Homology inference of protein-protein interactions via conserved binding sites

The coverage and reliability of protein-protein interactions determined by high-throughput experiments still needs to be improved, especially for higher organisms, therefore the question persists, how interactions can be verified and predicted by computational approaches using available data on protein structural complexes. Recently we developed an approach called IBIS (Inferred Biomolecular Interaction Server) to predict and annotate protein-protein binding sites and interaction partners, which is based on the assumption that the structural location and sequence patterns of protein-protein binding sites are conserved between close homologs. In this study first we confirmed high accuracy of our method and found that its accuracy depends critically on the usage of all available data on structures of homologous complexes, compared to the approaches where only a non-redundant set of complexes is employed. Second we showed that there exists a trade-off between specificity and sensitivity if we employ in the prediction only evolutionarily conserved binding site clusters or clusters supported by only one observation (singletons). Finally we addressed the question of identifying the biologically relevant interactions using the homology inference approach and demonstrated that a large majority of crystal packing interactions can be correctly identified and filtered by our algorithm. At the same time, about half of biological interfaces that are not present in the protein crystallographic asymmetric unit can be reconstructed by IBIS from homologous complexes without the prior knowledge of crystal parameters of the query protein.     

Mitochondrial mitophagic mechanisms of myocardial matrix metabolism and remodelling

High levels of homocysteine (Hcy), known as hyperhomocysteinmia (HHcy), are correlated with an increase in extracellular matrix remodelling (ECM) via the matrix metalloproteinases (MMPs) and plasminogen/plasmin system. This results in an increase deposition of collagen that leads to endothelial-myocyte (EM) and myocyte-myocyte (MM) uncoupling; the physiological consequences are a plethora of cardiovascular pathologies. Homocysteine-induced increase in intracellular and mitochondrial Ca(2+) plays an important role in increasing reactive oxygen species (ROS) within mitochondria and instigating mitophagy within the cell. This occurs via several Hcy-mitigated processes: agonizing N-methyl-d-aspartate receptor-1 (NMDA-R1), decreasing expression of peroxisome proliferator activator receptor (PPAR) [thereby increasing oxidation], impairing Ca(2+) handling via Na(+)/Ca(2+) exchanger (NCX1) and Sarco endoplasmic reticulum Ca(2+) ATPase (SERCA-2a). The end result is an increase in ROS that directly or indirectly lead to MMP activation within mitochondria or the cytoplasm. Hcy induces a mitochondrial permeability transition that allows MMPs to be released from mitochondria thereby metabolizing matrix and impairing cardiac function. Further work remains to be elucidated concerning the specific mitochondrial mitophagic mechanisms under which matrix metabolism and remodelling occurs. Moreover, the therapeutic implications of NMDA and PPAR ligands are some promise to patient.     

Autophagy and Heart Failure: A Possible Role for Homocysteine

Autophagy is a process used for intracellular digestion of organelles and proteins and has special relevance to the long-lived cardiomyocytes in heart disease. The pathway for autophagy and all its mediators remain to be elucidated, but involve such proteins as Atg, Beclin-1, LAMP-2, BH3, Bcl2, PI3K Kinase as well as a plethora of others. It is still not entirely clear whether autophagy is destructive or beneficial to the cell; evidence suggests that the answer is case-specific. For instance, autophagy appears to preserve cell life under cases of ischemia in I/R injury, but is detrimental during reperfusion. High levels of homocysteine (Hcy), a sulfur-containing amino acid, have been shown to be an independent risk factor for chronic heart failure. There are several links to induction and repression of autophagy and Hcy; the following connections to Hcy and autophagy have been made: intracellular nitrous oxide production, intracellular calcium production, and reactive oxygen species production. Further work remains to be elucidated concerning the specific mechanisms under which autophagy occurs and possible Hcy-mediated connections. Moreover, the therapeutic implications might be of some promise to patients.     

Hydrogen sulfide mitigates cardiac remodeling during myocardial infarction via improvement of angiogenesis

