we placed particular emphasis on the S102P mutation
within the transcription factor GATAD1, a mutation associated with dilated
cardiomyopathy. Our screening demonstrated that this mutation disrupts a crucial
phosphorylation site responsible for 14-3-3 protein binding. To delve deeper into
this interaction, we conducted a thorough investigation, employing techniques such
as isothermal titration calorimetry, X-ray crystallography, and alanine scanning
coupled with mass spectrometry. Our meticulous analyses hinted at the regulatory
role of 14-3-3 binding in GATAD1's nucleocytoplasmic transport, achieved by
masking its nuclear localization signal.
In conclusion, this doctoral thesis focuses on the profound impact of pathogenic
mutations within human phosphorylation sites on protein-protein interactions. The
insights from our research shed fresh light on potential molecular mechanisms
underpinning the development of various human diseases, offering a promising
avenue for further investigation and therapeutic exploration. Less
Despite their lack of a defined D structure intrinsically disordered regions IDRs of proteins play important biological roles Many IDRs contain short linear motifs SLiMs that mediate protein-protein interactions PPIs which can be regulated by post-translational modifications like phosphorylation of pathogenic missense mutations are found in IDRs and understanding how such mutations affect PPIs is essential for unraveling disease mechanisms Here we employ peptide-based interaction proteomics to investigate disease-associated mutations affecting phosphorylation sites Our results unveil significant differences in interactomes between phosphorylated and non-phosphorylated peptides often due to disrupted phosphorylation-dependent SLiMs We focused on a mutation of a serine phosphorylation ... More
Despite their lack of a defined 3D structure, intrinsically disordered regions (IDRs) of proteins play important biological roles. Many IDRs contain short linear motifs (SLiMs) that mediate protein-protein interactions (PPIs), which can be regulated by post-translational modifications like phosphorylation. 20% of pathogenic missense mutations are found in IDRs, and understanding how such mutations affect PPIs is essential for unraveling disease mechanisms. Here, we employ peptide-based interaction proteomics to investigate 36 disease-associated mutations affecting phosphorylation sites. Our results unveil significant differences in interactomes between phosphorylated and non-phosphorylated peptides, often due to disrupted phosphorylation-dependent SLiMs. We focused on a mutation of a serine phosphorylation site in the transcription factor GATAD1, which causes dilated cardiomyopathy. We find that this phosphorylation site mediates interaction with 14-3-3 family proteins. Follow-up experiments reveal the structural basis of this interaction and suggest that 14-3-3 binding affects GATAD1 nucleocytoplasmic transport by masking a nuclear localisation signal. Our results demonstrate that pathogenic mutations of human phosphorylation sites can significantly impact protein-protein interactions, offering insights into potential molecular mechanisms underlying pathogenesis. Less
Light-driven sodium pumps NaRs are unique ion-transporting microbial rhodopsins The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base We thoroughly characterize a member of this subgroup namely the protein ErNaR from Erythrobacter sp HL- and show that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity which is in contrast to previously studied NaRs ... More
Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterize a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and show that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics. Less
Nowadays an efficient and robust virtual screening procedure is crucial in the drug discovery process especially when performed on large and chemically diverse databases Virtual screening methods like molecular docking and classic QSAR models are limited in their ability to handle vast numbers of compounds and to learn from scarce data respectively In this study we introduce a universal methodology that uses a machine learning-based approach to predict docking scores without the need for time-consuming molecular docking procedures The developed protocol yielded times faster binding energy predictions than classical docking-based screening The proposed predictive model learns from docking results allowing ... More
Nowadays, an efficient and robust virtual screening procedure is crucial in the drug discovery process, especially when performed on large and chemically diverse databases. Virtual screening methods, like molecular docking and classic QSAR models, are limited in their ability to handle vast numbers of compounds and to learn from scarce data, respectively. In this study, we introduce a universal methodology that uses a machine learning-based approach to predict docking scores without the need for time-consuming molecular docking procedures. The developed protocol yielded 1000 times faster binding energy predictions than classical docking-based screening. The proposed predictive model learns from docking results, allowing users to choose their preferred docking software without relying on insufficient and incoherent experimental activity data. The methodology described employs multiple types of molecular fingerprints and descriptors to construct an ensemble model that further reduces prediction errors and is capable of delivering highly precise docking score values for monoamine oxidase ligands, enabling faster identification of promising compounds. An extensive pharmacophore-constrained screening of the ZINC database resulted in a selection of 24 compounds that were synthesized and evaluated for their biological activity. A preliminary screen discovered weak inhibitors of MAO-A with a percentage efficiency index close to a known drug at the lowest tested concentration. The approach presented here can be successfully applied to other biological targets as target-specific knowledge is not incorporated at the screening phase. Less
