Valuable Contributions and Lessons Learned from Proteomics and Metabolomics Studies of COVID-19.

The COVID-19 pandemic caused by the SARS-CoV-2 virus infected more than 775,686,716 humans and was responsible for the death of more than 7,054,093 individuals. COVID-19 has taught us that the development of vaccines, repurposing of drugs, and understanding the mechanism of a disease can be done within a short time. The COVID-19 proteomics and metabolomics has contributed to its diagnosis, understanding of its progression, host-virus interaction, disease mechanism, and also in the search of suitable anti-COVID therapeutics. Mass spectrometry based proteomics was used to find the potential biomarkers of different stages of COVID-19 including severe and nonsevere cases in the blood serum. Notably, protein-protein interaction techniques to understand host-virus interactions were also significantly useful. The single-cell proteomics studies were carried out to ascertain the changes in immune cell composition and its activation in mild COVID-19 patients versus severe COVID-19 patients using whole-blood and peripheral-blood mononuclear cells. Modern technologies were helpful to deal with the pandemic; however, there is still scope for further development. Further, attempts were made to understand the protein-protein, metabolite-metabolite, and protein-metabolite interactomes, derived from proteins and metabolite fingerprints of COVID-19 patients by reanalysis of COVID-19 public mass spectrometry based proteomics and metabolomics studies. Further, some of these interactions were supported by the literature as validations in the COVID-19 studies.

Proteins and metabolites fingerprints of gestational diabetes mellitus forming protein-metabolite interactomes are its potential biomarkers.

Gestational diabetes mellitus (GDM) is a consequence of glucose intolerance with an inadequate production of insulin that happens during pregnancy and leads to adverse health consequences for both mother and fetus. GDM patients are at higher risk for preeclampsia, and developing diabetes mellitus type 2 in later life, while the child born to GDM mothers are more prone to macrosomia, and hypoglycemia. The universally accepted diagnostic criteria for GDM are lacking, therefore there is a need for a diagnosis of GDM that can identify GDM at its early stage (first trimester). We have reviewed the literature on proteins and metabolites fingerprints of GDM. Further, we have performed protein-protein, metabolite-metabolite, and protein-metabolite interaction network studies on GDM proteins and metabolites fingerprints. Notably, some proteins and metabolites fingerprints are forming strong interaction networks at high confidence scores. Therefore, we have suggested that those proteins and metabolites that are forming protein-metabolite interactomes are the potential biomarkers of GDM. The protein-metabolite biomarkers interactome may help in a deep understanding of the prognosis, pathogenesis of GDM, and also detection of GDM. The protein-metabolites interactome may be further applied in planning future therapeutic strategies to promote long-term health benefits in GDM mothers and their children.

An insight into major signaling pathways and protein-protein interaction networks involved in the pathogenesis of gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is associated with the increase of glucose in the blood rather than being absorbed by the cells. A better understanding of the signaling pathways is necessary to understand the pathophysiology of GDM. This study provides details about a series of signaling pathways and protein-protein interactions involved in the pathogenesis of GDM and their evaluations in GDM development. Protein-protein interactions were found between proteins of several signaling pathways that suggest interlink between these signaling pathways. Protein-protein interactions were generated with high confidence interaction scores based on textmining, cooccurrence, coexpression, neighborhood, gene fusion, experiments, and databases. The dysregulation of signaling pathways may also contribute to the increased risk of complications associated with GDM in the mother and child. Further, studies on signaling pathways involved in the pathogenesis of GDM would help in the development of an effective intervention to prevent GDM along with the identification of key targets for effective therapies in the future.

Comparison of Nuclear Matrix and Mitotic Chromosome Scaffold Proteins in Drosophila S2 Cells-Transmission of Hallmarks of Nuclear Organization Through Mitosis.

