Protein and Peptide Letters - Volume 20, Issue 9, 2013

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known to interact with different biomolecules and was implicated in many novel cellular activities including programmed cell death, nuclear RNA transport unrelated to the commonly known carbohydrate metabolism. We reported here the purification of GAPDH from Chironomidae larvae (Insecta, Diptera) that showed different biologic activity towards heavy metals. It was inhibited by copper, cobalt nickel, iron and lead but was activated by zinc. The GAPDH was purified by ammonium sulphate fractionation and Chelating Sepharose CL-6B chromatography followed by Blue Sepharose CL-6B chromatography. The 150-kDa tetrameric GAPDH showed optimal activity at pH 8.5 and 37 °C. The multiple alignment of sequence of the Chironomidae GAPDH with other known species showed 78 - 88 % identity to the conserved regions of the GADPH. Bioinformatic analysis unveils substantial N-terminal sequence similarity of GAPDH of Chironomidae larvae to mammalian GADPHs. However, the GADPH of Chironomidae larvae showed different biologic activities and cytotoxicity towards heavy metals. The GAPDH enzyme would undergo adaptive molecular changes through binding at the active site leading to higher tolerance to heavy metals.

There are accumulating evidences suggesting that ferrochelatase is involved in different cellular functions other than heme biosynthesis. The carboxyl-terminal extension of the enzyme may play a role associated with its stability and signaling process. Two transcript variants encoding different isoforms have been found for this ferrochelatase-coding gene. Variants encoding different isoforms have been found for this gene and were implicated in novel cellular activities including gene expression. Deficiency in ferrochelatase variants is believed to be associated with its stability. In this study, mutagenesis study of the lysine residues at 357, 360 and 365 was conducted. The relative change in activity by measurement of fluorescence intensity of the mutant enzyme revealed that K365L retained > 40% activity, while the mutant K365I retained 24% activity. Other mutants with K357S and K360Q showed little enzyme activity (< 4%). The change in the tryptophan fluorescence intensity after Guanidinium chloride-induced denaturation of the mutants indicated that the enzyme become unfolded under the assay condition. The K365L mutation suggests a structural role of tryptophan in the enzyme. The stability of the enzyme attributed to conservation of lysine residues in the C-terminal extension of the enzyme. The lysine and tryptophan residues serve as a structure determinant of ferrochelatase.

In tumorigenesis, cancer genetics and the related mutations have been the main topic of study these days. Caspases have been found to be actively involved in the process of apoptosis. Malfunction of apoptosis is one of the causes for cancerous tumors and different caspase mutations are related to that process. It has been found that two groups of caspases involved in this process apoptosis which are initiator caspases and executioner caspases. SNPs have been extensively studied over the last decade, due to their association with a number of genetic diseases. Human SNPs have always been a source of information related to the complex changes associated with their origin. SNPs which can change the resulting amino acid i.e., nonsynonymous SNPs (nsSNPs) are of prime concern these days because of their direct relation with the disease or the respective individual. In this study our focus is not only to detect the nsSNPs available in the human caspase data but to further evaluate the potentially damaging nsSNPs. Using the computational approach we have been able to obtain almost seventy eight nsSNPs, among these few of the nsSNPs seem to have serious consequences, as they have been cross verified from a variety of SNP prediction tools. The functional as well as structural impact of the nsSNPs is determined and discussed. Our predicted nsSNPs on human caspases may be associated with cancer risk.

Circadian rhythms have been widely observed in plants, animals, fungi and cyanobacteria. The periodic rhythm is roughly 24-hour cycle in biochemical, physiological, or behavioral processes in organisms. Although circadian rhythms are endogenously-driven, an organism’s circadian clock is adjusted to the environmental stimuli and must maintain the periodicity of biologic rhythm despite the changing kinetics in cellular activities. Some cellular proteins are believed to be associated with circadian rhythms. In this study, deficiency of a serum protein in human subjects was detected in individuals diagnosed with sleep disorders. N-terminal sequence analysis of the short peptide with BLAST sequence alignment software on NCBI indicated that it might be related to the protein named Hlark (human lark protein). Lark is required for embryonic development, and is, in fact, RBM4, a splicing regulator. RBM4 is crucial for targeting to speckles. This finding is consistent with the potential existence of a novel sub-nuclear targeting pathway. RBM4 and Lark is probably the same protein and are localized in speckles and nucleoli, but can redistribute to peri-nucleolar clusters, consistent with a novel sub-nuclear pathway.

