DescribePROT in 2023: more, higher-quality and experimental annotations and improved data download options.

The DescribePROT database of amino acid-level descriptors of protein structures and functions was substantially expanded since its release in 2020. This expansion includes substantial increase in the size, scope, and quality of the underlying data, the addition of experimental structural information, the inclusion of new data download options, and an upgraded graphical interface. DescribePROT currently covers 19 structural and functional descriptors for proteins in 273 reference proteomes generated by 11 accurate and complementary predictive tools. Users can search our resource in multiple ways, interact with the data using the graphical interface, and download data at various scales including individual proteins, entire proteomes, and whole database. The annotations in DescribePROT are useful for a broad spectrum of studies that include investigations of protein structure and function, development and validation of predictive tools, and to support efforts in understanding molecular underpinnings of diseases and development of therapeutics. DescribePROT can be freely accessed at http://biomine.cs.vcu.edu/servers/DESCRIBEPROT/.

Exploring an Alternative Cysteine-Reactive Chemistry to Enable Proteome-Wide PPI Analysis by Cross-Linking Mass Spectrometry.

The development of MS-cleavable cross-linking mass spectrometry (XL-MS) has enabled the effective capture and identification of endogenous protein-protein interactions (PPIs) and their residue contacts at the global scale without cell engineering. So far, only lysine-reactive cross-linkers have been successfully applied for proteome-wide PPI profiling. However, lysine cross-linkers alone cannot uncover the complete PPI map in cells. Previously, we have developed a maleimide-based cysteine-reactive MS-cleavable cross-linker (bismaleimide sulfoxide (BMSO)) that is effective for mapping PPIs of protein complexes to yield interaction contacts complementary to lysine-reactive reagents. While successful, the hydrolysis and limited selectivity of maleimides at physiological pH make their applications in proteome-wide XL-MS challenging. To enable global PPI mapping, we have explored an alternative cysteine-labeling chemistry and thus designed and synthesized a sulfoxide-containing MS-cleavable haloacetamide-based cross-linker, Dibromoacetamide sulfoxide (DBrASO). Our results have demonstrated that DBrASO cross-linked peptides display the same fragmentation characteristics as other sulfoxide-containing MS-cleavable cross-linkers, permitting their unambiguous identification by MSn. In combination with a newly developed two-dimensional peptide fractionation method, we have successfully performed DBrASO-based XL-MS analysis of HEK293 cell lysates and demonstrated its capability to complement lysine-reactive reagents and expand PPI coverage at the systems-level.

Oxidant-Induced Bioconjugation for Protein Labeling in Live Cells.

Chemical proteomics is a powerful technology that can be used in the studies of the functions of uncharacterized proteins in the human proteome. It relies on a suitable bioconjugation strategy for protein labeling. This could be either a UV-responsive photo-crosslinker or an electrophilic warhead embedded in chemical probes that can form covalent bonds with target proteins. Here, we report a new protein-labeling strategy in which a nitrile oxide, a highly reactive intermediate that reacts with proteins, can be efficiently generated by the treatment of oximes with a water-soluble and a minimally toxic oxidant, phenyliodine bis (trifluoroacetate) (PIFA). The resulting intermediate can rapidly bioconjugate with amino acid residues of target proteins, thus enabling target identification of oxime-containing bioactive molecules. Excellent chemoselectivity of cysteine residues by the nitrile oxide was observed, and over 4000 reactive and/or accessible cysteines, including KRAS G12C, have been successfully characterized by quantitative chemical proteomics. Some of these residues could not be detected by conventional cysteine reagents, thus demonstrating the complementary utility of this method.

Network pharmacology analysis and molecular docking to unveil the potential mechanisms of San-Huang-Chai-Zhu formula treating cholestasis.

