Human IFIT3 Modulates IFIT1 RNA Binding Specificity and Protein Stability.

Although interferon-induced proteins with tetratricopeptide repeats (IFIT proteins) inhibit infection of many viruses by recognizing their RNA, the regulatory mechanisms involved remain unclear. Here we report a crystal structure of cap 0 (m7GpppN) RNA bound to human IFIT1 in complex with the C-terminal domain of human IFIT3. Structural, biochemical, and genetic studies suggest that IFIT3 binding to IFIT1 has dual regulatory functions: (1) extending the half-life of IFIT1 and thereby increasing its steady-state amounts in cells; and (2) allosterically regulating the IFIT1 RNA-binding channel, thereby enhancing the specificity of recognition for cap 0 but not cap 1 (m7GpppNm) or 5'-ppp RNA. Mouse Ifit3 lacks this key C-terminal domain and does not bind mouse Ifit1. The IFIT3 interaction with IFIT1 is important for restricting infection of viruses lacking 2'-O methylation in their RNA cap structures. Our experiments establish differences in the regulation of IFIT1 orthologs and define targets for modulation of human IFIT protein activity.

Calcium Binding to the Innate Immune Protein Human Calprotectin Revealed by Integrated Mass Spectrometry.

Although knowledge of the coordination chemistry and metal-withholding function of the innate immune protein human calprotectin (hCP) has broadened in recent years, understanding of its Ca2+-binding properties in solution remains incomplete. In particular, the molecular basis by which Ca2+ binding affects structure and enhances the functional properties of this remarkable transition-metal-sequestering protein has remained enigmatic. To achieve a molecular picture of how Ca2+ binding triggers hCP oligomerization, increases protease stability, and enhances antimicrobial activity, we implemented a new integrated mass spectrometry (MS)-based approach that can be readily generalized to study other protein-metal and protein-ligand interactions. Three MS-based methods (hydrogen/deuterium exchange MS kinetics; protein-ligand interactions in solution by MS, titration, and H/D exchange (PLIMSTEX); and native MS) provided a comprehensive analysis of Ca2+ binding and oligomerization to hCP without modifying the protein in any way. Integration of these methods allowed us to (i) observe the four regions of hCP that serve as Ca2+-binding sites, (ii) determine the binding stoichiometry to be four Ca2+ per CP heterodimer and eight Ca2+ per CP heterotetramer, (iii) establish the protein-to-Ca2+ molar ratio that causes the dimer-to-tetramer transition, and (iv) calculate the binding affinities associated with the four Ca2+-binding sites per heterodimer. These quantitative results support a model in which hCP exists in its heterodimeric form and is at most half-bound to Ca2+ in the cytoplasm of resting cells. With release into the extracellular space, hCP encounters elevated Ca2+ concentrations and binds more Ca2+ ions, forming a heterotetramer that is poised to compete with microbial pathogens for essential metal nutrients.

Moderately Neutralizing Epitopes in Nonfunctional Regions Dominate the Antibody Response to Plasmodium falciparum EBA-140.

Plasmodium falciparum erythrocyte-binding antigen 140 (EBA-140) plays a role in tight junction formation during parasite invasion of red blood cells and is a potential vaccine candidate for malaria. Individuals in areas where malaria is endemic possess EBA-140-specific antibodies, and individuals with high antibody titers to this protein have a lower rate of reinfection by parasites. The red blood cell binding segment of EBA-140 is comprised of two Duffy-binding-like domains, called F1 and F2, that together create region II. The sialic acid-binding pocket of F1 is essential for binding, whereas the sialic acid-binding pocket in F2 appears dispensable. Here, we show that immunization of mice with the complete region II results in poorly neutralizing antibodies. In contrast, immunization of mice with the functionally relevant F1 domain of region II results in antibodies that confer a 2-fold increase in parasite neutralization compared to that of the F2 domain. Epitope mapping of diverse F1 and F2 monoclonal antibodies revealed that the functionally relevant F1 sialic acid-binding pocket is a privileged site inaccessible to antibodies, that the F2 sialic acid-binding pocket contains a nonneutralizing epitope, and that two additional epitopes reside in F1 on the opposite face from the sialic acid-binding pocket. These studies indicate that focusing the immune response to the functionally important F1 sialic acid binding pocket improves the protective immune response of EBA-140. These results have implications for improving future vaccine designs and emphasize the importance of structural vaccinology for malaria.

