Function Of Specific Proteins Research Articles

Complete Genome Sequence and Characterization of a Protein-Glutaminase Producing Strain, Chryseobacterium proteolyticum QSH1265.

Recently, an enzyme named protein-glutaminase (PG) has been identified as a new type of enzyme with significant potential for deamidation of food proteins. The enzyme is shown to be expressed as a pre-pro-protein with a putative signal peptide of 21 amino acids, a pro-sequence of 114 amino acids, and a mature PG of 185 amino acids. The microbial enzyme PG specifically catalyzes deamidation of proteins without protein hydrolysis pretreatment and only reacts with glutamine residues in the side-chains of proteins or long peptides. All these attributes suggest that it has a great potential for food industrial applications. However, until recently, there have been relatively few studies of the PG-producing strains. A strain named Chryseobacterium proteolyticum QSH1265 which can produce PG was isolated from a soil sample collected in Songjiang, Shanghai, China. Its enzyme activity was about 0.34 ± 0.01 U/mL when using carboxybenzoxy-Gln-Gly as a substrate. The strain can produce acid from D-glucose, maltose, L-arabinose sucrose, glycerol, and mannitol but not fructose, and it is also positive for indole production and urease. Here we describe the complete genome sequence of this strain via PacBio RSII sequencing. The C. proteolyticum QSH1265 genome consists of a circular chromosome with total length of 4,849,803 bp without any plasmids. All of 4563 genes were predicted including 4459 genes for protein-coding and 104 RNA-relative genes with an average G+C content of 36.16%. The KEGG and COG annotation provide information for the specific function of proteins encoded in the genome, such as proteases, chromoproteins, stress proteins, antiporters, etc. A highly conserved hypothetical protein shares a promoter with the gene encoding the protein-glutaminase enzyme. The genome sequence and preliminary annotation provide valuable genetic information for further study of C. proteolyticum.

Reframing the Biological Basis of Neuroprotection Using Functional Genomics: Differentially Weighted, Time-Dependent Multifactor Pathogenesis of Human Ischemic Brain Damage.

Background: Neuroprotection studies are generally unable to demonstrate efficacy in humans. Our specific hypothesis is that multiple pathophysiologic pathways, of variable importance, contribute to ischemic brain damage. As a corollary to this, we discuss the broad hypothesis that a multifaceted approach will improve the probability of efficacious neuroprotection. But to properly test this hypothesis the nature and importance of the multiple contributing pathways needs elucidation. Our aim is to demonstrate, using functional genomics, in human cardiac surgery procedures associated with cerebral ischemia, that the pathogenesis of perioperative human ischemic brain damage involves the function of multiple variably weighted proteins involving several pathways. We then use these data and literature to develop a proposal for rational design of human neuroprotection protocols.Methods: Ninety-four patients undergoing deep hypothermic circulatory arrest (DHCA) and/or aortic valve replacement surgery had brain damage biomarkers, S100β and neurofilament H (NFH), assessed at baseline, 1 and 24 h post-cardiopulmonary bypass (CPB) with analysis for association with 92 single nucleotide polymorphisms (SNPs) (selected by co-author WAK) related to important proteins involved in pathogenesis of cerebral ischemia.Results: At the nominal significance level of 0.05, changes in S100β and in NFH at 1 and 24 h post-CPB were associated with multiple SNPs involving several prospectively determined pathophysiologic pathways, but were not individually significant after multiple comparison adjustments. Variable weights for the several evaluated SNPs are apparent on regression analysis and, notably, are dissimilar related to the two biomarkers and over time post CPB. Based on our step-wise regression model, at 1 h post-CPB, SOD2, SUMO4, and GP6 are related to relative change of NFH while TNF, CAPN10, NPPB, and SERPINE1 are related to the relative change of S100B. At 24 h post-CPB, ADRA2A, SELE, and BAX are related to the relative change of NFH while SLC4A7, HSPA1B, and FGA are related to S100B.Conclusions: In support of the proposed hypothesis, association SNP data suggest function of specific disparate proteins, as reflected by genetic variation, may be more important than others with variation at different post-insult times after human brain ischemia. Such information may support rational design of post-insult time-sensitive multifaceted neuroprotective therapies.

Potent and specific Atg8-targeting autophagy inhibitory peptides from giant ankyrins.

