Predisposing deleterious variants in the cancer-associated human kinases in the global populations.

Human kinases play essential and diverse roles in the cellular activities of maintaining homeostasis and growth. Genetic mutations in the genes encoding the kinases (or phosphotransferases) have been linked with various types of cancers. In this study, we cataloged mutations in 500 kinases genes in >65,000 individuals of global populations from the Human Genetic Diversity Project (HGDP) and ExAC databases, and assessed their potentially deleterious impact by using the in silico tools SIFT, Polyphen2, and CADD. The analysis highlighted 35 deleterious non-synonymous SNVs in the ExAC and 5 SNVs in the HGDP project. Notably, a higher number of deleterious mutations was observed in the Non-Finnish Europeans (26 SNVs), followed by the Africans (14 SNVs), East Asians (13 SNVs), and South Asians (12 SNVs). The gene set enrichment analysis highlighted NTRK1 and FGFR3 being most significantly enriched among the kinases. The gene expression analysis revealed over-expression of NTRK1 in liver cancer, whereas, FGFR3 was found over-expressed in lung, breast, and liver cancers compared to their expression in the respective normal tissues. Also, 13 potential drugs were identified that target the NTRK1 protein, whereas 6 potential drugs for the FGFR3 target were identified. Taken together, the study provides a framework for exploring the predisposing germline mutations in kinases to suggest the underlying pathogenic mechanisms in cancers. The potential drugs are also suggested for personalized cancer management.

Overview of Lignocellulolytic Enzyme Systems with Special Reference to Valorization of Lignocellulosic Biomass.

Lignocellulosic biomass, one of the most valuable natural resources, is abundantly present on earth. Being a renewable feedstock, it harbors a great potential to be exploited as a raw material, to produce various value-added products. Lignocellulolytic microorganisms hold a unique position regarding the valorization of lignocellulosic biomass as they contain efficient enzyme systems capable of degrading this biomass. The ubiquitous nature of these microorganisms and their survival under extreme conditions have enabled their use as an effective producer of lignocellulolytic enzymes with improved biochemical features crucial to industrial bioconversion processes. These enzymes can prove to be an exquisite tool when it comes to the eco-friendly manufacturing of value-added products using waste material. This review focuses on highlighting the significance of lignocellulosic biomass, microbial sources of lignocellulolytic enzymes and their use in the formation of useful products.

Distinct genetic landscape and a low response to doxorubicin in a luminal-A breast cancer cell line of Pakistani origin.

BACKGROUND: Breast cancers exhibit genetic heterogeneity which causes differential responses to various chemotherapy agents. Given the unique demographic and genomic background in South Asia, genetic architecture in breast cancers is not fully explored. METHODS AND RESULTS: In this study, we determined the genetic landscape of our previously established luminal-A subtype breast cancer cell line (BC-PAK1), and compared it with a Caucasian origin MCF7 breast cancer cell line of the same molecular subtype. Deep whole-exome sequencing (100X) was performed from early passages of the primary cancer cells using the Illumina NextSeq500. Data analysis with in silico tools showed novel non-silent somatic mutations previously not described in breast cancers, including a frameshift insertion (p.Ala1591AlafsTer28) in CIC, and a frameshift deletion (p.Lys333LysfsTer21) in PABPC1. Five genes CDC27, PIK3CG, ARAP3, RAPGEF1, and EFNA3, related with cell cycle pathway (hsa04110), ErbB signaling pathway (hsa04012), Ras signaling pathway (hsa04014), and Rap1 signaling pathway (hsa04015) were found to have recurrent non-silent somatic mutations. Further, the major contribution of COSMIC signatures 3 (failure of DNA double-strand break repair by homologous recombination), and 12 (transcriptional strand-bias for T>C substitutions) was observed. Also, the somatic mutations landscape in BC-PAK1 was found to be different as compared to the MCF7 cell line. The unique genetic landscape of BC-PAK1 might be responsible for significantly reduced response to doxorubicin than the MCF7 cell line. CONCLUSION: This study presents a distinct genetic architecture in luminal-A breast cancer potentially responsible for differential response to chemotherapy. Further studies on large cohorts of breast cancer patients are suggested for implementation in personalized medicine.