MurG is an essential bacterial glycosyltransferase enzyme in Pseudomonas aeruginosa performing one of the key membrane steps of peptidoglycan synthesis catalyzing the transfer of N-acetyl glucosamine (GlcNAc) from its donor substrate, UDP-GlcNAc, to the acceptor substrate Lipid I. We have solved the crystal structure of the complex between Pseudomonas aeruginosa MurG and UDP-GlcNAc and compared it with the previously solved complex from E. coli. The structure reveals a large-scale conformational change in the relative orientations of the N- and C-terminal domains, which has the effect of widening the cofactor binding site and displacing the UDP-GlcNAc donor. These results suggest new opportunities to design potent inhibitors of peptidoglycan biosynthesis.

The receptor-type protein-tyrosine phosphatase (RPTP) phogrin is localized at the membrane of secretory granules of pancreatic islet β-cells and, similarly to the closely related ICA512, plays a role in the regulation of insulin secretion, in ensuring proper granulogenesis and stability, and in the regulation of β-cell growth. The mature membraneproximal ectodomain of phogrin (MPE phogrin) was produced as a recombinant protein and characterized. CD, fluorescence, controlled proteolysis, size-exclusion chromatography, and multi-angle light scattering showed that it is a properlyfolded monomeric domain. Equilibrium experiments, in the presence of guanidinium chloride and thermal unfolding, suggest a two-state mechanism with a ΔG of 2.3-3.3 kcal/mol, respectively. The study establishes common features and differences of MPE phogrin and the homologous ectodomain of ICA512. A homology model of phogrin was built based in the x-ray structure of MPE ICA512. The model is a starting point for modeling the entire receptor and for testing the quaternary structure and interactions of this protein in vivo. A description of the membrane insertion mode and putative interacting surfaces of this large protein is fundamental for the understanding of its biological function.

Many important protein interactions related to cell signaling networks and post-translational modification events are mediated by the binding of a globular domain in one protein to a short peptide stretch in another. In the current study, we describe a structure-level protocol to realize the quantitative prediction of weak affinity in such interactions. This method uses the crystal structure of CAL PDZ domain complexed with a CFTR C-terminus mimic peptide as the template to construct other congeneric domain-peptide complex structure models. Subsequently, independent residue-pair interactions between the domain and peptide in constructed complexes are computed and correlated with experimentally measured affinity of 80 CAL PDZ binders by using partial least squares (PLS) and random forest (RF). We demonstrate that (a) the nonlinear RF is time-consuming but performs much well as compared to linear PLS in modeling and predicting the binding affinity of domain-peptide interactions, (b) the proposed structure-based strategy is more effective and accurate than those of traditional sequence-based methods in capturing the binding behavior and interaction information of domain with peptide, and (c) only very few residue-pairs at complex interface contribute significantly to domain-peptide binding.

The end-point of diabetic renal disease is the accumulation of excess collagen (fibrosis/sclerosis). A number of studies have shown that the hormone relaxin (RLX) ameliorates progression of renal and non-renal fibrosis. This study assessed the anti-fibrotic potential of RLX in streptozotocin (STZ)-treated transgenic mRen-2 rats, an accelerated model of type 1 diabetes. Eight-week old hyperglycaemic (STZ-treated at week-6) and normoglycaemic (STZ-untreated) animals were treated with or without recombinant human gene-2 (H2) RLX for 4-weeks (by osmotic mini-pumps) and assessed for various parameters at 12-weeks of age. Hyperglycaemic mRen-2 rats had elevated kidney weight/body weight ratio, glomerular filtration rate (GFR), albumin excretion rate (AER), interstitial collagen I and glomerulosclerosis (all p<0.05 vs non-diabetic controls). H2 RLX infusion had no effect on any of these parameters. Increased MMP-2 levels in RLX-treated rats demonstrated that the hormone was administered and active in this model. The inability of H2 RLX to slow glomerulopathy in diabetic mRen-2 rats could be in part due to the absence of its receptor, RXFP1, in rat mesangial cells, a primary mediator of diabetic glomerulosclerosis and/or the lack of any effect on TGF-β1/Smad2 signalling, a well described mediator of RLX activity. These findings highlight the cell specific actions of RLX, the dissociation of anti-fibrogenic (collagen synthesis) and antifibrolytic (MMP mediated collagen degradation) properties, and the central involvement of TGF-β1 in its actions.

Protein tyrosine phosphatase (PTP) catalytic domains undergo a series of conformational changes in order to mediate dephosphorylation of their tyrosine phosphorylated substrates. An important conformational change occurs in the Tryptophan-Proline-Aspartic acid (WPD) loop, which contains the conserved catalytic aspartate. Upon substrate binding, the WPD loop transitions from the ‘open’ to the ‘closed’ state, thus allowing optimal positioning of the catalytic aspartate for substrate dephosphorylation. The dynamics of WPD loop conformational changes have previously been studied for PTP1B, HePTP, and the bacterial phosphatase YopH, but have not yet been comprehensively studied for the nonreceptor tyrosine phosphatase SHP-1 (PTPN6). To structurally describe the changes in WPD loop conformation in SHP-1, we have determined the 1.4 Å crystal structure of the catalytic domain of SHP-1 in the Apo state and the 1.8 &Å crystal structure of the SHP-1 catalytic domain in complex with a phosphate ion. We provide structural analysis for the WPD loop closed state of SHP phosphatases and the conformational changes that occur upon WPD loop closure.

