Department of Bioscience & Bioengineering

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IIT Jodhpur

In living cells, polypeptide chains emerging from ribosomes and preexisting polypeptide chains face constant threat of misfolding and aggregation. To prevent protein aggregation and to fulfill their biological activity, generally, protein must fold into its proper threedimensional structure throughout their lifetimes. Eukaryotic cell possesses a quality control (QC) system to contend the problem of protein misfolding and aggregation. Cells achieve this functional QC system with the help of molecular chaperones and ubiquitin-proteasome system (UPS). The well-conserved UPS regulates the stability of various proteins and maintains all essential cellular function through intracellular protein degradation. E3 ubiquitin ligase enzyme determines specificity for degradation of certain substrates via UPS. New emerging evidences have provided considerable information that various E3 ubiquitin ligases play a major role in cellular QC mechanism and principally designated as QC E3 ubiquitin ligases. Nevertheless, very little is known about how E3 ubiquitin ligase maintains QC mechanism against abnormal proteins under various stress conditions. Here in this review, we highlight and discuss the functions of various E3 ubiquitin ligases implicated in protein QC mechanism. Improving our knowledge about such processes may provide opportunities to modulate protein QC mechanism in age-of-onset diseases that are caused by protein aggregation. © Springer Science+Business Media, LLC 2012.

World University root node

Molecular Neurobiology

E3 ubiquitin ligases in protein quality control mechanism

Objective: Inflammatory bowel diseases (IBDs), namely ulcerative colitis (UC) and Crohn's disease (CD), are characterized by chronic and idiopathic inflammatory conditions of gastrointestinal tract that are immunologically mediated. Single nucleotide polymorphisms in cytokine genes have been reported to modulate inflammation. Therefore, we analyzed the association of pro/anti-inflammatory cytokine genes polymorphism with IBD susceptibility. Methods: Genotyping of interleukin (IL)-4 repeat polymorphism in intron-3, IL-10 (G-1082A and C-819T), and tumor necrosis factor-alpha (TNF-A) (-1031 T>C, -863 C>A, and -857 C>T) was performed in 153 patients with IBD and in 207 controls. Results: TNF-A -863 AA genotype was associated with enhanced IBD susceptibility (odds ratio (OR), 4.82; 95% confidence interval (CI), 2.60-8.96), more so for UC (OR, 5.79; 95% CI, 2.99-11.21), Crohn's disease [CD] (OR, 3.13; 95% CI, 1.16-8.47). TNF-A T/C/T (OR, 4.40; 95% CI, 1.64-11.81) and C/A/C (OR, 4.15; 95% CI, 2.48-6.96) haplotypes were associated with increased IBD risk. The frequency of IL-4, B2 carrier (B1/B2+B2/B2) was significantly lower in left-sided UC (17.1%) than proctosigmoiditis (47.6%); p, 0.016. In contrast, TNF-A -863 AA genotype frequency was much higher in pancolitis (45.5) than in proctosigmoiditis (14.2); p, 0.037. Variant genotypes of IL-4 (B1/B2+B2/B2) were absent in colonic type CD. IL-10 polymorphisms did not demonstrate any association with IBD. None of the polymorphisms were associated with steroid treatment and surgery. Conclusion: The present study depicts that high-producing genotype of TNF-A (-863 AA) was associated with increased risk of IBD more so with UC. Similarly, combined effect of TNF-A polymorphisms in haplotype analysis demonstrated additively increased risk of IBD. © Springer Science+Business Media, LLC 2011.

Journal of gastrointestinal cancer

Role of tumor necrosis factor-alpha (C-863A) polymorphism in pathogenesis of inflammatory bowel disease in Northern India

Stability and induction of the lysogenic state of bacteriophage $\lambda$ are balanced by a complex regulatory network. A key feature of this network is the mutually exclusive cooperative binding of a repressor dimer (CI) to one of two pairs of binding sites, O R1-O R2 or O R2-O R3. The structural features that underpin the mutually exclusive binding mode are not well understood. Recent studies have demonstrated that CI is an asymmetric dimer. The functional importance of the asymmetry is not fully clear. Due to the asymmetric nature of the CI dimer as well as its binding sites, there are two possible bound orientations. By fluorescence resonance energy transfer measurements we showed that CI prefers one bound orientation. We also demonstrated that the relative configuration of the binding sites is important for CI dimer-dimer interactions and consequent cooperative binding. We proposed that the operator configuration dictates the orientations of the bound CI molecules, which in turn dictates CI cooperative interaction between the O R1-O R2 or O R2-O R3, but not both. Modeling suggests that the relative orientation of the C- and N-terminal domains may play an important role in the mutually exclusive nature of the cooperative binding. This work correlates unique structural features of a transcription regulatory protein with the functional properties of a gene regulatory network. {\textcopyright} 2012 Biophysical Society.

