Biochemical characterization of C4 protein of cotton leaf curl Kokhran virus-Dabawali

Background: Cotton leaf curl Kokhran Virus-Dabawali (CLCuKV-Dab) is a monopartite begomovirus encoding two proteins V1 and V2 in the virion sense and four proteins Cl, C2, C3 and C4 in the complementary sense. The C4 protein of monopartite begomoviruses has been implicated to play a role in symptom determination and virus movement. The present work aims at the biochemical characterization of this protein. Methods: The C4 protein of CLCuKV-Dab was purified in fusion with GST and tested for the ability to hydrolyze ATP and other phosphate containing compounds. ATPase activity was assayed by using radiolabeled gamma-32P]-ATP and separating the product of reaction by thin layer chromatography. The hydrolysis of other compounds was monitored by the formation of a blue colored phosphomolybdate complex which was estimated by measuring the absorbance at 655 nm. Results: The purified GST-C4 protein exhibited metal ion dependent ATPase and inorganic pyrophosphatase activities. Deletion of a sequence resembling the catalytic motif present in phosphotyrosine phosphatases resulted in 70% reduction in both the activities. Mutational analysis suggested arginine 13 to be catalytically important for the ATPase and cysteine 8 for the pyrophosphatase activity of GST-C4. Interaction of V2 with GST-C4 resulted in an increase in both the enzymatic activities of GST-C4. Conclusions: The residues important for the enzymatic activities of GST-C4 are present in a motif different from the classical Walker motifs and the non-classical ATP binding motifs reported so far. General significance: The C4 protein of CLCuKV-Dab, a putative natively unfolded protein, exhibits enzymatic activities.

Human la protein interaction with GCAC near the initiator AUG enhances hepatitis C virus RNA replication by promoting linkage between 5 ` and 3 ` untranslated regions

Human La protein has been implicated in facilitating the internal initiation of translation as well as replication of hepatitis C virus (HCV) RNA. Previously, we demonstrated that La interacts with the HCV internal ribosome entry site (IRES) around the GCAC motif near the initiator AUG within stem-loop IV by its RNA recognition motif (RRM) (residues 112 to 184) and influences HCV translation. In this study, we have deciphered the role of this interaction in HCV replication in a hepatocellular carcinoma cell culture system. We incorporated mutation of the GCAC motif in an HCV monocistronic subgenomic replicon and a pJFH1 construct which altered the binding of La and checked HCV RNA replication by reverse transcriptase PCR (RT-PCR). The mutation drastically affected HCV replication. Furthermore, to address whether the decrease in replication is a consequence of translation inhibition or not, we incorporated the same mutation into a bicistronic replicon and observed a substantial decrease in HCV RNA levels. Interestingly, La overexpression rescued this inhibition of replication. More importantly, we observed that the mutation reduced the association between La and NS5B. The effect of the GCAC mutation on the translation-to-replication switch, which is regulated by the interplay between NS3 and La, was further investigated. Additionally, our analyses of point mutations in the GCAC motif revealed distinct roles of each nucleotide in HCV replication and translation. Finally, we showed that a specific interaction of the GCAC motif with human La protein is crucial for linking 5' and 3' ends of the HCV genome. Taken together, our results demonstrate the mechanism of regulation of HCV replication by interaction of the cis-acting element GCAC within the HCV IRES with human La protein.

Cyclic AMP-dependent protein lysine acylation in mycobacteria regulates fatty acid and propionate metabolism

Acetylation of lysine residues is a posttranslational modification that is used by both eukaryotes and prokaryotes to regulate a variety of biological processes. Here we identify multiple substrates for the cAMP-dependent protein lysine acetyltransferase from Mycobacterium tuberculosis (KATmt). We demonstrate that a catalytically important lysine residue in a number of FadD (fatty acyl CoA synthetase) enzymes is acetylated by KATmt in a cAMP-dependent manner and that acetylation inhibits the activity of FadD enzymes. A sirtuin-like enzyme can deacetylate multiple FadDs, thus completing the regulatory cycle. Using a strain deleted for the KATmt ortholog in Mycobacterium bovis Bacillus Calmette-Guerin (BCG), we show for the first time that acetylation is dependent on intracellular cAMP levels. KATmt can utilize propionyl CoA as a substrate and, therefore, plays a critical role in alleviating propionyl CoA toxicity in mycobacteria by inactivating acyl CoA synthetase (ACS). The precision by which mycobacteria can regulate the metabolism of fatty acids in a cAMP-dependent manner appears to be unparalleled in other biological organisms and is ideally suited to adapt to the complex environment that pathogenic mycobacteria experience in the host.