The mammalian Atg8 family proteins are central drivers of autophagy and contain six members, classified into the LC3 and GABARAP subfamilies. Due to their high sequence similarity and consequent functional overlaps, it is difficult to delineate specific functions of Atg8 proteins in autophagy. Here we discover a super-strong GABARAP-selective inhibitory peptide harbored in 270/480 kDa ankyrin-G and a super-potent pan-Atg8 inhibitory peptide from 440 kDa ankyrin-B. Structural studies elucidate the mechanism governing the Atg8 binding potency and selectivity of the peptides, reveal a general Atg8-binding sequence motif, and allow development of a more GABARAP-selective inhibitory peptide. These peptides effectively blocked autophagy when expressed in cultured cells. Expression of these ankyrin-derived peptides in Caenorhabditis elegans also inhibited autophagy, causing accumulation of the p62 homolog SQST-1, delayed development and shortened life span. Thus, these genetically encodable autophagy inhibitory peptides can be used to occlude autophagy spatiotemporally in living animals.

Shewanella oneidensis as a living electrode for controlled radical polymerization

Metabolic engineering has facilitated the production of pharmaceuticals, fuels, and soft materials but is generally limited to optimizing well-defined metabolic pathways. We hypothesized that the reaction space available to metabolic engineering could be expanded by coupling extracellular electron transfer to the performance of an exogenous redox-active metal catalyst. Here we demonstrate that the electroactive bacterium Shewanella oneidensis can control the activity of a copper catalyst in atom-transfer radical polymerization (ATRP) via extracellular electron transfer. Using S. oneidensis, we achieved precise control over the molecular weight and polydispersity of a bioorthogonal polymer while similar organisms, such as Escherichia coli, showed no significant activity. We found that catalyst performance was a strong function of bacterial metabolism and specific electron transport proteins, both of which offer potential biological targets for future applications. Overall, our results suggest that manipulating extracellular electron transport pathways may be a general strategy for incorporating organometallic catalysis into the repertoire of metabolically controlled transformations.

Commonly used trafficking blocks disrupt ARF1 activation and the localization and function of specific Golgi proteins

Cold temperature blocks used to synchronize protein trafficking inhibit GBF1 function, leading to a decrease in ARF1-GTP levels and mislocalization of the ARF1 effector golgin-160. Several other, but not all, Golgi proteins including ARL1 also mislocalize. ARF1 activity and golgin-160 localization require more than 30 min to recover from these blocks.

Hsc70-2 is required for Beet black scorch virus infection through interaction with replication and capsid proteins

Dissecting the complex molecular interplay between the host plant and invading virus improves our understanding of the mechanisms underlying viral pathogenesis. In this study, immunoprecipitation together with the mass spectrometry analysis revealed that the heat shock protein 70 (Hsp70) family homolog, Hsc70-2, was co-purified with beet black scorch virus (BBSV) replication protein p23 and coat protein (CP), respectively. Further experiments demonstrated that Hsc70-2 interacts directly with both p23 and CP, whereas there is no interaction between p23 and CP. Hsc70-2 expression is induced slightly during BBSV infection of Nicotiana benthamiana, and overexpression of Hsc70-2 promotes BBSV accumulation, while knockdown of Hsc70-2 in N. benthamiana leads to drastic reduction of BBSV accumulation. Infection experiments revealed that CP negatively regulates BBSV replication, which can be mitigated by overexpression of Hsc70-2. Further experiments indicate that CP impairs the interaction between Hsc70-2 and p23 in a dose-dependent manner. Altogether, we provide evidence that besides specific functions of Hsp70 family proteins in certain aspects of viral infection, they can serve as a mediator for the orchestration of virus infection by interacting with different viral components. Our results provide new insight into the role of Hsp70 family proteins in virus infection.

Fabrication of three-dimensional proteinaceous micro- and nano-structures by femtosecond laser cross-linking

Proteins are a class of biomaterials having a vast array of functions, including the catalysis of metabolic reactions, DNA replication, stimuli response and transportation of molecules. Recent progress in laser-based fabrication technologies has enabled the formation of three-dimensional (3D) proteinaceous micro- and nano-structures by femtosecond laser cross-linking, which has expanded the possible applications of proteins. This article reviews the current knowledge and recent advancements in the femtosecond laser cross-linking of proteins. An overview of previous studies related to fabrication using a variety of proteins and detailed discussions of the associated mechanisms are provided. In addition, advances and applications utilizing specific protein functions are introduced. This review thus provides a valuable summary of the 3D micro- and nano-fabrication of proteins for biological and medical applications.