An Integrated Approach for Determining a Protein-Protein Binding Interface in Solution and an Evaluation of Hydrogen-Deuterium Exchange Kinetics for Adjudicating Candidate Docking Models.

We describe an integrated approach of using hydrogen-deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking mass spectrometry (XL-MS), and molecular docking to characterize the binding interface and to predict the three-dimensional quaternary structure of a protein-protein complex in solution. Interleukin 7 (IL-7) and its α-receptor, IL-7Rα, serving as essential mediators in the immune system, are the model system. HDX kinetics reports widespread protection on IL-7Rα but shows no differential evidence of binding-induced protection or remote conformational change. Cross-linking with reagents that differ in spacer lengths and targeting residues increases the spatial resolution. Using five cross-links as distance restraints for protein-protein docking, we generated a high-confidence model of the IL-7/IL-7Rα complex. Both the predicted binding interface and regions with direct contacts agree well with those in the solid-state structure, as confirmed by previous X-ray crystallography. An additional binding region was revealed to be the C-terminus of helix B of IL-7, highlighting the value of solution-based characterization. To generalize the integrated approach, protein-protein docking was executed with a different number of cross-links. Combining cluster analysis and HDX kinetics adjudication, we found that two intermolecular cross-link-derived restraints are sufficient to generate a high-confidence model with root-mean-square distance (rmsd) value of all alpha carbons below 2.0 Å relative to the crystal structure. The remarkable results of binding-interface determination and quaternary structure prediction highlight the effectiveness and capability of the integrated approach, which will allow more efficient and comprehensive analysis of interprotein interactions with broad applications in the multiple stages of design, implementation, and evaluation for protein therapeutics.

StableIsotope Labeling with Amino Acids in Cell Culture (SILAC)-based strategy for proteome-wide thermodynamic analysis of protein-ligand binding interactions.

Described here is a quantitative mass spectrometry-based proteomics method for the large-scale thermodynamic analysis of protein-ligand binding interactions. The methodology utilizes a chemical modification strategy termed, Stability of Proteins from Rates of Oxidation (SPROX), in combination with a Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) approach to compare the equilibrium folding/unfolding properties of proteins in the absence and presence of target ligands. The method, which is general with respect to ligand, measures the ligand-induced changes in protein stability associated with protein-ligand binding. The methodology is demonstrated in a proof-of-principle study in which the well-characterized protein-drug interaction between cyclosporine A (CsA) and cyclophilin A was successfully analyzed in the context of a yeast cell lysate. A control experiment was also performed to assess the method's false positive rate of ligand discovery, which was found to be on the order of 0.4 - 3.5%. The new method was utilized to characterize the adenosine triphosphate (ATP)-interactome in Saccharomyces cerevisiae using the nonhydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP), and the proteins in a yeast cell lysate. The new methodology enabled the interrogation of 526 yeast proteins for interactions with ATP using 2035 peptide probes. Ultimately, 325 peptide hits from 139 different proteins were identified. Approximately 70% of the hit proteins identified in this work were not previously annotated as ATP binding proteins. However, nearly two-thirds of the newly discovered ATP interacting proteins have known interactions with other nucleotides and co-factors (e.g. NAD and GTP), DNA, and RNA based on GO-term analyses. The current work is the first proteome-wide profile of the yeast ATP-interactome, and it is the largest proteome-wide profile of any ATP-interactome generated, to date, using an energetics-based method. The data is available via ProteomeXchange with identifiers PXD000858, DOI 10.6019/PXD000858, and PXD000860.

Global analysis of protein folding thermodynamics for disease state characterization.

