Therapeutic alphavirus cross-reactive E1 human antibodies inhibit viral egress.

Alphaviruses cause severe arthritogenic or encephalitic disease. The E1 structural glycoprotein is highly conserved in these viruses and mediates viral fusion with host cells. However, the role of antibody responses to the E1 protein in immunity is poorly understood. We isolated E1-specific human monoclonal antibodies (mAbs) with diverse patterns of recognition for alphaviruses (ranging from Eastern equine encephalitis virus [EEEV]-specific to alphavirus cross-reactive) from survivors of natural EEEV infection. Antibody binding patterns and epitope mapping experiments identified differences in E1 reactivity based on exposure of epitopes on the glycoprotein through pH-dependent mechanisms or presentation on the cell surface prior to virus egress. Therapeutic efficacy in vivo of these mAbs corresponded with potency of virus egress inhibition in vitro and did not require Fc-mediated effector functions for treatment against subcutaneous EEEV challenge. These studies reveal the molecular basis for broad and protective antibody responses to alphavirus E1 proteins.

PAX3-FOXO1 coordinates enhancer architecture, eRNA transcription, and RNA polymerase pause release at select gene targets.

Transcriptional control is a highly dynamic process that changes rapidly in response to various cellular and extracellular cues, making it difficult to define the mechanism of transcription factor function using slow genetic methods. We used a chemical-genetic approach to rapidly degrade a canonical transcriptional activator, PAX3-FOXO1, to define the mechanism by which it regulates gene expression programs. By coupling rapid protein degradation with the analysis of nascent transcription over short time courses and integrating CUT&RUN, ATAC-seq, and eRNA analysis with deep proteomic analysis, we defined PAX3-FOXO1 function at a small network of direct transcriptional targets. PAX3-FOXO1 degradation impaired RNA polymerase pause release and transcription elongation at most regulated gene targets. Moreover, the activity of PAX3-FOXO1 at enhancers controlling this core network was surprisingly selective, affecting single elements in super-enhancers. This combinatorial analysis indicated that PAX3-FOXO1 was continuously required to maintain chromatin accessibility and enhancer architecture at regulated enhancers.

Discovery of Widespread Host Protein Interactions with the Pre-replicated Genome of CHIKV Using VIR-CLASP.

The primary interactions between incoming viral RNA genomes and host proteins are crucial to infection and immunity. Until now, the ability to study these events was lacking. We developed viral cross-linking and solid-phase purification (VIR-CLASP) to characterize the earliest interactions between viral RNA and cellular proteins. We investigated the infection of human cells using Chikungunya virus (CHIKV) and influenza A virus and identified hundreds of direct RNA-protein interactions. Here, we explore the biological impact of three protein classes that bind CHIKV RNA within minutes of infection. We find CHIKV RNA binds and hijacks the lipid-modifying enzyme fatty acid synthase (FASN) for pro-viral activity. We show that CHIKV genomes are N6-methyladenosine modified, and YTHDF1 binds and suppresses CHIKV replication. Finally, we find that the innate immune DNA sensor IFI16 associates with CHIKV RNA, reducing viral replication and maturation. Our findings have direct applicability to the investigation of potentially all RNA viruses.

Inhibition of mRNA nuclear export promotes SARS-CoV-2 pathogenesis.

The nonstructural protein 1 (Nsp1) of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a virulence factor that targets multiple cellular pathways to inhibit host gene expression and antiviral response. However, the underlying mechanisms of the various Nsp1-mediated functions and their contributions to SARS-CoV-2 virulence remain unclear. Among the targets of Nsp1 is the mRNA (messenger ribonucleic acid) export receptor NXF1-NXT1, which mediates nuclear export of mRNAs from the nucleus to the cytoplasm. Based on Nsp1 crystal structure, we generated mutants on Nsp1 surfaces and identified an acidic N-terminal patch that is critical for interaction with NXF1-NXT1. Photoactivatable Nsp1 probe reveals the RNA Recognition Motif (RRM) domain of NXF1 as an Nsp1 N-terminal binding site. By mutating the Nsp1 N-terminal acidic patch, we identified a separation-of-function mutant of Nsp1 that retains its translation inhibitory function but substantially loses its interaction with NXF1 and reverts Nsp1-mediated mRNA export inhibition. We then generated a recombinant (r)SARS-CoV-2 mutant on the Nsp1 N-terminal acidic patch and found that this surface is key to promote NXF1 binding and inhibition of host mRNA nuclear export, viral replication, and pathogenicity in vivo. Thus, these findings provide a mechanistic understanding of Nsp1-mediated mRNA export inhibition and establish the importance of this pathway in the virulence of SARS-CoV-2.

