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.