Fulltext

Biophysical Journal

A genetic network that balances two outcomes utilizes asymmetric recognition of operator sites

Clustering technique is one of the useful tools to elucidate similar patterns across large number of transcripts and to identify likely co-regulated genes. It attempts to partition the genes into groups exhibiting similar patterns of variation in expression level. An application of rough-fuzzy c-means (RFCM) algorithm is presented in this paper to discover co-expressed gene clusters. Selection of initial prototypes of different clusters is one of the major issues of the RFCM based microarray data clustering. The pearson correlation based initialization method is used to address this limitation. It enables the RFCM algorithm to discover co-expressed gene clusters. The effectiveness of the RFCM algorithm and the initialization method, along with a comparison with other related methods, is demonstrated on five yeast gene expression data sets using standard cluster validity indices and gene ontology based analysis. © 2012 Springer-Verlag.

Lecture Notes in Computer Science (including subseries Lecture Notes in Artificial Intelligence and Lecture Notes in Bioinformatics)

Rough-fuzzy c-means for clustering microarray gene expression data

Background: Laccases are blue multi-copper oxidases and catalyze the oxidation of phenolic and non-phenolic compounds. There is considerable interest in using these enzymes for dye degradation as well as for synthesis of aromatic compounds. Laccases are produced at relatively low levels and, sometimes, as isozymes in the native fungi. The investigation of properties of individual enzymes therefore becomes difficult. The goal of this study was to over-produce a previously reported laccase from Cyathus bulleri using the well-established expression system of Pichia pastoris and examine and compare the properties of the recombinant enzyme with that of the native laccase.Results: In this study, complete cDNA encoding laccase (Lac) from white rot fungus Cyathus bulleri was amplified by RACE-PCR, cloned and expressed in the culture supernatant of Pichia pastoris under the control of the alcohol oxidase (AOX)1 promoter. The coding region consisted of 1,542 bp and encodes a protein of 513 amino acids with a signal peptide of 16 amino acids. The deduced amino acid sequence of the matured protein displayed high homology with laccases from Trametes versicolor and Coprinus cinereus. The sequence analysis indicated the presence of Glu 460 and Ser 113 and LEL tripeptide at the position known to influence redox potential of laccases placing this enzyme as a high redox enzyme. Addition of copper sulfate to the production medium enhanced the level of laccase by about 12-fold to a final activity of 7200 U L-1. The recombinant laccase (rLac) was purified by ~4-fold to a specific activity of ~85 U mg-1 protein. A detailed study of thermostability, chloride and solvent tolerance of the rLac indicated improvement in the first two properties when compared to the native laccase (nLac). Altered glycosylation pattern, identified by peptide mass finger printing, was proposed to contribute to altered properties of the rLac.Conclusion: Laccase of C. bulleri was successfully produced extra-cellularly to a high level of 7200 U L-1 in P. pastoris under the control of the AOX1 promoter and purified by a simple three-step procedure to homogeneity. The kinetic parameters against ABTS, Guaiacol and Pyrogallol were similar with the nLac and the rLac. Tryptic finger print analysis of the nLac and the rLac indicated altered glycosylation patterns. Increased thermo-stability and salt tolerance of the rLac was attributed to this changed pattern of glycosylation. © 2012 Garg et al.; licensee BioMed Central Ltd.

BMC Biotechnology

Cloning, sequence analysis, expression of Cyathus bulleri laccase in Pichia pastoris and characterization of recombinant laccase

Here, we present the genome sequence of Enterobacter cloacae GS1. This strain proficiently colonizes rice roots and promotes plant growth by improving plant nutrition. Analyses of the E. cloacae GS1 genome will throw light on the genetic factors involved in root colonization, growth promotion, and ecological success of this rhizobacterium. © 2012, American Society for Microbiology.

Journal of Bacteriology

Genome sequence of the plant growth-promoting bacterium enterobacter cloacae GS1

Here we report the genome sequence of a plant growth-promoting rhizobacterium, Pseudomonas putida S11. The length of the draft genome sequence is approximately 5,970,799 bp, with a G+C content of 62.4%. The genome contains 6,076 protein-coding sequences. © 2012, American Society for Microbiology.