Chromatin condenses several folds to form mitotic chromosomes during cell division and decondenses post-mitotically to reoccupy their nuclear territory and regain their specific transcriptional profile in a precisely lineage specific manner. This necessitates that the features of nuclear architecture and DNA topology persist through mitosis. We compared the proteome of nuclease and high salt resistant fraction of interphase nucleus known as nuclear matrix (NuMat) and an equivalent biochemical fraction in the mitotic chromosome known as mitotic chromosome scaffold (MiCS). Our study elucidates that as much as 67% of the NuMat proteins are retained in the MiCS indicating that the features of nuclear architecture in interphase nucleus are retained on the mitotic chromosomes. Proteins of the NuMat/MiCS have large dynamic range of MS signal and were detected in sub-femtomolar amounts. Chromatin/RNA binding proteins with hydrolase and helicase activity are highly enriched in NuMat as well as MiCS. Although several transcription factors involved in functioning of interphase nucleus are present exclusively in NuMat, protein components responsible for assembly of membrane-less nuclear bodies are uniquely retained in MiCS. Our study clearly indicates that the features of nuclear architecture, in the structural context of NuMat, are retained in MiCS and possibly play an important role in maintenance of cell lineage specific transcriptional status during cell division and thereby, serve as components of cellular memory.

An Insight into Powdery Mildew-Infected, Susceptible, Resistant, and Immune Sunflower Genotypes.

Powdery mildew (PM, caused by Golovinomyces orontii) is one of the major diseases on sunflower that causes severe yield losses in the tropics. Sources of resistance to PM are reported in an exotic accession and some wild Helianthus species. The present study aims at quantitative proteomic analysis of susceptible, resistant, and immune genotypes of sunflower in response to PM infection at 3, 7, 10 days post infection. The majority of differentially expressed proteins in the resistant genotype belonged to oxidative stress (catalase, ATP-sulfurylase, and formate dehydrogenase), defense (HSP-70, heat shock transcription factors), and photosynthesis (LHCB3). In case of immune genotype, 50% of proteins are related to photosynthesis, which play a key role in plant immunity, whereas a few similar proteins are also expressed in the susceptible genotype, but in their reduced abundance besides being inadequate in timing of expression probably leading to its susceptibility to PM. KEGG enrichment analysis shows that carbon metabolism (6-phosphogluconate dehydrogenase, pyruvate dehydrogenase, glutamine synthetase), photosynthesis, and plant-pathogen protein pathways are key pathways governing the resistance. The transcriptional expression of eight of nine differentially expressed proteins are in agreement with the expression of proteins at the corresponding time. The present study provides information on the key proteins that are upregulated in resistant and immune genotypes which restrict the disease progression and constitutes the first quantitative proteomic data of sunflower-PM infection process.

Investigation of post-translational modifications in type 2 diabetes.

The investigation of post-translational modifications (PTMs) plays an important role for the study of type 2 diabetes. The importance of PTMs has been realized with the advancement of analytical techniques. The challenging detection and analysis of post-translational modifications is eased by different enrichment methods and by high throughput mass spectrometry based proteomics studies. This technology along with different quantitation methods provide accurate knowledge about the changes happening in disease conditions as well as in normal conditions. In this review, we have discussed PTMs such as phosphorylation, N-glycosylation, O-GlcNAcylation, acetylation and advanced glycation end products in type 2 diabetes which have been characterized by high throughput mass spectrometry based proteomics analysis.

Proteomics in India: A Report on a Brainstorming Meeting at Hyderabad, India.

The Centre for Cellular and Molecular Biology, Hyderabad, India, was host for an international forum, or "brainstorming meeting," on proteomics held in November 2014, which provided the opportunity to showcase proteomic science in India and to allow discussions between Indian scientists and students and several international visitors. This provided an amalgamation of speakers and participants whose interests lay mainly in developing and using mass-spectrometry-based proteomics to advance their research work. A week-long workshop with hands-on training in proteomic methodology followed the meeting.

Identification of Components of the SUMOylation Machinery in Candida glabrata: ROLE OF THE DESUMOYLATION PEPTIDASE CgUlp2 IN VIRULENCE.

Regulation of protein function by reversible post-translational modification, SUMOylation, is widely conserved in the eukaryotic kingdom. SUMOylation is essential for cell growth, division, and adaptation to stress in most organisms, including fungi. As these are key factors in determination of fungal virulence, in this study, we have investigated the importance of SUMOylation in the human pathogen, Candida glabrata We identified the enzymes involved in small ubiquitin-like modifier conjugation and show that there is strong conservation between Saccharomyces cerevisiae and C. glabrata We demonstrate that SUMOylation is an essential process and that adaptation to stress involves changes in global SUMOylation in C. glabrata Importantly, loss of the deSUMOylating enzyme CgUlp2 leads to highly reduced small ubiquitin-like modifier protein levels, and impaired growth, sensitivity to multiple stress conditions, reduced adherence to epithelial cells, and poor colonization of specific tissues in mice. Our study thus demonstrates a key role for protein SUMOylation in the life cycle and pathobiology of C. glabrata.

