Proteogenomic analysis of Mycobacterium tuberculosis by high resolution mass spectrometry

The genome sequencing of H37Rv strain of Mycobacterium tuberculosis was completed in 1998 followed by the whole genome sequencing of a clinical isolate, CDC1551 in 2002. Since then, the genomic sequences of a number of other strains have become available making it one of the better studied pathogenic bacterial species at the genomic level. However, annotation of its genome remains challenging because of high GC content and dissimilarity to other model prokaryotes. To this end, we carried out an in-depth proteogenomic analysis of the M. tuberculosis H37Rv strain using Fourier transform mass spectrometry with high resolution at both MS and tandem MS levels. In all, we identified 3176 proteins from Mycobacterium tuberculosis representing ~80% of its total predicted gene count. In addition to protein database search, we carried out a genome database search, which led to identification of ~250 novel peptides. Based on these novel genome search-specific peptides, we discovered 41 novel protein coding genes in the H37Rv genome. Using peptide evidence and alternative gene prediction tools, we also corrected 79 gene models. Finally, mass spectrometric data from N terminus-derived peptides confirmed 727 existing annotations for translational start sites while correcting those for 33 proteins. We report creation of a high confidence set of protein coding regions in Mycobacterium tuberculosis genome obtained by high resolution tandem mass-spectrometry at both precursor and fragment detection steps for the first time. This proteogenomic approach should be generally applicable to other organisms whose genomes have already been sequenced for obtaining a more accurate catalogue of protein-coding genes.     

Loop 1 of transducer region in mammalian class I myosin, Myo1b, modulates actin affinity, ATPase activity, and nucleotide access

Loop 1, a flexible surface loop in the myosin motor domain, comprises in part the transducer region that lies near the nucleotide-binding site and is proposed from structural studies to be responsible for the kinetic tuning of product release following ATP hydrolysis (1). Biochemical studies have shown that loop 1 affects the affinity of actin-myosin-II for ADP, motility and the V(max) of the actin-activated Mg2+-ATPase activity, possibly through P(i) release (2-8). To test the influence of loop 1 on the mammalian class I myosin, Myo1b, chimeric molecules in which (i) loop 1 of a truncated form of Myo1b, Myo1b1IQ, was replaced with either loop 1 from other myosins; (ii) loop 1 was replaced with glycine; or (iii) some amino acids in the loop were substituted with alanine and were expressed in baculovirus, and their interactions with actin and nucleotide were evaluated. The steady-state actin-activated ATPase activity; rate of ATP-induced dissociation of actin from Myo1b1IQ; rate of ADP release from actin-Myo1b1IQ; and the affinity of actin for Myo1b1IQ and Myo1b1IQ.ADP differed in the chimeras versus wild type, indicating that loop 1 has a much wider range of effects on the coupling between actin and nucleotide binding events than previously thought. In particular, the biphasic ATP-induced dissociation of actin from actin-Myo1b1IQ was significantly altered in the chimeras. This provided evidence that loop 1 contributes to the accessibility of the nucleotide pocket and is involved in the integration of information from the actin-, nucleotide-, gamma-P(i)-, and calmodulin-binding sites and predicts that loop 1 modulates the load dependence of the motor.     

Platelet storage under in vitro condition is associated with calcium-dependent apoptosis-like lesions and novel reorganization in platelet cytoskeleton

Platelets are cleared from circulation after a life span of 8-10 days. The molecular mechanisms underlying platelet senescence remain poorly characterized. Here we report that, progressive functional impairment in the platelets incubated in vitro in a plasma-free isotonic medium for up to 24 h at 37 degrees C is associated with release of cytochrome c from platelet mitochondria and cleavage of procaspase-9, but without evidence of caspase-3 activation. Concomitantly, there was proteolysis of survival proteins like focal adhesion kinase, Src, gelsolin, and specific cytoskeleton-associated peptides, in a manner regulated by extracellular calcium and calpain activity. Cytoskeleton played a critical role as evidenced from the association of these proteins and their degradation products, as well as procaspase-3 and the actin regulatory small GTPase, CDC42Hs, with the cytoskeleton of the stored platelets. The cytoskeletal enrichment with specific proteins was not associated with increase in the content of F-actin and was cytochalasin-resistant, thus signifying a novel mechanism of interaction of the translocating proteins with the pre-existing cytoskeleton. There was progressive exposure of phosphatidylserine on the outer leaflet of platelet membrane and specific electron microscopic changes suggestive of apoptotic lesions. Based on these observations we discuss the caspase-independent but calpain-mediated signaling events in the stored platelets resembling the features of apoptosis in the nucleated cells.     

