Crystal structures of SCP2-thiolases of Trypanosomatidae, human pathogens causing widespread tropical diseases: the importance for catalysis of the cysteine of the unique HDCF loop

Thiolases are essential CoA-dependent enzymes in lipid metabolism. In the present study we report the crystal structures of trypanosomal and leishmanial SCP2 (sterol carrier protein, type-2)-thiolases. Trypanosomatidae cause various widespread devastating (sub)-tropical diseases, for which adequate treatment is lacking. The structures reveal the unique geometry of the active site of this poorly characterized subfamily of thiolases. The key catalytic residues of the classical thiolases are two cysteine residues, functioning as a nucleophile and an acid/base respectively. The latter cysteine residue is part of a CxG motif. Interestingly, this cysteine residue is not conserved in SCP2-thiolases. The structural comparisons now show that in SCP2-thiolases the catalytic acid/base is provided by the cysteine residue of the HDCF motif, which is unique for this thiolase subfamily. This HDCF cysteine residue is spatially equivalent to the CxG cysteine residue of classical thiolases. The HDCF cysteine residue is activated for acid/base catalysis by two main chain NH-atoms, instead of two water molecules, as present in the CxG active site. The structural results have been complemented with enzyme activity data, confirming the importance of the HDCF cysteine residue for catalysis. The data obtained suggest that these trypanosomatid SCP2-thiolases are biosynthetic thiolases. These findings provide promise for drug discovery as biosynthetic thiolases catalyse the first step of the sterol biosynthesis pathway that is essential in several of these parasites.

Predicting gene ontology annotations of orphan GWAS genes using protein-protein interactions

Background: The number of genome-wide association studies (GWAS) has increased rapidly in the past couple of years, resulting in the identification of genes associated with different diseases. The next step in translating these findings into biomedically useful information is to find out the mechanism of the action of these genes. However, GWAS studies often implicate genes whose functions are currently unknown; for example, MYEOV, ANKLE1, TMEM45B and ORAOV1 are found to be associated with breast cancer, but their molecular function is unknown. Results: We carried out Bayesian inference of Gene Ontology (GO) term annotations of genes by employing the directed acyclic graph structure of GO and the network of protein-protein interactions (PPIs). The approach is designed based on the fact that two proteins that interact biophysically would be in physical proximity of each other, would possess complementary molecular function, and play role in related biological processes. Predicted GO terms were ranked according to their relative association scores and the approach was evaluated quantitatively by plotting the precision versus recall values and F-scores (the harmonic mean of precision and recall) versus varying thresholds. Precisions of similar to 58% and similar to 40% for localization and functions respectively of proteins were determined at a threshold of similar to 30 (top 30 GO terms in the ranked list). Comparison with function prediction based on semantic similarity among nodes in an ontology and incorporation of those similarities in a k nearest neighbor classifier confirmed that our results compared favorably. Conclusions: This approach was applied to predict the cellular component and molecular function GO terms of all human proteins that have interacting partners possessing at least one known GO annotation. The list of predictions is available at http://severus.dbmi.pitt.edu/engo/GOPRED.html. We present the algorithm, evaluations and the results of the computational predictions, especially for genes identified in GWAS studies to be associated with diseases, which are of translational interest.

The Presence of Multiple Cellular Defects Associated with a Novel G50E Iron-Sulfur Cluster Scaffold Protein ( ISCU) Mutation Leads to Development of Mitochondrial Myopathy

Background: Muscle-specific deficiency of iron-sulfur (Fe-S) cluster scaffold protein (ISCU) leads to myopathy. Results: Cells carrying the myopathy-associated G50E ISCU mutation demonstrate impaired Fe-S cluster biogenesis and mitochondrial dysfunction. Conclusion: Reduced mitochondrial respiration as a result of diminished Fe-S cluster synthesis results in muscle weakness in myopathy patients. Significance: The molecular mechanism behind disease progression should provide invaluable information to combat ISCU myopathy. Iron-sulfur (Fe-S) clusters are versatile cofactors involved in regulating multiple physiological activities, including energy generation through cellular respiration. Initially, the Fe-S clusters are assembled on a conserved scaffold protein, iron-sulfur cluster scaffold protein (ISCU), in coordination with iron and sulfur donor proteins in human mitochondria. Loss of ISCU function leads to myopathy, characterized by muscle wasting and cardiac hypertrophy. In addition to the homozygous ISCU mutation (g.7044GC), compound heterozygous patients with severe myopathy have been identified to carry the c.149GA missense mutation converting the glycine 50 residue to glutamate. However, the physiological defects and molecular mechanism associated with G50E mutation have not been elucidated. In this report, we uncover mechanistic insights concerning how the G50E ISCU mutation in humans leads to the development of severe ISCU myopathy, using a human cell line and yeast as the model systems. The biochemical results highlight that the G50E mutation results in compromised interaction with the sulfur donor NFS1 and the J-protein HSCB, thus impairing the rate of Fe-S cluster synthesis. As a result, electron transport chain complexes show significant reduction in their redox properties, leading to loss of cellular respiration. Furthermore, the G50E mutant mitochondria display enhancement in iron level and reactive oxygen species, thereby causing oxidative stress leading to impairment in the mitochondrial functions. Thus, our findings provide compelling evidence that the respiration defect due to impaired biogenesis of Fe-S clusters in myopathy patients leads to manifestation of complex clinical symptoms.