Spatial heterogeneity is ubiquitous across life and the universe the same is true for phase separating pharmaceutical formulations cells and tissues To interrogate these spatially-varying complicated samples simple analysis techniques such as fluorescence recovery after photobleaching FRAP can provide information on molecular transport Conventional FRAP approaches localize analysis to small spots which may not be representative of trends across the full field of view Taking advantage of strategies used for structures illumination an approach has been developed to use patterned illumination in combination with FRAP for probing large fields of view while representatively sampling Patterned illumination is used to establish ... More
Spatial heterogeneity is ubiquitous across life and the universe; the same is true for phase separating pharmaceutical formulations, cells, and tissues. To interrogate these spatially-varying complicated samples, simple analysis techniques such as fluorescence recovery after
photobleaching (FRAP) can provide information on molecular transport. Conventional FRAP
approaches localize analysis to small spots, which may not be representative of trends across the full field of view. Taking advantage of strategies used for structures illumination, an approach has been developed to use patterned illumination in combination with FRAP for probing large fields of view while representatively sampling. Patterned illumination is used to establish a concentration
gradient across a sample by irreversibly photobleaching fluorophores, such as with the simple comb pattern photobleach presented in Chapters 1 and 4. Patterned photobleaching allows spatial Fourier-domain analysis of multiple spatial harmonics simultaneously. In the spatial FT-domain the real-space photobleach signal is integrated into puncta, greatly increasing the signal to noise ratio compared to conventional point-bleach FRAP. The order of the spatial harmonic is directly related to the length scale of translational diffusion measured, with a series of harmonics accessing
diffusion over many length scales in a single experiment. Measurements of diffusion at multiple length scales informs on the diffusion mechanism by sensitively reporting on deviations away from normal diffusion. Complementing the physical hardware for inducing patterned illumination, this dissertation
introduces novel algorithms for reconstructing spatially-resolved diffusion maps in heterogeneous materials by combining Fourier domain analysis with patterned photobleaching. FT-FRAP is introduced in Chapter 1 for interrogating phase-separating samples using beam-scanning instrumentation for comb-bleach illumination. This analysis allowed disentangling separate contributions to diffusion from normal bulk diffusion and an interfacial exchange mechanism only available due to multi-harmonic analysis. The introduction of a dot-array bleach pattern using widefield microscopy is presented in Chapter 2 for high-throughput detection of mobility in simple binary systems as well as for segmentation in phase-separating pharmaceutical formulations. The analysis becomes more complicated as more components are added to the system such as a surfactant. Introduced in chapter 3, FT-FRAP with dot-array photobleaching was shown to be
useful for characterizing diffusion of phase-separating micro-domain smaller than a single pixel of the camera. Supported by simulations, a biexponential fitting model was developed for
quantification of diffusion by multiple species simultaneously. Chapter 4 introduces imaging
inside of 3D particles comprised of an active pharmaceutical ingredient (API) in
microencapsulated agglomerates which exhibited strong interfacial exchange. Multi-photon excited fluorescence enabled imaging a small focal volume within the particles. Less
photobleaching (FRAP) can provide information on molecular transport. Conventional FRAP
approaches localize analysis to small spots, which may not be representative of trends across the full field of view. Taking advantage of strategies used for structures illumination, an approach has been developed to use patterned illumination in combination with FRAP for probing large fields of view while representatively sampling. Patterned illumination is used to establish a concentration
gradient across a sample by irreversibly photobleaching fluorophores, such as with the simple comb pattern photobleach presented in Chapters 1 and 4. Patterned photobleaching allows spatial Fourier-domain analysis of multiple spatial harmonics simultaneously. In the spatial FT-domain the real-space photobleach signal is integrated into puncta, greatly increasing the signal to noise ratio compared to conventional point-bleach FRAP. The order of the spatial harmonic is directly related to the length scale of translational diffusion measured, with a series of harmonics accessing
diffusion over many length scales in a single experiment. Measurements of diffusion at multiple length scales informs on the diffusion mechanism by sensitively reporting on deviations away from normal diffusion. Complementing the physical hardware for inducing patterned illumination, this dissertation
introduces novel algorithms for reconstructing spatially-resolved diffusion maps in heterogeneous materials by combining Fourier domain analysis with patterned photobleaching. FT-FRAP is introduced in Chapter 1 for interrogating phase-separating samples using beam-scanning instrumentation for comb-bleach illumination. This analysis allowed disentangling separate contributions to diffusion from normal bulk diffusion and an interfacial exchange mechanism only available due to multi-harmonic analysis. The introduction of a dot-array bleach pattern using widefield microscopy is presented in Chapter 2 for high-throughput detection of mobility in simple binary systems as well as for segmentation in phase-separating pharmaceutical formulations. The analysis becomes more complicated as more components are added to the system such as a surfactant. Introduced in chapter 3, FT-FRAP with dot-array photobleaching was shown to be
useful for characterizing diffusion of phase-separating micro-domain smaller than a single pixel of the camera. Supported by simulations, a biexponential fitting model was developed for