Exogenous hydrogen sulfide (H2S) leads to down-regulation of inflammatory responses and provides myocardial protection during acute ischemia/reperfusion injury; however its role during chronic heart failure (CHF) due to myocardial infarction (MI) is yet to be unveiled. We previously reported that H2S inhibits antiangiogenic factors such, as endostatin and angiostatin, but a little is known about its effect on parstatin (a fragment of proteinase-activated receptor-1, PAR-1). We hypothesize that H2S inhibits parstatin formation and promotes VEGF activation, thus promoting angiogenesis and significantly limiting the extent of MI injury. To verify this hypothesis MI was created in 12 week-old male mice by ligation of left anterior descending artery (LAD). Sham surgery was performed except LAD ligation. After the surgery mice were treated with sodium hydrogen sulfide (30 μmol/l NaHS, a donor for H2S, in drinking water) for 4 weeks. The LV tissue was analyzed for VEGF, flk-1 and flt-1, endostatin, angiostatin and parstatin. The expression of VEGF, flk-1 and flt-1 were significantly increased in treated mice while the level of endostatin, angiostatin and parstatin were decreased compared to in untreated mice. The echocardiography in mice treated with H2S showed the improvement of heart function compared to in untreated mice. The X-ray and Doppler blood flow measurements showed enhancement of cardiac-angiogenesis in mice treated with H2S. This observed cytoprotection was associated with an inhibition of anti-angiogenic proteins and stimulation of angiogenic factors. We established that administration of H2S at the time of MI ameliorated infarct size and preserved LV function during development of MI in mice. These results suggest that H2S is cytoprotective and angioprotective during evolution of MI.     

Large-scale mapping of human protein interactome using structural complexes

Although the identification of protein interactions by high-throughput (HTP) methods progresses at a fast pace, 'interactome' data sets still suffer from high rates of false positives and low coverage. To map the human protein interactome, we describe a new framework that uses experimental evidence on structural complexes, the atomic details of binding interfaces and evolutionary conservation. The structurally inferred interaction network is highly modular and more functionally coherent compared with experimental interaction networks derived from multiple literature citations. Moreover, structurally inferred and high-confidence HTP networks complement each other well, allowing us to construct a merged network to generate testable hypotheses and provide valuable experimental leads.     

Urinary metabolomic phenotyping of nickel induced acute toxicity in rat: an NMR spectroscopy approach

The metabolomic approach has been widely used in toxicology to investigate mechanisms of toxicity. To understand the mammalian system??s response to nickel exposure, we analysed the NiCl₂ induced metabolomic changes in urine of rats using ¹H nuclear magnetic resonance (¹H NMR) spectroscopy together with clinically relevant biochemical parameters. Male Sprague?CDawley rats were administered intraperitoneally with NiCl₂ at doses of 4, 10 and 20?mg/kg body weight. Urine samples were collected at 8, 16, 24, 72, 96 and 120?h post treatment. The metabolomic profile of rat urine showed prominent changes in citrate, dimethylamine, creatinine, choline, trimethylamine oxide (TMAO), phenyl alanine and hippurate at all doses. Principal component analysis of urine ¹H NMR spectra demonstrated the dose and time dependent development of toxicity. The metabolomic time trajectory, based on pattern recognition analysis of ¹H NMR spectra of urine, illustrated clear separation of pre and post treatments (temporal). Only animals treated with a low dose of NiCl₂ returned to normal physiology. The ¹H NMR spectral data correlated well with the clinically relevant nephrotoxic biomarkers. The urinary metabolomic phenotyping for NiCl₂ induced nephrotoxicity was defined according to the predictive ability of the known metabolite biomarkers, creatinine, citrate and TMAO. The current approach demonstrates that metabolomics, one of the most important platform in system biology, may be a promising tool for identifying and characterizing biochemical responses to toxicity.     

Genome-wide investigation and expression analysis suggest diverse roles of auxin-responsive <Emphasis Type="Italic">GH3</Emphasis> genes during development and response to different stimuli in tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>)

In plants, auxin-mediated responses are regulated by diverse proteins. One such class of proteins, i.e. GH3, is involved in the conjugation of IAA to amino acids and provides a negative feedback loop to control auxin homoeostasis. In order to have a better understanding of the mechanism of the auxin action, 15 genes encoding GH3 members were identified using existing EST databases of tomato. Their orthologs were identified from tobacco, potato, N. benthemiana, pepper, and petunia. Phylogenetic analysis of AtGH3, SlGH3, and their Solanaceae orthologs provided insights into various orthologous relationships among these proteins. These genes were found to be responsive to a variety of signals including, phytohormones and environmental stresses. Analysis of AuxRE elements in their promoters showed variability in the sequence as well as number of this element. Up-regulation of only 11 SlGH3 genes, in response to exogenous auxin, suggested possible relationship between the diversity in the sequence and number of AuxRE element with the auxin inducibility. Expression analysis of SlGH3 genes in different vegetative and reproductive tissues/stages suggested limited or no role for most of the SlGH3 genes at the initiation of fruit ripening. However, up-regulation of SlGH3-1 and -2 at the onset of fruit ripening indicates that these genes could have a role in fruit ripening. The present study characterizes GH3 gene family of tomato and its evolutionary relationship with members of this family from other Solanaceae species and Arabidopsis. It could help in the identification of GH3 genes and revelation of their function during vegetative/reproductive development stages from other Solanaceae members.