Ceramide plays a prominent role in MDA‐7/IL‐24‐induced cancer‐specific apoptosis

Melanoma differentiation associated gene‐7/interleukin‐24 (mda‐7/IL‐24) uniquely displays broad cancer‐specific apoptosis‐inducing activity through induction of endoplasmic reticulum (ER) stress. We hypothesize that ceramide, a promoter of apoptosis, might contribute to mda‐7/IL‐24 induction of apoptosis. Ad.mda‐7‐infected tumor cells, but not normal cells, showed increased ceramide accumulation. Infection with Ad.mda‐7 induced a marked increase in various ceramides (C16, C24, C24:1) selectively in prostate cancer cells. Inhibiting the enzyme serine palmitoyltransferase (SPT) using the potent SPT inhibitor myriocin (ISP1), impaired mda‐7/IL‐24‐induced apoptosis and ceramide production, suggesting that ceramide formation caused by Ad.mda‐7 occurs through de novo synthesis of ceramide and that ceramide is required for mda‐7/IL‐24‐induced cell death. Fumonisin B1 (FB1) elevated ceramide formation as well as apoptosis induced by Ad.mda‐7, suggesting that ceramide formation may also occur through the salvage pathway. Additionally, Ad.mda‐7 infection enhanced expression of acid sphingomyelinase (ASMase) with a concomitant increase in ASMase activity and decreased sphingomyelin in cancer cells. ASMase silencing by RNA interference inhibited the decreased cell viability and ceramide formation after Ad.mda‐7 infection. Ad.mda‐7 activated protein phosphatase 2A (PP2A) and promoted dephosphorylation of the anti‐apoptotic molecule BCL‐2, a downstream ceramide‐mediated pathway of mda‐7/IL‐24 action. Pretreatment of cells with FB1 or ISP‐1 abolished the induction of ER stress markers (BiP/GRP78, GADD153 and pospho‐eIF2α) triggered by Ad.mda‐7 infection indicating that ceramide mediates ER stress induction by Ad.mda‐7. Additionally, recombinant MDA‐7/IL‐24 protein induced cancer‐specific production of ceramide. These studies define ceramide as a key mediator of an ER stress pathway that may underlie mda‐7/IL‐24 induction of cancer‐specific killing. J. Cell. Physiol. 222: 546–555, 2010. © 2009 Wiley‐Liss, Inc.     

In silico modeling and hydrogen peroxide binding study of rice catalase

Homology modeling of the catalase, CatC cloned and sequenced from rice (Oryza sativa L., cv Ratna an Indica cultivar) has been performed based on the crystal structure of the catalase CatF (PDB code 1m7s) by using the software MODELLER. With the aid of molecular mechanics and molecular dynamics methods, the final model is obtained and is further assessed by PROCHECK and VERIFY - 3D graph, which show that the final refined model is reliable. With this model, a flexible docking study with the hydrogen peroxide, the substrate for catalase, is performed and the results indicate that Arg310, Asp343 and Arg346 in catalase are three important determinant residues in binding as they have strong hydrogen bonding contacts with the substrate. These hydrogen-bonding interactions play an important role for the stability of the complex. Our results may be helpful for further experimental investigations.     

Effects of poly-ether B on proteome and phosphoproteome expression in biofouling Balanus amphitrite cyprids

Biofouling is ubiquitous in marine environments, and the barnacle Balanus amphitrite is one of the most recalcitrant and aggressive biofoulers in tropical waters. Several natural antifoulants that were claimed to be non-toxic have been isolated in recent years, although the mechanism by which they inhibit fouling is yet to be investigated. Poly-ether B has shown promise in the non-toxic inhibition of larval barnacle attachment. Hence, in this study, multiplex two-dimensional electrophoresis (2-DE) was applied in conjunction with mass spectrometry to investigate the effects of poly-ether B on barnacle larvae at the molecular level. The cyprid proteome response to poly-ether B treatment was analyzed at the total proteome and phosphoproteome levels, with 65 protein and 19 phosphoprotein spots found to be up- or down-regulated. The proteins were found to be related to energy-metabolism, oxidative stress, and molecular chaperones, thus indicating that poly-ether B may interfere with the redox-regulatory mechanisms governing the settlement of barnacle larvae. The results of this study demonstrate the usefulness of the proteomic technique in revealing the working mechanisms of antifouling compounds.     

mda-7/IL-24 differentially regulates soluble and nuclear clusterin in prostate cancer

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), a unique member of the IL-10 gene family, displays a broad range of antitumor properties including cancer-specific induction of apoptosis, inhibition of tumor angiogenesis, and modulation of anti-tumor immune responses. Here, we identify clusterin (CLU) as a MDA-7/IL-24 interacting protein in DU-145 cells and investigate the role of MDA-7/IL-24 in regulating CLU expression and mediating the antitumor properties of mda-7/IL-24 in prostate cancer. Ad.mda-7 decreased expression of soluble CLU (sCLU) and increased expression of nuclear CLU (nCLU). In the initial phase of Ad.mda-7 infection sCLU expression increased and CLU interacted with MDA-7/IL-24 producing a cytoprotective effect. Infection of stable clones of DU-145 prostate cancer cells expressing sCLU with Ad.mda-7 resulted in generation of nCLU that correlated with decreased cell viability and increased apoptosis. In the presence of mda-7/IL-24, sCLU-DU-145 cells displayed G(2)/M phase arrest followed by apoptosis. Similarly, Ad.mda-7 infection decreased cell migration by altering cytoskeleton in sCLU-DU-145 cells. Ad.mda-7-treated sCLU-DU-145 cells displayed a significant reduction in tumor growth in mouse xenograft models and reduced angiogenesis when compared to the vector control group. Tumor tissue lysates demonstrated enhanced nCLU generated from sCLU with increased apoptosis in the presence of MDA-7/IL-24. Our findings reveal novel aspects relative to the role of sCLU/nCLU in regulating the anticancer properties of MDA-7/IL-24 that may be exploited for developing enhanced therapies for prostate cancer.