De novo sequencing and construction of a unique antibody for the recognition of alternative conformations of cytochrome c in cells.

Cytochrome c (cyt c) can undergo reversible conformational changes under biologically relevant conditions. Revealing these alternative cyt c conformers at the cell and tissue level is challenging. A monoclonal antibody (mAb) identifying a key conformational change in cyt c was previously reported, but the hybridoma was rendered nonviable. To resurrect the mAb in a recombinant form, the amino-acid sequences of the heavy and light chains were determined by peptide mapping-mass spectrometry-bioinformatic analysis and used to construct plasmids encoding the full-length chains. The recombinant mAb (R1D3) was shown to perform similarly to the original mAb in antigen-binding assays. The mAb bound to a variety of oxidatively modified cyt c species (e.g., nitrated at Tyr74 or oxidized at Met80), which lose the sixth heme ligation (Fe-Met80); it did not bind to several cyt c phospho- and acetyl-mimetics. Peptide competition assays together with molecular dynamic studies support that R1D3 binds a neoepitope within the loop 40-57. R1D3 was employed to identify alternative conformations of cyt c in cells under oxidant- or senescence-induced challenge as confirmed by immunocytochemistry and immunoaffinity studies. Alternative conformers translocated to the nuclei without causing apoptosis, an observation that was further confirmed after pinocytic loading of oxidatively modified cyt c to B16-F1 cells. Thus, alternative cyt c conformers, known to gain peroxidatic function, may represent redox messengers at the cell nuclei. The availability and properties of R1D3 open avenues of interrogation regarding the presence and biological functions of alternative conformations of cyt c in mammalian cells and tissues.

Collagen IV of basement membranes: IV. Adaptive mechanism of collagen IV scaffold assembly in Drosophila.

Collagen IV is an essential structural protein in all metazoans. It provides a scaffold for the assembly of basement membranes, a specialized form of extracellular matrix, which anchors and signals cells and provides microscale tensile strength. Defective scaffolds cause basement membrane destabilization and tissue dysfunction. Scaffolds are composed of α-chains that coassemble into triple-helical protomers of distinct chain compositions, which in turn oligomerize into supramolecular scaffolds. Chloride ions mediate the oligomerization via NC1 trimeric domains, forming an NC1 hexamer at the protomer-protomer interface. The chloride concentration-"chloride pressure"-on the outside of cells is a primordial innovation that drives the assembly and dynamic stabilization of collagen IV scaffolds. However, a Cl-independent mechanism is operative in Ctenophora, Ecdysozoa, and Rotifera, which suggests evolutionary adaptations to environmental or tissue conditions. An understanding of these exceptions, such as the example of Drosophila, could shed light on the fundamentals of how NC1 trimers direct the oligomerization of protomers into scaffolds. Here, we investigated the NC1 assembly of Drosophila. We solved the crystal structure of the NC1 hexamer, determined the chain composition of protomers, and found that Drosophila adapted an evolutionarily unique mechanism of scaffold assembly that requires divalent cations. By studying the Drosophila case we highlighted the mechanistic role of chloride pressure for maintaining functionality of the NC1 domain in humans. Moreover, we discovered that the NC1 trimers encode information for homing protomers to distant tissue locations, providing clues for the development of protein replacement therapy for collagen IV genetic diseases.

LRRC8A anion channels modulate vascular reactivity via association with myosin phosphatase rho interacting protein.

