Cell-Type-Specific Profiling of Alternative Translation Identifies Regulated Protein Isoform Variation in the Mouse Brain

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Ac2PIM-responsive miR-150 and miR-143 Target Receptor-interacting Protein Kinase 2 and Transforming Growth Factor Beta-activated Kinase 1 to Suppress NOD2-induced Immunomodulators

Specific and coordinated regulation of innate immune receptor-driven signaling networks often determines the net outcome of the immune responses. Here, we investigated the cross-regulation of toll-like receptor (TLR)2 and nucleotide-binding oligomerization domain (NOD)2 pathways mediated by Ac₂PIM, a tetra-acylated form of mycobacterial cell wall component and muramyl dipeptide (MDP), a peptidoglycan derivative respectively. While Ac₂PIM treatment of macrophages compromised their ability to induce NOD2-dependent immunomodulators like cyclooxygenase (COX)-2, suppressor of cytokine signaling (SOCS)-3, and matrix metalloproteinase (MMP)-9, no change in the NOD2-responsive NO, TNF-α, VEGF-A, and IL-12 levels was observed. Further, genome-wide microRNA expression profiling identified Ac₂PIM-responsive miR-150 and miR-143 to target NOD2 signaling adaptors, RIP2 and TAK1, respectively. Interestingly, Ac₂PIM was found to activate the SRC-FAK-PYK2-CREB cascade via TLR2 to recruit CBP/P300 at the promoters of miR-150 and miR-143 and epigenetically induce their expression. Loss-of-function studies utilizing specific miRNA inhibitors establish that Ac₂PIM, via the miRNAs, abrogate NOD2-induced PI3K-PKCδ-MAPK pathway to suppress β-catenin-mediated expression of COX-2, SOCS-3, and MMP-9. Our investigation has thus underscored the negative regulatory role of Ac₂PIM-TLR2 signaling on NOD2 pathway which could broaden our understanding on vaccine potential or adjuvant utilities of Ac₂PIM and/or MDP.     

The WNT Signaling Pathway Contributes to Dectin-1-Dependent Inhibition of Toll-Like Receptor-Induced Inflammatory Signature

Macrophages regulate cell fate decisions during microbial challenges by carefully titrating signaling events activated by innate receptors such as dectin-1 or Toll-like receptors (TLRs). Here, we demonstrate that dectin-1 activation robustly dampens TLR-induced proinflammatory signature in macrophages. Dectin-1 induced the stabilization of β-catenin via spleen tyrosine kinase (Syk)-reactive oxygen species (ROS) signals, contributing to the expression of WNT5A. Subsequently, WNT5A-responsive protein inhibitors of activated STAT (PIAS-1) and suppressor of cytokine signaling 1 (SOCS-1) mediate the downregulation of IRAK-1, IRAK-4, and MyD88, resulting in decreased expression of interleukin 12 (IL-12), IL-1β, and tumor necrosis factor alpha (TNF-α). In vivo activation of dectin-1 with pathogenic fungi or ligand resulted in an increased bacterial burden of Mycobacteria, Klebsiella, Staphylococcus, or Escherichia, with a concomitant decrease in TLR-triggered proinflammatory cytokines. All together, our study establishes a new role for dectin-1-responsive inhibitory mechanisms employed by virulent fungi to limit the proinflammatory environment of the host.     

CLAP: A web-server for automatic classification of proteins with special reference to multi-domain proteins

Background

The function of a protein can be deciphered with higher accuracy from its structure than from its amino acid sequence. Due to the huge gap in the available protein sequence and structural space, tools that can generate functionally homogeneous clusters using only the sequence information, hold great importance. For this, traditional alignment-based tools work well in most cases and clustering is performed on the basis of sequence similarity. But, in the case of multi-domain proteins, the alignment quality might be poor due to varied lengths of the proteins, domain shuffling or circular permutations. Multi-domain proteins are ubiquitous in nature, hence alignment-free tools, which overcome the shortcomings of alignment-based protein comparison methods, are required. Further, existing tools classify proteins using only domain-level information and hence miss out on the information encoded in the tethered regions or accessory domains. Our method, on the other hand, takes into account the full-length sequence of a protein, consolidating the complete sequence information to understand a given protein better.