Trans-spliced Heat Shock Protein 90 Modulates Encystation in Giardia lamblia

Background: Hsp90 from Giardia lamblia is expressed by splicing of two independently transcribed RNA molecules, coded by genes named HspN and HspC located 777 kb apart. The reasons underlying such unique trans-splicing based generation of GlHsp90 remain unclear. Principle Finding: In this study using mass-spectrometry we identify the sequence of the unique, junctional peptide contributed by the 5' UTR of HspC ORF. This peptide is critical for the catalytic function of Hsp90 as it harbours an essential ``Arg'' in its sequence. We also show that full length GlHsp90 possesses all the functional hall marks of a canonical Hsp90 including its ability to bind and hydrolyze ATP. Using qRT-PCR as well as western blotting approach we find the reconstructed Hsp90 to be induced in response to heat shock. On the contrary we find GlHsp90 to be down regulated during transition from proliferative trophozoites to environmentally resistant cysts. This down regulation of GlHsp90 appears to be mechanistically linked to the encystation process as we find pharmacological inhibition of GlHsp90 function to specifically induce encystation. Significance: Our results implicate the trans-spliced GlHsp90 from Giardia lamblia to regulate an essential stage transition in the life cycle of this important human parasite.

The Glycosylated Rv1860 Protein of Mycobacterium tuberculosis Inhibits Dendritic Cell Mediated TH1 and TH17 Polarization of T Cells and Abrogates Protective Immunity Conferred by BCG

We previously reported interferon gamma secretion by human CD4(+) and CD8(+) T cells in response to recombinant E. coli-expressed Rv1860 protein of Mycobacterium tuberculosis (MTB) as well as protection of guinea pigs against a challenge with virulent MTB following prime-boost immunization with DNA vaccine and poxvirus expressing Rv1860. In contrast, a Statens Serum Institute Mycobacterium bovis BCG (BCG-SSI) recombinant expressing MTB Rv1860 (BCG-TB1860) showed loss of protective ability compared to the parent BCG strain expressing the control GFP protein (BCG-GFP). Since Rv1860 is a secreted mannosylated protein of MTB and BCG, we investigated the effect of BCG-TB1860 on innate immunity. Relative to BCG-GFP, BCG-TB1860 effected a significant near total reduction both in secretion of cytokines IL-2, IL-12p40, IL-12p70, TNF-alpha, IL-6 and IL-10, and up regulation of co-stimulatory molecules MHC-II, CD40, CD54, CD80 and CD86 by infected bone marrow derived dendritic cells (BMDC), while leaving secreted levels of TGF-beta unchanged. These effects were mimicked by BCG-TB1860His which carried a 6-Histidine tag at the C-terminus of Rv1860, killed sonicated preparations of BCG-TB1860 and purified H37Rv-derived Rv1860 glycoprotein added to BCG-GFP, but not by E. coli-expressed recombinant Rv1860. Most importantly, BMDC exposed to BCG-TB1860 failed to polarize allogeneic as well as syngeneic T cells to secrete IFN-gamma and IL-17 relative to BCG-GFP. Splenocytes from mice infected with BCG-SSI showed significantly less proliferation and secretion of IL-2, IFN-gamma and IL-17, but secreted higher levels of IL-10 in response to in vitro restimulation with BCG-TB1860 compared to BCG-GFP. Spleens from mice infected with BCG-TB1860 also harboured significantly fewer DC expressing MHC-II, IL-12, IL-2 and TNF-alpha compared to mice infected with BCG-GFP. Glycoproteins of MTB, through their deleterious effects on DC may thus contribute to suppress the generation of a TH1- and TH17-dominated adaptive immune response that is vital for protection against tuberculosis.