Finding inhibitors for PCSK9 using computational methods.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is one of the key targets for atherosclerosis drug development as its binding with low-density lipoprotein receptor leads to atherosclerosis. The protein-ligand interaction helps to understand the actual mechanism for the pharmacological action. This research aims to discover the best inhibitory candidates targeting PCSK9. To start with, reported ACE inhibitors were incorporated into pharmacophore designing using PharmaGist to produce pharmacophore models. Selected models were later screened against the ZINC database using ZINCPHARMER to define potential drug candidates that were docked with the target protein to understand their interactions. Molecular docking revealed the top 10 drug candidates against PCSK9, with binding energies ranging from -9.8 kcal·mol-1 to -8.2 kcal·mol-1, which were analyzed for their pharmacokinetic properties and oral bioavailability. Some compounds were identified as plant-derived compounds like (S)-canadine, hesperetin or labetalol (an antihypertensive drug). Molecular dynamics results showed that these substances formed stable protein-ligand complexes. (S)-canadine-PCSK9 complex was the most stable with the lowest RMSD. It was concluded that (S)-canadine may act as a potential inhibitor against atherosclerosis for the development of new PCSK9 inhibitory drugs in future in vitro research.

Small organic molecules accelerate the expansion of regulatory T cells.

The regulatory T cells (Treg cells) expressing CD4 + CD25 + FOXP3 + markers are indispensable for the initiation of immune homeostasis and tolerance to self-antigens in both mice and humans. A decrease in regulatory T cells leads to various autoimmune pathologies. Herein, we report three low molecular weight, small organic molecules as a new series of Treg proliferators TRP-1-3. These small molecules were tested for their proliferative effect on regulatory T cells. It was found that TRP-1 (Oleracein E) strongly accelerates the Treg proliferation in vitro in a concentration-dependent manner. The effect was evident for all subsets of Treg cells tested, including naturally occurring, thymus-derived and peripherally-induced or adaptive Treg, indicating an effect independent of the maturation site. Importantly, increased Treg cells numbers by TRP-1 correlated with improved CD4 + CD25 + FOXP3 + expression in vitro, while propidium iodide-based staining showed low TRP-1-induced cytotoxicity. Molecular docking plus simulation studies of these TRP-1-3 with IL-2R, mTOR and TCR receptors suggest a TCR-based Treg cells activation mechanism. Because of its high Treg cells activities and low cellular cytotoxicity, TRP-1-3 may be useful in stimulating ex-vivo/in-vivo, Treg cell-specific responses for therapeutic applications.

Phosphorylated and O-GlcNAc Modified IRS-1 (Ser1101) and -2 (Ser1149) Contribute to Human Diabetes Type II.

BACKGROUND: The prevalence of the chronic metabolic disorder Type 2 diabetes mellitus (T2DM) is increasing steadily, and has even turned into an epidemic in some countries. T2DM results from defective responses to insulin and obesity is a major factor behind insulin resistance in T2DM. Insulin receptor substrate (IRS) proteins are adaptor proteins in the insulin receptor signalling pathway. The insulin signalling is controlled through tyrosine phosphorylation of IRS-1 and IRS-2, and dysregulation of IRS proteins signalling may lead to glucose intolerance and eventually insulin resistance. OBJECTIVE: In this work, we suggest that both glycosylation (O-GlcNAc modification) and phosphorylation of IRS-1 and -2 are involved in the pathogenesis of T2DM. METHODS: Phosphorylation and O-GlcNAc modifications (Ser1101 in IRS-1 and Ser1149 in IRS-2) proteins were determined experimentally by sandwich ELISA with specific antibodies and with bioinformatics tools. RESULTS: When IRS-1 (on Ser1101) and IRS-2 (Ser1149) become glycosylated following an increase in UDP-GlcNAc pools, it may contribute to insulin resistance. Whereas when the same (IRS-1 on Ser1101 and IRS-2 on Ser1149) are phosphorylated, the insulin signalling is inhibited. DISCUSSION: In this work OGlcNAc-modified proteins were specifically detected using O-Glc- NAc-specific antibodies, suggesting that elevated levels of O-GlcNAc-modified proteins are found, independently of their possible involvement in Advanced Glycation End products (AGEs). CONCLUSION: This study suggests a mechanism, which is controlled by posttranslational modifications, and may contribute to the pathogenesis of type II diabetes.