Results

Our web-server, CLAP (Classification of Proteins), is one such alignment-free software for automatic classification of protein sequences. It utilizes a pattern-matching algorithm that assigns local matching scores (LMS) to residues that are a part of the matched patterns between two sequences being compared. CLAP works on full-length sequences and does not require prior domain definitions.Pilot studies undertaken previously on protein kinases and immunoglobulins have shown that CLAP yields clusters, which have high functional and domain architectural similarity. Moreover, parsing at a statistically determined cut-off resulted in clusters that corroborated with the sub-family level classification of that particular domain family.

Conclusions

CLAP is a useful protein-clustering tool, independent of domain assignment, domain order, sequence length and domain diversity. Our method can be used for any set of protein sequences, yielding functionally relevant clusters with high domain architectural homogeneity. The CLAP web server is freely available for academic use at http://nslab.mbu.iisc.ernet.in/clap/.     

Loss of Urokinase Receptor Sensitizes Cells to DNA Damage and Delays DNA Repair

DNA damage induced by numerous exogenous or endogenous factors may have irreversible consequences on the cell leading to cell cycle arrest, senescence and cell death. The DNA damage response (DDR) is powerful signaling machinery triggered in response to DNA damage, to provide DNA damage recognition, signaling and repair. Most anticancer drugs induce DNA damage, and DNA repair in turn attenuates therapeutic efficiency of those drugs. Approaches delaying DNA repair are often used to increase efficiency of treatment. Recent data show that ubiquitin-proteasome system is essential for signaling and repair of DNA damage. However, mechanisms providing regulation of proteasome intracellular localization, activity, and recruitment to DNA damage sites are elusive. Even less investigated are the roles of extranuclear signaling proteins in these processes. In this study, we report the involvement of the serine protease urokinase-type plasminogen activator receptor (uPAR) in DDR-associated regulation of proteasome. We show that in vascular smooth muscle cells (VSMC) uPAR activates DNA single strand break repair signaling pathway. We provide evidence that uPAR is essential for functional assembly of the 26S proteasome. We further demonstrate that uPAR mediates DNA damage-induced phosphorylation, nuclear import, and recruitment of the regulatory subunit PSMD6 to proteasome. We found that deficiency of uPAR and PSMD6 delays DNA repair and leads to decreased cell survival. These data may offer new therapeutic approaches for diseases such as cancer, cardiovascular and neurodegenerative disorders.     

Hybrid and Rogue Kinases Encoded in the Genomes of Model Eukaryotes

The highly modular nature of protein kinases generates diverse functional roles mediated by evolutionary events such as domain recombination, insertion and deletion of domains. Usually domain architecture of a kinase is related to the subfamily to which the kinase catalytic domain belongs. However outlier kinases with unusual domain architectures serve in the expansion of the functional space of the protein kinase family. For example, Src kinases are made-up of SH2 and SH3 domains in addition to the kinase catalytic domain. A kinase which lacks these two domains but retains sequence characteristics within the kinase catalytic domain is an outlier that is likely to have modes of regulation different from classical src kinases. This study defines two types of outlier kinases: hybrids and rogues depending on the nature of domain recombination. Hybrid kinases are those where the catalytic kinase domain belongs to a kinase subfamily but the domain architecture is typical of another kinase subfamily. Rogue kinases are those with kinase catalytic domain characteristic of a kinase subfamily but the domain architecture is typical of neither that subfamily nor any other kinase subfamily. This report provides a consolidated set of such hybrid and rogue kinases gleaned from six eukaryotic genomes–S.cerevisiae, D. melanogaster, C.elegans, M.musculus, T.rubripes and H.sapiens–and discusses their functions. The presence of such kinases necessitates a revisiting of the classification scheme of the protein kinase family using full length sequences apart from classical classification using solely the sequences of kinase catalytic domains. The study of these kinases provides a good insight in engineering signalling pathways for a desired output. Lastly, identification of hybrids and rogues in pathogenic protozoa such as P.falciparum sheds light on possible strategies in host-pathogen interactions.     