Current methods for the large-scale characterization of disease states generally rely on the analysis of gene and/or protein expression levels. These existing methods fail to detect proteins with disease-related functions and unaltered expression levels. Here we describe the large-scale use of thermodynamic measurements of protein folding and stability for the characterization of disease states. Using the Stable Isotope Labeling with Amino Acids in Cell Culture and Stability of Proteins from Rates of Oxidation (SILAC-SPROX) technique, we assayed ∼800 proteins for protein folding and stability changes in three different cell culture models of breast cancer including the MCF-10A, MCF-7, and MDA-MB-231 cell lines. The thermodynamic stability profiles generated here created distinct molecular markers to differentiate the three cell lines, and a significant fraction (∼45%) of the differentially stabilized proteins did not have altered expression levels. Thus, the differential thermodynamic profiling strategy reported here created novel molecular signatures of breast cancer and provided additional insight into the molecular basis of the disease. Our results establish the utility of protein folding and stability measurements for the study of disease processes, and they suggest that such measurements may be useful for biomarker discovery in disease.

Use of Statin and Target Low-density lipoprotein cholesterol attainment among post-ST elevation myocardial infarction patients in Shahid Gangalal National Heart Centre, Kathmandu, Nepal.

BACKGROUND: and objective: Lipid-lowering is an important intervention to reduce cardiovascular morbidity and mortality in the secondary prevention of STEMI. There is no study to analyze the use of statin and LDL-C treatment target attainment among STEMI patients in Nepal. This study aims to assess the use of statin and LDL-C treatment target attainment among STEMI patients. METHODS: It was a prospective observational single-center study conducted at the Shahid Gangalal National Heart Centre, Kathmandu, Nepal outpatient department. An outpatient department-based survey was conducted among STEMI patients who have lipid profile levels at the time of admission for STEMI and after 4-13 weeks of the index event. Lipid profile levels, diagnosis, and risk factors were collected during the outpatient follow-up. RESULTS: Our study included 280 post-STEMI patients; the mean age was 57.5±11.7 years with the majority being male. The mean duration of follow-up was 6.7 ± 0.1 weeks. Rosuvastatin was the preferred statin with 82.1%. The most common dose of statin used was Rosuvastatin 20mg (70%), followed by Atorvastatin 40mg (12.5%). LDL-C levels of <1.4mmol/l were achieved in 44.6% of cases and LDL levels of <1.8mmol/l in 71.8% of cases. In 36.8% of the study population, there was a greater than 50% decline in LDL-C levels. Diabetic patients (55.1% and 83.1%) only have the significant achievement of LDL goal of both <1.4mmol/l and <1.8mmol/l respectively, when compared to those without diabetes (44.9% and 16.9%). CONCLUSIONS: Most of the post-STEMI patients were treated with high doses of statins and achieved the target LDL-C levels.

Epitope Mapping of Japanese Encephalitis Virus Neutralizing Antibodies by Native Mass Spectrometry and Hydrogen/Deuterium Exchange.

Japanese encephalitis virus (JEV) remains a global public health concern due to its epidemiological distribution and the existence of multiple strains. Neutralizing antibodies against this infection have shown efficacy in in vivo studies. Thus, elucidation of the epitopes of neutralizing antibodies can aid in the design and development of effective vaccines against different strains of JEV. Here, we describe a combination of native mass spectrometry (native-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) to complete screening of eight mouse monoclonal antibodies (MAbs) against JEV E-DIII to identify epitope regions. Native-MS was used as a first pass to identify the antibodies that formed a complex with the target antigen, and it revealed that seven of the eight monoclonal antibodies underwent binding. Native mass spectra of a MAb (JEV-27) known to be non-binding showed broad native-MS peaks and poor signal, suggesting the protein is a mixture or that there are impurities in the sample. We followed native-MS with HDX-MS to locate the binding sites for several of the complex-forming antibodies. This combination of two mass spectrometry-based approaches should be generally applicable and particularly suitable for screening of antigen-antibody and other protein-protein interactions when other traditional approaches give unclear results or are difficult, unavailable, or need to be validated.

Predicting the structural basis of targeted protein degradation by integrating molecular dynamics simulations with structural mass spectrometry.