quantification of diffusion by multiple species simultaneously. Chapter 4 introduces imaging
inside of 3D particles comprised of an active pharmaceutical ingredient (API) in
microencapsulated agglomerates which exhibited strong interfacial exchange. Multi-photon excited fluorescence enabled imaging a small focal volume within the particles. Less
Dysregulation of phosphorylation-dependent signaling is a hallmark of tumorigenesis Protein phosphatase PP A is an essential regulator of cell growth One scaffold subunit A binds to a catalytic subunit C to form a core AC heterodimer which together with one of many regulatory B subunits forms the active trimeric enzyme The combinatorial number of distinct PP A complexes is large which results in diverse substrate specificity and subcellular localization The detailed mechanism of PP A assembly and regulation remains elusive and reports about an important role of methylation of the carboxy terminus of PP A C are conflicting A better ... More
Dysregulation of phosphorylation-dependent signaling is a hallmark of tumorigenesis. Protein phosphatase 2 (PP2A) is an essential regulator of cell growth. One scaffold subunit (A) binds to a catalytic subunit (C) to form a core AC heterodimer, which together with one of many regulatory (B) subunits forms the active trimeric enzyme. The combinatorial number of distinct PP2A complexes is large, which results in diverse substrate specificity and subcellular localization. The detailed mechanism of PP2A assembly and regulation remains elusive and reports about an important role of methylation of the carboxy terminus of PP2A C are conflicting. A better understanding of the molecular underpinnings of PP2A assembly and regulation is critical to dissect PP2A function in physiology and disease. Here, we combined biochemical reconstitution, mass spectrometry, X-ray crystallography and functional assays to characterize the assembly of trimeric PP2A. In vitro studies demonstrated that methylation of the carboxy-terminus of PP2A C was dispensable for PP2A assembly in vitro. To corroborate these findings, we determined the X-ray crystal structure of the unmethylated PP2A Aα-B56ε-Cα trimer complex to 3.1 Å resolution. The experimental structure superimposed well with an Alphafold2Multimer prediction of the PP2A trimer. We then predicted models of all canonical PP2A complexes providing a framework for structural analysis of PP2A. In conclusion, methylation was dispensable for trimeric PP2A assembly and integrative structural biology studies of PP2A offered predictive models for all canonical PP2A complexes. Less
Sphingolipid activator protein B saposin B SapB is an essential activator of globotriaosylceramide Gb catabolism by -galactosidase A However the manner by which SapB stimulates -galactosidase A activity remains unknown To uncover the molecular mechanism of SapB presenting Gb to -galactosidase A we subjected the fluorescent substrate globotriaosylceramide-nitrobenzoxidazole Gb -NBD to a series of biochemical and structural assays involving SapB First we showed that SapB stably binds Gb -NBD using a fluorescence equilibrium binding assay isolates Gb -NBD from micelles and facilitates -galactosidase A cleavage of Gb -NBD in vitro Second we crystallized SapB in the presence of Gb -NBD ... More
Sphingolipid activator protein B (saposin B; SapB) is an essential activator of globotriaosylceramide (Gb3) catabolism by α-galactosidase A. However, the manner by which SapB stimulates α-galactosidase A activity remains unknown. To uncover the molecular mechanism of SapB presenting Gb3 to α-galactosidase A, we subjected the fluorescent substrate globotriaosylceramide-nitrobenzoxidazole (Gb3-NBD) to a series of biochemical and structural assays involving SapB. First, we showed that SapB stably binds Gb3-NBD using a fluorescence equilibrium binding assay, isolates Gb3-NBD from micelles, and facilitates α-galactosidase A cleavage of Gb3-NBD in vitro. Second, we crystallized SapB in the presence of Gb3-NBD and validated the ligand-bound assembly. Third, we captured transient interactions between SapB and α-galactosidase A by chemical cross-linking. Finally, we determined the crystal structure of SapB bound to α-galactosidase A. These findings establish general principles for molecular recognition in saposin:hydrolase complexes and highlight the utility of NBD reporter lipids in saposin biochemistry and structural biology. Less
Carbohydrate-active enzymes from the glycoside hydrolase family GH play a key role in processing lignocellulosic biomass Although the structural features of some GH enzymes are known the molecular mechanisms that drive their interactions with cellulosic substrates remain unclear To investigate the molecular mechanisms that the two-domain Bacillus licheniformis BlCel A enzyme utilizes to depolymerize cellulosic substrates we used a combination of biochemical assays X-ray crystallography small-angle X-ray scattering and molecular dynamics simulations The results reveal that BlCel A breaks down cellulosic substrates releasing cellobiose and glucose as the major products but is highly inefficient in cleaving oligosaccharides shorter than cellotetraose ... More
Carbohydrate-active enzymes from the glycoside hydrolase family 9 (GH9) play a key role in processing lignocellulosic biomass. Although the structural features of some GH9 enzymes are known, the molecular mechanisms that drive their interactions with cellulosic substrates remain unclear. To investigate the molecular mechanisms that the two-domain Bacillus licheniformis BlCel9A enzyme utilizes to depolymerize cellulosic substrates, we used a combination of biochemical assays, X-ray crystallography, small-angle X-ray scattering, and molecular dynamics simulations. The results reveal that BlCel9A breaks down cellulosic substrates, releasing cellobiose and glucose as the major products, but is highly inefficient in cleaving oligosaccharides shorter than cellotetraose. In addition, fungal lytic polysaccharide oxygenase (LPMO) TtLPMO9H enhances depolymerization of crystalline cellulose by BlCel9A, while exhibiting minimal impact on amorphous cellulose. The crystal structures of BlCel9A in both apo form and bound to cellotriose and cellohexaose were elucidated, unveiling the interactions of BlCel9A with the ligands and their contribution to substrate binding and products release. MD simulation analysis reveals that BlCel9A exhibits higher interdomain flexibility under acidic conditions, and SAXS experiments indicate that the enzyme flexibility is induced by pH and/or temperature. Our findings provide new insights into BlCel9A substrate specificity and binding, and synergy with the LPMOs. Less