Leucine-rich repeat containing 8A (LRRC8A) volume regulated anion channels (VRACs) are activated by inflammatory and pro-contractile stimuli including tumor necrosis factor alpha (TNFα), angiotensin II and stretch. LRRC8A associates with NADPH oxidase 1 (Nox1) and supports extracellular superoxide production. We tested the hypothesis that VRACs modulate TNFα signaling and vasomotor function in mice lacking LRRC8A exclusively in vascular smooth muscle cells (VSMCs, Sm22α-Cre, Knockout). Knockout (KO) mesenteric vessels contracted normally but relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) was enhanced compared to wild type (WT). Forty-eight hours of ex vivo exposure to TNFα (10 ng/mL) enhanced contraction to norepinephrine (NE) and markedly impaired dilation to ACh and SNP in WT but not KO vessels. VRAC blockade (carbenoxolone, CBX, 100 µM, 20 min) enhanced dilation of control rings and restored impaired dilation following TNFα exposure. Myogenic tone was absent in KO rings. LRRC8A immunoprecipitation followed by mass spectroscopy identified 33 proteins that interacted with LRRC8A. Among them, the myosin phosphatase rho-interacting protein (MPRIP) links RhoA, MYPT1 and actin. LRRC8A-MPRIP co-localization was confirmed by confocal imaging of tagged proteins, Proximity Ligation Assays, and IP/western blots. siLRRC8A or CBX treatment decreased RhoA activity in VSMCs, and MYPT1 phosphorylation was reduced in KO mesenteries suggesting that reduced ROCK activity contributes to enhanced relaxation. MPRIP was a target of redox modification, becoming oxidized (sulfenylated) after TNFα exposure. Interaction of LRRC8A with MPRIP may allow redox regulation of the cytoskeleton by linking Nox1 activation to impaired vasodilation. This identifies VRACs as potential targets for treatment or prevention of vascular disease.

Structure and activation mechanism of the yeast RNA Pol II CTD kinase CTDK-1 complex.

The C-terminal domain (CTD) kinase I (CTDK-1) complex is the primary RNA Polymerase II (Pol II) CTD Ser2 kinase in budding yeast. CTDK-1 consists of a cyclin-dependent kinase (CDK) Ctk1, a cyclin Ctk2, and a unique subunit Ctk3 required for CTDK-1 activity. Here, we present a crystal structure of CTDK-1 at 1.85-Å resolution. The structure reveals that, compared to the canonical two-component CDK-cyclin system, the third component Ctk3 of CTDK-1 plays a critical role in Ctk1 activation by stabilizing a key element of CDK regulation, the T-loop, in an active conformation. In addition, Ctk3 contributes to the assembly of CTDK-1 through extensive interactions with both Ctk1 and Ctk2. We also demonstrate that CTDK-1 physically and genetically interacts with the serine/arginine-like protein Gbp2. Together, the data in our work reveal a regulatory mechanism of CDK complexes.

Identification of an Essential LolD-Like Protein in Helicobacter pylori.

The localization of lipoprotein (Lol) system is used by Gram-negative bacteria to export lipoproteins to the outer membrane. Lol proteins and models of how Lol transfers lipoproteins from the inner to the outer membrane have been extensively characterized in the model organism Escherichia coli, but in numerous bacterial species, lipoprotein synthesis and export pathways deviate from the E. coli paradigm. For example, in the human gastric bacterium Helicobacter pylori, a homolog of the E. coli outer membrane component LolB is not found, E. coli LolC and LolE correspond to a single inner membrane component (LolF), and a homolog of the E. coli cytoplasmic ATPase LolD has not been identified. In the present study, we sought to identify a LolD-like protein in H. pylori. We used affinity-purification mass spectrometry to identify interaction partners of the H. pylori ATP-binding cassette (ABC) family permease LolF and identified the ABC family ATP-binding protein HP0179 as its interaction partner. We engineered H. pylori to conditionally express HP0179 and showed that HP0179 and its conserved ATP binding and ATP hydrolysis motifs are essential for H. pylori growth. We then performed affinity purification-mass spectrometry using HP0179 as the bait and identified LolF as its interaction partner. These results indicate that H. pylori HP0179 is a LolD-like protein and provide a more complete understanding of lipoprotein localization processes in H. pylori, a bacterium in which the Lol system deviates from the E. coli paradigm. IMPORTANCE Lipoproteins are critical in Gram-negative-bacteria for cell surface assembly of LPS, insertion of outer membrane proteins, and sensing envelope stress. Lipoproteins also contribute to bacterial pathogenesis. For many of these functions, lipoproteins must localize to the Gram-negative outer membrane. Transporting lipoproteins to the outer membrane involves the Lol sorting pathway. Detailed analyses of the Lol pathway have been performed in the model organism Escherichia coli, but many bacteria utilize altered components or are missing essential components of the E. coli Lol pathway. Identifying a LolD-like protein in Helicobacter pylori is important to better understand the Lol pathway in diverse bacterial classes. This becomes particularly relevant as lipoprotein localization is targeted for antimicrobial development.

