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.

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.

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.

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.

In silico approaches to study the human asparagine synthetase: An insight of the interaction between the enzyme active sites and its substrates.

Cancer is a leading concern and important cause of death worldwide. Cancer is a non-communicable illness defined as uncontrolled division of cells. It can develop into metastatic cancer when tumor cells migrate to other organs. In recent years evidence has emerged that the bioavailability of Asn play a crucial role in cancer metastasis. Asn is a non-essential amino acid formed from an ATP dependent catalyzed reaction by the enzyme asparagine synthetase (ASNS), where Asp and Gln are converted to Asn and Glu, respectively. The human ASNS enzyme consist of 561 amino acids, with a molecular weight of 64 KDa. ASNS governs the activation of transcriptional factors that regulate the process of metastasis. In this work the 3D model of ASNS in E. coli (AS-B) and the human ASNS docked with its different ligands have been used to study the 3D mechanism of the conversion of Asp and Gln to Asn and Glu, in human ASNS. The stability evaluation of the docked complexes was checked by molecular dynamic simulation through the bioinformatic tool Desmond. The binding residues and their interactions can be exploited for the development of inhibitors, as well as for finding new drug molecules against ASNS and prevention of metastatic cancer.

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.

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.