Glutathiolation Triggers Proteins for Degradation by the Ubiquitin- Proteasome Pathway.

Glutathione is a small antioxidant peptide in cells and it plays an important role in maintaining a reducing intracellular environment. Glutathione is also involved in the dynamic regulation of specific protein functions by reversible glutathiolation of certain proteins in response to oxidative stress. The purpose of this work is to mechanistically investigate the effects of glutathiolation on the susceptibility of proteins to degradation by the ubiquitinproteasome pathway (UPP). The data show that γC-crystallin and carbonic anhydrase III were barely degraded by the UPP without modifications, but both were rapidly degraded by the UPP after glutathiolation. Modifications of sulfhydryls by other thiol-modification reagents, such as iodoacetamide, also increased the degradation of γC-crystallin, but not as effectively as glutathiolation. Biophysical analysis showed that glutathiolation caused reversible conformational changes of these proteins, including a significant increase in protein surface hydrophobicity and a decrease in thermal stability. The modified protein regained its native conformation and its resistance to degradation upon removal of the glutathione moiety. A cataract-causing T5P mutant γC-crystallin shares many biophysical characteristics as glutathiolated γC-crystallin, including increased surface hydrophobicity and decreased thermal stability. T5P mutant γC-crystallin was also rapidly degraded. Comparison of the conformational changes and the susceptibility to degradation of glutathiolated γC-crystallin with other forms of modified γC-crystallin suggests that the glutathiolation-induced exposure of hydrophobic patches, rather than the modification per se, serves as the signal for degradation by the UPP. Consistent with this hypothesis, masking the surface hydrophobicity of glutathiolated and T5P mutant γC-crystallins significantly reduced their susceptibility to degradation by the UPP. This work demonstrates that glutathiolation is a novel mechanism for the UPP to recognize substrates in response to oxidative stress.

Characterization of cysteine thiol modifications based on protein microenvironments and local secondary structures.

We have demonstrated earlier that protein microenvironments were conserved around disulfide-bridged cystine motifs with similar functions, irrespective of diversity in protein sequences. Here, cysteine thiol modifications were characterized based on protein microenvironments, secondary structures and specific protein functions. Protein microenvironment around an amino acid was defined as the summation of hydrophobic contributions from the surrounding protein fragments and the solvent molecules present within its first contact shell. Cysteine functions (modifications) were grouped into enzymatic and non-enzymatic classes. Modifications studied were-disulfide formation, thio-ether formation, metal-binding, nitrosylation, acylation, selenylation, glutathionylation, sulfenylation, and ribosylation. 1079 enzymatic proteins were reported from high-resolution crystal structures. Protein microenvironments around cysteine thiol, derived from above crystal structures, were clustered into 3 groups-buried-hydrophobic, intermediate and exposed-hydrophilic clusters. Characterization of cysteine functions were statistically meaningful for 4 modifications (disulfide formation, thioether formation, sulfenylation, and iron/zinc binding) those have sufficient amount of data in the current dataset. Results showed that protein microenvironment, secondary structure and protein functions were conserved for enzymatic cysteine functions, in contrast to the same function from non-enzymatic cysteines. Disulfide forming enzymatic cysteines were tightly packed within intermediate protein microenvironment cluster, have alpha-helical conformation and mostly belonged to CxxC motif of electron transport proteins. Disulfide forming non-enzymatic cysteines did not belong to conserved motif and have variable secondary structures. Similarly, enzymatic thioether forming cysteines have conserved microenvironment compared to non-enzymatic cystienes. Based on the compatibility between protein microenvironment and cysteine modifications, more efficient drug molecules could be designed against cysteine-related diseases.

A Mucin-Like Protein of Planthopper Is Required for Feeding and Induces Immunity Response in Plants.