The activities of the phospholipase C gamma PLC and enzymes are essential for numerous cellular processes Unsurprisingly dysregulation of PLC or PLC activity is associated with multiple maladies including immune disorders cancers and neurodegenerative diseases Therefore the modulation of either of these two enzymes has been suggested as a therapeutic strategy to combat these diseases To aid in the discovery of PLC family enzyme modulators that could be developed into therapeutic agents we have synthesized a high-throughput screening-amenable micellular fluorogenic substrate called C CF -coumarin Herein the ability of PLC and PLC to enzymatically process C CF -coumarin was confirmed ... More
The activities of the phospholipase C gamma (PLCγ) 1 and 2 enzymes are essential for numerous cellular processes. Unsurprisingly, dysregulation of PLCγ1 or PLCγ2 activity is associated with multiple maladies including immune disorders, cancers, and neurodegenerative diseases. Therefore, the modulation of either of these two enzymes has been suggested as a therapeutic strategy to combat these diseases. To aid in the discovery of PLCγ family enzyme modulators that could be developed into therapeutic agents, we have synthesized a high-throughput screening-amenable micellular fluorogenic substrate called C16CF3-coumarin. Herein, the ability of PLCγ1 and PLCγ2 to enzymatically process C16CF3-coumarin was confirmed, the micellular assay conditions were optimized, and the kinetics of the reaction were determined. A proof-of-principle pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) was performed. This new substrate allows for an additional screening methodology to identify modulators of the PLCγ family of enzymes. Less
Genetic studies have identified thousands of individual disease-associated non-coding alleles but identification of the causal alleles and their functions remain critical bottlenecks Even though CRISPR-Cas editing has enabled targeted modification of DNA inefficient editing leads to heterogeneous outcomes across individual cells limiting the ability to detect functional consequences of disease alleles To overcome these challenges we present a multi-omic single cell sequencing approach that directly identifies genomic DNA edits assays the transcriptome and measures cell surface protein expression We apply this approach to investigate the effects of gene disruption deletions in regulatory regions and non-coding single nucleotide polymorphisms We identify ... More
Genetic studies have identified thousands of individual disease-associated non-coding alleles, but identification of the causal alleles and their functions remain critical bottlenecks. Even though CRISPR-Cas editing has enabled targeted modification of DNA, inefficient editing leads to heterogeneous outcomes across individual cells, limiting the ability to detect functional consequences of disease alleles. To overcome these challenges, we present a multi-omic single cell sequencing approach that directly identifies genomic DNA edits, assays the transcriptome, and measures cell surface protein expression. We apply this approach to investigate the effects of gene disruption, deletions in regulatory regions, and non-coding single nucleotide polymorphisms. We identify the specific effects of individual SNPs, including the state-specific effects of an IL2RA autoimmune variant in primary human T cells. Multimodal functional genomic single cell assays including DNA sequencing bridge a crucial gap in our understanding of complex human diseases by directly identifying causal variation in primary human cells. Less
Corticospinal neurons CSNs synapse directly on spinal neurons a diverse assortment of cells with unique structural and functional properties necessary for body movements CSNs modulating forelimb behavior fractionate into caudal forelimb area CFA and rostral forelimb area RFA motor cortical populations Despite their prominence the full diversity of spinal neurons targeted by CFA and RFA CSNs is uncharted Here we use anatomical and RNA sequencing methods to show that CSNs synapse onto a remarkably selective group of spinal cell types favoring inhibitory populations that regulate motoneuron activity and gate sensory feedback CFA and RFA CSNs target similar spinal neuron types ... More
Corticospinal neurons (CSNs) synapse directly on spinal neurons, a diverse assortment of cells with unique structural and functional properties necessary for body movements. CSNs modulating forelimb behavior fractionate into caudal forelimb area (CFA) and rostral forelimb area (RFA) motor cortical populations. Despite their prominence, the full diversity of spinal neurons targeted by CFA and RFA CSNs is uncharted. Here, we use anatomical and RNA sequencing methods to show that CSNs synapse onto a remarkably selective group of spinal cell types, favoring inhibitory populations that regulate motoneuron activity and gate sensory feedback. CFA and RFA CSNs target similar spinal neuron types, with notable exceptions that suggest that these populations differ in how they influence behavior. Finally, axon collaterals of CFA and RFA CSNs target similar brain regions yet receive highly divergent inputs. These results detail the rules of CSN connectivity throughout the brain and spinal cord for two regions critical for forelimb behavior. Less