Identification of an exported heat shock protein 70 in Plasmodium falciparum

Host cell remodelling is a hallmark of malaria pathogenesis. It involves protein folding, unfolding and trafficking events and thus participation of chaperones such as Hsp70s and Hsp40s is well speculated. Until recently, only Hsp40s were thought to be the sole representative of the parasite chaperones in the exportome. However, based on the re-annotated Plasmodium falciparum genome sequence, a putative candidate for exported Hsp70 has been reported, which otherwise was known to be a pseudogene. We raised a specific antiserum against a C-terminal peptide uniquely present in PfHsp70-x. Immunoblotting and immunofluorescence-based approaches in combination with sub-cellular fractionation by saponin and streptolysin-O have been taken to determine the expression and localization of PfHsp70-x in infected erythrocyte. The re-annotated sequence of PfHsp70-x reveals it to be a functional protein with an endoplasmic reticulum signal peptide. It gets maximally expressed at the schizont stage of intra-erythrocytic life cycle. Majority of the protein localizes to the parasitophorous vacuole and some of it gets exported to the erythrocyte compartment where it associates with Maurer's clefts. The identification of an exported parasite Hsp70 chaperone presents us with the fact that the parasite has evolved customized chaperones which might be playing crucial roles in aspects of trafficking and host cell remodelling.

Network properties of decoys and CASP predicted models: a comparison with native protein structures

Protein structure space is believed to consist of a finite set of discrete folds, unlike the protein sequence space which is astronomically large, indicating that proteins from the available sequence space are likely to adopt one of the many folds already observed. In spite of extensive sequence-structure correlation data, protein structure prediction still remains an open question with researchers having tried different approaches (experimental as well as computational). One of the challenges of protein structure prediction is to identify the native protein structures from a milieu of decoys/models. In this work, a rigorous investigation of Protein Structure Networks (PSNs) has been performed to detect native structures from decoys/ models. Ninety four parameters obtained from network studies have been optimally combined with Support Vector Machines (SVM) to derive a general metric to distinguish decoys/models from the native protein structures with an accuracy of 94.11%. Recently, for the first time in the literature we had shown that PSN has the capability to distinguish native proteins from decoys. A major difference between the present work and the previous study is to explore the transition profiles at different strengths of non-covalent interactions and SVM has indeed identified this as an important parameter. Additionally, the SVM trained algorithm is also applied to the recent CASP10 predicted models. The novelty of the network approach is that it is based on general network properties of native protein structures and that a given model can be assessed independent of any reference structure. Thus, the approach presented in this paper can be valuable in validating the predicted structures. A web-server has been developed for this purpose and is freely available at http://vishgraph.mbu.iisc.ernet.in/GraProStr/PSN-QA.html.

Bactericidal/permeability increasing protein: A multifaceted protein with functions beyond LPS neutralization

Bactericidal permeability increasing protein (BPI), a 55-60kDa protein, first reported in 1975, has gone a long way as a protein with multifunctional roles. Its classical role in neutralizing endotoxin (LPS) raised high hopes among septic shock patients. Today, BPI is not just a LPS-neutralizing protein, but a protein with diverse functions. These functions can be as varied as inhibition of endothelial cell growth and inhibition of dendritic cell maturation, or as an anti-angiogenic, chemoattractant or opsonization agent. Though the literature available is extremely limited, it is fascinating to look into how BPI is gaining major importance as a signalling molecule. In this review, we briefly summarize the recent research focused on the multiple roles of BPI and its use as a therapeutic.

Nitric oxide and KLF4 protein epigenetically modify class II transactivator to repress Major histocompatibility complex II expression during Mycobacterium bovis Bacillus Calmette-Guerin infection

Pathogenic mycobacteria employ several immune evasion strategies such as inhibition of class II transactivator (CIITA) and MHC-II expression, to survive and persist in host macrophages. However, precise roles for specific signaling components executing down-regulation of CIITA/MHC-II have not been adequately addressed. Here, we demonstrate that Mycobacterium bovis bacillus Calmette-Guerin (BCG)-mediated TLR2 signaling-induced iNOS/NO expression is obligatory for the suppression of IFN-gamma-induced CIITA/MHC-II functions. Significantly, NOTCH/PKC/MAPK-triggered signaling cross-talk was found critical for iNOS/NO production. NO responsive recruitment of a bifunctional transcription factor, KLF4, to the promoter of CIITA during M. bovis BCG infection of macrophages was essential to orchestrate the epigenetic modifications mediated by histone methyltransferase EZH2 or miR-150 and thus calibrate CIITA/MHC-II expression. NO-dependent KLF4 regulated the processing and presentation of ovalbumin by infected macrophages to reactive T cells. Altogether, our study delineates a novel role for iNOS/NO/KLF4 in dictating the mycobacterial capacity to inhibit CIITA/MHC-II-mediated antigen presentation by infected macrophages and thereby elude immune surveillance.