Genome sequence of the plant growth-promoting rhizobacterium pseudomonas putida S11

Oxygen-sensing prolyl-hydroxylase (PHD)-2 negatively regulates hypoxia-inducible factor (HIF)1-α and suppresses the hypoxic response. Hypoxia signaling is thought to be proinflammatory but also attenuates cellular injury and apoptosis. Although increased hypoxic response has been noted in asthma, its functional relevance is unknown. The objectives of this study were to dissect the mechanisms and role of the hypoxic response in asthma pathophysiology. Experimental studies were conducted in mice using acute and chronic allergic models of asthma. The hypoxic response in allergically inflamed lungs was modulated by using pharmacologic PHD inhibitors (ethyl-3-4- dihydroxybenzoic acid [DHB], 1-10 mg/kg) or siRNA-mediated genetic knockdowns. Increased hypoxia response led to exacerbation of the asthma phenotype, with HIF-1α knockdown being beneficial. Chronically inflamed lungs from mice treated with 10 mg/kg DHB showed diffuse up-regulation of the hypoxia response, severe airway remodeling, and inflammation. Fatal asphyxiation during methacholine challenge was noted. However, bronchial epithelium restricted up-regulation of the hypoxia response seen with low-dose DHB (1 mg/kg) reduced epithelial injury and attenuated the asthmatic phenotype. Up-regulation of the hypoxia response was associated with increased expression of CX3CR1, a lymphocyte survival factor, and increased inflammatory cell infiltrate. This study shows that an exaggerated hypoxia response may contribute to airway inflammation, remodeling, and the development of asthma. However, the hypoxia response may also be protective of epithelial apoptosis at lower levels, and the net effects of modulating the hypoxia response may vary based on the context. Copyright © 2012 by the American Thoracic Society.

American Journal of Respiratory Cell and Molecular Biology

Hypoxia response in asthma: Differential modulation on inflammation and epithelial injury

Collagens presently used in tissue engineering are primarily of bovine or porcine origin. However, a risk of a spongiform encephalopathy epidemic has limited the use of collagen from these sources. Keeping the aforementioned perspective in mind, we explored the possibility of using domestic goat available in the subcontinent as a potential source of collagen for tissue-engineering application. This article delineates the isolation, physico-chemical characterization, biocompatibility study and wound healing application of acid soluble caprine (goat) tendon collagen (GTC). Physico-chemical characterization of 1% acetic acid extracted GTC was done by SDS-PAGE, amino-acid composition analysis, FT-IR and CD spectroscopy. Results revealed that GTC was comprised of type-I collagen. Biocompatibility study showed that GTC augmented cell adhesion, cell cycle progression and proliferation. Immuno-cytochemical analysis in conjugation with traction force microscopy further confirmed a superior focal adhesion complex mediated cell-substrate interaction in GTC. Finally, in vivo study in mice model revealed that GTC has low immunogenicity and it augments healing process significantly. Throughout the study, calf skin collagen (CSC) was used as standard for comparative evaluation. In conclusion, it can be said that GTC may find its application as biomaterial in skin tissue engineering. © 2012 VSP.

Journal of Biomaterials Science, Polymer Edition

Caprine (Goat) collagen: A potential biomaterial for skin tissue engineering

MicroRNAs (miRNAs) are short, endogenous RNAs having ability to regulate gene expression at the post-transcriptional level. Various studies have revealed that miRNAs tend to cluster on chromosomes. Members of a cluster that are at close proximity on chromosome are highly likely to be processed as cotranscribed units. Therefore, a large proportion of miRNAs are co-expressed. Expression profiling of miRNAs generates a huge volume of data. Complicated networks of miRNA-mRNA interaction create a big challenge for scientists to decipher this huge expression data. In order to extract meaningful information from expression data, this paper presents the application of robust rough-fuzzy c-means (rRFCM) algorithm to discover co-expressed miRNA clusters. The rRFCM algorithm comprises a judicious integration of rough sets, fuzzy sets, and c-means algorithm. The effectiveness of the rRFCM algorithm and different initialization methods, along with a comparison with other related methods, is demonstrated on three miRNA microarray expression data sets using Silhouette index, Davies-Bouldin index, Dunn index, β index, and gene ontology based analysis. © 2012 IEEE.