HupB, a Nucleoid-Associated Protein of Mycobacterium tuberculosis, Is Modified by Serine/Threonine Protein Kinases In Vivo

HU, a widely conserved bacterial histone-like protein, regulates many genes, including those involved in stress response and virulence. Whereas ample data are available on HU-DNA communication, the knowledge on how HU perceives a signal and transmit it to DNA remains limited. In this study, we identify HupB, the HU homolog of the human pathogen Mycobacterium tuberculosis, as a component of serine/threonine protein kinase (STPK) signaling. HupB is extracted in its native state from the exponentially growing cells of M. tuberculosis H37Ra and is shown to be phosphorylated on both serine and threonine residues. The STPKs capable of modifying HupB are determined in vitro and the residues modified by the STPKs are identified for both in vivo and the in vitro proteins through mass spectrometry. Of the identified phosphosites, Thr(65) and Thr(74) in the DNA-embracing beta-strand of the N-terminal domain of HupB (N-HupB) are shown to be crucial for its interaction with DNA. In addition, Arg(55) is also identified as an important residue for N-HupB-DNA interaction. N-HupB is shown to have a diminished interaction with DNA after phosphorylation. Furthermore, hupB is shown to be maximally expressed during the stationary phase in M. tuberculosis H37Ra, while HupB kinases were found to be constitutively expressed (PknE and PknF) or most abundant during the exponential phase (PknB). In conclusion, HupB, a DNA-binding protein, with an ability to modulate chromatin structure is proposed to work in a growth-phase-dependent manner through its phosphorylation carried out by the mycobacterial STPKs.

Heat shock protein 90 as a potential drug target against surra

Trypanosomiasis is caused by Trypanosoma species which affect both human and animal populations and pose a major threat to developing countries. The incidence of animal trypanosomiasis is on the rise. Surra is a type of animal trypanosomiasis, caused by Trypanosoma evansi, and has been included in priority list B of significant diseases by the World Organization of Animal Health (OIE). Control of surra has been a challenge due to the lack of effective drugs and vaccines and emergence of resistance towards existing drugs. Our laboratory has previously implicated Heat shock protein 90 (Hsp90) from protozoan parasites as a potential drug target and successfully demonstrated efficacy of an Hsp90 inhibitor in cell culture as well as a pre-clinical mouse model of trypanosomiasis. This article explores the role of Hsp90 in the Trypanosoma life cycle and its potential as a drug target. It appears plausible that the repertoire of Hsp90 inhibitors available in academia and industry may have value for treatment of surra and other animal trypanosomiasis.

Re-analysis of cryoEM data on HCV IRES bound to 40S subunit of human ribosome integrated with recent structural information suggests new contact regions between ribosomal proteins and HCV RNA

In this study, we combine available high resolution structural information on eukaryotic ribosomes with low resolution cryo-EM data on the Hepatitis C Viral RNA (IRES) human ribosome complex. Aided further by the prediction of RNA-protein interactions and restrained docking studies, we gain insights on their interaction at the residue level. We identified the components involved at the major and minor contact regions, and propose that there are energetically favorable local interactions between 40S ribosomal proteins and IRES domains. Domain II of the IRES interacts with ribosomal proteins S5 and S25 while the pseudoknot and the downstream domain IV region bind to ribosomal proteins S26, S28 and S5. We also provide support using UV cross-linking studies to validate our proposition of interaction between the S5 and IRES domains II and IV. We found that domain IIIe makes contact with the ribosomal protein S3a (S1e). Our model also suggests that the ribosomal protein S27 interacts with domain IIIc while S7 has a weak contact with a single base RNA bulge between junction IIIabc and IIId. The interacting residues are highly conserved among mammalian homologs while IRES RNA bases involved in contact do not show strict conservation. IRES RNA binding sites for S25 and S3a show the best conservation among related viral IRESs. The new contacts identified between ribosomal proteins and RNA are consistent with previous independent studies on RNA-binding properties of ribosomal proteins reported in literature, though information at the residue level is not available in previous studies.

Infection of human amniotic and endothelial cells by Japanese encephalitis virus: Increased expression of HLA-F

Productive infection of human amniotic and endothelial cell lines with Japanese encephalitis virus (JEV) was established leading to the induction of NF kappa B and HLA-F, a non-classical MHC molecule. Induction of the HLA-F gene and protein in JEV-infected cells was shown to be NF kappa B dependent since it was blocked by inhibitors of NF kappa B activation. ShRNA targeting lentivirus-mediated stable knockdown of the p65 subunit of NF kappa B inhibited JEV-mediated induction of HLA-F both in the amniotic cell line, AV-3 as well as the human brain microendothelial cell line, HBMEC. The induction of HLA-F by treatment of AV-3 with TNF-alpha was also inhibited by ShRNA mediated knockdown of NF kappa B. TNF-alpha treatment of HEK293T cells that were transfected with reporter plasmids under the control of HLA-F enhancer A elements resulted in significant transactivation of the luciferase reporter gene. NF kappa B-mediated induction of HLA-F following JEV infection and TNF-alpha exposure is being suggested for the first time. (C) 2014 Elsevier Inc. All rights reserved.

