Elayanambi Sundaramoorthy

Mining literature for a comprehensive pathway analysis: A case study for retrieval of homocysteine related genes for genetic and epigenetic studies

Homocysteine is an independent risk factor for cardiovascular diseases. It is also known to be associated with a variety of complex disorders. While there are a large number of independent studies implicating homocysteine in isolated pathways, the mechanism of homocysteine induced adverse effects are not clear. Homocysteine-induced modulation of gene expression through alteration of methylation status or by hitherto unknown mechanisms is predicted to lead to several pathological conditions either directly or indirectly. In the present manuscript, using literature mining approach, we have identified the genes that are modulated directly or indirectly by an elevated level of homocysteine. These genes were then placed in appropriate pathways in an attempt to understand the molecular basis of homocysteine induced complex disorders and to provide a resource for selection of genes for polymorphism screening and analysis of mutations as well as epigenetic modifications in relation to hyperhomocysteinemia. We have identified 135 genes in 1137 abstracts that either modulate the levels of homocysteine or are modulated by elevated levels of homocysteine. Mapping the genes to their respective pathways revealed that an elevated level of homocysteine leads to the atherosclerosis either by directly affecting lipid metabolism and transport or via oxidative stress and/or Endoplasmic Reticulum (ER) stress. Elevated levels of homocysteine also decreases the bioavailability of nitric oxide and modulates the levels of other metabolites including S-adenosyl methionine and S-adenosyl homocysteine which may result in cardiovascular or neurological disorders. The ER stress emerges as the common pathway that relates to apoptosis, atherosclerosis and neurological disorders and is modulated by levels of homocysteine. The comprehensive network collated has lead to the identification of genes that are modulated by homocysteine indicating that homocysteine exerts its effect not only through modulating the substrate levels for various catalytic processes but also through regulation of expression of genes involved in complex diseases.

A cancer-associated BRCA2 mutation reveals masked nuclear export signals controlling localization

Germline missense mutations affecting a single BRCA2 allele predispose humans to cancer. Here we identify a protein-targeting mechanism that is disrupted by the cancer-associated mutation, BRCA2D2723H, and that controls the nuclear localization of BRCA2 and its cargo, the recombination enzyme RAD51. A nuclear export signal (NES) in BRCA2 is masked by its interaction with a partner protein, DSS1, such that point mutations impairing BRCA2-DSS1 binding render BRCA2 cytoplasmic. In turn, cytoplasmic mislocalization of mutant BRCA2 inhibits the nuclear retention of RAD51 by exposing a similar NES in RAD51 that is usually obscured by the BRCA2-RAD51 interaction. Thus, a series of NES-masking interactions localizes BRCA2 and RAD51 in the nucleus. Notably, BRCA2D2723H decreases RAD51 nuclear retention even when wild-type BRCA2 is also present. Our findings suggest a mechanism for the regulation of the nucleocytoplasmic distribution of BRCA2 and RAD51 and its impairment by a heterozygous disease-associated mutation.

A DNA-Damage Selective Role for BRCA1 E3 Ligase in Claspin Ubiquitylation, CHK1 Activation, and DNA Repair

BACKGROUND The breast and ovarian cancer suppressor BRCA1 is essential for cellular responses to DNA damage. It heterodimerizes with BARD1 to acquire an E3 ubiquitin (Ub) ligase activity that is often compromised by cancer-associated mutations. Neither the significance of this activity to damage responses, nor a relevant in vivo substrate, is clear. RESULTS We have separated DNA-damage responses requiring the BRCA1 E3 ligase from those independent of it, using a gene-targeted point mutation in vertebrate DT40 cells that abrogates BRCA1s catalytic activity without perturbing BARD1 binding. We show that BRCA1 ubiquitylates claspin, an essential coactivator of the CHK1 checkpoint kinase, after topoisomerase inhibition, but not DNA crosslinking by mitomycin C. BRCA1 E3 inactivation decreases chromatin-bound claspin levels and impairs homology-directed DNA repair by interrupting signal transduction from the damage-activated ATR kinase to its effector, CHK1. CONCLUSIONS Our findings identify claspin as an in vivo substrate for the BRCA1 E3 ligase and suggest that its modification selectively triggers CHK1 activation for the homology-directed repair of a subset of genotoxic lesions. This mechanism unexpectedly defines an essential but selective function for BRCA1 E3 ligase activity in cellular responses to DNA damage.