OBJECTIVE: Chinese medicine formulae possess the potential for cholestasis treatment. This study aimed to explore the underlying mechanisms of San-Huang-Chai-Zhu formula (SHCZF) against cholestasis. METHODS: The major chemical compounds of SHCZF were identified by high-performance liquid chromatography. The bioactive compounds and targets of SHCZF, and cholestasis-related targets were obtained from public databases. Intersected targets of SHCZF and cholestasis were visualized by Venn diagram. The protein-protein interaction and compound-target networks were established by Cytoscape according to the STRING database. The biological functions and pathways of potential targets were characterized by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis. The biological process-target-pathway network was constructed by Cytoscape. Finally, the interactions between biological compounds and hub target proteins were validated via molecular docking. RESULTS: There 7 major chemical compounds in SHCZF. A total of 141 bioactive compounds and 83 potential targets were screened for SHCZF against cholestasis. The process of SHCZF against cholestasis was mainly involved in AGE-RAGE signaling pathway in diabetic complications, fluid shear stress and atherosclerosis, and drug metabolism-cytochrome P450. ALB, IL6, AKT1, TP53, TNF, MAPK3, APOE, IL1B, PPARG, and PPARA were the top 10 hub targets. Molecular docking showed that bioactive compounds of SHCZF had a good binding affinity with hub targets. CONCLUSIONS: This study predicted that the mechanisms of SHCZF against cholestasis mainly involved in AGE-RAGE signaling pathway in diabetic complications, fluid shear stress and atherosclerosis, and drug metabolism-cytochrome P450. Moreover, APOE, AKT1, and TP53 were the critical hub targets for bioactive compounds of SHCZF.

Tandem mass tag-based quantitative proteomic analysis of the liver reveals potential protein targets of Xiaochaihutang in CUMS model of depression.

Depression is a global mental disorder disease and greatly threatened human health. Xiaochaihutang (XCHT) has been used successfully in treatment of depression for many years in China, but the mechanism is unclear. Using the chronic unpredictable mild stress (CUMS) mice model of depression, the present study aimed to reveal possible antidepressant mechanisms of XCHT from the perspective of liver by analyzing hepatic proteomics in mice. Bioinformatics analysis identified 31 differentially expressed proteins (DEPs), including 5 upregulated and 26 downregulated proteins, between the CUMS model and XCHT groups. The bile secretion pathway was found by KEGG pathway analysis of these DEPs. Four of the 31 differentially expressed proteins, including 2 active proteins involved in bile secretion, carbonic anhydrase 2 (CA2) and cystic fibrosis transmembrane conductance regulator (CFTR), were selected to verify their genes. Four genes (Cyp7a1, Fxr, Shp and Ntcp) related to bile acid synthesis and transport were further investigated by quantitative real-time polymerase chain reaction (qRT-PCR). Both biochemical tests and gene studies demonstrated that CUMS affected bile acid synthesis and transport, while XCHT regulated this pathway. The results indicated that there may be a potential relationship between CUMS induced depression and hepatic injury caused by increased bile acid, and also provide a novel insight to understand the underlying anti-depression mechanisms of XCHT.

Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV.

The emergence and global spread of SARS-CoV-2 has resulted in the urgent need for an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several individual omics studies have extended our knowledge of COVID-19 pathophysiology1-10. Integration of such datasets to obtain a holistic view of virus-host interactions and to define the pathogenic properties of SARS-CoV-2 is limited by the heterogeneity of the experimental systems. Here we report a concurrent multi-omics study of SARS-CoV-2 and SARS-CoV. Using state-of-the-art proteomics, we profiled the interactomes of both viruses, as well as their influence on the transcriptome, proteome, ubiquitinome and phosphoproteome of a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed crosstalk between the perturbations taking place upon infection with SARS-CoV-2 and SARS-CoV at different levels and enabled identification of distinct and common molecular mechanisms of these closely related coronaviruses. The TGF-ß pathway, known for its involvement in tissue fibrosis, was specifically dysregulated by SARS-CoV-2 ORF8 and autophagy was specifically dysregulated by SARS-CoV-2 ORF3. The extensive dataset (available at https://covinet.innatelab.org ) highlights many hotspots that could be targeted by existing drugs and may be used to guide rational design of virus- and host-directed therapies, which we exemplify by identifying inhibitors of kinases and matrix metalloproteases with potent antiviral effects against SARS-CoV-2.

DescribePROT: database of amino acid-level protein structure and function predictions.

