The Candida albicans transcription factor Cas5 couples stress responses, drug resistance and cell cycle regulation

We thank Cowen lab members for helpful discussions. We also thank David Rogers (University of Tennessee) for sharing microarray analysis of the CAS5 homozygous mutant, and Li Ang (University of Macau) for assistance in optimizing the ChIP-Seq experiments. J.L.X. is supported by a Canadian Institutes of Health Research Doctoral award and M.D.L. is supported by a Sir Henry Wellcome Postdoctoral Fellowship (Wellcome Trust 096072). B.T.G. holds an Ontario Graduate Scholarship. C.B. and B.J.A. are supported by the Canadian Institutes of Health Research Foundation Grants (FDN-143264 and -143265). D.J.K. is supported by a National Institute of Allergy and Infectious Diseases grant (1R01AI098450) and J.D.L.C.D. is supported by the University of Rochester School of Dentistry and Medicine PREP program (R25 GM064133). A.S. is supported by the Creighton University and the Nebraska Department of Health and Human Services (LB506-2017-55). K.H.W. is supported by the Science and Technology Development Fund of Macau S.A.R. (FDCT; 085/2014/A2). L.E.C. is supported by the Canadian Institutes of Health Research Operating Grants (MOP-86452 and MOP-119520), the Natural Sciences and Engineering Council (NSERC) of Canada Discovery Grants (06261 and 462167), and an NSERC E.W.R. Steacie Memorial Fellowship (477598).Peer reviewedPublisher PD

Tankyrase represses autoinflammation through the attenuation of TLR2 signaling

Dysregulation of Toll-like receptor (TLR) signaling contributes to the pathogenesis of autoimmune diseases. Here, we provide genetic evidence that tankyrase, a member of the poly(ADP-ribose) polymerase (PARP) family, negatively regulates TLR2 signaling. We show that mice lacking tankyrase in myeloid cells developed severe systemic inflammation with high serum inflammatory cytokine levels. We provide mechanistic evidence that tankyrase deficiency resulted in tyrosine phosphorylation and activation of TLR2 and show that phosphorylation of tyrosine 647 within the TIR domain by SRC and SYK kinases was critical for TLR2 stabilization and signaling. Last, we show that the elevated cytokine production and inflammation observed in mice lacking tankyrase in myeloid cells were dependent on the adaptor protein 3BP2, which is required for SRC and SYK activation. These data demonstrate that tankyrase provides a checkpoint on the TLR-mediated innate immune response

Engineered SH2 domains with tailored specificities and enhanced affinities for phosphoproteome analysis

Protein phosphorylation is the most abundant post-translational modification in cells. Src homology 2 (SH2) domains specifically recognize phosphorylated tyrosine (pTyr) residues to mediate signaling cascades. A conserved pocket in the SH2 domain binds the pTyr side chain and the EF and BG loops determine binding specificity. By using large phage-displayed libraries, we engineered the EF and BG loops of the Fyn SH2 domain to alter specificity. Engineered SH2 variants exhibited distinct specificity profiles and were able to bind pTyr sites on the epidermal growth factor receptor, which were not recognized by the wild-type Fyn SH2 domain. Furthermore, mass spectrometry showed that SH2 variants with additional mutations in the pTyr-binding pocket that enhanced affinity were highly effective for enrichment of diverse pTyr peptides within the human proteome. These results showed that engineering of the EF and BG loops could be used to tailor SH2 domain specificity, and SH2 variants with diverse specificities and high affinities for pTyr residues enabled more comprehensive analysis of the human phosphoproteome. Statement: Src Homology 2 (SH2) domains are modular domains that recognize phosphorylated tyrosine embedded in proteins, transducing these post-translational modifications into cellular responses. Here we used phage display to engineer hundreds of SH2 domain variants with altered binding specificities and enhanced affinities, which enabled efficient and differential enrichment of the human phosphoproteome for analysis by mass spectrometry. These engineered SH2 domain variants will be useful tools for elucidating the molecular determinants governing SH2 domains binding specificity and for enhancing analysis and understanding of the human phosphoproteome