Charge and Polarity Preferences for N-Glycosylation: A Genome-Wide In Silico Study and Its Implications Regarding Constitutive Proliferation and Adhesion of Carcinoma Cells.

The structural and functional diversity of the human proteome is mediated by N- and O-linked glycosylations that define the individual properties of extracellular and membrane-associated proteins. In this study, we utilized different computational tools to perform in silico based genome-wide mapping of 1,117 human proteins and unravel the contribution of both penultimate and vicinal amino acids for the asparagine-based, site-specific N-glycosylation. Our results correlate the non-canonical involvement of charge and polarity environment of classified amino acids (designated as L, O, A, P, and N groups) in the N-glycosylation process, as validated by NetNGlyc predictions, and 130 literature-reported human proteins. From our results, particular charge and polarity combinations of non-polar aliphatic, acidic, basic, and aromatic polar side chain environment of both penultimate and vicinal amino acids were found to promote the N-glycosylation process. However, the alteration in side-chain charge and polarity environment of genetic variants, particularly in the vicinity of Asn-containing epitope, may induce constitutive glycosylation (e.g., aberrant glycosylation at preferred and non-preferred sites) of membrane proteins causing constitutive proliferation and triggering epithelial-to-mesenchymal transition. The current genome-wide mapping of 1,117 proteins (2,909 asparagine residues) was used to explore charge- and polarity-based mechanistic constraints in N-glycosylation, and discuss alterations of the neoplastic phenotype that can be ascribed to N-glycosylation at preferred and non-preferred sites.

N-acetylgalactosaminyltransferases in cancer.

Aberrant mucin-type O-glycosylation by glycosyltransferases is a well-described hallmark of many cancers and is also associated with additional non-cancerous developmental and metabolic disorders. The current review focuses on N-acetylgalactosaminyltransferase genes (GALNTs) and proteins (GalNAcTs) to illustrate their importance in cancer biology. Aberrant O-glycosylation by GalNAcTs activates a wide range of proteins that carry out interactions of sessile and motile cells affecting organogenesis, responses to agonists and stimulating hyperproliferation and metastatisation of neoplastic cells. As genome-wide analyses have provided abundant clues regarding under- or over-expressed genes that characterize different types of cancers, GALNTs and their transferase products have attracted attention by being unexpected actors in neoplastic contexts. We intend to review the current knowledge on GALNTs and their encoded transferases in cancer and suggest what could be the significance of such information in cancer pathogenesis and management.

Mechanistic understanding and significance of small peptides interaction with MHC class II molecules for therapeutic applications.

Major histocompatibility complex (MHC) class II molecules are expressed by antigen-presenting cells and stimulate CD4(+) T cells, which initiate humoral immune responses. Over the past decade, interest has developed to therapeutically impact the peptides to be exposed to CD4(+) T cells. Structurally diverse small molecules have been discovered that act on the endogenous peptide exchanger HLA-DM by different mechanisms. Exogenously delivered peptides are highly susceptible to proteolytic cleavage in vivo; however, it is only when successfully incorporated into stable MHC II-peptide complexes that these peptides can induce an immune response. Many of the small molecules so far discovered have highlighted the molecular interactions mediating the formation of MHC II-peptide complexes. As potential drugs, these small molecules open new therapeutic approaches to modulate MHC II antigen presentation pathways and influence the quality and specificity of immune responses. This review briefly introduces how CD4(+) T cells recognize antigen when displayed by MHC class II molecules, as well as MHC class II-peptide-loading pathways, structural basis of peptide binding and stabilization of the peptide-MHC complexes. We discuss the concept of MHC-loading enhancers, how they could modulate immune responses and how these molecules have been identified. Finally, we suggest mechanisms whereby MHC-loading enhancers could act upon MHC class II molecules.

BRAF gene: From human cancers to developmental syndromes.