Evolution leads to conservation of amino acid residues in protein families Conserved proline residues are usually considered to ensure the correct folding and to stabilize the three-dimensional structure Surprisingly proline residues that are highly conserved in class A -lactamases were found to tolerate various substitutions without large losses in enzyme activity We investigated the roles of three conserved prolines at positions and in the -lactamase BlaC from Mycobacterium tuberculosis and found that mutations can lead to dimerization of the enzyme and an overall less stable protein that is prone to aggregate over time For the variant Pro Thr the crystal ... More
Evolution leads to conservation of amino acid residues in protein families. Conserved proline residues are usually considered to ensure the correct folding and to stabilize the three-dimensional structure. Surprisingly, proline residues that are highly conserved in class A β-lactamases were found to tolerate various substitutions without large losses in enzyme activity. We investigated the roles of three conserved prolines at positions 107, 226, and 258 in the β-lactamase BlaC from Mycobacterium tuberculosis and found that mutations can lead to dimerization of the enzyme and an overall less stable protein that is prone to aggregate over time. For the variant Pro107Thr, the crystal structure shows dimer formation resembling domain swapping. It is concluded that the proline substitutions loosen the structure, enhancing multimerization. Even though the enzyme does not lose its properties without the conserved proline residues, the prolines ensure the long-term structural integrity of the enzyme. Less
A novel monoacylglycerol MAG has been produced for use in the in meso lipid cubic phase crystallization of membrane proteins and complexes MAG differs from monoolein the most extensively used lipid for in meso crystallization in that it is shorter in chain length by one methylene and its cis olefinic bond is two carbons closer to the glycerol headgroup These changes in structure alter the phase behavior of the hydrated lipid and the microstructure of the corresponding mesophases formed Temperature composition phase diagrams for MAG have been constructed using small- and wide-angle X-ray scattering over a range of temperatures and ... More
A novel monoacylglycerol, 7.10 MAG, has been produced for use in the in meso (lipid cubic phase) crystallization of membrane proteins and complexes. 7.10 MAG differs from monoolein, the most extensively used lipid for in meso crystallization, in that it is shorter in chain length by one methylene and its cis olefinic bond is two carbons closer to the glycerol headgroup. These changes in structure alter the phase behavior of the hydrated lipid and the microstructure of the corresponding mesophases formed. Temperature–composition phase diagrams for 7.10 MAG have been constructed using small- and wide-angle X-ray scattering over a range of temperatures and hydration levels that span those used for crystallization. The phase diagrams include lamellar crystalline, fluid isotropic, lamellar liquid-crystalline, cubic-Ia3d, and cubic-Pn3m phases, as observed with monoolein. Conspicuous by its absence is the inverted hexagonal phase which is rationalized on the basis of 7.10 MAG’s chemical constitution. The cubic phase prepared with the new lipid facilitates the growth of crystals that were used to generate high-resolution structures of intramembrane β-barrel and α-helical proteins. Compatibility of fully hydrated 7.10 MAG with cholesterol and phosphatidylcholine means that these two lipids can be used as additives to optimize crystallogenesis in screening trials with 7.10 MAG as the host lipid. Less
Proteins usually execute their biological functions through interactions with other proteins and by forming macromolecular complexes but global profiling of protein complexes directly from human tissue samples has been limited In this study we utilized co-fractionation mass spectrometry CF-MS to map protein complexes within the post-mortem human brain with experimental replicates First we used concatenated anion and cation Ion Exchange Chromatography IEX to separate native protein complexes in fractions then proceeded with Data-Independent Acquisition DIA mass spectrometry to analyze the proteins in each fraction quantifying a total of proteins with overlapping in both replicates We improved DIA quantitative accuracy by ... More
Proteins usually execute their biological functions through interactions with other proteins and by forming macromolecular complexes, but global profiling of protein complexes directly from human tissue samples has been limited. In this study, we utilized co-fractionation mass spectrometry (CF-MS) to map protein complexes within the post-mortem human brain with experimental replicates. First, we used concatenated anion and cation Ion Exchange Chromatography (IEX) to separate native protein complexes in 192 fractions, then proceeded with Data-Independent Acquisition (DIA) mass spectrometry to analyze the proteins in each fraction, quantifying a total of 4,804 proteins with 3,260 overlapping in both replicates. We improved DIA quantitative accuracy by implementing a constant amount of bovine serum albumin (BSA) in each fraction as an internal standard. Next, advanced computational pipelines, which integrate both a database-based complex analysis and an unbiased protein-protein interaction (PPI) search, were applied to identify protein complexes and construct protein-protein interaction networks in the human brain. Our study led to the identification of 486 protein complexes and 10,054 binary protein-protein interactions, which represents the first global profiling of human brain PPIs using CF-MS. Overall, this study offers a resource and tool for a wide range of human brain research, including the identification of disease-specific protein complexes in the future. Less
The mammalian orthoreovirus reovirus NS protein is required for formation of replication compartments that support viral genome replication and capsid assembly Despite its functional importance a mechanistic understanding of NS is lacking We conducted structural and biochemical analyses of a NS mutant that forms dimers instead of the higher-order oligomers formed by wildtype WT NS The crystal structure shows that dimers interact with each other using N-terminal arms to form a helical assembly resembling WT NS filaments in complex with RNA observed using cryo-EM The interior of the helical assembly is of appropriate diameter to bind RNA The helical assembly ... More