Defects in the Ca²§+ release channel of skeletal muscle sarcoplasmic reticulum that are associated with malignant hyperthermia and central core disease

grantor: University of TorontoMalignant hyperthermia (MH) and central core disease (CCD) are autosomal dominant disorders of skeletal muscle in which a potentially fatal hypermetabolic crisis can be triggered by commonly used anesthetic agents. To date, seventeen mutations in the human RYR1 gene encoding the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (the ryanodine receptor or RyR1) have been associated with MH and/or CCD. The hypotheses tested in this thesis was that MH and CCD mutations increase the sensitivity of mutant Ca2+ release channels to caffeine and halothane; that MH and CCD mutations increase the permeability (leakiness) of the mutant channels; and that CCD mutant channels are more leaky than MH channels. Sixteen mutations corresponding to human MH or CCD mutations were made in three distinct regions in a full length rabbit RYR1 cDNA. Ca 2+ release, monitored by Fura-2, in HEK-293 cells expressing MH or CCD mutant ryanodine receptors was more sensitive to low concentrations of caffeine and halothane than Ca2+ release in cells expressing wild-type receptors. Linear regression analysis showed a strong correlation (r = 0.95, p < 0.001) between caffeine sensitivity of different RYR1 mutants measured by the cellular Ca2+ photometry assay and by the clinical ' in vitro' caffeine halothane contracture test (IVCT). Resting Ca 2+ concentrations were higher and maximal peak amplitudes of caffeine-induced Ca2+ release were lower in HEK-293 cells expressing homotetrameric CCD mutant RyR1 than in cells expressing homotetrameric MH mutant RyR1, supporting the hypothesis that CCD mutant channels are more leaky. A CCD mutation in the transmembrane/lumenal domain of RyR1, 14897T, was so permeable that its altered function could only be demonstrated in heterotetrameric form. The content of endogenous sarco(endo)plasmic reticulum Ca2+ ATPase isoform 2b (SERCA2b), measured by ELISA, 45Ca 2+ uptake and confocal microscopy, was increased in HEK-293 cells expressing wild type or mutant RyR1, supporting the view that ER Ca 2+ storage capacity is increased as a compensatory response to an enhanced Ca2+ leak. These results support the hypothesis that MH/CCD mutant channels are more sensitive to triggering anesthetics than wild type channel, and that CCD mutant channels have higher Ca2+ permeability than MH mutant channels.Ph.D

Emerging applications for phospho-proteomics in cancer molecular therapeutics

Protein phosphorylation is a key mechanism of cell regulation in normal and cancer cells. Various new cancer drugs and drug candidates are aimed at protein kinase targets. However, selecting patients likely to respond to these treatments, even among individuals with tumors expressing validated kinase targets remains a major challenge. There exists a need for biomarkers to facilitate the monitoring of modulation of drug-targeted kinase pathways. Phospho-proteomics involves the enrichment of phosphorylated proteins from tissue, and the application of technologies such as mass spectrometry (MS) for the identification and quantification of protein phosphorylation sites. It has potential to provide pharmacodynamic readouts of disease states and cellular drug responses in tumor samples, but technical hurdles and bioinformatics challenges will need to be addressed

Tyrosine Phosphorylation of the Lyn Src Homology 2 (SH2) Domain Modulates Its Binding Affinity and Specificity

Typical iron balconies in the French Quarter; Views of the traditional (19th and 20th century) cast iron balconies in New Orleans

Odin (ANKS1A) Modulates EGF Receptor Recycling and Stability

<div><p>The ANKS1A gene product, also known as Odin, was first identified as a tyrosine-phosphorylated component of the epidermal growth factor receptor network. Here we show that Odin functions as an effector of EGFR recycling. In EGF-stimulated HEK293 cells tyrosine phosphorylation of Odin was induced prior to EGFR internalization and independent of EGFR-to-ERK signaling. Over-expression of Odin increased EGF-induced EGFR trafficking to recycling endosomes and recycling back to the cell surface, and decreased trafficking to lysosomes and degradation. Conversely, Odin knockdown in both HEK293 and the non-small cell lung carcinoma line RVH6849, which expresses roughly 10-fold more EGF receptors than HEK293, caused decreased EGFR recycling and accelerated trafficking to the lysosome and degradation. By governing the endocytic fate of internalized receptors, Odin may provide a layer of regulation that enables cells to contend with receptor cell densities and ligand concentration gradients that are physiologically and pathologically highly variable.</p></div