We present DescribePROT, the database of predicted amino acid-level descriptors of structure and function of proteins. DescribePROT delivers a comprehensive collection of 13 complementary descriptors predicted using 10 popular and accurate algorithms for 83 complete proteomes that cover key model organisms. The current version includes 7.8 billion predictions for close to 600 million amino acids in 1.4 million proteins. The descriptors encompass sequence conservation, position specific scoring matrix, secondary structure, solvent accessibility, intrinsic disorder, disordered linkers, signal peptides, MoRFs and interactions with proteins, DNA and RNAs. Users can search DescribePROT by the amino acid sequence and the UniProt accession number and entry name. The pre-computed results are made available instantaneously. The predictions can be accesses via an interactive graphical interface that allows simultaneous analysis of multiple descriptors and can be also downloaded in structured formats at the protein, proteome and whole database scale. The putative annotations included by DescriPROT are useful for a broad range of studies, including: investigations of protein function, applied projects focusing on therapeutics and diseases, and in the development of predictors for other protein sequence descriptors. Future releases will expand the coverage of DescribePROT. DescribePROT can be accessed at http://biomine.cs.vcu.edu/servers/DESCRIBEPROT/.

Differences in cryostimulation and sauna effects on post-exercise changes in blood serum of athletes.

OBJECTIVES: There is a growing body of evidence supporting the role of whole-body cryostimulation (WBC) and sauna - bathing as treatments for relaxation, mental well-being and several health problems. Despite their polar opposite temperatures, both of these treatments come with a dose of similar health benefits. This study is designed to compare effects of WBC and sauna application on the athletes' response to exercise. DESIGN: The blood samples were collected from 10 professional cross-country skiers at four stages: before exercise, after exercise, at 1-h recovery and after 24 h of rest in sessions before and after 10 thermal treatments. Differential scanning calorimetry (DSC) was used to examine the process of serum denaturation. The parameters of endothermic transition were compared at various stages of each exercise session. RESULTS: Post-exercise changes in DSC profiles of athlete's blood serum are similar in character but clearly stronger in the session held after sauna treatments and slightly weaker after WBC than those in the session not preceded by treatments. These changes can be, at least in part, explained by the exercise induced increase in the concentration of oxidized albumin. A return of serum denaturation transition to pre-exercise shape has been observed within a few hours of rest. It suggests relatively quick restoration of a fraction of non-oxidized albumin molecules during the recovery period. CONCLUSIONS: An exercise performed by athletes after a series of sauna treatments leads to temporary greater modification of the blood serum proteome than the similar exercise during the session preceded by WBC treatments.

Proteomic Characterisation of Lupin (Lupinus angustifolius) Milk as Influenced by Extraction Techniques, Seed Coat and Cultivars.

Lupin seeds are rich in proteins and other essential ingredients that can help to improve human health. The protein contents in both whole and split seeds of two lupin cultivars (Mandleup and PBA Jurien) were used to produce the lupin milk using the cheesecloth and centrifuge method. Proteins were extracted from the lupin milk using thiourea/urea solubilization. The proteins were separated by a two-dimensional polyacrylamide gel electrophoresis and then identified with mass spectrometry. A total of 230 protein spots were identified, 60 of which showed differential abundances. The cheesecloth separation showed protein extractability much better than that of the centrifuge method for both the cultivars. The results from this study could offer guidance for future comparative analysis and identification of lupin milk protein and provide effective separation technique to determine specific proteins in the cheese-making process.

[A Qualitative Proteome-Wide Lysine Succinylation Profiling of Tea Revealed its Involvement in Primary Metabolism].

Lysine succinylation of proteins has potential impacts on protein structure and function, which occurs on post-translation level. However, the information about the succinylation of proteins in tea plants is limited. In the present study, the significant signal of succinylation in tea plants was found by western blot. Subsequently, we performed a qualitative analysis to globally identify the lysine succinylation of proteins using high accuracy nano LC-MS/MS combined with affinity purification. As a result, a total of 142 lysine succinylation sites were identified on 86 proteins in tea leaves. The identified succinylated proteins were involved in various biological processes and a large proportion of the succinylation sites were presented on proteins in the primary metabolism, including glyoxylate and dicarboxylate metabolism, TCA cycle and glycine, serine and threonine metabolism. Moreover, 10 new succinylation sites were detected on histones in tea leaves. The results suggest that succinylated proteins in tea plants might play critical regulatory roles in biological processes, especially in the primary metabolism. This study not only comprehensively analyzed the lysine succinylome in tea plants, but also provided valuable information for further investigating the functions of lysine succinylation in tea plants.