Withania somnifera reverses Alzheimer's disease pathology by enhancing low-density lipoprotein receptor-related protein in liver.

A 30-d course of oral administration of a semipurified extract of the root of Withania somnifera consisting predominantly of withanolides and withanosides reversed behavioral deficits, plaque pathology, accumulation of ß-amyloid peptides (Aß) and oligomers in the brains of middle-aged and old APP/PS1 Alzheimer's disease transgenic mice. It was similarly effective in reversing behavioral deficits and plaque load in APPSwInd mice (line J20). The temporal sequence involved an increase in plasma Aß and a decrease in brain Aß monomer after 7 d, indicating increased transport of Aß from the brain to the periphery. Enhanced expression of low-density lipoprotein receptor-related protein (LRP) in brain microvessels and the Aß-degrading protease neprilysin (NEP) occurred 14-21 d after a substantial decrease in brain Aß levels. However, significant increase in liver LRP and NEP occurred much earlier, at 7 d, and were accompanied by a rise in plasma sLRP, a peripheral sink for brain Aß. In WT mice, the extract induced liver, but not brain, LRP and NEP and decreased plasma and brain Aß, indicating that increase in liver LRP and sLRP occurring independent of Aß concentration could result in clearance of Aß. Selective down-regulation of liver LRP, but not NEP, abrogated the therapeutic effects of the extract. The remarkable therapeutic effect of W. somnifera mediated through up-regulation of liver LRP indicates that targeting the periphery offers a unique mechanism for Aß clearance and reverses the behavioral deficits and pathology seen in Alzheimer's disease models.

miRNA-protein-metabolite interaction network reveals the regulatory network and players of pregnancy regulation in dairy cows.

Pregnancy is a complex process involving complex molecular interaction networks, such as between miRNA-protein, protein-protein, metabolite-metabolite, and protein-metabolite interactions. Advances in technology have led to the identification of many pregnancy-associated microRNA (miRNA), protein, and metabolite fingerprints in dairy cows. An array of miRNA, protein, and metabolite fingerprints produced during the early pregnancy of dairy cows were described. We have found the in silico interaction networks between miRNA-protein, protein-protein, metabolite-metabolite, and protein-metabolite. We have manually constructed miRNA-protein-metabolite interaction networks such as bta-miR-423-3p-IGFBP2-PGF2α interactomes. This interactome is obtained by manually combining the interaction network formed between bta-miR-423-3p-IGFBP2 and the interaction network between IGFBP2-PGF2α with IGFBP2 as a common interactor with bta-miR-423-3p and PGF2α with the provided sources of evidence. The interaction between bta-miR-423-3p and IGFBP2 has many sources of evidence including a high miRanda score of 169, minimum free energy (MFE) score of -25.14, binding probability (p) of 1, and energy of -25.5. The interaction between IGFBP2 and PGF2α occurs at high confidence scores (≥0.7 or 70%). Interestingly, PGF2α is also found to interact with different metabolites, such as PGF2α-PGD2, PGF2α-thromboxane B2, PGF2α-PGE2, and PGF2α-6-keto-PGF1α at high confidence scores (≥0.7 or 70%). Furthermore, the interactions between C3-PGE2, C3-PGD2, PGE2-PGD2, PGD2-thromboxane B2, PGE2-thromboxane B2, 6-keto-PGF1α-thromboxane B2, and PGE2-6-keto-PGF1α were also obtained at high confidence scores (≥0.7 or 70%). Therefore, we propose that miRNA-protein-metabolite interactomes involving miRNA, protein, and metabolite fingerprints of early pregnancy of dairy cows such as bta-miR-423-3p, IGFBP2, PGF2α, PGD2, C3, PGE2, 6-keto-PGF1 alpha, and thromboxane B2 may form the key regulatory networks and players of pregnancy regulation in dairy cows. This is the first study involving miRNA-protein-metabolite interactomes obtained in the early pregnancy stage of dairy cows.