Targeted protein degradation (TPD) is a promising approach in drug discovery for degrading proteins implicated in diseases. A key step in this process is the formation of a ternary complex where a heterobifunctional molecule induces proximity of an E3 ligase to a protein of interest (POI), thus facilitating ubiquitin transfer to the POI. In this work, we characterize 3 steps in the TPD process. (1) We simulate the ternary complex formation of SMARCA2 bromodomain and VHL E3 ligase by combining hydrogen-deuterium exchange mass spectrometry with weighted ensemble molecular dynamics (MD). (2) We characterize the conformational heterogeneity of the ternary complex using Hamiltonian replica exchange simulations and small-angle X-ray scattering. (3) We assess the ubiquitination of the POI in the context of the full Cullin-RING Ligase, confirming experimental ubiquitinomics results. Differences in degradation efficiency can be explained by the proximity of lysine residues on the POI relative to ubiquitin.

Hydrogen-deuterium exchange mass spectrometry identifies spatially distinct antibody epitopes on domain III of the Zika virus envelope protein.

Zika Virus (ZIKV) has become a global public health concern because it causes fetal microcephaly and other neurological complications in humans. Currently, there are no approved treatments or vaccines for ZIKV infection. We describe here the detailed epitopes for six monoclonal antibodies (mAbs) that bind to domain III of the envelope protein of ZIKV, some of which have therapeutic potential. We show that by using hydrogen-deuterium exchange mass spectrometry (HDX-MS), we can identify three spatially distinct epitopes for the six mAbs investigated. The HDX-MS approach identified epitopes for three mAbs that agreed well with recently reported X-ray crystallography data. The HDX-MS determined epitopes for the other three anti-ZIKV mAbs for which there were no crystal structures, and the epitopes were confirmed by structure-guided mutagenesis and biolayer interferometry (BLI) competition binding assay. Our results have implications for the design of vaccine and antibody therapeutics against ZIKV and demonstrate the use of HDX-MS as a rapid and valid approach for epitope mapping.

Atrial Septal Defect Size and Rims on Transesophageal Echocardiogram.

Background and aims:Rims and size of atrial septal defect (ASD) are crucial for the success of transcatheter ASD closure. The maximal diameter and dimensions of various rims of the ASD are essential for sizing and optimal placement of the device. We aimed to study the size and rims of ASD in our patients. Methods:This was a prospective study that was done at Shahid Gangalal National Heart Centre. All patients aged over 18 and referred to a unit IV in the Department of Cardiology for ASD device closure were included in the study. The study duration was six months, from April to September 2018. The size and rims of ASD were evaluated by transesophageal echocardiogram. Results:During the study, 173 patients underwent transesophageal echocardiogram. Most of them [122 (70.1%)] were women. Age ranged from 18 to 68 (mean, 35 years). The most common symptom was shortness of breath. Twenty-one (12.1%) patients were incidentally detected with ASDs. Sinus rhythm with right bundle branch block was present in 148 (85.5%) subjects. Right atrium and right ventricle were dilated in 162 (93.6%) patients. One patient had dextrocardia with situs inversus. More than half of all patients (54.9%) had mild tricuspid regurgitation. Mean tricuspid regurgitation pressure gradient was 39.5±16.8 mm Hg. More than one ASD was present in 11 (6.3%) patients. ASD size ranged from 2 mm to 43 mm in 4-chamber view, 2 mm to 44 mm in short axis view, and 2 mm to 47 mm in bicaval view. The mean ASD size was 18.6±7.7 mm in 4-chamber view, 19.6±8.5 mm in short axis view, and 18.7±8.0 mm in bicaval view. In only 11 (6.4%) patients, all rims were present and not floppy, while in other 11 (6.4%) subjects all rims were present, but floppy. With the exception of aortic rim, all other rims were present and good in 55 (33.9%) patients, while in 45 (27.7%) patients, other rims were present but floppy. Conclusion:Many ASD have absent, inadequate and floppy rims.

Near-atomic structure of a giant virus.