The proteomic profiling of multiple tissue damage in chickens for a selenium deficiency biomarker discovery.

Over the past decades, substantial advances have been made in both the early diagnosis and accurate prognosis of numerous cancers because of the impressive development of novel proteomic strategies. Selenium (Se) is an essential trace element in humans and animals. Se deficiency could lead to Keshan disease in humans, mulberry heart disease in pigs and damage of tissues including cardiac injury, apoptosis in the liver, reduction in the immune responses in spleen and cerebral lesions in chickens. However, it is well know that plasma biomarkers are not specific and also show alterations in various diseases including those caused by Se deficiency. Therefore, new definition biomarkers are needed to improve disease surveillance and reduce unnecessary chicken losses due to Se deficiency. To identify new biomarkers for Se deficiency, we performed exploratory heart, liver, spleen, muscle, vein, and artery proteomic screens to further validate the biomarkers using Venn analysis, GO enrichment, heatmap analysis, and IPA analysis. Based on the bioinformatics methods mentioned above, we found that differentially expressed genes and proteins are enriched to the PI3K/AKT/mTOR signal pathway and insulin pathway. We further used western blot to detect the expression of proteins related to the two pathways. Results showed that the components of the PI3K/AKT/mTOR signal pathway were definitely decreased in heart, liver, spleen, muscle, vein and artery tissues in the Se deficient group. Expression IGF and IGFBP2 of the insulin pathway were differentially increased in the heart, liver, and spleen in Se deficient group samples and decreased in muscle and artery. In conclusion, 5 proteins, namely PI3K, AKT, mTOR, IGF, and IGFBP2, were differentially expressed, which could be potentially useful Se deficient biomarkers. In the present study, proteomic profiling was used to elucidate protein biomarkers that distinguished Se deficient samples from the controls, which might provide a new direction for the diagnosis and targeted treatment induced by Se deficiency in chickens.

Bioactive proteins in bovine colostrum and effects of heating, drying and irradiation.

Bovine colostrum (BC) contains bioactive proteins, such as immunoglobulin G (IgG), lactoferrin (LF) and lactoperoxidase (LP). BC was subjected to low-temperature, long-time pasteurization (LTLT, 63 °C, 30 min) or high-temperature, short-time pasteurization (HTST, 72 °C, 15 s) and spray-drying (SD), with or without γ-irradiation (GI, ∼14 kGy) to remove microbial contamination. Relative to unpasteurized liquid BC, SD plus GI increased protein denaturation by 6 and 11%, respectively, increasing to 19 and 27% after LTLT and to 48% after HTST, with no further effects after GI (all P < 0.05). LTLT, without or with GI, resulted in 15 or 29% denaturation of IgG, compared with non-pasteurized BC, and 34 or 58% for HTST treatment (all P < 0.05, except LTLT without GI). For IgG, only GI, not SD or LTLT, increased denaturation (30-38%, P < 0.05) but HTST increased denaturation to 40%, with further increases after GI (60%, P < 0.05). LTLT and HTST reduced LP levels (56 and 81% respectively) and LTLT reduced LF levels (21%), especially together with GI (47%, P < 0.05). Denaturation of BSA, ß-LgA, ß-LgB and α-La were similar to IgG. Methionine, a protective amino acid against free oxygen radicals, was oxidised by LTLT + GI (P < 0.05) while LTLT and HTST had no effect. Many anti-inflammatory proteins, including serpin anti-proteinases were highly sensitive to HTST and GI but preserved after LTLT pasteurization. LTLT, followed by SD is an optimal processing technique preserving bioactive proteins when powdered BC is used as a diet supplement for sensitive patients.

Phosphoproteomics of Acute Cell Stressors Targeting Exercise Signaling Networks Reveal Drug Interactions Regulating Protein Secretion.