The brown planthopper, Nilaparvata lugens, is a pest that threatens rice (Oryza sativa) production worldwide. While feeding on rice plants, planthoppers secrete saliva, which plays crucial roles in nutrient ingestion and modulating plant defense responses, although the specific functions of salivary proteins remain largely unknown. We identified an N. lugens-secreted mucin-like protein (NlMLP) by transcriptome and proteome analyses and characterized its function, both in brown planthopper and in plants. NlMLP is highly expressed in salivary glands and is secreted into rice during feeding. Inhibition of NlMLP expression in planthoppers disturbs the formation of salivary sheaths, thereby reducing their performance. In plants, NlMLP induces cell death, the expression of defense-related genes, and callose deposition. These defense responses are related to Ca2+ mobilization and the MEK2 MAP kinase and jasmonic acid signaling pathways. The active region of NlMLP that elicits plant responses is located in its carboxyl terminus. Our work provides a detailed characterization of a salivary protein from a piercing-sucking insect other than aphids. Our finding that the protein functions in plant immune responses offers new insights into the mechanism underlying interactions between plants and herbivorous insects.

Molecular cloning and biophysical characterization of CXCL3 chemokine

CXCL3 is a neutrophil activating chemokine that belongs to GRO subfamily of CXC chemokines. GRO chemokine family comprises of three chemokines GRO α (CXCL1), GROβ (CXCL2), and GRO γ (CXCL3), which arose as a result of gene duplication events during the course of chemokine evolution. Although primary sequences of GRO chemokines are highly similar, they performs several protein specific functions in addition to their common property of neutrophil trafficking. However, the molecular basis for their differential functions has not well understood. Although structural details are available for CXCL1 and CXCL2, no such information regarding CXCL3 is available till date. In the present study, we have successfully cloned, expressed, and purified the recombinant CXCL3. Around 15mg/L of pure recombinant CXCL3 protein was obtained. Further, we investigated its functional divergence and biophysical characteristics such as oligomerization, thermal stability and heparin binding etc., and compared all these features with its closest paralog CXCL2. Our studies revealed that, although overall structural and oligomerization features of CXCL3 and CXCL2 are similar, prominent differences were observed in their surface characteristics, thus implicating for a functional divergence.

The in situ bacterial production of fluorescent organic matter; an investigation at a species level

Aquatic dissolved organic matter (DOM) plays an essential role in biogeochemical cycling and transport of organic matter throughout the hydrological continuum. To characterise microbially-derived organic matter (OM) from common environmental microorganisms (Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa), excitation-emission matrix (EEM) fluorescence spectroscopy was employed. This work shows that bacterial organisms can produce fluorescent organic matter (FOM) in situ and, furthermore, that the production of FOM differs at a bacterial species level. This production can be attributed to structural biological compounds, specific functional proteins (e.g. pyoverdine production by P. aeruginosa), and/or metabolic by-products. Bacterial growth curve data demonstrates that the production of FOM is fundamentally related to microbial metabolism. For example, the majority of Peak T fluorescence (> 75%) is shown to be intracellular in origin, as a result of the building of proteins for growth and metabolism. This underpins the use of Peak T as a measure of microbial activity, as opposed to bacterial enumeration as has been previously suggested. This study shows that different bacterial species produce a range of FOM that has historically been attributed to high molecular weight allochthonous material or the degradation of terrestrial FOM. We provide definitive evidence that, in fact, it can be produced by microbes within a model system (autochthonous), providing new insights into the possible origin of allochthonous and autochthonous organic material present in aquatic systems.

Telomere and centromere—DNA tandem arrays on the chromosome

The eukaryotic genome contains a large amount of tandem repeated sequences. Among these repeats, telomere and the centromere are two unique structures on a chromosome, which play an important role in maintaining the stability and function of the genome. A telomere is repetitive nucleotide sequences that located at the ends of chromosomes, which protects the chromosome from deterioration or fusion with another chromosome. Although the repetitive sequences of telomeres in different species are different, telomeres share the same structure. The typical structure of telomere consists of the D-loop and T-loop, which are protected by Shelterin complex. The telomere length is preserved by the enzyme telomerase which elongates telomeres through its reverse transcription activity. It has been widely accepted that telomere attrition is related to aging and cancer. The centromere is a region of the chromosome, which ensures correct separation of daughter chromosomes during cell division. According to the position on the chromosome, the centromere is classified into metacentric, submetacentric, acrocentric, telocentric and holocentric. It can also be divided into point centromere, regional centromere and holocentromere due to the characteristics of the repetitive sequences, in line with the characteristics of the centromeric repetitive sequences. The damaged centromere can cause a large number of chromosomal anomalies such as chromosome rearrangements and breaks, multi-branched chromosomes, and whole-arm deletions, etc. These abnormal chromosomes will lead to serious diseases, such as cancer, ICF syndrome, even cell death. Both the telomere and centromere are composed of highly repetitive sequences and combined with a variety of proteins to form a complex structure. Because of their numerous repetitive DNA sequences, accurate DNA sequencing of telomere and centromere is technically difficult. So there are still many unanswered questions regarding their functions and their working mechanisms. This paper briefly reviews the history of discovering telomere and centromere, the structural characteristics of the telomere and centromere, including the difference of telomere and centromere DNA sequences among different species, the functions of specific proteins binding in these regions. This review also emphasizes the relationships between the repetitive sequences and aging, ICF syndrome or the occurrence of diseases, especially cancer. At last, the paper highlights and prospects the hot research topics in telomere and centromere studies, such as the methods of telomere length measurement, studies on the characteristics of telomerase, and research about centromere repetitive sequence. Hopefully, the fast development of research technology including sequencing technics will help to further understand the biological roles of telomere and centromere, thus facilitate tumor treatment and aging research.