From Workstations to Workbenches: Towards Predicting Physicochemically Viable Protein-Protein Interactions Across a Host and a Pathogen

The understanding of protein-protein interactions is indispensable in comprehending most of the biological processes in a cell. Small-scale experiments as well as large-scale high-throughput techniques over the past few decades have facilitated identification and analysis of protein-protein interactions which form the basis of much of our knowledge on functional and regulatory aspects of proteins. However, such rich catalog of interaction data should be used with caution when establishing protein-protein interactions in silico, as the high-throughput datasets are prone to false positives. Numerous computational means developed to pursue genome-wide studies on protein-protein interactions at times overlook the mechanistic and molecular details, thus questioning the reliability of predicted protein-protein interactions. We review the development, advantages, and shortcomings of varied approaches and demonstrate that by providing a structural viewpoint in terms of shape complementarity and interaction energies at protein-protein interfaces coupled with information on expression and localization of proteins homologous to an interacting pair, it is possible to assess the credibility of predicted interactions in biological context. With a focus on human pathogen Mycobacterium tuberculosis H37Rv, we show that such scrupulous use of details at the molecular level can predict physicochemically viable protein-protein interactions across host and pathogen. Such predicted interactions have the potential to provide molecular basis of probable mechanisms of pathogenesis and hence open up ways to explore their usefulness as targets in the light of drug discovery. (c) 2014 IUBMB Life, 66(11):759-774, 2014

Mycobacterium tuberculosis Cell Division Protein, FtsE, is an ATPase in Dimeric Form

FtsE is one of the earliest cell division proteins that assembles along with FtsX at the mid-cell site during cell division in Escherichia coli. Both these proteins are highly conserved across diverse bacterial genera and are predicted to constitute an ABC transporter type complex, in which FtsE is predicted to bind ATP and hydrolyse it, and FtsX is predicted to be an integral membrane protein. We had earlier reported that the MtFtsE of the human pathogen, Mycobacterium tuberculosis, binds ATP and interacts with MtFtsX on the cell membrane of M. tuberculosis and E. coli. In this study, we demonstrate that MtFtsE is an ATPase, the active form of which is a dimer, wherein the participating monomers are held together by non-covalent interactions, with the Cys84 of each monomer present at the dimer interface. Under oxidising environment, the dimer gets stabilised by the formation of Cys84-Cys84 disulphide bond. While the recombinant MtFtsE forms a dimer on the membrane of E. coli, the native MtFtsE seems to be in a different conformation in the M. tuberculosis membrane. Although disulphide bridges were not observed on the cytoplasmic side (reducing environment) of the membrane, the two participating monomers could be isolated as dimers held together by non-covalent interactions. Taken together, these findings show that MtFtsE is an ATPase in the non-covalent dimer form, with the Cys84 of each monomer present in the reduced form at the dimer interface, without participating in the dimerisation or the catalytic activity of the protein.

REPERTOIRE OF PROTEIN KINASES ENCODED IN THE GENOME OF ZEBRAFISH SHOWS REMARKABLY LARGE POPULATION OF PIM KINASES

In recent times, zebrafish has garnered lot of popularity as model organism to study human cancers. Despite high evolutionary divergence from humans, zebrafish develops almost all types of human tumors when induced. However, mechanistic details of tumor formation have remained largely unknown. Present study is aimed at analysis of repertoire of kinases in zebrafish proteome to provide insights into various cellular components. Annotation using highly sensitive remote homology detection methods revealed ``substantial expansion'' of Ser/Thr/Tyr kinase family in zebrafish compared to humans, constituting over 3% of proteome. Subsequent classification of kinases into subfamilies revealed presence of large number of CAMK group of kinases, with massive representation of PIM kinases, important for cell cycle regulation and growth. Extensive sequence comparison between human and zebrafish PIM kinases revealed high conservation of functionally important residues with a few organism specific variations. There are about 300 PIM kinases in zebrafish kinome, while human genome codes for only about 500 kinases altogether. PIM kinases have been implicated in various human cancers and are currently being targeted to explore their therapeutic potentials. Hence, in depth analysis of PIM kinases in zebrafish has opened up new avenues of research to verify the model organism status of zebrafish.