Exercise engages signaling networks to control the release of circulating factors beneficial to health. However, the nature of these networks remains undefined. Using high-throughput phosphoproteomics, we quantify 20,249 phosphorylation sites in skeletal muscle-like myotube cells and monitor their responses to a panel of cell stressors targeting aspects of exercise signaling in vivo. Integrating these in-depth phosphoproteomes with the phosphoproteome of acute aerobic exercise in human skeletal muscle suggests that co-administration of ß-adrenergic and calcium agonists would activate complementary signaling relevant to this exercise context. The phosphoproteome of cells treated with this combination reveals a surprising divergence in signaling from the individual treatments. Remarkably, only the combination treatment promotes multisite phosphorylation of SERBP1, a regulator of Serpine1 mRNA stability, a pro-fibrotic secreted protein. Secretome analysis reveals that the combined treatments decrease secretion of SERPINE1 and other deleterious factors. This study provides a framework for dissecting phosphorylation-based signaling relevant to acute exercise.

Proteomic Sample Preparation through Extraction by Unspecific Adsorption on Silica Beads for ArgC-like Digestion.

Sample preparation for mass-spectrometry-based proteomic analyses usually requires intricate, multistep workflows that are often limited in capacity or suffer from sample loss. Here, we introduce a lean adsorption-based protocol (ABP) for the extraction of proteins from fresh cell lysates that enables us to modify and tag protein samples under harsh conditions, such as organic solvents, high salt concentrations, or low pH values. This offers high versatility while also reducing the required steps in the preparation process significantly. Protein identifications are slightly increased compared to traditional acetone precipitation followed by an in-solution digestion (AP/IS) or filter aided sample preparation (FASP) and proved complementary to both methods regarding proteome coverage. When combined with ArgC-like digestion, this approach delivered 5386 uniquely identified proteins, a substantial increase of 18.27% over tryptic digestion (4554), while decreasing spectra complexity due to a lower number of peptide to spectra matches per protein and the number of missed cleaved peptides. In addition, an increased number of identified membrane proteins and histones as well as improved fragmentation and intensity coverage were observed through comprehensive data analysis.

Ginsentides: Cysteine and Glycine-rich Peptides from the Ginseng Family with Unusual Disulfide Connectivity.

Ginseng, a popular and valuable traditional medicine, has been used for centuries to maintain health and treat disease. Here we report the discovery and characterization of ginsentides, a novel family of cysteine and glycine-rich peptides derived from the three most widely-used ginseng species: Panax ginseng, Panax quinquefolius, and Panax notoginseng. Using proteomic and transcriptomic methods, we identified 14 ginsentides, TP1-TP14 which consist of 31-33 amino acids and whose expression profiles are species- and tissues-dependent. Ginsentides have an eight-cysteine motif typical of the eight-cysteine-hevein-like peptides (8C-HLP) commonly found in medicinal herbs, but lack a chitin-binding domain. Transcriptomic analysis showed that the three-domain biosynthetic precursors of ginsentides differ from known 8C-HLP precursors in architecture and the absence of a C-terminal protein-cargo domain. A database search revealed an additional 50 ginsentide-like precursors from both gymnosperms and angiosperms. Disulfide mapping and structure determination of the ginsentide TP1 revealed a novel disulfide connectivity that differs from the 8C-HLPs. The structure of ginsentide TP1 is highly compact, with the N- and C-termini topologically fixed by disulfide bonds to form a pseudocyclic structure that confers resistance to heat, proteolysis, and acid and serum-mediated degradation. Together, our results expand the chemical space of natural products found in ginseng and highlight the occurrence, distribution, disulfide connectivity, and precursor architectures of cysteine- and glycine-rich ginsentides as a class of novel non-chitin-binding, non-cargo-carrying 8C-HLPs.

Combined Proteome and Toxicology Approach Reveals the Lethality of Venom Toxins from Jellyfish Cyanea nozakii.