The mammalian orthoreovirus (reovirus) σNS protein is required for formation of replication compartments that support viral genome replication and capsid assembly. Despite its functional importance, a mechanistic understanding of σNS is lacking. We conducted structural and biochemical analyses of a σNS mutant that forms dimers instead of the higher-order oligomers formed by wildtype (WT) σNS. The crystal structure shows that dimers interact with each other using N-terminal arms to form a helical assembly resembling WT σNS filaments in complex with RNA observed using cryo-EM. The interior of the helical assembly is of appropriate diameter to bind RNA. The helical assembly is disrupted by bile acids, which bind to the same site as the N-terminal arm. This finding suggests that the N-terminal arm functions in conferring context-dependent oligomeric states of σNS, which is supported by the structure of σNS lacking an N-terminal arm. We further observed that σNS has RNA chaperone activity likely essential for presenting mRNA to the viral polymerase for genome replication. This activity is reduced by bile acids and abolished by N-terminal arm deletion, suggesting that the activity requires formation of σNS oligomers. Our studies provide structural and mechanistic insights into the function of σNS in reovirus replication. Less
Cellulosomes are intricate cellulose-degrading multi-enzymatic complexes produced by anaerobic bacteria which are valuable for bioenergy development and biotechnology Cellulosome assembly relies on the selective interaction between cohesin modules in structural scaffolding proteins scaffoldins and dockerin modules in enzymes Although the number of tandem cohesins in the scaffoldins is believed to determine the complexity of the cellulosomes tandem dockerins also exist albeit very rare in some cellulosomal components whose assembly and functional roles are currently unclear In this study we characterized the structure and mode of assembly of a tandem bimodular double-dockerin which is connected to a putative S protease in ... More
Cellulosomes are intricate cellulose-degrading multi-enzymatic complexes produced by anaerobic bacteria, which are valuable for bioenergy development and biotechnology. Cellulosome assembly relies on the selective interaction between cohesin modules in structural scaffolding proteins (scaffoldins) and dockerin modules in enzymes. Although the number of tandem cohesins in the scaffoldins is believed to determine the complexity of the cellulosomes, tandem dockerins also exist, albeit very rare, in some cellulosomal components whose assembly and functional roles are currently unclear. In this study, we characterized the structure and mode of assembly of a tandem bimodular double-dockerin, which is connected to a putative S8 protease in the cellulosome-producing bacterium, Clostridium thermocellum. Crystal and NMR structures of the double-dockerin revealed two typical type I dockerin folds with significant interactions between them. Interaction analysis by isothermal titration calorimetry and NMR titration experiments revealed that the double-dockerin displays a preference for binding to the cell-wall anchoring scaffoldin ScaD through the first dockerin with a canonical dual-binding mode, while the second dockerin module was unable to bind to any of the tested cohesins. Surprisingly, the double-dockerin showed a much higher affinity to a cohesin from the CipC scaffoldin of Clostridium cellulolyticum than to the resident cohesins from C. thermocellum. These results contribute valuable insights into the structure and assembly of the double-dockerin module, and provide the basis for further functional studies on multiple-dockerin modules and cellulosomal proteases, thus highlighting the complexity and diversity of cellulosomal components. Less
Protease inhibitor drug discovery is challenged by the lack of cellular and oral permeability selectivity metabolic stability and rapid clearance of peptides Here we describe the rational design synthesis and evaluation of peptidomimetic side-chain-cyclized macrocycles which we converted into covalent serine protease inhibitors with the addition of an electrophilic ketone warhead We have identified potent and selective inhibitors of TMPRSS matriptase hepsin and HGFA and demonstrated their improved protease selectivity metabolic stability and pharmacokinetic PK properties We obtained an X-ray crystal structure of phenyl ether-cyclized tripeptide VD b bound to matriptase revealing an unexpected binding conformation Cyclic biphenyl ether VD ... More
Protease inhibitor drug discovery is challenged by the lack of cellular and oral permeability, selectivity, metabolic stability, and rapid clearance of peptides. Here, we describe the rational design, synthesis, and evaluation of peptidomimetic side-chain-cyclized macrocycles which we converted into covalent serine protease inhibitors with the addition of an electrophilic ketone warhead. We have identified potent and selective inhibitors of TMPRSS2, matriptase, hepsin, and HGFA and demonstrated their improved protease selectivity, metabolic stability, and pharmacokinetic (PK) properties. We obtained an X-ray crystal structure of phenyl ether-cyclized tripeptide VD4162 (8b) bound to matriptase, revealing an unexpected binding conformation. Cyclic biphenyl ether VD5123 (11) displayed the best PK properties in mice with a half-life of 4.5 h and compound exposure beyond 24 h. These new cyclic tripeptide scaffolds can be used as easily modifiable templates providing a new strategy to overcoming the obstacles presented by linear acyclic peptides in protease inhibitor drug discovery. Less
X-ray crystallography is the most commonly employed technique to discern macromolecular structures but the crucial step of crystallizing a protein into an ordered lattice amenable to diffraction remains challenging The crystallization of biomolecules is largely experimentally defined and this process can be labor-intensive and prohibitive to researchers at resource-limited institutions At the National High-Throughput Crystallization HTX Center highly reproducible methods have been implemented to facilitate crystal growth including an automated high-throughput -well microbatch-under-oil plate setup designed to sample a wide breadth of crystallization parameters Plates are monitored using state-of-the-art imaging modalities over the course of weeks to provide insight into ... More