Genome-wide analysis of VQ motif-containing proteins in Moso bamboo (Phyllostachys edulis).

29 Moso bamboo VQ proteins were genome-wide identified for the first time, and bioinformatics analysis was performed to investigate phylogenetic relationships and evolutionary divergence. The qRT-PCR data show that PeVQ genes response to different stress treatments. Accumulating evidence suggests that VQ motif-containing proteins in rice (Oryza sativa), Arabidopsis (Arabidopsis thaliana), and maize (Zea mays) play fundamental roles in response to various biotic and abiotic stresses. However, little is known about the functions of VQ family proteins in Moso bamboo (Phyllostachys edulis). In this study, we performed a genome-wide bioinformatic analysis and expression profiling of PeVQ genes. A total of 29 VQ genes was identified and divided into seven subgroups (I-VII) based on phylogenetic analysis. Gene structure and conserved motif analysis revealed that 25 of 29 VQ genes contained no introns. Multiple sequence alignment showed that Moso bamboo VQ motif-containing proteins contained five variations of the conserved motif. The time of duplication and divergence of Moso bamboo from rice and maize was calculated using K s analysis. A heat map was generated using microarray data from 29 Moso bamboo VQ genes suggesting that these genes were expressed in different tissues or developmental stages. Quantitative real-time PCR (qRT-PCR) and promoter analysis indicated that PeVQ genes were differentially regulated following treatment with polyethylene glycol, abscisic acid and salicylic acid. Our results provide a solid foundation for further research of the specific functions of VQ motif-containing proteins in Moso bamboo.

The effect of Rho drugs on mast cell activation and degranulation.

Mast cells are tissue-resident immune cells that produce potent proinflammatory mediators, which are stored in cytoplasmic granules. Stimulation triggers degranulation, a process that mobilizes granules to dock and fuse to the plasma membrane, releasing mediators. Mast cell degranulation has an important role in immunity but can also intensify inflammation and contribute to allergic disorders. Hence, it is important to understand signaling pathways that regulate mast cell degranulation. Here, we examined the role of Rho proteins in regulating mast cell activation leading to degranulation. RBL-2H3 cells and bone marrow-derived mast cells (BMMCs) were stimulated through aggregation of FcεRI receptors. Stimulated cells showed a large increase in the levels of activated Rac and, to a lesser extent, RhoA. Drugs were used to acutely inhibit the function of specific Rho proteins. The Rac inhibitor EHT-1864 and the RhoA inhibitor rhosin inhibited degranulation. Microscopic characterization showed that, upon stimulation, RBL-2H3 cells formed surface ridges that grew into large protrusions reminiscent of circular dorsal ruffles, which flattened into large lamellipodia. LysoTracker-labeled cells showed granules stream into peripheral protrusions. EHT-1864 reduced granule motility, whereas rhosin increased motility; both drugs affected the formation of peripheral protrusions. These results showed that, in response to stimuli, Rho proteins control discrete cytoskeletal remodeling processes that are needed for granule exocytosis. Rac is required to stimulate the remodeling of mast cells, triggering actin-mediated flattening of the cell periphery to create an active degranulation zone, whereas RhoA controls the streaming of highly motile granules into the active zone.