Delineation of the pH-Responsive Regulon Controlled by the Helicobacter pylori ArsRS Two-Component System.

Helicobacter pylori encounters a wide range of pH within the human stomach. In a comparison of H. pylori cultured in vitro under neutral or acidic conditions, about 15% of genes are differentially expressed, and corresponding changes are detectable for many of the encoded proteins. The ArsRS two-component system (TCS), comprised of the sensor kinase ArsS and its cognate response regulator ArsR, has an important role in mediating pH-responsive changes in H. pylori gene expression. In this study, we sought to delineate the pH-responsive ArsRS regulon and further define the role of ArsR in pH-responsive gene expression. We compared H. pylori strains containing an intact ArsRS system with an arsS null mutant or strains containing site-specific mutations of a conserved aspartate residue (D52) in ArsR, which is phosphorylated in response to signals relayed by the cognate sensor kinase ArsS. We identified 178 genes that were pH-responsive in strains containing an intact ArsRS system but not in ΔarsS or arsR mutants. These constituents of the pH-responsive ArsRS regulon include genes involved in acid acclimatization (ureAB, amidases), oxidative stress responses (katA, sodB), transcriptional regulation related to iron or nickel homeostasis (fur, nikR), and genes encoding outer membrane proteins (including sabA, alpA, alpB, hopD [labA], and horA). When comparing H. pylori strains containing an intact ArsRS TCS with arsRS mutants, each cultured at neutral pH, relatively few genes are differentially expressed. Collectively, these data suggest that ArsRS-mediated gene regulation has an important role in H. pylori adaptation to changing pH conditions.

The 15-Amino Acid Repeat Region of Adenomatous Polyposis Coli Is Intrinsically Disordered and Retains Conformational Flexibility upon Binding ß-Catenin.

The tumor suppressor Adenomatous polyposis coli (APC) is a large, multidomain protein with many identified cellular functions. The best characterized role of APC is to scaffold a protein complex that negatively regulates Wnt signaling via ß-catenin destruction. This destruction is mediated by ß-catenin binding to centrally located 15- and 20-amino acid repeat regions of APC. More than 80% of cancers of the colon and rectum present with an APC mutation. Most carcinomas with mutant APC express a truncated APC protein that retains the ∼200-amino acid long' 15-amino acid repeat region'. This study demonstrates that the 15-amino acid repeat region of APC is intrinsically disordered. We investigated the backbone dynamics in the presence of ß-catenin and predicted residues that may contribute to transient secondary features. This study reveals that the 15-amino acid region of APC retains flexibility upon binding ß-catenin and that APC does not have a single, observable "highest-affinity" binding site for ß-catenin. This flexibility potentially allows ß-catenin to be more readily captured by APC and then remain accessible to other elements of the destruction complex for subsequent processing.

Bacterial Energetic Requirements for Helicobacter pylori Cag Type IV Secretion System-Dependent Alterations in Gastric Epithelial Cells.