Jellyfish are a type of poisonous cnidarian invertebrate that secrete lethal venom for predation or defense. Human beings often become victims of jellyfish stings accidentally while swimming or fishing and suffer severe pain, itching, swelling, inflammation, shock, and even death. Jellyfish venom is composed of various toxins, and the lethal toxin is the most toxic and hazardous component of the venom, which is responsible for deaths caused by jellyfish stings and envenomation. Our previous study revealed many toxins in jellyfish venom, including phospholipase A2, metalloproteinase, and protease inhibitors. However, it is still unknown which type of toxin is lethal and how it works. Herein a combined toxicology analysis, proteome strategy, and purification approach was employed to investigate the lethality of the venom of the jellyfish Cyanea nozakii. Toxicity analysis revealed that cardiotoxicity including acute myocardial infarction and a significant decrease in both heart rate and blood pressure is the primary cause of death. Purified lethal toxin containing a fraction of jellyfish venom was subsequently subjected to proteome analysis and bioinformation analysis. A total of 316 and 374 homologous proteins were identified, including phospholipase A2-like toxins and metalloprotease-like toxins. Furthermore, we confirmed that the lethality of the jellyfish venom is related to metalloproteinase activity but without any phospholipase A2 activity or hemolytic activity. Altogether, this study not only provides a comprehensive understanding of the lethal mechanism of jellyfish venom but also provides very useful information for the therapeutic or rescue strategy for severe jellyfish stings.

Novel low-abundance allergens from mango via combinatorial peptide libraries treatment: A proteomics study.

Mango allergy is a rare condition, which may cause severe hypersensitivity reactions, such as anaphylaxis, angioedema, asthma and contact dermatitis. By exploiting the combinatorial peptide ligand library (CPLL) technology, mango proteomes have been extracted and the presence of traces of allergens assessed via Western blot analysis two-dimensional maps. Upon reactive spot elution and mass spectrometry analyses, four major mango allergens could be identified for the first time and shown to be in common with three of the five known banana species. These allergens include: Mus a 1, Mus a 2 and Mus a 5. Additional mango allergens detected do not seem to be in common with the banana species. In particular, a pectinesterase and a superoxide dismutase, both widely described as allergens, could be identified in mango extracts. Conversely, plain mango extracts not treated with CPLLs did not exhibit any reactive spots in Western blot analysis.

Manganese is a Deinococcus radiodurans growth limiting factor in rich culture medium.

To understand the effects triggered by Mn2+ on Deinococcus radiodurans, the proteome patterns associated with different growth phases were investigated. In particular, under physiological conditions we tested the growth rate and the biomass yield of D. radiodurans cultured in rich medium supplemented or not with MnCl2. The addition of 2.5-5.0 µM MnCl2 to the medium neither altered the growth rate nor the lag phase, but significantly increased the biomass yield. When higher MnCl2 concentrations were used (10-250 µM), biomass was again found to be positively affected, although we did observe a concentration-dependent lag phase increase. The in vivo concentration of Mn2+ was determined in cells grown in rich medium supplemented or not with 5 µM MnCl2. By atomic absorption spectroscopy, we estimated 0.2 and 0.75 mM Mn2+ concentrations in cells grown in control and enriched medium, respectively. We qualitatively confirmed this observation using a fluorescent turn-on sensor designed to selectively detect Mn2+in vivo. Finally, we investigated the proteome composition of cells grown for 15 or 19 h in medium to which 5 µM MnCl2 was added, and we compared these proteomes with those of cells grown in the control medium. The presence of 5 µM MnCl2 in the culture medium was found to alter the pI of some proteins, suggesting that manganese affects post-translational modifications. Further, we observed that Mn2+ represses enzymes linked to nucleotide recycling, and triggers overexpression of proteases and enzymes linked to the metabolism of amino acids.

Phosphorus-Based Probes as Molecular Tools for Proteome Studies: Recent Advances in Probe Development and Applications.

Studies on the human proteome have engaged diverse techniques; however, none of them represent a predominant approach. Chemical biology has made a major contribution to our understanding of human biology, stimulating the generation of biological hypotheses. Tools such as functional probes have advanced studies on biological mechanisms and helped in elucidating off-target reactivity and potential toxicities of drugs and drug candidates. Here, we accentuate the recent developments in the design and applications of phosph(on)ate-containing probes. Phosphate esters and anhydrides are present in a number of vital cell constituents, and their significance can be reflected by a number of biological processes that involve phosphorus-bearing molecules. We discuss the use of phosph(on)ate-derived probes for (1) the identification of phosphate-requiring enzymes, their substrates, interacting partners; (2) developing screening assays; and (3) their potential as diagnostics. Limitations that as yet need to be overcome and possible measures to be undertaken will also be addressed.