Vitamin B12 deficiency is associated with coronary artery disease in an Indian population

Abstract Background: The incidence of coronary artery disease (CAD) is increasing at an alarming rate, especially in developing countries, such as India. It is often advocated that a vegetarian lifestyle could reduce the burden of CAD. However, in spite of a majority of Indians being vegetarians, the incidence of CAD is highest in this population. This may be due to deficiency of vitamin B12, a micronutrient, sourced only from animal products. Methods: Herein, we assessed the effect of vitamin B12 with respect to CAD in 816 individuals (368 CAD patients and 448 controls) recruited from a tertiary care center in New Delhi, India. Results: We found that vitamin B12 levels were significantly lower in CAD patients than in controls (p<0.0001). Also, vegetarians were found to have significantly lower vitamin B12 concentrations (p=0.0001) and higher incidence of CAD (p=0.01). Interestingly, elevated homocysteine levels, a hallmark of vitamin B12 deficiency, was not associated with CAD. In contrast, cysteine levels were significantly higher in CAD patients than in controls (p=0.004). Conclusions: We believe that, when vitamin B12 is deficient, homocysteine is rapidly metabolized via the transsulfuration pathway leading to increased cysteine levels. Clin Chem Lab Med 2009;47:334–8.

Single nucleotide polymorphisms in homocysteine metabolism pathway genes: association of CHDH A119C and MTHFR C677T with hyperhomocysteinemia

Background— An elevated level of homocysteine (hyperhomocysteinemia) has been implicated as an independent risk factor for cardiovascular diseases. Deficiency of dietary factors like vitamin B12, folate, and genetic variations can cause hyperhomocysteinemia. The prevalence of hyperhomocysteinemia in the Indian population is likely to be high because most Indians adhere to a vegetarian diet, deficient in vitamin B12. In the background of vitamin B12 deficiency, variations in genes involved in homocysteine metabolism might have a greater impact on homocysteine levels. Methods and Results— We genotyped 44 nonsynonymous single-nucleotide polymorphisms (nsSNPs) from 11 genes involved in homocysteine metabolism and found only 14 to be polymorphic. These 14 nsSNPs were genotyped in 546 individuals recruited from a tertiary care center in New Delhi, India, and it was found that choline dehydrogenase ( CHDH A119C) and methylenetetrahydrofolate reductase ( MTHFR C677T) were significantly associated with plasma total homocysteine levels ( P =0.009 and P =0.001, respectively). These 2 SNPs were further genotyped in 330 individuals recruited from the same center, and the association remained significant even after increasing the sample size. Furthermore, we found the possibility of a significant interaction between vegetarian diet and the 2 polymorphisms that could explain the variation of homocysteine levels. We also genotyped all the polymorphic nsSNPs in apparently healthy individuals recruited from 24 different subpopulations (based on their linguistic lineage) spread across the country to determine their basal frequencies. The frequencies of these SNPs varied significantly between linguistic groups. Conclusion— Vegetarian diet along with CHDH A119C and MTHFR C677T play an important role in modulating the homocysteine levels in Indian population. Received July 16, 2008; accepted August 21, 2009. # CLINICAL PERSPECTIVE {#article-title-2}Background— An elevated level of homocysteine (hyperhomocysteinemia) has been implicated as an independent risk factor for cardiovascular diseases. Deficiency of dietary factors like vitamin B12, folate, and genetic variations can cause hyperhomocysteinemia. The prevalence of hyperhomocysteinemia in the Indian population is likely to be high because most Indians adhere to a vegetarian diet, deficient in vitamin B12. In the background of vitamin B12 deficiency, variations in genes involved in homocysteine metabolism might have a greater impact on homocysteine levels. Methods and Results— We genotyped 44 nonsynonymous single-nucleotide polymorphisms (nsSNPs) from 11 genes involved in homocysteine metabolism and found only 14 to be polymorphic. These 14 nsSNPs were genotyped in 546 individuals recruited from a tertiary care center in New Delhi, India, and it was found that choline dehydrogenase (CHDH A119C) and methylenetetrahydrofolate reductase (MTHFR C677T) were significantly associated with plasma total homocysteine levels (P=0.009 and P=0.001, respectively). These 2 SNPs were further genotyped in 330 individuals recruited from the same center, and the association remained significant even after increasing the sample size. Furthermore, we found the possibility of a significant interaction between vegetarian diet and the 2 polymorphisms that could explain the variation of homocysteine levels. We also genotyped all the polymorphic nsSNPs in apparently healthy individuals recruited from 24 different subpopulations (based on their linguistic lineage) spread across the country to determine their basal frequencies. The frequencies of these SNPs varied significantly between linguistic groups. Conclusion— Vegetarian diet along with CHDH A119C and MTHFR C677T play an important role in modulating the homocysteine levels in Indian population.