X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into an ordered lattice amenable to diffraction remains challenging. The crystallization of biomolecules is largely experimentally defined, and this process can be labor-intensive and prohibitive to researchers at resource-limited institutions. At the National High-Throughput Crystallization (HTX) Center, highly reproducible methods have been implemented to facilitate crystal growth, including an automated high-throughput 1,536-well microbatch-under-oil plate setup designed to sample a wide breadth of crystallization parameters. Plates are monitored using state-of-the-art imaging modalities over the course of 6 weeks to provide insight into crystal growth, as well as to accurately distinguish valuable crystal hits. Furthermore, the implementation of a trained artificial intelligence scoring algorithm for identifying crystal hits, coupled with an open-source, user-friendly interface for viewing experimental images, streamlines the process of analyzing crystal growth images. Here, the key procedures and instrumentation are described for the preparation of the cocktails and crystallization plates, imaging the plates, and identifying hits in a way that ensures reproducibility and increases the likelihood of successful crystallization. Less
Protocols for robotic protein crystallization using the Crystallization Facility at Harwell and in situ room temperature data collection from crystallization plates at Diamond Light Source beamline VMXi are described This approach enables high-quality room-temperature crystal structures to be determined from multiple crystals in a straightforward manner and provides very rapid feedback on the results of crystallization trials as well as enabling serial crystallography The value of room temperature structures in understanding protein structure ligand binding and dynamics is becoming increasingly recognized in the structural biology community This pipeline is accessible to users from all over the world with several available ... More
Protocols for robotic protein crystallization using the Crystallization Facility at Harwell and in situ room temperature data collection from crystallization plates at Diamond Light Source beamline VMXi are described. This approach enables high-quality room-temperature crystal structures to be determined from multiple crystals in a straightforward manner and provides very rapid feedback on the results of crystallization trials as well as enabling serial crystallography. The value of room temperature structures in understanding protein structure, ligand binding, and dynamics is becoming increasingly recognized in the structural biology community. This pipeline is accessible to users from all over the world with several available modes of access. Crystallization experiments that are set up can be imaged and viewed remotely with crystals identified automatically using a machine learning tool. Data are measured in a queue-based system with up to 60° rotation datasets from user-selected crystals in a plate. Data from all the crystals within a particular well or sample group are automatically merged using xia2.multiplex with the outputs straightforwardly accessed via a web browser interface. Less
The CD IGFBP axis proteins are key factors expressed in endothelial cells EC that mediate EC angiogenesis and migration Their upregulation contributes to tumor vascular abnormality and a blockade of this interaction promotes a favorable tumor microenvironment for therapeutic interventions However the interactions of these proteins with each other remain unclear In this study we determined a partial structure of the human CD IGFBP complex comprising the EGF domain of CD and the IB domain of IGFBP Mutagenesis studies confirmed interactions and specificities Cellular and mouse tumor studies demonstrated the physiological relevance of the CD IGFBP interaction in EC angiogenesis ... More
The CD93/IGFBP7 axis proteins are key factors expressed in endothelial cells (EC) that mediate EC angiogenesis and migration. Their upregulation contributes to tumor vascular abnormality and a blockade of this interaction promotes a favorable tumor microenvironment for therapeutic interventions. However, the interactions of these proteins with each other remain unclear. In this study, we determined a partial structure of the human CD93–IGFBP7 complex comprising the EGF1 domain of CD93 and the IB domain of IGFBP7. Mutagenesis studies confirmed interactions and specificities. Cellular and mouse tumor studies demonstrated the physiological relevance of the CD93–IGFBP7 interaction in EC angiogenesis. Our study provides leads for the development of therapeutic agents to precisely disrupt unwanted CD93–IGFBP7 signaling in the tumor microenvironment. Additionally, analysis of the CD93 full-length architecture provides insights into how CD93 protrudes on the cell surface and forms a flexible platform for binding to IGFBP7 and other ligands. Less
Cancer cells often exhibit DNA copy number aberrations and can vary widely in their ploidy Correct estimation of the ploidy of single-cell genomes is paramount for downstream analysis Based only on single-cell DNA sequencing information scAbsolute achieves accurate and unbiased measurement of single-cell ploidy and replication status including whole-genome duplications We demonstrate scAbsolute s capabilities using experimental cell multiplets a FUCCI cell cycle expression system and a benchmark against state-of-the-art methods scAbsolute provides a robust foundation for single-cell DNA sequencing analysis across different technologies and has the potential to enable improvements in a number of downstream analyses
