13 research outputs found

    CDK-Dependent Hsp70 Phosphorylation Controls G1 Cyclin Abundance and Cell-Cycle Progression

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    In budding yeast, the essential functions of Hsp70 chaperones Ssa1–4 are regulated through expression level, isoform specificity, and cochaperone activity. Suggesting a novel regulatory paradigm, we find that phosphorylation of Ssa1 T36 within a cyclin-dependent kinase (CDK) consensus site conserved among Hsp70 proteins alters cochaperone and client interactions. T36 phosphorylation triggers displacement of Ydj1, allowing Ssa1 to bind the G1 cyclin Cln3 and promote its degradation. The stress CDK Pho85 phosphorylates T36 upon nitrogen starvation or pheromone stimulation, destabilizing Cln3 to delay onset of S phase. In turn, the mitotic CDK Cdk1 phosphorylates T36 to block Cln3 accumulation in G2/M. Suggesting broad conservation from yeast to human, CDK-dependent phosphorylation of Hsc70 T38 similarly regulates Cyclin D1 binding and stability. These results establish an active role for Hsp70 chaperones as signal transducers mediating growth control of G1 cyclin abundance and activity

    CDK-dependent Hsp70 phosphorylation controls G1 cyclin abundance and cell-cycle progression

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    In budding yeast, the essential functions of Hsp70 chaperones Ssa1-4 are regulated through expression level, isoform specificity, and cochaperone activity. Suggesting a novel regulatory paradigm, we find that phosphorylation of Ssa1 T36 within a cyclin-dependent kinase (CDK) consensus site conserved among Hsp70 proteins alters cochaperone and client interactions. T36 phosphorylation triggers displacement of Ydj1, allowing Ssa1 to bind the G1 cyclin Cln3 and promote its degradation. The stress CDK Pho85 phosphorylates T36 upon nitrogen starvation or pheromone stimulation, destabilizing Cln3 to delay onset of S phase. In turn, the mitotic CDK Cdk1 phosphorylates T36 to block Cln3 accumulation in G2/M. Suggesting broad conservation from yeast to human, CDK-dependent phosphorylation of Hsc70 T38 similarly regulates Cyclin D1 binding and stability. These results establish an active role for Hsp70 chaperones as signal transducers mediating growth control of G1 cyclin abundance and activity

    SIRT3 Deacetylates and Activates OPA1 To Regulate Mitochondrial Dynamics during Stress

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    Mitochondrial morphology is regulated by the balance between two counteracting mitochondrial processes of fusion and fission. There is significant evidence suggesting a stringent association between morphology and bioenergetics of mitochondria. Morphological alterations in mitochondria are linked to several pathological disorders, including cardiovascular diseases. The consequences of stress-induced acetylation of mitochondrial proteins on the organelle morphology remain largely unexplored. Here we report that OPA1, a mitochondrial fusion protein, was hyperacetylated in hearts under pathological stress and this posttranslational modification reduced the GTPase activity of the protein. The mitochondrial deacetylase SIRT3 was capable of deacetylating OPA1 and elevating its GTPase activity. Mass spectrometry and mutagenesis analyses indicated that in SIRT3-deficient cells OPA1 was acetylated at lysine 926 and 931 residues. Overexpression of a deacetylation-mimetic version of OPA1 recovered the mitochondrial functions of OPA1-null cells, thus demonstrating the functional significance of K926/931 acetylation in regulating OPA1 activity. Moreover, SIRT3-dependent activation of OPA1 contributed to the preservation of mitochondrial networking and protection of cardiomyocytes from doxorubicin-mediated cell death. In summary, these data indicated that SIRT3 promotes mitochondrial function not only by regulating activity of metabolic enzymes, as previously reported, but also by regulating mitochondrial dynamics by targeting OPA1

    Differential Growth of Francisella tularensis, Which Alters Expression of Virulence Factors, Dominant Antigens, and Surface-Carbohydrate Synthases, Governs the Apparent Virulence of Ft SchuS4 to Immunized Animals