Pleiotropic roles of a ribosomal protein in Dictyostelium discoideum

The cell cycle phase at starvation influences post-starvation differentiation and morphogenesis in Dictyostelium discoideum. We found that when expressed in Saccharomyces cerevisiae, a D. discoideum cDNA that encodes the ribosomal protein S4 (DdS4) rescues mutations in the cell cycle genes cdc24, cdc42 and bem1. The products of these genes affect morphogenesis in yeast via a coordinated moulding of the cytoskeleton during bud site selection. D. discoideum cells that over- or under-expressed DdS4 did not show detectable changes in protein synthesis but displayed similar developmental aberrations whose intensity was graded with the extent of over- or under-expression. This suggested that DdS4 might influence morphogenesis via a stoichiometric effect--specifically, by taking part in a multimeric complex similar to the one involving Cdc24p, Cdc42p and Bem1p in yeast. In support of the hypothesis, the S. cerevisiae proteins Cdc24p, Cdc42p and Bem1p as well as their D. discoideum cognates could be co-precipitated with antibodies to DdS4. Computational analysis and mutational studies explained these findings: a C-terminal domain of DdS4 is the functional equivalent of an SH3 domain in the yeast scaffold protein Bem1p that is central to constructing the bud site selection complex. Thus in addition to being part of the ribosome, DdS4 has a second function, also as part of a multi-protein complex. We speculate that the existence of the second role can act as a safeguard against perturbations to ribosome function caused by spontaneous variations in DdS4 levels.     

Serodiagnosis of Tuberculosis in Asian Elephants (Elephas maximus) in Southern India: A Latent Class Analysis

Background

Mycobacterium tuberculosis, a causative agent of chronic tuberculosis disease, is widespread among some animal species too. There is paucity of information on the distribution, prevalence and true disease status of tuberculosis in Asian elephants (Elephas maximus). The aim of this study was to estimate the sensitivity and specificity of serological tests to diagnose M. tuberculosis infection in captive elephants in southern India while simultaneously estimating sero-prevalence.

Methodology/Principal Findings

Health assessment of 600 elephants was carried out and their sera screened with a commercially available rapid serum test. Trunk wash culture of select rapid serum test positive animals yielded no animal positive for M. tuberculosis isolation. Under Indian field conditions where the true disease status is unknown, we used a latent class model to estimate the diagnostic characteristics of an existing (rapid serum test) and new (four in-house ELISA) tests. One hundred and seventy nine sera were randomly selected for screening in the five tests. Diagnostic sensitivities of the four ELISAs were 91.3–97.6% (95% Credible Interval (CI): 74.8–99.9) and diagnostic specificity were 89.6–98.5% (95% CI: 79.4–99.9) based on the model we assumed. We estimate that 53.6% (95% CI: 44.6–62.8) of the samples tested were free from infection with M. tuberculosis and 15.9% (97.5% CI: 9.8 - to 24.0) tested positive on all five tests.

Conclusions/Significance

Our results provide evidence for high prevalence of asymptomatic M. tuberculosis infection in Asian elephants in a captive Indian setting. Further validation of these tests would be important in formulating area-specific effective surveillance and control measures.     

Characteristics of bifunctional acidic endoglucanase (Cel5B) from Gloeophyllum trabeum

The endoglucanase (Cel5B) from the filamentous fungus Gloeophyllum trabeum was cloned and expressed without a signal peptide, and alanine residue 22 converted to glutamine in Pichia pastoris GS115. The DNA sequence of Cel5B had an open reading frame of 1,077 bp, encoding a protein of 359 amino acid residues with a molecular weight of 47 kDa. On the basis of sequence similarity, Cel5B displayed active site residues at Glu-175 and Glu-287. Both residues lost full hydrolytic activity when replaced with alanine through point mutation. The purified recombinant Cel5B showed very high specific activity, about 80- to 1,000-fold and 13- to 70-fold in comparison with other endoglucanases and cellobiohydrolase, on carboxymethylcellulose and filter paper, respectively, at pH 3.5 and 55°C. Cel5B displayed bifunctional characteristics under acidic conditions. The kinetic properties of the enzyme determined using a Lineweaver-Burk plot indicated that Cel5B is a catalytically efficient cellulolytic enzyme. These results suggest that Cel5B has high bifunctional endo- and exoglucanase activity under acidic conditions and is a good candidate for bioconversion of lignocellulose.