Introduction Type diabetes mellitus T DM is a prevalent form of diabetes that affects - of all diabetic patients Insulin sensitizers and insulin exogenous supply could temporarily ameliorate hyperglycaemia however they are accompanied by side effects As a result new approaches are required to address insulin resistance and regenerate beta cells simultaneously The secretome of hypoxic mesenchymal stem cells SH-MSCs contains various growth factors and anti-inflammatory cytokines that could potentially enhance insulin resistance and improve pancreatic function Objectives In this study we performed SH-MSCs infusion to ameliorate HFD-induced hyperglycaemia in T DM rats Methods We created a T DM rat ... More
Introduction: Type 2 diabetes mellitus (T2DM) is a prevalent form of diabetes that affects 90 - 95 % of all diabetic patients. Insulin sensitizers and insulin exogenous supply could temporarily ameliorate hyperglycaemia; however, they are accompanied by side effects. As a result, new approaches are required to address insulin resistance and regenerate beta cells simultaneously. The secretome of hypoxic mesenchymal stem cells (SH-MSCs) contains various growth factors and anti-inflammatory cytokines that could potentially enhance insulin resistance and improve pancreatic function. Objectives: In this study, we performed SH-MSCs infusion to ameliorate HFD-induced hyperglycaemia in T2DM rats. Methods: We created a T2DM rat model using a combination of a high-fat diet (HFD) and streptozotocin (STZ) administration. Then, we administered SH-MSCs injection at doses of 250 and 500 µL and assessed the therapeutic effects of SH-MSCs. We also investigated the potential underlying mechanisms involved. Results: The administration of SH-MSCs improved hyperglycemia in rats with T2DM. Infusion of SH-MSCs at 500 µL dose decreased homeostatic model assessment for insulin resistance (HOMA-IR). Histological analysis revealed that injection of SH-MSCs alleviated morphological damage of pancreas. SH-MSCs administration also inhibit the level of IL-6 and promote the expression of CD163 type 2 macrophage. Conclusion: The results of our study indicate that SH-MSCs have the potential to improve hyperglycemia and exert a protective effect on T2DM rats. Less
Pacak-Zhuang syndrome is caused by mutations in the EPAS gene which encodes for one of the three hypoxia-inducible factor alpha HIF paralogs HIF and is associated with defined but varied phenotypic presentations including neuroendocrine tumors and polycythemia However the mechanisms underlying the complex genotype-phenotype correlations remain incompletely understood Here we devised a quantitative method for determining the dissociation constant Kd of the HIF peptides containing disease-associated mutations and the catalytic domain of prolyl-hydroxylase PHD using microscale thermophoresis MST and showed that neuroendocrine-associated Class HIF mutants have distinctly higher Kd than the exclusively polycythemia-associated Class HIF mutants Based on the co-crystal ... More
Pacak-Zhuang syndrome is caused by mutations in the EPAS1 gene, which encodes for one of the three hypoxia-inducible factor alpha (HIFα) paralogs HIF2α and is associated with defined but varied phenotypic presentations including neuroendocrine tumors and polycythemia. However, the mechanisms underlying the complex genotype-phenotype correlations remain incompletely understood. Here, we devised a quantitative method for determining the dissociation constant (Kd) of the HIF2α peptides containing disease-associated mutations and the catalytic domain of prolyl-hydroxylase (PHD2) using microscale thermophoresis (MST) and showed that neuroendocrine-associated Class 1 HIF2α mutants have distinctly higher Kd than the exclusively polycythemia-associated Class 2 HIF2α mutants. Based on the co-crystal structure of PHD2/HIF2α peptide complex at 1.8 Å resolution, we showed that the Class 1 mutated residues are localized to the critical interface between HIF2α and PHD2, adjacent to the PHD2 active catalytic site, while Class 2 mutated residues are localized to the more flexible region of HIF2α that makes less contact with PHD2. Concordantly, Class 1 mutations were found to significantly increase HIF2α-mediated transcriptional activation in cellulo compared to Class 2 counterparts. These results reveal a structural mechanism in which the strength of the interaction between HIF2α and PHD2 is at the root of the general genotype-phenotype correlations observed in Pacak-Zhuang syndrome. Less
Menstrual toxic shock syndrome mTSS is a rare but severe disorder associated with the use of menstrual products such as high-absorbency tampons and is caused by Staphylococcus aureus strains that produce the toxic shock syndrome toxin- TSST- superantigen Herein we screened a library of small bioactive molecules for the ability to inhibit transcription of the TSST- gene without inhibiting growth of S aureus The dominant positive regulator of TSST- is the SaeRS two-component system TCS and we identified phenazopyridine hydrochloride PP-HCl that repressed production of TSST- by inhibiting the kinase function of SaeS PP-HCl competed with ATP for binding of ... More
Menstrual toxic shock syndrome (mTSS) is a rare but severe disorder associated with the use of menstrual products such as high-absorbency tampons and is caused by Staphylococcus aureus strains that produce the toxic shock syndrome toxin-1 (TSST-1) superantigen. Herein, we screened a library of 3920 small bioactive molecules for the ability to inhibit transcription of the TSST-1 gene without inhibiting growth of S. aureus. The dominant positive regulator of TSST-1 is the SaeRS two-component system (TCS), and we identified phenazopyridine hydrochloride (PP-HCl) that repressed production of TSST-1 by inhibiting the kinase function of SaeS. PP-HCl competed with ATP for binding of the kinase SaeS leading to decreased phosphorylation of SaeR and reduced expression of TSST-1 as well as several other secreted virulence factors known to be regulated by SaeRS. PP-HCl targets virulence of S. aureus, but it also decreases the impact of TSST-1 on human lymphocytes without affecting the healthy vaginal microbiota. Our findings demonstrate the promising potential of PP-HCl as a therapeutic strategy against mTSS. Less