Although the nucleocytoplasmic large DNA viruses (NCLDVs) are one of the largest group of viruses that infect many eukaryotic hosts, the near-atomic resolution structures of these viruses have remained unknown. Here we describe a 3.5 Å resolution icosahedrally averaged capsid structure of Paramecium bursaria chlorella virus 1 (PBCV-1). This structure consists of 5040 copies of the major capsid protein, 60 copies of the penton protein and 1800 minor capsid proteins of which there are 13 different types. The minor capsid proteins form a hexagonal network below the outer capsid shell, stabilizing the capsid by binding neighboring capsomers together. The size of the viral capsid is determined by a tape-measure, minor capsid protein of which there are 60 copies in the virion. Homologs of the tape-measure protein and some of the other minor capsid proteins exist in other NCLDVs. Thus, a similar capsid assembly pathway might be used by other NCLDVs.

Mouse and Human Monoclonal Antibodies Protect against Infection by Multiple Genotypes of Japanese Encephalitis Virus.

Japanese encephalitis virus (JEV) remains a leading cause of viral encephalitis worldwide. Although JEV-specific antibodies have been described, an assessment of their ability to neutralize multiple genotypes of JEV has been limited. Here, we describe the development of a panel of mouse and human neutralizing monoclonal antibodies (MAbs) that inhibit infection in cell culture of four different JEV genotypes tested. Mechanism-of-action studies showed that many of these MAbs inhibited infection at a postattachment step, including blockade of virus fusion. Mapping studies using site-directed mutagenesis and hydrogen-deuterium exchange with mass spectrometry revealed that the lateral ridge on domain III of the envelope protein was a primary recognition epitope for our panel of strongly neutralizing MAbs. Therapeutic studies in mice demonstrated protection against lethality caused by genotype I and III strains when MAbs were administered as a single dose even 5 days after infection. This information may inform the development of vaccines and therapeutic antibodies as emerging strains and genotypic shifts become more prevalent.IMPORTANCE Although Japanese encephalitis virus (JEV) is a vaccine-preventable cause of viral encephalitis, the inactivated and live attenuated platforms available are derived from strains belonging to a single genotype (GIII) due to its historical prevalence in areas of JEV epidemics. Related to this, studies with vaccines and antibodies have focused on assessing the in vitro and in vivo protective responses to homologous or heterologous GIII strains. An epidemiological shift in JEV genotype distribution warrants the induction of broadly neutralizing antibody responses that inhibit infection of multiple JEV genotypes. Here, we generated a panel of mouse and human neutralizing monoclonal antibodies and evaluated their inhibitory activity, epitope location, and capacity for protection against multiple JEV genotypes in mice.

Kinetic Isotope Effects and Hydrogen/Deuterium Exchange Reveal Large Conformational Changes During the Catalysis of the Clostridium acetobutylicum Spore Photoproduct Lyase.

Spore photoproduct lyase (SPL) catalyzes the direct reversal of a thymine dimer 5-thyminyl-5,6-dihydrothymine (i.e. the spore photoproduct (SP)) to two thymine residues in germinating endospores. Previous studies suggest that SPL from the bacterium Bacillus subtilis (Bs) harbors an unprecedented radical-transfer pathway starting with cysteine 141 proceeding through tyrosine 99. However, in SPL from the bacterium Clostridium acetobutylicum (Ca), the cysteine (at position 74) and the tyrosine are located on the opposite sides of a substrate-binding pocket that has to collapse to bring the two residues into proximity, enabling the C→Y radical passage as implied in SPL(Bs) . To test this hypothesis, we adopted hydrogen/deuterium exchange mass spectrometry (HDX-MS) to show that C74(Ca) is located at a highly flexible region. The repair of dinucleotide SP TpT by SPL(Ca) is eight-fold to 10-fold slower than that by SPL(Bs) ; the process also generates a large portion of the aborted product TpTSO2- . SPL(Ca) exhibits apparent (D V) kinetic isotope effects (KIEs) of ~6 and abnormally large competitive (D V/K) KIEs (~20), both of which are much larger than the KIEs observed for SPL(Bs) . All these observations indicate that SPL(Ca) possesses a flexible active site and readily undergoes conformational changes during catalysis.

Dimerization Controls Marburg Virus VP24-dependent Modulation of Host Antioxidative Stress Responses.