ITRAQ-based quantitative proteomic analysis reveals proteomic changes in three fenoxaprop-P-ethyl-resistant Beckmannia syzigachne biotypes with differing ACCase mutations

American sloughgrass (Beckmannia syzigachne Steud.) is a weed widely distributed in wheat fields of China. In recent years, the evolution of herbicide (fenoxaprop-P-ethyl)-resistant populations has decreased the susceptibility of B. syzigachne. This study compared 4 B. syzigachne populations (3 resistant and 1 susceptible) using iTRAQ to characterize fenoxaprop-P-ethyl resistance in B. syzigachne at the proteomic level. Through searching the UniProt database, 3104 protein species were identified from 13,335 unique peptides. Approximately 2834 protein species were assigned to 23 functional classifications provided by the COG database. Among these, 2299 protein species were assigned to 125 predicted pathways. The resistant biotype contained 8 protein species that changed in abundance relative to the susceptible biotype; they were involved in photosynthesis, oxidative phosphorylation, and fatty acid biosynthesis pathways. In contrast to previous studies comparing only 1 resistant and 1 susceptible population, our use of 3 fenoxaprop-resistant B. syzigachne populations with different genetic backgrounds minimized irrelevant differential expression and eliminated false positives. Therefore, we could more confidently link the differentially expressed proteins to herbicide resistance. Proteomic analysis demonstrated that fenoxaprop-P-ethyl resistance is associated with photosynthetic capacity, a connection that might be related to the target-site mutations in resistant B. syzigachne. This is the first large-scale proteomics study examining herbicide stress responses in different B. syzigachne biotypes. Biological significanceThis study has biological relevance because it is the first to employ proteomic analysis for understanding the mechanisms underlying Beckmannia syzigachne herbicide resistance. The plant is a major weed in China and negatively affects crop yield, but has developed considerable resistance to the most common herbicide, fenoxaprop-P-ethyl. Through comparisons of resistant and sensitive biotypes, our study identified multiple proteins (involved in photosynthesis, oxidative phosphorylation, and fatty acid biosynthesis) that are putatively linked to B. syzigachne herbicide response. This large-scale proteomics study, sorely lacking in weed science, contributes valuable data that can be applied to more fine-tuned analyses on the functions of specific proteins in herbicide resistance.

Increased rates of protein evolution and asymmetric deceleration after the whole-genome duplication in yeasts

BackgroundWhole-genome duplication (WGD) events have shaped the genomes of eukaryotic organisms. Relaxed selection after duplication along with inherent functional constraints are thought to determine the fate of the paralogs and, ultimately, the evolution of gene function. Here, we investigated the rate of protein evolution (as measured by dN/dS ratios) before and after the WGD in the hemiascomycete yeasts, and the way in which changes in such rates relate to molecular and biological function.ResultsFor most groups of orthologous genes (81%) we observed a change in the rates of evolution after genome duplication. Genes with atypically-low dN/dS ratio before the WGD were prone to increase their rates of evolution after duplication. Importantly, the paralogs were often different in their rates of evolution after the WGD (50% cases), however, this was more consistent with an asymmetric deceleration in the protein-evolution rates, rather than an asymmetric increase of the initial rates. Functional-category analysis showed that regulatory proteins such as protein kinases and transcription factors were enriched in genes that increase their rates of evolution after the WGD. While changes in the rate of protein-sequence evolution were associated to protein abundance, content of disordered regions, and contribution to fitness, these features were an attribute of specific functional classes.ConclusionsOur results indicate that strong purifying selection in ancestral pre-duplication sequences is a strong predictor of increased rates after the duplication in yeasts and that asymmetry in evolution rate is established during the deceleration phase. In addition, changes in the rates at which paralogous sequences evolve before and after WGD are different for specific protein functions; increased rates of protein evolution after duplication occur preferentially in specific protein functions.

Detecting Protein Sulfenylation in Cells Exposed to a Toxicant.

Protein sulfenylation is a post-translational modification that is linked to many cell signaling networks and specific protein functions, thus the detection of any sulfenylated protein after a toxicological exposure is of importance. Specifically, the detection of protein sulfenylation can provide multiple levels of mechanistic insight towards understanding the impact of a toxicological exposure. For instance, sulfenylation is caused by only a handful of reactive chemical species. Any altered sulfenylation suggests a change in cellular health, and the elucidation of the specific protein target that undergoes sulfenylation can help ascertain downstream targets and associated adverse outcomes. This document describes straightforward approaches to detect protein sulfenylation of total protein as well as individual proteins of interest with a focus on immunoblotting approaches. © 2017 by John Wiley & Sons, Inc.