Cryogenically cured hydroxyapatite-gelatin nanobiocomposite for bovine serum albumin protein adsorption and release

This research paper presents the first results on the protein adsorption and release kinetics and in vitro biodegradability of cryogenically cured hydroxyapatite-gelatin based micro/macroporous scaffolds (CHAMPS). While the adsorption and release of bovine serum albumin (BSA) protein exhibits steady state behavior over an incubation period of up to 10 days, Fourier transform infrared (FT-IR) analysis importantly confirms the absence of any change in the secondary structure of BSA proteins due to interaction with the CHAMPS scaffold. The compression properties of the CHAMPS scaffold with interconnected porosity (pore size similar to 50-200 mm) is characterized by a non-linear stress-strain response with a strength close to 5 MPa and a maximum strain of up to 24%. The slow but systematic increase in weight loss over a period of 7 days as well as apatite layer formation indicates its good bioactivity. The extensive micro-computed tomography (micro-CT) analysis establishes cancellous bone-like highly interconnected and complex porous architecture of the CHAMPS scaffold. Importantly, the excellent adsorption (up to 50%) and release (up to 60% of adsorbed protein) of BSA has been uniquely attributed to the inherent porous microstructure of the CHAMPS scaffold. Overall, the present study provides an assessment of the interaction of protein with the gelatin-hydroxyapatite macroporous scaffold in vitro, as well as reporting for the first time the efficacy of such scaffolds to release 60% of BSA loaded onto the scaffold in vitro, which is significantly higher than earlier literature reports.

Redox regulation of protein tyrosine phosphatase 1B (PTP1B): Importance of steric and electronic effects on the unusual cyclization of the sulfenic acid intermediate to a sulfenyl amide

The redox regulation of protein tyrosine phosphatase 1B (PTP1B) via the unusual transformation of its sulfenic acid (PTP1B-SOH) to a cyclic sulfenyl amide intermediate is studied by using small molecule chemical models. These studies suggest that the sulfenic acids derived from the H2O2-mediated reactions o-amido thiophenols do not efficiently cyclize to sulfenyl amides and the sulfenic acids produced in situ can be trapped by using methyl iodide. Theoretical calculations suggest that the most stable conformer of such sulfenic acids are stabilized by n(O) -> sigma* (S-OH) orbital interactions, which force the -OH group to adopt a position trans to the S center dot center dot center dot O interaction, leading to an almost linear arrangement of the O center dot center dot center dot S-O moiety and this may be the reason for the slow cyclization of such sulfenic acids to their corresponding sulfenyl amides. On the other hand, additional substituents at the 6-position of o-amido phenylsulfenic acids that can induce steric environment and alter the electronic properties around the sulfenic acid moiety by S center dot center dot center dot N or S center dot center dot center dot O nonbonded interactions destabilize the sulfenic acids by inducing strain in the molecule. This may lead to efficient the cyclization of such sulfenic acids. This model study suggests that the amino acid residues in the close proximity of the sulfenic acid moiety in PTP1B may play an important role in the cyclization of PTP1B-SOH to produce the corresponding sulfenyl amide.

N-Terminal disordered domain of saccharomyces cerevisiae Hop1 protein Is dispensable for DNA binding, bridging, and synapsis of double-stranded DNA molecules but is ecessary for spore formation

The cytological architecture of the synaptonemal complex (SC), a meiosis-specific proteinaceous structure, is evolutionarily conserved among eukaryotes. However, little is known about the biochemical properties of SC components or the mechanisms underlying their roles in meiotic chromosome synapsis and recombination. Functional analysis of Saccharomyces cerevisiae Hop1, a key structural component of SC, has begun to reveal important insights into its function in interhomolog recombination. Previously, we showed that Hop1 is a structure-specific DNA-binding protein, exhibits higher binding affinity for the Holliday junction, and induces structural distortion at the core of the junction. Furthermore, Hop1 promotes DNA condensation and intra- and intermolecular synapsis between duplex DNA molecules. Here, we show that Hop1 possesses a modular domain organization, consisting of an intrinsically disordered N-terminal domain and a protease-resistant C-terminal domain (Hop1CTD). Furthermore, we found that Hop1CTD exhibits strong homotypic as well as heterotypic protein protein interactions, and its biochemical activities were similar to those of the full-length Hop1 protein. However, Hop1CTD failed to complement the meiotic recombination defects of the Delta hop1 strain, indicating that both N- and C-terminal domains of Hop1 are essential for meiosis and spore formation. Altogether, our findings reveal novel insights into the structure-function relationships of Hop1 and help to further our understanding of its role in meiotic chromosome synapsis and recombination.