Helicobacter pylori colonizes the stomach in about half of the world's population. H. pylori strains containing the cag pathogenicity island (cag PAI) are associated with a higher risk of gastric adenocarcinoma or peptic ulcer disease than cag PAI-negative strains. The cag PAI encodes a type IV secretion system (T4SS) that mediates delivery of the CagA effector protein as well as nonprotein bacterial constituents into gastric epithelial cells. H. pylori-induced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and interleukin-8 (IL-8) secretion are attributed to T4SS-dependent delivery of lipopolysaccharide metabolites and peptidoglycan into host cells, and Toll-like receptor 9 (TLR9) activation is attributed to delivery of bacterial DNA. In this study, we analyzed the bacterial energetic requirements associated with these cellular alterations. Mutant strains lacking Cagα, Cagß, or CagE (putative ATPases corresponding to VirB11, VirD4, and VirB4 in prototypical T4SSs) were capable of T4SS core complex assembly but defective in CagA translocation into host cells. Thus, the three Cag ATPases are not functionally redundant. Cagα and CagE were required for H. pylori-induced NF-κB activation, IL-8 secretion, and TLR9 activation, but Cagß was dispensable for these responses. We identified putative ATP-binding motifs (Walker-A and Walker-B) in each of the ATPases and generated mutant strains in which these motifs were altered. Each of the Walker box mutant strains exhibited properties identical to those of the corresponding deletion mutant strains. These data suggest that Cag T4SS-dependent delivery of nonprotein bacterial constituents into host cells occurs through mechanisms different from those used for recruitment and delivery of CagA into host cells.

Negative regulation of Candida glabrata Pdr1 by the deubiquitinase subunit Bre5 occurs in a ubiquitin independent manner.

The primary route for development of azole resistance in the fungal pathogen Candida glabrata is acquisition of a point mutation in the PDR1 gene. This locus encodes a transcription factor that upon mutation drives high level expression of a range of genes including the ATP-binding cassette transporter-encoding gene CDR1. Pdr1 activity is also elevated in cells that lack the mitochondrial genome (ρ° cells), with associated high expression of CDR1 driving azole resistance. To gain insight into the mechanisms controlling activity of Pdr1, we expressed a tandem affinity purification (TAP)-tagged form of Pdr1 in both wild-type (ρ+ ) and ρ° cells. Purified proteins were analyzed by multidimensional protein identification technology mass spectrometry identifying a protein called Bre5 as a factor that co-purified with TAP-Pdr1. In Saccharomyces cerevisiae, Bre5 is part of a deubiquitinase complex formed by association with the ubiquitin-specific protease Ubp3. Genetic analyses in C. glabrata revealed that loss of BRE5, but not UBP3, led to an increase in expression of PDR1 and CDR1 at the transcriptional level. These studies support the view that Bre5 acts as a negative regulator of Pdr1 transcriptional activity and behaves as a C. glabrata-specific modulator of azole resistance.

Novel Method for Noninvasive Sampling of the Distal Airspace in Acute Respiratory Distress Syndrome.

RATIONALE: A major barrier to a more complete understanding of acute respiratory distress syndrome (ARDS) pathophysiology is the inability to sample the distal airspace of patients with ARDS. The heat moisture exchanger (HME) filter is an inline bacteriostatic sponge that collects exhaled moisture from the lungs of mechanically ventilated patients. OBJECTIVES: To test the hypothesis that HME filter fluid (HMEF) represents the distal airspace fluid in patients with ARDS. METHODS: Samples of HMEF were collected from 37 patients with acute pulmonary edema (either from ARDS or hydrostatic causes [HYDRO; control subjects]). Concurrent undiluted pulmonary edema fluid (EF) and HMEF were collected from six patients. HMEF from 11 patients (8 ARDS and 3 HYDRO) were analyzed by liquid chromatography-coupled tandem mass spectometry. Total protein (bicinchoninic acid assay), MMP-9 (matrix metalloproteinase-9), and MPO (myeloperoxidase) (ELISA) were measured in 29 subjects with ARDS and 5 subjects with HYDRO. SP-D (surfactant protein-D), RAGE (receptor for advanced glycation end-products) (ELISA), and cytokines (IL-1ß, IL-6, IL-8, and tumor necrosis factor-α) (electrochemiluminescent assays) were measured in six concurrent HMEF and EF samples. MEASUREMENTS AND MAIN RESULTS: Liquid chromatography-coupled tandem mass spectrometry on concurrent EF and HMEF samples from four patients revealed similar base peak intensities and m/z values indicating similar protein composition. There were 21 significantly elevated proteins in HMEF from patients with ARDS versus HYDRO. Eight proteins measured in concurrent EF and HMEF from six patients were highly correlated. In HMEF, total protein and MMP-9 were significantly higher in ARDS than in HYDRO. CONCLUSIONS: These data suggest that HMEF is a novel, noninvasive method to accurately sample the distal airspace in patients with ARDS.

Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric gene silencing.

Ubiquitin, and components of the ubiquitin-proteasome system, feature extensively in the regulation of gene transcription. Although there are many examples of how ubiquitin controls the activity of transcriptional regulators and coregulators, there are few examples of core components of the transcriptional machinery that are directly controlled by ubiquitin-dependent processes. The budding yeast protein Asr1 is the prototypical member of the RPC (RING, PHD, CBD) family of ubiquitin-ligases, characterized by the presence of amino-terminal RING (really interesting new gene) and PHD (plant homeo domain) fingers and a carboxyl-terminal domain that directly binds the largest subunit of RNA polymerase II (pol II), Rpb1, in response to phosphorylation events tied to the initiation of transcription. Asr1-mediated oligo-ubiquitylation of pol II leads to ejection of two core subunits of the enzyme and is associated with inhibition of polymerase function. Here, we present evidence that Asr1-mediated ubiquitylation of pol II is required for silencing of subtelomeric gene transcription. We show that Asr1 associates with telomere-proximal chromatin and that disruption of the ubiquitin-ligase activity of Asr1--or mutation of ubiquitylation sites within Rpb1--induces transcription of silenced gene sequences. In addition, we report that Asr1 associates with the Ubp3 deubiquitylase and that Asr1 and Ubp3 play antagonistic roles in setting transcription levels from silenced genes. We suggest that control of pol II by nonproteolytic ubiquitylation provides a mechanism to enforce silencing by transient and reversible inhibition of pol II activity at subtelomeric chromatin.

Dephosphorylation of F-BAR protein Cdc15 modulates its conformation and stimulates its scaffolding activity at the cell division site.

Cytokinesis in Schizosaccharomyces pombe requires the function of Cdc15, the founding member of the pombe cdc15 homology (PCH) family of proteins. As an early, abundant contractile ring component with multiple binding partners, Cdc15 plays a key role in organizing the ring. We demonstrate that Cdc15 phosphorylation at many sites generates a closed conformation, inhibits Cdc15 assembly at the division site in interphase, and precludes interaction of Cdc15 with its binding partners. Cdc15 dephosphorylation induces an open conformation, oligomerization, and scaffolding activity during mitosis. Cdc15 mutants with reduced phosphorylation precociously appear at the division site in filament-like structures and display increased association with protein partners and the membrane. Our results indicate that Cdc15 phosphoregulation impels both assembly and disassembly of the contractile apparatus and suggest a regulatory strategy that PCH family and BAR superfamily members might broadly employ to achieve temporal specificity in their roles as linkers between membrane and cytoskeleton.

Quantitative Multiple-Reaction Monitoring Proteomic Analysis of Gß and Gγ Subunits in C57Bl6/J Brain Synaptosomes.

Gßγ dimers are one of the essential signaling units of activated G protein-coupled receptors (GPCRs). There are five Gß and 12 Gγ subunits in humans; numerous studies have demonstrated that different Gß and Gγ subunits selectively interact to form unique Gßγ dimers, which in turn may target specific receptors and effectors. Perturbation of Gßγ signaling can lead to impaired physiological responses. Moreover, previous targeted multiple-reaction monitoring (MRM) studies of Gß and Gγ subunits have shown distinct regional and subcellular localization patterns in four brain regions. Nevertheless, no studies have quantified or compared their individual protein levels. In this study, we have developed a quantitative MRM method not only to quantify but also to compare the protein abundance of neuronal Gß and Gγ subunits. In whole and fractionated crude synaptosomes, we were able to identify the most abundant neuronal Gß and Gγ subunits and their subcellular localizations. For example, Gß1 was mostly localized at the membrane while Gß2 was evenly distributed throughout synaptosomal fractions. The protein expression levels and subcellular localizations of Gß and Gγ subunits may affect the Gßγ dimerization and Gßγ-effector interactions. This study offers not only a new tool for quantifying and comparing Gß and Gγ subunits but also new insights into the in vivo distribution of Gß and Gγ subunits, and Gßγ dimer assembly in normal brain function.

