Identification of substrates of MBL Associated Serine Protease-1 (MASP-1) from human plasma using N-terminomics strategy.

MBL Associated Serine Protease-1 (MASP-1) is an abundant enzyme of the lectin complement pathway. MASP-1 cleaves numerous substrates like MASP-2, MASP-3, C2, C3i, fibrinogen, FXIII and prothrombin. It has thrombin-like specificity and can cleave thrombin substrates. Owing to its high concentration and relaxed substrate specificity, MASP-1 has substrates outside the complement system and can influence other proteolytic cascades and physiological processes. The unidentified substrates may assist us to ascertain the role(s) of MASP-1. In this study, we used a high-throughput N-terminomics method to identify substrates of MASP-1 from human plasma. We have identified 35 putative substrates of MASP-1. Among the identified proteins, alpha 2-antiplasmin, alpha-1-acid glycoprotein, antithrombin III, and siglec-6 were demonstrated to be cleaved by MASP-1. We have discussed the physiological relevance of cleavage of these substrates by MASP-1. The expression of Siglec-6 and MASP-1 has been reported in the B cells. Alpha-1-acid glycoprotein cleavage by MASP-1 may occur in the acute phase as it is known to be an inhibitor of platelet aggregation, whereas MASP-1 triggers platelet aggregation. The cleavage alpha2 antiplasmin by MASP-1 implies that MASP-1 may be promoting plasmin-mediated fibrinolysis. Our study supports that MASP-1 may be implicated in thrombosis as well as thrombolysis.

Regulatory role of Non-canonical DNA Polymorphisms in human genome and their relevance in Cancer.

DNA has the ability to form polymorphic structures like canonical duplex DNA and non-canonical triplex DNA, Cruciform, Z-DNA, G-quadruplex (G4), i-motifs, and hairpin structures. The alteration in the form of DNA polymorphism in the response to environmental changes influences the gene expression. Non-canonical structures are engaged in various biological functions, including chromatin epigenetic and gene expression regulation via transcription and translation, as well as DNA repair and recombination. The presence of non-canonical structures in the regulatory region of the gene alters the gene expression and affects the cellular machinery. Formation of non-canonical structure in the regulatory site of cancer-related genes either inhibits or dysregulate the gene function and promote tumour formation. In the current article, we review the influence of non-canonical structure on the regulatory mechanisms in human genome. Moreover, we have also discussed the relevance of non-canonical structures in cancer and provided information on the drugs used for their treatment by targeting these structures.

Identification of unexplored substrates of the serine protease, thrombin, using N-terminomics strategy.

The function and regulation of thrombin is a complex as well as an intriguing aspect of evolution and has captured the interest of many investigators over the years. The reported substrates of thrombin are coagulation factors V, VIII, XI, XIII, protein C and fibrinogen. However, these may not be all the substrate of thrombin and therefore its functional role(s), may not have been completely comprehended. The purpose of our study was to identify hitherto unreported substrates of thrombin from human plasma using a N-terminomics protease substrate identification method. We identified 54 putative substrates of thrombin of which 12 are already known and 42 are being reported for the first time. Amongst the proteins identified, recombinant siglec-6 and purified serum alpha-1-acid glycoprotein were validated by cleavage with thrombin. We have discussed the probable relevance of siglec-6 cleavage by thrombin in human placenta mostly because an upregulation in the expression of siglec-6 and thrombin has been reported in the placenta of preeclampsia patients. We also speculate the role of alpha-1-acid glycoprotein cleavage by thrombin in the acute phase as alpha-1-acid glycoprotein is known to be an inhibitor of platelet aggregation whereas thrombin is known to trigger platelet aggregation.

Exploring the Potential of Small Molecule-Based Therapeutic Approaches for Targeting Trinucleotide Repeat Disorders.

In recent years, neurological disorders have globally become a leading cause of disability and death. Neurological disorders are very common in both high- and low-income countries, and the number of patients is predicted to increase in the coming decades. Disorders caused by the expanded trinucleotide repeats (CAG, CGG, CCG, CTG, CUG, GAA, and GCN) in the genome, also described as trinucleotide repeat expansion disorders (TREDs), comprise of the major class of neurological diseases. Various TREDs have different modes of pathogenesis, but the severity and time of onset of disease depends on the trinucleotide repeat numbers. Numerous therapeutic strategies, including symptomatic treatment, blockage of mutant protein synthesis, targeting the toxic protein aggregates and degradation of RNA transcripts have been developed for the treatment of these diseases. However, various limitations to these therapeutic strategies have been reported, and therefore, researchers are exploring different avenues of therapeutics development. One of the recent developments include targeting the expanded repeats with small molecules. Small molecule binds with the secondary/tertiary structure of RNA (like bulges, loops, and hairpins) irrespective of its sequences. Altogether, small molecule-based therapeutics may have the advantage over others to be able to overcome the hurdles of the blood-brain barrier, poor absorption, and allergic reactions. In this review, we have summarized various TREDs and envisage the potential of small molecule-based therapeutics for targeting these hitherto incurable neurological disorders.

Analytical techniques for characterization of biological molecules - proteins and aptamers/oligonucleotides.