Hypothalamic protein profiling from mice subjected to social defeat stress.

The Hypothalmic-Pituitary-Adrenal axis also known as the HPA axis is central to stress response. It also acts as the relay center between the body and the brain. We analysed hypothalamic proteome from mice subjected to chronic social defeat paradigm using iTRAQ based quantitative proteomics to identify changes associated with stress response. We identified greater than 2000 proteins after processing our samples analysed through Q-Exactive (Thermo) and Orbitrap Velos (Thermo) at 5% FDR. Analysis of data procured from the runs showed that the proteins whose levels were affected belonged primarily to mitochondrial and metabolic processes, translation, complement pathway among others. We also found increased levels of fibrinogen, myelin basic protein (MBP) and neurofilaments (NEFL, NEFM, NEFH) in the hypothalamus from socially defeated mice. Interestingly, research indicates that these proteins are upregulated in blood and CSF of subjects exposed to trauma and stress. Since hypothalamus secreted proteins can be found in blood and CSF, their utility as biomarkers in depression holds an impressive probability and should be validated in clinical samples.

Deep and highly sensitive proteome coverage by LC-MS/MS without prefractionation.

In-depth MS-based proteomics has necessitated fractionation of either proteins or peptides or both, often requiring considerable analysis time. Here we employ long liquid chromatography runs with high resolution coupled to an instrument with fast sequencing speed to investigate how much of the proteome is directly accessible to liquid chromatography-tandem MS characterization without any prefractionation steps. Triplicate single-run analyses identified 2990 yeast proteins, 68% of the total measured in a comprehensive yeast proteome. Among them, we covered the enzymes of the glycolysis and gluconeogenesis pathway targeted in a recent multiple reaction monitoring study. In a mammalian cell line, we identified 5376 proteins in a triplicate run, including representatives of 173 out of 200 KEGG metabolic and signaling pathways. Remarkably, the majority of proteins could be detected in the samples at sub-femtomole amounts and many in the low attomole range, in agreement with absolute abundance estimation done in previous works (Picotti et al. Cell, 138, 795-806, 2009). Our results imply an unexpectedly large dynamic range of the MS signal and sensitivity for liquid chromatography-tandem MS alone. With further development, single-run analysis has the potential to radically simplify many proteomic studies while maintaining a systems-wide view of the proteome.

Role of 16S ribosomal RNA methylations in translation initiation in Escherichia coli.

Translation initiation from the ribosomal P-site is the specialty of the initiator tRNAs (tRNA(fMet)). Presence of the three consecutive G-C base pairs (G29-C41, G30-C40 and G31-C39) in their anticodon stems, a highly conserved feature of the initiator tRNAs across the three kingdoms of life, has been implicated in their preferential binding to the P-site. How this feature is exploited by ribosomes has remained unclear. Using a genetic screen, we have isolated an Escherichia coli strain, carrying a G122D mutation in folD, which allows initiation with the tRNA(fMet) containing mutations in one, two or all the three G-C base pairs. The strain shows a severe deficiency of methionine and S-adenosylmethionine, and lacks nucleoside methylations in rRNA. Targeted mutations in the methyltransferase genes have revealed a connection between the rRNA modifications and the fundamental process of the initiator tRNA selection by the ribosome.

Remdesivir and Its Combination With Repurposed Drugs as COVID-19 Therapeutics.

The SARS-CoV-2 virus needs multiple copies for its multiplication using an enzyme RNA-dependent RNA polymerase (RdRp). Remdesivir inhibits viral RdRp, controls the multiplication of the virus, and protects patients. However, treatment of COVID-19 with remdesivir involves adverse effects. Many ongoing clinical trials are exploring the potential of the combination of remdesivir with repurposed drugs by targeting multiple targets of virus and host human simultaneously. Better results were obtained with the remdesivir-baricitinib combination treatment for COVID-19 compared to the treatment with remdesivir alone. Notably, recovery from COVID-19 was found to be 8 days less via the remdesivir-baricitinib combination treatment as compared to remdesivir treatment alone. Furthermore, the mortality rate via the remdesivir-baricitinib combination treatment was lower compared to the remdesivir-only treatment. Remdesivir targets the SARS-CoV-2 enzyme while baricitinib targets the host human enzyme. Simultaneously, remdesivir and baricitinib as a combination inhibit their target viral RdRp and human Janus kinase, respectively. Ongoing trials for the combination of drugs will suggest in the future whether they may reduce the recovery time, reduce the mortality rate, and improve patient clinical status for noninvasive ventilation. In the future, simultaneously targeting virus replication enzymes and host human kinases may be the strategy for SARS-CoV-2 therapeutics.