The BRAF gene encodes for a serine/threonine protein kinase that participates in the MAPK/ERK signalling pathway and plays a vital role in cancers and developmental syndromes (RASopathies). The current review discusses the clinical significance of the BRAF gene and other members of RAS/RAF cascade in human cancers and RAS/MAPK syndromes, and focuses the molecular basis and clinical genetics of BRAF to better understand its parallel involvement in both tumourigenesis and RAS/MAPK syndromes-Noonan syndrome, cardio-facio-cutaneous syndrome and LEOPARD syndrome.

Exploring the sequence context of phosphorylatable amino acids: the contribution of the upgraded MAPRes tool.

Several models that predict where post-translational modifications are likely to occur and formulate the corresponding association rules are available to analyze the functional potential of a protein sequence, but an algorithm incorporating the functional groups of the involved amino acids in the sequence analyses process is not yet available. In its previous version, MAPRes was utilized to investigate the influence of the surrounding amino acids of post- translationally and co-translationally modifiable sites. The MAPRes has been upgraded to take into account the different biophysical and biochemical properties of the amino acids that have the potential to influence different post- translational modifications (PTMs). In the present study, the upgraded version of MAPRes was implemented on phosphorylated Ser/Thr/Tyr data by considering the polarity and charge of the surrounding amino acids. The patterns mined by MAPRes incorporating structural information on polarity and charge of amino acids suggest distinct structure-function relationships for phosphorylated serines in a multifunctional protein such as the insulin-receptor substrate-1 (IRS-1) protein. The new version of MAPRes is freely available at http://www.imsb.edu.pk/Database.htm.

Post-translational modifications in activation and inhibition of Oct-1-DNA binding complex in H2B and other diverse gene regulation: prediction of interplay sites.

Octamer DNA binding transcription factors play important roles in housekeeping and specific gene regulations. Octamer DNA binding transcription factor-1 (Oct-1), expressed ubiquitously, is a multifunctional molecule. The binding sites of Oct-1 are the promoters of H2B gene and the genes of snRNA, U2, U6, and 7SK, yet Oct-1 has been described as constitutively expressed transcription factor regulating the expression of housekeeping genes. Diverse tissue-specific genes regulations by Oct-1 include genes for interleukins (IL) 2, 3, 5; the granulocyte-macrophagal colony-stimulating factor, immunoglobulins α, ß, Ly9; the endocrine-associated Pit-1 gene; the genes for gonadoliberin, prolactin, the thyroid transcription factor, and thyrotropin. The most interesting aspect of the gene regulations of Oct-1 includes both activation and inhibition of transcription. These opposite regulations of Oct-1 have been described through presence/absence of a post-translational modification (PTM) in its different domains. We propose a mechanism of interplay of different PTMs or presence/absence of PTMs in the different domains of Oct-1. We also suggest that the absence of phosphorylation and acetylation in G1 and S phases of the cell cycle is associated with interplay of methylation and O-GlcNAc modification. This interplay of O-GlcNAc modification with the phosphorylation and methylation with acetylation in POU sub-domain of Oct-1 may facilitate the formation of Oct-1-DNA complex, consequently activating H2B gene transcription. Whereas, in G2 and M phases these sites are occupied by phosphate resulting in inhibition of Oct-1-DNA complex formation leading to the suppression of H2B gene transcription.

Influence of the sequence environment and properties of neighboring amino acids on amino-acetylation: relevance for structure-function analysis.

Proteins function is regulated by co-translational modifications and post-translational modifications (PTMs) such as phosphorylation, glycosylation, and acetylation, which induce proteins to perform multiple tasks in a specified environment. Acetylation takes place post-translationally on the ε-amino group of Lys in histone proteins, allowing regulation of gene expression. Furthermore, amino group acetylation also occurs co-translationally on Ser, Thr, Gly, Met, and Ala, possibly contributing to the stability of proteins. In this work, the influence of amino acids next to acetylated sites has been investigated by using MAPRes (Mining Association Patterns among preferred amino acid residues in the vicinity of amino acids targeted for PTMs). MAPRes was utilized to examine the sequence patterns vicinal to modified and non-modified residues, taking into account their charge and polarity. The PTMs data were further sub-divided according to their sub-cellular location (nuclear, mitochondrial, and cytoplasmic), and their association patterns were mined. The association patterns mined by MAPRes for acetylated and non-acetylated residues are consistent with the existing literature but also revealed novel patterns. These rules have been utilized to describe the acetylation and its effects on the protein structure-function relationship.