Characterization of Precursor PfHsp60 in Plasmodium falciparum Cytosol during Its Asexual Development in Human Erythrocytes

Mitochondrial heat shock protein 60 (Hsp60) is a nuclear encoded gene product that gets post-translationally translocated into the mitochondria. Using multiple approaches such as immunofluorescence experiments, isoelectric point analysis with two-dimensional gel electrophoresis, and mass spectrometric identification of the signal peptide, we show that Hsp60 from Plasmodium falciparum (PfHsp60) accumulates in the parasite cytoplasm during the ring, trophozoite, and schizont stages of parasite development before being imported into the parasite mitochondria. Using co-immunoprecipitation experiments with antibodies specific to cytoplasmic PfHsp90, PfHsp70-1, and PfHsp60, we show association of precursor PfHsp60 with cytoplasmic chaperone machinery. Metabolic labeling involving pulse and chase indicates translocation of the precursor pool into the parasite mitochondrion during chase. Analysis of results obtained with Geldanamycin treatment confirmed precursor PfHsp60 to be one of the clients for PfHsp90. Cytosolic chaperones bind precursor PfHsp60 prior to its import into the mitochondrion of the parasite. Our data suggests an inefficient co-ordination in the synthesis and translocation of mitochondrial PfHsp60 during asexual growth of malaria parasite in human erythrocytes.

Glioblastoma-derived Macrophage Colony-stimulating Factor (MCSF) Induces Microglial Release of Insulin-like Growth Factor-binding Protein 1 (IGFBP1) to Promote Angiogenesis

Glioblastoma (grade IV glioma/GBM) is the most common primary adult malignant brain tumor with poor prognosis. To characterize molecular determinants of tumor-stroma interaction in GBM, we profiled 48 serum cytokines and identified macrophage colony-stimulating factor (MCSF) as one of the elevated cytokines in sera from GBM patients. Both MCSF transcript and protein were up-regulated in GBM tissue samples through a spleen tyrosine kinase (SYK)-dependent activation of the PI3K-NF kappa B pathway. Ectopic overexpression and silencing experiments revealed that glioma-secreted MCSF has no role in autocrine functions and M2 polarization of macrophages. In contrast, silencing expression of MCSF in glioma cells prevented tube formation of human umbilical vein endothelial cells elicited by the supernatant from monocytes/microglial cells treated with conditioned medium from glioma cells. Quantitative proteomics based on stable isotope labeling by amino acids in cell culture showed that glioma-derived MCSF induces changes in microglial secretome and identified insulin-like growth factor-binding protein 1 (IGFBP1) as one of the MCSF-regulated proteins secreted by microglia. Silencing IGFBP1 expression in microglial cells or its neutralization by an antibody reduced the ability of supernatants derived from microglial cells treated with glioma cell-conditioned medium to induce angiogenesis. In conclusion, this study shows up-regulation of MCSF in GBM via a SYK-PI3K-NF kappa B-dependent mechanism and identifies IGFBP1 released by microglial cells as a novel mediator of MCSF-induced angiogenesis, of potential interest for developing targeted therapy to prevent GBM progression.

Mycobacterium tuberculosis RecG Protein but Not RuvAB or RecA Protein Is Efficient at Remodeling the Stalled Replication Forks IMPLICATIONS FOR MULTIPLE MECHANISMS OF REPLICATION RESTART IN MYCOBACTERIA

Aberrant DNA replication, defects in the protection, and restart of stalled replication forks are major causes of genome instability in all organisms. Replication fork reversal is emerging as an evolutionarily conserved physiological response for restart of stalled forks. Escherichia coli RecG, RuvAB, and RecA proteins have been shown to reverse the model replication fork structures in vitro. However, the pathways and the mechanisms by which Mycobacterium tuberculosis, a slow growing human pathogen, responds to different types of replication stress and DNA damage are unclear. Here, we show that M. tuberculosis RecG rescues E. coli Delta recG cells from replicative stress. The purified M. tuberculosis RecG (MtRecG) and RuvAB(MtRuvAB) proteins catalyze fork reversal of model replication fork structures with and without a leading strand single-stranded DNA gap. Interestingly, single-stranded DNA-binding protein suppresses the MtRecG- and MtRuvAB-mediated fork reversal with substrates that contain lagging strand gap. Notably, our comparative studies with fork structures containing template damage and template switching mechanism of lesion bypass reveal that MtRecG but not MtRuvAB or MtRecA is proficient in driving the fork reversal. Finally, unlike MtRuvAB, we find that MtRecG drives efficient reversal of forks when fork structures are tightly bound by protein. These results provide direct evidence and valuable insights into the underlying mechanism of MtRecG-catalyzed replication fork remodeling and restart pathways in vivo.