SARS-CoV-2 Infection Triggers Phosphorylation: Potential Target for Anti-COVID-19 Therapeutics.

The SARS-CoV-2 infection triggers host kinases and is responsible for heavy phosphorylation in the host and also in the virus. Notably, phosphorylations in virus were achieved using the host enzyme for its better survival and further mutations. We have attempted to study and understand the changes that happened in phosphorylation during and post SARS-CoV-2 infection. There were about 70 phosphorylation sites detected in SARS-CoV-2 viral proteins including N, M, S, 3a, and 9b. Furthermore, more than 15,000 host phosphorylation sites were observed in SARS-CoV-2-infected cells. SARS-CoV-2 affects several kinases including CMGC, CK2, CDK, PKC, PIKFYVE, and EIF2AK2. Furthermore, SARS-CoV-2 regulates various signaling pathways including MAPK, GFR signaling, TGF-ß, autophagy, and AKT. These elevated kinases and signaling pathways can be potential therapeutic targets for anti-COVID-19 drug discovery. Specific inhibitors of these kinases and interconnected signaling proteins have great potential to cure COVID-19 patients and slow down the ongoing COVID-19 pandemic.

Diverse vaccine platforms safeguarding against SARS-CoV-2 and its variants.

INTRODUCTION: Appearances of SARS-CoV-2 variants have created havoc and additional challenges for the ongoing vaccination drive against pandemic COVID-19. Interestingly, several vaccine platforms are showing great potential to produce successful vaccines against SARS-CoV-2 and its variants. Billions of COVID-19 vaccine doses have been administered worldwide. Mix-and-Match COVID-19 vaccines involving the mixing of the same platform vaccines and also two different vaccine platforms may provide greater protection against SARS-CoV-2 and its variants. COVID-19 vaccines have become one of the most important tools to mitigate the ongoing pandemic COVID-19. AREAS COVERED: We describe SARS-Cov-2 variants, their impact on the population, COVID-19 vaccines, diverse vaccine platforms, doses of vaccines, the efficacy of vaccines against SARS-CoV-2 and its variants, mitigation of the COVID-19 transmission- alternatives to vaccines. EXPERT OPINION: Diverse vaccine platforms may safeguard against ongoing, deadly SARS-CoV-2 and its infectious variants. The efficacies of COVID-19 vaccines are significantly high after the administration of the second dose. Further, it protects individuals including vulnerable patients with co-morbidities from SARS-CoV-2 and its variants. The hospitalizations and deaths of the individuals may be prevented by COVID-19 vaccines.

ACE2 as a potential therapeutic target for pandemic COVID-19.

SARS-CoV-2 virus invades the host through angiotensin-converting enzyme 2 (ACE2) receptors by decreasing the ACE2 expression of the host. This disturbs the dynamic equilibrium between the ACE/Ang II/AT1R axis and ACE2/Ang (1-7)/Mas receptor axis. Therefore, the clinically approved drugs belonging to (i) angiotensin converting enzyme (ACE) inhibitors such as captopril, and enalaprilat, (ii) angiotensin-receptor blockers (ARBs) such as losartan, candesartan, olmesartan, azilsartan, irbesartan, and telmisartan and (iii) the combination of ACE inhibitors and ARBs such as losartan with lisinopril and captopril with losartan, and (iv) recombinant ACE2, were studied for their ability to activate ACE2 in different medical conditions including hypertension, inflammation, cardiovascular, renal and lung diseases. These clinically approved drugs were found to activate ACE2 that had been downregulated in different medical conditions including hypertension, inflammation, cardiovascular, renal and lung diseases. Therefore, these drugs may be repurposed to re-activate the downregulated ACE2 of COVID-19 patients. These drugs either alone or in combination may be repurposed as prophylactics and therapeutics against SARS-CoV-2 virus.