O-GlcNAc modification of the anti-malarial vaccine candidate PfAMA1: in silico-defined structural changes and potential to generate a better vaccine.

The complex life cycle of plasmodial parasites makes the selection of a single subunit protein a less than optimal strategy to generate an efficient vaccinal protection against malaria. Moreover, the full protection afforded by malarial proteins carried by intact parasites implies that immune responses against different antigens expressed in different phases of the cycle are required, but also suggests that native malarial antigens are presented to the host immune system in a manner that recombinant proteins do not achieve. The malarial apical membrane antigen 1 (AMA1) represents a suitable vaccine candidate because AMA1 is expressed on sporozoites and merozoites and allows them to invade hepatocytes and erythrocytes, respectively. Anti-AMA1 antibodies and cytotoxic T-cells are therefore expected to interfere both with the primary invasion of hepatocytes by sporozoites and with the later propagation of merozoites in erythrocytes, and thus efficiently counteract parasite development in its human host. AMA1 bears potential glycosylation sites and the human erythrocytic O-linked N-acetylglucosamine transferase (OGT) could glycosylate AMA1 through combinatorial metabolism. This hypothesis was tested in silico by developing binding models of AMA1 with human OGT complexed with UDP-GlcNc, and followed by the binding of O-GlcNAc with the hydroxyl group of AMA1 serine and threonine residues. Our results suggests that AMA1 shows potential for glycosylation at Thr517 and Ser498 and that O-GlcNAc AMA1 may constitute a conformationally more appropriate antigen for developing a protective anti-malarial immune response.

Membrane-associated signaling in human B-lymphoma lines.

In B-non-Hodgkin lymphomas, Lyn and Cbp/PAG constitute the core of an oncogenic signalosome that captures the Phosphatidylinositol-3-kinase, the Spleen tyrosine kinase and the Signal transducer and activator of transcription-3 to generate pro-survival and proliferative signals. Lymphoma lines corresponding to follicular, mantle-cell and Burkitt-derived lymphomas display type-specific signalosome organizations that differentially activate PI3K, Syk and STAT3. In the follicular lymphoma line, PI3K, Syk and STAT3 were optimally activated upon association with the Lyn-Cbp/PAG signalosome, while in the Burkitt lymphoma-derived line, the association with Cbp/PAG and activation of PI3K were interfered with by the latent membrane proteins encoded by the Epstein-Barr virus. In the Jeko-1 mantle-cell line, a weak association of Syk with the Lyn-Cbp/PAG signalosome resulted in poor activation of Syk, but in those cells, as in the follicular and Burkitt-derived lines, efficient apoptosis induction by the Syk inhibitor R406 indicated that Syk is nonetheless an important prosurvival element and therefore a valuable therapeutic target. In all configurations described herein is the Lyn-Cbp/PAG signalosome independent of external signals and provides efficient means of activation for its associated lipid and protein kinases. In follicular and Burkitt-derived lines, Syk appears to be activated following binding to Cbp/PAG and no longer requires B-cell receptor-associated activation motifs for activation. Assessment of the different modalities of Lyn-Cbp/PAG signalosome organization could help in selecting the appropriate combination of kinase inhibitors to eliminate a particular type of lymphoma cells.

CREB in long-term potentiation in hippocampus: role of post-translational modifications-studies In silico.

The multifunctionality of proteins is dictated by post-translational modifications (PTMs) which involve the attachment of small functional groups such as phosphate and acetate, as well as carbohydrate moieties. These functional groups make the protein perform various functions in different environments. PTMs play a crucial role in memory and learning. Phosphorylation of synaptic proteins and transcription factors regulate the generation and storage of memory. Among these is the cAMP-regulated element binding protein CREB that regulates CRE containing genes like c-fos. Both phosphorylation and acetylation control the function of CREB as a transcription factor. CREB is also susceptible to O-GlcNAc modification, which inhibits its activity. O-GlcNAc modification occurs on the same or neighboring Ser/Thr residues akin to phosphorylation. An interplay between these modifications was shown to operate in nuclear and cytoplasmic proteins. In this study computational methods were utilized to predict different modification sites in CREB. These in silico results suggest that phosphorylation, O-GlcNAc modification and acetylation modulate the transcriptional activity of CREB and thus dictate its contribution to synaptic plasticity.