With the advent of the high-throughput technologies and exciting times for biology, the discipline of analytical methodology is experiencing a surge in the growth and the scope. Over the years, multitude of analytical techniques have evolved from a work-intensive, low sensitivity and high volume of reagent and sample consumption endeavor to automated, better selectivity, lower limit of quantification and cost-effective techniques for biological research. In this review, we give an overview of the currently available wide range of cell-based and noncell based and structural based analytical techniques, their principle and biological applications. The analytical techniques discussed in this paper includes surface plasmon resonance, electrophoresis, enzyme linked immunosorbent assay, Western blotting, flow cytometry, fluorescence activated cell sorting, mass spectrometry, nuclear magnetic resonance and x-ray crystallography.

Proteolysis to Identify Protease Substrates: Cleave to Decipher.

Proteolysis is an irreversible post-translational modification process, characterized by highly precise yet stable cleavage of proteins. Downstream events in signaling processes are reliant on proteolysis triggered by the protease activity. Studies indicate that abnormal proteolytic activity may lead to the manifestation of diseased conditions. Therefore, characterization of proteases may provide clues to understand their role in fundamental cellular processes like cellular growth, differentiation, apoptosis, and survival. The relevance of proteases and their substrates as clinical targets are being studied. Understanding the mechanism of proteolytic activity, the identity, and the role of repertoire of its substrates in a physiological pathway has opened avenues for novel drug designing. However, only a limited knowledge of protease substrates is currently available. In this review, the authors recapitulate the library screening, proteomics, and bioinformatics based approaches that have been employed for the identification of protease substrates.

Cell surfactomes of two endometrial epithelial cell lines that differ in their adhesiveness to embryonic cells.

Adhesiveness of the endometrial epithelium to an embryo plays a critical role in the initiation of pregnancy. Loss or gain of adhesiveness also dictates the potential of endometrial epithelial cells to metastasize, an event that can result from certain genetic insults. A proteomics-based exploration of the "adhesiveness" these epithelial cells was employed that could identify targets that could disrupt embryo-endometrium interactions and/or metastasis of endometrial cancer cells. The present study defined the surfactomes of two human endometrial epithelial cell lines known for their differential adhesiveness to embryonic cells. Comparative, two-dimensional electrophoretic analysis of the surfactomes of RL95-2 (exhibiting higher adhesiveness to the embryonic cell line JAr) and HEC-1A (exhibiting reduced adhesiveness to JAr cells) revealed 55 differentially enriched proteins. Of these, 10 proteins were identified by MALDI-TOF/TOF or LC-MS/MS. TUBB2C, ADAMTS3, and elongation factor beta were more abundant on the HEC-1A cell surface whereas HSP27, HSPA9, GP96, CRT, Tapasin-ERP57, PDI, and ß-actin were more abundant on the RL95-2 cell surface. Nano LC-MS/MS was also employed to generate a more comprehensive surfactomes of RL95-2 and HEC-1A. The study also demonstrated a pro-adhesive role of CRT and HSPA9 and an anti-adhesive role of TUBB2C populations found on the cell surface. In brief, this study identifies the cell-surface protein complements of two human endometrial epithelial cell lines, and reveals the role of three proteins in heterotypic cell adhesion.

Endometrial receptivity: a revisit to functional genomics studies on human endometrium and creation of HGEx-ERdb.

BACKGROUND: Endometrium acquires structural and functional competence for embryo implantation only during the receptive phase of menstrual cycle in fertile women. Sizeable data are available to indicate that this ability is acquired by modulation in the expression of several genes/gene products. However, there exists little consensus on the identity, number of expressed/not-detected genes and their pattern of expression (up or down regulation). METHODS: Literature search was carried out to retrieve the data on endometrial expression of genes/proteins in various conditions. Data were compiled to generate a comprehensive database, Human Gene Expression Endometrial Receptivity database (HGEx-ERdb). The database was used to identify the Receptivity Associated Genes (RAGs) which display the similar pattern of expression across different investigations. Transcript levels of select RAGs encoding cell adhesion proteins were compared between two human endometrial epithelial cell lines; RL95-2 and HEC-1-A by quantitative real time polymerase chain reaction (q-RT-PCR). Further select RAGs were investigated for their expression in pre-receptive (n = 4) and receptive phase (n = 4) human endometrial tissues by immunohistochemical studies. JAr spheroid attachment assays were carried out to assess the functional significance of two RAGs. RESULTS: HGEx-ERdb (http://resource.ibab.ac.in/HGEx-ERdb/) helped identification of 179 RAGs, of which 151 genes were consistently expressed and upregulated and 28 consistently not-detected and downregulated in receptive phase as compared to pre-receptive phase. q-RT-PCR confirmed significantly higher (p<0.005) expression of Thrombospondin1 (THBS1), CD36 and Mucin 16 transcripts, in RL95-2 as compared to HEC-1-A. Further, the pretreatment with antibodies against CD36 and COMP led to a reduction in the percentage of JAr spheroids attached to RL95-2. Immunohistochemical studies demonstrated significantly higher (p<0.05) expression of endometrial THBS1, Cartilage Oligomeric Matrix Protein (COMP) and CD36 in the receptive phase as compared to pre-receptive phase human endometrial tissues. CONCLUSION: HGEx-ERdb is a catalogue of 19,285 genes, reported for their expression in human endometrium. Further 179 genes were identified as the RAGs. Expression analysis of some RAGs validated the utility of approach employed in creation of HGEx-ERdb. Studies aimed towards defining the specific functions of RAGs and their potential networks may yield relevant information about the major 'nodes' which regulate endometrial receptivity.