DNA and protein targeting 1,2,4-triazole based water soluble dinickel(II) complexes enhances antiproliferation and lactate dehydrogenase inhibition

Water soluble dinickel(II) complexes Ni-2(L)(2)(1-2)](NO3)(4) (1-2), where L1-2 are triazole based dinucleating ligands, were synthesized and characterized. The DNA binding, protein binding, DNA hydrolysis and anticancer properties were investigated. The interactions of complexes 1 and 2 with calf thymus DNA were studied by spectroscopic techniques, including absorption and fluorescence spectroscopy. The DNA binding constant values of the complexes 1 and 2 were found to be 2.36 x 10(5) and 4.87 x 10(5) M-1 and the binding affinities are in the following order: 2 > 1. Both the dinickel(II) complexes 1 and 2, promoted the hydrolytic cleavage of plasmid pBR322 DNA under both anaerobic and aerobic conditions. Kinetic data for DNA hydrolysis promoted by 1 and 2 under physiological conditions give the observed rate constants (k(obs)) of 5.05 +/- 0.2 and 5.65 +/- 0.1 h(-1), respectively, which shows 10(8)-fold rate acceleration over the uncatalyzed reaction of ds-DNA. Meanwhile, the interactions of the complex with BSA have also been studied by spectroscopy. Both the complexes 1 and 2 display strong binding propensity and the binding constant (K-b), number of binding sites (n) were obtained are 0.71 x 10(6) 1.47] and 5.62 x 10(6) 1.98] M-1, respectively. The complexes 1 and 2 also promoted the apoptosis against human carcinoma (HeLa, and BeWo) cancer cells. Cytotoxicity of the complexes was further confirmed by lactate dehydrogenase enzyme level in cancer cell lysate and content media. (c) 2013 Elsevier Ltd. All rights reserved.

Maltose Binding Protein Is Partially Structured in Its Molten Globule State

Seven double cysteine mutants of maltose binding protein (MBP) were generated with one each in the active cleft at position 298 and the second cysteine distributed over both domains of the protein. These cysteines were spin labeled and distances between the labels in biradical pairs determined by pulsed double electron-electron resonance (DEER) measurements. The values were compared with theoretical predictions of distances between the labels in biradicals constructed by molecular modeling from the crystal structure of MBP without maltose and were found to be in excellent agreement. MBP is in a molten globule state at pH 3.3 and is known to still bind its substrate maltose. The nitroxide spin label was sufficiently stable under these conditions. In preliminary experiments, DEER measurements were carried out with one of the mutants yielding a broad distance distribution as was to be expected if there is no explicit tertiary structure and the individual helices pointing into all possible directions.

An automated approach to network features of protein structure ensembles

Network theory applied to protein structures provides insights into numerous problems of biological relevance. The explosion in structural data available from PDB and simulations establishes a need to introduce a standalone-efficient program that assembles network concepts/parameters under one hood in an automated manner. Herein, we discuss the development/application of an exhaustive, user-friendly, standalone program package named PSN-Ensemble, which can handle structural ensembles generated through molecular dynamics (MD) simulation/NMR studies or from multiple X-ray structures. The novelty in network construction lies in the explicit consideration of side-chain interactions among amino acids. The program evaluates network parameters dealing with topological organization and long-range allosteric communication. The introduction of a flexible weighing scheme in terms of residue pairwise cross-correlation/interaction energy in PSN-Ensemble brings in dynamical/chemical knowledge into the network representation. Also, the results are mapped on a graphical display of the structure, allowing an easy access of network analysis to a general biological community. The potential of PSN-Ensemble toward examining structural ensemble is exemplified using MD trajectories of an ubiquitin-conjugating enzyme (UbcH5b). Furthermore, insights derived from network parameters evaluated using PSN-Ensemble for single-static structures of active/inactive states of 2-adrenergic receptor and the ternary tRNA complexes of tyrosyl tRNA synthetases (from organisms across kingdoms) are discussed. PSN-Ensemble is freely available from http://vishgraph.mbu.iisc.ernet.in/PSN-Ensemble/psn_index.html.