Binding of the Covalent Flavin Assembly Factor to the Flavoprotein Subunit of Complex II.

Escherichia coli harbors two highly conserved homologs of the essential mitochondrial respiratory complex II (succinate:ubiquinone oxidoreductase). Aerobically the bacterium synthesizes succinate:quinone reductase as part of its respiratory chain, whereas under microaerophilic conditions, the quinol:fumarate reductase can be utilized. All complex II enzymes harbor a covalently bound FAD co-factor that is essential for their ability to oxidize succinate. In eukaryotes and many bacteria, assembly of the covalent flavin linkage is facilitated by a small protein assembly factor, termed SdhE in E. coli. How SdhE assists with formation of the covalent flavin bond and how it binds the flavoprotein subunit of complex II remain unknown. Using photo-cross-linking, we report the interaction site between the flavoprotein of complex II and the SdhE assembly factor. These data indicate that SdhE binds to the flavoprotein between two independently folded domains and that this binding mode likely influences the interdomain orientation. In so doing, SdhE likely orients amino acid residues near the dicarboxylate and FAD binding site, which facilitates formation of the covalent flavin linkage. These studies identify how the conserved SdhE assembly factor and its homologs participate in complex II maturation.

A unique covalent bond in basement membrane is a primordial innovation for tissue evolution.

Basement membrane, a specialized ECM that underlies polarized epithelium of eumetazoans, provides signaling cues that regulate cell behavior and function in tissue genesis and homeostasis. A collagen IV scaffold, a major component, is essential for tissues and dysfunctional in several diseases. Studies of bovine and Drosophila tissues reveal that the scaffold is stabilized by sulfilimine chemical bonds (S = N) that covalently cross-link methionine and hydroxylysine residues at the interface of adjoining triple helical protomers. Peroxidasin, a heme peroxidase embedded in the basement membrane, produces hypohalous acid intermediates that oxidize methionine, forming the sulfilimine cross-link. We explored whether the sulfilimine cross-link is a fundamental requirement in the genesis and evolution of epithelial tissues by determining its occurrence and evolutionary origin in Eumetazoa and its essentiality in zebrafish development; 31 species, spanning 11 major phyla, were investigated for the occurrence of the sulfilimine cross-link by electrophoresis, MS, and multiple sequence alignment of de novo transcriptome and available genomic data for collagen IV and peroxidasin. The results show that the cross-link is conserved throughout Eumetazoa and arose at the divergence of Porifera and Cnidaria over 500 Mya. Also, peroxidasin, the enzyme that forms the bond, is evolutionarily conserved throughout Metazoa. Morpholino knockdown of peroxidasin in zebrafish revealed that the cross-link is essential for organogenesis. Collectively, our findings establish that the triad-a collagen IV scaffold with sulfilimine cross-links, peroxidasin, and hypohalous acids-is a primordial innovation of the ECM essential for organogenesis and tissue evolution.

Proteome informatics research group (iPRG)_2012: a study on detecting modified peptides in a complex mixture.

The proteome informatics research group of the Association of Biomolecular Resource Facilities conducted a study to assess the community's ability to detect and characterize peptides bearing a range of biologically occurring post-translational modifications when present in a complex peptide background. A data set derived from a mixture of synthetic peptides with biologically occurring modifications combined with a yeast whole cell lysate as background was distributed to a large group of researchers and their results were collectively analyzed. The results from the twenty-four participants, who represented a broad spectrum of experience levels with this type of data analysis, produced several important observations. First, there is significantly more variability in the ability to assess whether a results is significant than there is to determine the correct answer. Second, labile post-translational modifications, particularly tyrosine sulfation, present a challenge for most researchers. Finally, for modification site localization there are many tools being employed, but researchers are currently unsure of the reliability of the results these programs are producing.