Local Alignment of Ligand Binding Sites in Proteins for Polypharmacology and Drug Repositioning.

The administration of drugs is a key strategy in pharmacotherapy to treat diseases. Drugs are typically developed to modulate the function of specific proteins, which are directly associated with particular disease states. Nonetheless, recent studies suggest that protein-drug interactions are rather promiscuous and the majority of pharmaceuticals exhibit activity against multiple, often unrelated proteins. Certainly, the lack of selectivity often leads to drug side effects; on the other hand, these polypharmacological attributes can be used to develop drugs acting on multiple targets within a unique disease pathway, as well as to identify new targets for existing drugs, which is known as drug repositioning. To support drug development and repurposing, we developed eMatchSite, a new approach to detect those binding sites having the capability to bind similar compounds. eMatchSite is available as a standalone software and a webserver at http://www.brylinski.org/ematchsite .

Novel Sites of Gamma Carboxylation in Human Factor VII

IntroductionGamma- (γ-) carboxylation of glutamate residues is a vitamin K-dependent post-translational modification critical to the function of several plasma proteins. Found in the N-terminal domains of specific proteins, most of which are involved in hemostasis, these γ-carboxyglutamate residues (Gla) help mediate binding of divalent cations and are essential to protein function. Coagulation factor VII(a) bears 10 known Gla residues as characterized by N-terminal sequencing, yet Thim et al.( Biochemistry 27:7785 1988) used amino acid analysis to quantify 11.0 mol of Gla/mol of protein for plasma-derived factor VII(a). We used mass spectrometry to map and validate Gla residues of coagulation factor VII(a) to potentially identify Gla residues outside of the Gla domain.MethodsFour sources of factor VII(a) (2 plasma-derived and 2 recombinant) were extracted, digested, and analyzed by tandem mass spectrometry (LC-MS/MS). Each sample was digested with trypsin and chymotrypsin to provide orthogonal coverage. Peptides derived from factor VII(a) proteolysis were analyzed on a nanoLC coupled to a quadrupole-Orbitrap-quadrupole linear ion trap mass spectrometer (Orbitrap Fusion Lumos Thermo Scientific). Multiple fragmentation methods were used to map and validate the sites including collisional based dissociation (CAD), higher energy collisional activated dissociation (HCD), electron transfer dissociation (ETD), and electron-transfer/higher-energy electron transfer activation (EThcD). Tandem MS spectra were collected at resolution 30K at 200 m/z, and data were processed using MaxQuant, COMPASS, and Proteome Discoverer. All identified sites were validated through manual annotation of spectra. We estimate that any site with >1% occupancy will be identified as Gla with this method.To validate select sites of novel gamma carboxylation, synthetic peptides were made for 4 different sites that were consistently identified in factor VII(a) from all 4 sources. Synthetic peptides were analyzed using MS methods described above, generating "true positives" to match with peptides identified from the factor VII(a) sources. Spectra from the synthetic peptides and factor VII from each source were compared using manual spectral annotation.ResultsIn addition to identifying known Gla residues at positions 6, 7, 19, 20, 29, and 35 of factor VII(a), we detected and validated 9 novel Gla residues outside of the N-terminal Gla domain. Novel sites include residues 94, 116, 132, 219, 215, 229, 265, 196 and 385. Four of these residues (210, 220, 296 and 385) were identified as Gla in all 4 sources of factor VII(a) and were validated with synthetic peptides using a combination of fragmentation methods, providing high confidence in their characterization. Published crystallographic data suggest that residues 210 and 220 of factor VIIa-tissue factor are closely approximated to a Ca2+ ion complexed to the C-terminal protease domain; this is not the case for residues 296 and 385. We continue to refine the technique to map the Gla residues (novel and known), and to quantify the fraction of factor VII(a) molecules from each source that contain the modification at each site, in order to better incorporate our data with established studies showing >90% occupancy at each of the 10 Gla domain sites. These data suggest there is room to expand our understanding of how carboxylation contributes to specific protein function, in order to provide more comprehensive understanding of this post-translational modification, and refine our understanding of hemostatic mechanisms. DisclosuresNo relevant conflicts of interest to declare.