Hexavalent chromium [Cr(VI)] is a widespread environmental pollutant, arising as a by-product of numerous industrial processes. Bacteria can reduce toxic and carcinogenic Cr(VI) to insoluble and less toxic Cr(III), offering promise for an environmental friendly and affordable solution to chromate pollution. ChrR, a class I chromate-reducing flavoenzyme from Pseudomonas putida is an efficient chromate reducer. The crystal structure of ChrR is yet unknown to the scientific community, hence a three-dimensional (3D) structure is very essential for structural studies, protein – ligand interaction simulations and designing novel bioremediation strategies. The 3D model of the P. putida ChrR protein was predicted using in silico approach. Due to low percentage of sequence identity for homology modeling, I-TASSER was used for structure prediction which combines the methods of threading, ab initio modeling and structural refinement. The stereo chemical quality of the best model was validated with 90.6% residues under favored region from Ramachandran plot. The modeled protein was submitted to Protein Model Database and can be downloaded with the ID PM0077375. The degree of conservation was mapped onto the predicted model and ligand binding sites were found. The results of conservation analysis and binding site prediction were combined to show several highly conserved binding sites. Altogether, the structure for ChrR has been predicted. The work reveals novel universally conserved residues. These residues could be candidates for binding interactions and provide the basis for designing advanced chromium bioremediation strategies.

Bacterial ADP-ribosyltransferases (BADPRTs) are extensively contributed to determine the strain-specific virulence state and pathogenesis in human hosts. Understanding molecular evolution and functional diversity of the BADPRTs is an important standpoint to describe the fundamental behind in the vaccine designing for bacterial infections. In the present study, we have evaluated the origin and functional evolution of conserved domains within the BADPRTs by analyzing their sequence-function relationship. To represent the evolution history of BADPRTs, phylogenetic trees were constructed based on their protein sequence, structure and conserved domains using different evolutionary programs. Sequence divergence and genetic diversity were studied herein to deduce the functional evolution of conserved domains across the family and superfamily. The results of sequence similarity search have shown that three hypothetical proteins (above 90%) were identical to the members of BADPRTs and their functions were annotated by phylogenetic approach. Phylogenetic analysis of this study has revealed the family members of BADPRTs were phylogenetically related to one another, functionally diverged within the same family, and dispersed into closely related bacteria. The presence of core substitution pattern in the conserved domains would determine the family-specific function of BADPRTs. Functional diversity of the BADPRTs was exclusively distinguished by Darwinian positive selection (diphtheria toxin C and pertussis toxin S) and neutral selection (arginine ADP-ribosyltransferase, enterotoxin A and binary toxin A) acting on the existing domains. Many of the family members were sharing their sequence-specific features from members in the arginine ADPribosyltransferase family. Conservative functions of members in the BADPRTs have shown to be expanded only within closely related families, and retained as such in pathogenic bacteria by evolutionary process (domain duplication or recombination events). Hence, we conclude that evolutionary significance of the members in the BADPRTs would provide an insight for experimental set-up on site-directed mutagenesis and vaccine engineering.

The article describes the development of a robust pharmacophore model and the investigation of structure activity relationship analysis of 3-amino-4-(2-cyanopyrrolidide)pyrrolidinyl analogs reported for DPP-IV inhibition using PHASE module of Schrodinger software. The present works also encompass molecular interaction study of 3-amino-4-(2- cyanopyrrolidide)pyrrolidinyl analogs on maestro 8.5 workstation. The Phase study module comprises the five points pharmacophore model (AAHPR.617), consisting two hydrogen bond acceptor (A), one Hydrophobic (H), one Positive(P) and one aromatic ring (R) and with discrete geometries as pharmacophoric feature. The developed pharmacophore model was used to derive a predictive atom-based 3D QSAR model. The obtained 3D QSAR model has an excellent correlation coefficient value (r2=0.9926) along with good statistical significance as shown by high Fisher ratio (F=671.7). The model also exhibits good predictive power, which is confirmed by high value of cross validated correlation coefficient (q2 = 0.7311). The QSAR model suggests that hydrophobic and aromatic characters are crucial for the DPP-IV inhibitory activity. The QSAR model also suggests that the inclusion of hydrophobic substituents would enhance the DPP-IV inhibition. In addition to the hydrogen bond acceptor, hydrophobic character, electro withdrawing character positively contributes to the DPP-IV inhibition. This study provides a set of guidelines for designing compounds with better DPP-IV inhibitory potency.