The sphingolipid-rich rafts of ALK+ lymphomas downregulate the Lyn-Cbp/PAG signalosome.

Human anaplastic lymphoma kinase (ALK) + lymphomas express the constitutively active ALK as a fusion protein that drives several survival pathways. The catalytic domain of the anaplastic receptor tyrosine kinase is frequently fused with the nuclear localization protein nucleophosmin but may also fuse with other proteins that associate it with other subcellular structures. Similarly to other B human lymphomas, ALK+ lymphomas express the Cbp/PAG adaptor protein and the non-receptor Lyn kinase in the plasma membrane. In the majority of human lymphomas, the Cbp/PAG adaptor and the Lyn kinase constitute an oncogenic signalosome that serves as a membrane anchor for other signaling enzymes and transcription factors. We show that ALK+ lymphoma membranes harbor sphingolipid-rich microdomains (rafts) in which Lyn is poorly active. However, Lyn activity and consequently Cbp/PAG tyrosine phosphorylation can be restored by extracting sphingolipids from ALK+ lymphoma plasma membranes. In the membrane environment of ALK+ lymphoma rafts, where the glycosphingolipid to signaling protein ratio is higher than in B-NHL rafts, the Lyn activity is suboptimal and does not allow the formation of an efficient Lyn-Cbp/PAG signalosome.

The function of GluR1 and GluR2 in cerebellar and hippocampal LTP and LTD is regulated by interplay of phosphorylation and O-GlcNAc modification.

Long-term potentiation (LTP) and long-term depression (LTD) are the current models of synaptic plasticity and widely believed to explain how different kinds of memory are stored in different brain regions. Induction of LTP and LTD in different regions of brain undoubtedly involve trafficking of AMPA receptor to and from synapses. Hippocampal LTP involves phosphorylation of GluR1 subunit of AMPA receptor and its delivery to synapse whereas; LTD is the result of dephosphorylation and endocytosis of GluR1 containing AMPA receptor. Conversely the cerebellar LTD is maintained by the phosphorylation of GluR2 which promotes receptor endocytosis while dephosphorylation of GluR2 triggers receptor expression at the cell surface and results in LTP. The interplay of phosphorylation and O-GlcNAc modification is known as functional switch in many neuronal proteins. In this study it is hypothesized that a same phenomenon underlies as LTD and LTP switching, by predicting the potential of different Ser/Thr residues for phosphorylation, O-GlcNAc modification and their possible interplay. We suggest the involvement of O-GlcNAc modification of dephosphorylated GluR1 in maintaining the hippocampal LTD and that of dephosphorylated GluR2 in cerebral LTP.

Effect on the Ras/Raf signaling pathway of post-translational modifications of neurofibromin: in silico study of protein modification responsible for regulatory pathways.

Mapping and chemical characterization of post-translational modifications (PTMs) in proteins are critical to understand the regulatory mechanisms involving modified proteins and their role in disease. Neurofibromatosis type 1 (NF-1) is an autosomal dominantly inherited disorder, where NF1 mutations usually result in a reduced level of the tumor suppressor protein, neurofibromin (NF). NF is a multifunctional cytoplasmic protein that regulates microtubule dynamics and participates in several signaling pathways, particularly the RAS signaling pathway. NF is a Ras GTPase-activating protein (GAP) that prevents oncogenesis by converting GTP-Ras to GDP-Ras. This function of NF is regulated by phosphorylation. Interplay of phosphorylation with O-GlcNAc modification on the same or vicinal Ser/Thr residues, the Yin Yang sites, is well known in cytoplasmic and nuclear proteins. The dynamic aspects of PTMs and their interplay being difficult to follow in vivo, we undertook this in silico work to predict and define the possible role of Yin Yang sites in NF-1. Interplay of phosphorylation and O-GlcNAc modification is proposed as a mechanism controlling the Ras signaling pathway.