Marburg virus (MARV), a member of the Filoviridae family that also includes Ebola virus (EBOV), causes lethal hemorrhagic fever with case fatality rates that have exceeded 50% in some outbreaks. Within an infected cell, there are numerous host-viral interactions that contribute to the outcome of infection. Recent studies identified MARV protein 24 (mVP24) as a modulator of the host antioxidative responses, but the molecular mechanism remains unclear. Using a combination of biochemical and mass spectrometry studies, we show that mVP24 is a dimer in solution that directly binds to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) to regulate nuclear factor (erythroid-derived 2)-like 2 (Nrf2). This interaction between Keap1 and mVP24 occurs through the Kelch interaction loop (K-Loop) of mVP24 leading to upregulation of antioxidant response element transcription, which is distinct from other Kelch binders that regulate Nrf2 activity. N-terminal truncations disrupt mVP24 dimerization, allowing monomeric mVP24 to bind Kelch with higher affinity and stimulate higher antioxidative stress response element (ARE) reporter activity. Mass spectrometry-based mapping of the interface revealed overlapping binding sites on Kelch for mVP24 and the Nrf2 proteins. Substitution of conserved cysteines, C209 and C210, to alanine in the mVP24 K-Loop abrogates Kelch binding and ARE activation. Our studies identify a shift in the monomer-dimer equilibrium of MARV VP24, driven by its interaction with Keap1 Kelch domain, as a critical determinant that modulates host responses to pathogenic Marburg viral infections.

SILAC-pulse proteolysis: A mass spectrometry-based method for discovery and cross-validation in proteome-wide studies of ligand binding.

Reported here is the use of stable isotope labeling with amino acids in cell culture (SILAC) and pulse proteolysis (PP) for detection and quantitation of protein-ligand binding interactions on the proteomic scale. The incorporation of SILAC into PP enables the PP technique to be used for the unbiased detection and quantitation of protein-ligand binding interactions in complex biological mixtures (e.g., cell lysates) without the need for prefractionation. The SILAC-PP technique is demonstrated in two proof-of-principle experiments using proteins in a yeast cell lysate and two test ligands including a well-characterized drug, cyclosporine A (CsA), and a non-hydrolyzable adenosine triphosphate (ATP) analogue, adenylyl imidodiphosphate (AMP-PNP). The well-known tight-binding interaction between CsA and cyclophilin A was successfully detected and quantified in replicate analyses, and a total of 33 proteins from a yeast cell lysate were found to have AMP-PNP-induced stability changes. In control experiments, the method's false positive rate of protein target discovery was found to be in the range of 2.1% to 3.6%. SILAC-PP and the previously reported stability of protein from rates of oxidation (SPROX) technique both report on the same thermodynamic properties of proteins and protein-ligand complexes. However, they employ different probes and mass spectrometry-based readouts. This creates the opportunity to cross-validate SPROX results with SILAC-PP results, and vice-versa. As part of this work, the SILAC-PP results obtained here were cross-validated with previously reported SPROX results on the same model systems to help differentiate true positives from false positives in the two experiments.

Thermodynamic analysis of protein-ligand binding interactions in complex biological mixtures using the stability of proteins from rates of oxidation.

The detection and quantification of protein-ligand binding interactions is crucial in a number of different areas of biochemical research from fundamental studies of biological processes to drug discovery efforts. Described here is a protocol that can be used to identify the protein targets of biologically relevant ligands (e.g., drugs such as tamoxifen or cyclosporin A) in complex protein mixtures such as cell lysates. The protocol utilizes quantitative, bottom-up, shotgun proteomics technologies (isobaric mass tags for relative and absolute quantification, or iTRAQ) with a covalent labeling technique, termed stability of proteins from rates of oxidation (SPROX). In SPROX, the thermodynamic properties of proteins and protein-ligand complexes are assessed using the hydrogen peroxide-mediated oxidation of methionine residues as a function of the chemical denaturant (e.g., guanidine hydrochloride or urea) concentration. The proteome-wide SPROX experiments described here enable the ligand-binding properties of hundreds of proteins to be simultaneously assayed in the context of complex biological samples. The proteomic capabilities of the protocol render it amenable to the detection of both the on- and off-target effects of ligand binding. The protocol can be completed in 5 d.