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    The gram-negative bacterium Francisella tularensis (Ft) is both a potential biological weapon and a naturally occurring microbe that survives in arthropods, fresh water amoeba, and mammals with distinct phenotypes in various environments. Previously, we used a number of measurements to characterize Ft grown in Brain-Heart Infusion (BHI) broth as (1) more similar to infection-derived bacteria, and (2) slightly more virulent in naïve animals, compared to Ft grown in Mueller Hinton Broth (MHB). In these studies we observed that the free amino acids in MHB repress expression of select Ft virulence factors by an unknown mechanism. Here, we tested the hypotheses that Ft grown in BHI (BHI-Ft) accurately displays a full protein composition more similar to that reported for infection-derived Ft and that this similarity would make BHI-Ft more susceptible to pre-existing, vaccine-induced immunity than MHB-Ft. We performed comprehensive proteomic analysis of Ft grown in MHB, BHI, and BHI supplemented with casamino acids (BCA) and compared our findings to published “omics” data derived from Ft grown in vivo. Based on the abundance of ~1,000 proteins, the fingerprint of BHI-Ft is one of nutrient-deprived bacteria that—through induction of a stringent-starvation-like response—have induced the FevR regulon for expression of the bacterium's virulence factors, immuno-dominant antigens, and surface-carbohydrate synthases. To test the notion that increased abundance of dominant antigens expressed by BHI-Ft would render these bacteria more susceptible to pre-existing, vaccine-induced immunity, we employed a battery of LVS-vaccination and S4-challenge protocols using MHB- and BHI-grown Ft S4. Contrary to our hypothesis, these experiments reveal that LVS-immunization provides a barrier to infection that is significantly more effective against an MHB-S4 challenge than a BHI-S4 challenge. The differences in apparent virulence to immunized mice are profoundly greater than those observed with primary infection of naïve mice. Our findings suggest that tularemia vaccination studies should be critically evaluated in regard to the growth conditions of the challenge agent

    The quantitative changes in the yeast Hsp70 and Hsp90 interactomes upon DNA damage

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    The molecular chaperones Hsp70 and Hsp90 participate in many important cellular processes, including how cells respond to DNA damage. Here we show the results of applied quantitative affinity-purification mass spectrometry (AP-MS) proteomics to understand the protein network through which Hsp70 and Hsp90 exert their effects on the DNA damage response (DDR). We characterized the interactomes of the yeast Hsp70 isoform Ssa1 and Hsp90 isoform Hsp82 before and after exposure to methyl methanesulfonate. We identified 256 chaperone interactors, 146 of which are novel. Although the majority of chaperone interaction remained constant under DNA damage, 5 proteins (Coq5, Ast1, Cys3, Ydr210c and Rnr4) increased in interaction with Ssa1 and/or Hsp82. This data presented here are related to [1] (Truman et al., in press). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (Vizcaino et al. (2013) [2]) with the dataset identifier PXD001284

    The dynamic interactome of human Aha1 upon Y223 phosphorylation

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    Heat Shock Protein 90 (Hsp90) is an essential chaperone that supports the function of a wide range of signaling molecules. Hsp90 binds to a suite of co-chaperone proteins that regulate Hsp90 function through alteration of intrinsic ATPase activity. Several studies have determined Aha1 to be an important co-chaperone whose binding to Hsp90 is modulated by phosphorylation, acetylation and SUMOylation of Hsp90 [1,2]. In this study, we applied quantitative affinity-purification mass spectrometry (AP-MS) proteomics to understand how phosphorylation of hAha1 at Y223 altered global client/co-chaperone interaction [3]. Specifically, we characterized and compared the interactomes of Aha1–Y223F (phospho-mutant form) and Aha1–Y223E (phospho-mimic form). We identified 99 statistically significant interactors of hAha1, a high proportion of which (84%) demonstrated preferential binding to the phospho-mimic form of hAha1. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [4] with the dataset identifier PXD001737

    CDK-Dependent Hsp70 Phosphorylation Controls G1 Cyclin Abundance and Cell-Cycle Progression

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    In budding yeast, the essential functions of Hsp70 chaperones Ssa1–4 are regulated through expression level, isoform specificity, and cochaperone activity. Suggesting a novel regulatory paradigm, we find that phosphorylation of Ssa1 T36 within a cyclin-dependent kinase (CDK) consensus site conserved among Hsp70 proteins alters cochaperone and client interactions. T36 phosphorylation triggers displacement of Ydj1, allowing Ssa1 to bind the G1 cyclin Cln3 and promote its degradation. The stress CDK Pho85 phosphorylates T36 upon nitrogen starvation or pheromone stimulation, destabilizing Cln3 to delay onset of S phase. In turn, the mitotic CDK Cdk1 phosphorylates T36 to block Cln3 accumulation in G2/M. Suggesting broad conservation from yeast to human, CDK-dependent phosphorylation of Hsc70 T38 similarly regulates Cyclin D1 binding and stability. These results establish an active role for Hsp70 chaperones as signal transducers mediating growth control of G1 cyclin abundance and activity
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