ZNF598 and RACK1 Regulate Mammalian Ribosome-Associated Quality Control Function by Mediating Regulatory 40S Ribosomal Ubiquitylation

Ribosomes that experience terminal stalls during translation are resolved by ribosome-associated quality control (QC) pathways that oversee mRNA and nascent chain destruction and recycle ribosomal subunits. The proximal factors that sense stalled ribosomes and initiate mammalian ribosome-associated QC events remain undefined. We demonstrate that the ZNF598 ubiquitin ligase and the 40S ribosomal protein RACK1 help to resolve poly(A)-induced stalled ribosomes. They accomplish this by regulating distinct and overlapping regulatory 40S ribosomal ubiquitylation events. ZNF598 primarily mediates regulatory ubiquitylation of RPS10 and RPS20, whereas RACK1 regulates RPS2, RPS3, and RPS20 ubiquitylation. Gain or loss of ZNF598 function or mutations that block RPS10 or RPS20 ubiquitylation result in defective resolution of stalled ribosomes and subsequent readthrough of poly(A)-containing stall sequences. Together, our results indicate that ZNF598, RACK1, and 40S regulatory ubiquitylation plays a pivotal role in mammalian ribosome-associated QC pathways.

Progressive degradation of serum samples limits proteomic biomarker discovery.

Preanalytical variables play a key role in discovery of biomarkers. Although the effect of several preanalytical variables on the mass spectral profiles has been studied extensively, little is known about long-term storage of serum samples. This is important because samples used in case-control or epidemiological studies are usually stored for a long time before analysis. Here we evaluated long-term storage effects on mass spectral peak patterns of serum peptides extracted using weak cation exchange magnetic beads. For this, 20 serum samples stored at -80 degrees C were divided equally into two groups based on their storage time. We found that intensities of 26 mass spectral peaks significantly varied between these two groups. Intensities of these peaks significantly correlated with storage time. Genetic algorithm-based models generated using these 26 peaks could classify 63 additional samples into these two groups with 100% and 96% accuracy, respectively. We also show that storing samples for 10 months at -80 and -20 degrees C results in the appearance/disappearance or intensity variation of peaks, some of which were previously reported as disease biomarkers.

Predicting protein homocysteinylation targets based on dihedral strain energy and pKa of cysteines

A multitude of complex diseases have been linked to elevated homocysteine levels; however, till date there is no plausible explanation for a single amino acids involvement in so many diseases. Since homocysteine is a reactive thiol amino acid and the majority of plasma homocysteine is protein thiol bound, we hypothesized that homocysteine might bind to accessible cysteine residues in target proteins, thereby modulating its structure or function or both. The parameters that dictate homocysteine–protein interaction are not well understood, and the few known homocysteine binding proteins wereidentified by a candidate protein approach. In this study, we identified potential homocysteine interacting proteins based on cysteine content, solvent accessibility of cysteine residues, and dihedral strain energies and pKa of these cysteines. Pathway mapping of the cysteine‐rich proteins revealed that proteins in the coagulation cascade, notch receptor‐mediated signaling, LDL endocytosis, programmed cell death, and extracellular matrix proteins were significantly over‐represented with cysteine‐rich proteins, and we believe that homocysteine has a high probability to bind to proteins in these pathways. In fact, several clinical studies have implicated high homocysteine levels to be associated with diseases like thrombosis, neural tube defects, and so forth, which result from dysfunction of one or more of the proteins identified in our study. Further, we successfully validated our prediction parameters on the proteins that have already been experimentally shown to bind homocysteine, and our structural analysis argues a plausible explanation for these prior reported protein interactions with homocysteine that could not be previously explained. Proteins 2008.

USP21 and OTUD3 Antagonize Regulatory Ribosomal Ubiquitylation and Ribosome-Associated Quality Control

Defects within mRNAs or nascent chains that halt ribosomal progression can trigger ribosome-associated quality control (RQC) pathways that facilitate mRNA and nascent polypeptide destruction as well as ribosome recycling. Failure to remove defective mRNAs or nascent chains can lead to the accumulation of cytotoxic protein aggregates and proteotoxic stress. We previously established that the E3 ligase ZNF598 catalyzes regulatory ribosomal ubiquitylation of specific 40S ribosomal proteins required for downstream RQC events. Utilizing an optical RQC reporter we identify OTUD3 and USP21 as deubiquitylating enzymes that antagonize ZNF598-mediated 40S ubiquitylation and facilitate ribosomal deubiquitylation following RQC activation. Overexpression of either USP21 or OTUD3 enhances readthrough of stall-inducing sequences as compared to knock-in cells lacking individual RRub sites suggesting that combinatorial ubiquitylation of RPS10 (eS10) and RPS20 (uS10) is required for optimal resolution of RQC events and that deubiquitylating enzymes can limit RQC activation.

95 Identification of synthetic lethality compounds from natural products for cancers

L. Packer, S. Byron, C. Mahon, D. Loch, A. Wortmann, K. Nones, S. Grimmond, J. Pearson, N. Waddell, P. Pollock. Translational Research Institute, Queensland University of Technology, Brisbane Qld, Australia; Translational Genomics Research Institute, Phoenix, USA; Queensland University of Technology, Brisbane Qld, Australia; Institute of Molecular Biology, University of Queensland, Brisbane Qld, Australia; QIMR Berghofer Medical Research Institute, Brisbane Qld, Australia