Physiological IRE-1-XBP-1 and PEK-1 signaling in Caenorhabditis elegans larval development and immunity

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.Cataloged from PDF version of thesis.Includes bibliographical references (p. 27-29).The endoplasmic reticulum (ER) is responsible for the folding and processing of approximately one third of proteins in eukaryotic cells, and homeostasis in this compartment is tightly regulated. The Unfolded Protein Response, or UPR, is activated in response to perturbations in protein folding in the ER, collectively termed ER stress. This compensatory mechanism, mediated by IRE-1, PERK- 1/PEK-1 and ATF-6 in metazoans, resolves an overcrowded ER lumen in part through the increase of protein degradation and folding. Typical studies focus on activation of the UPR in response to characterized chemical agents that potently alter ER function or protein stability and folding, leaving physiological or native roles for the UPR relatively uncharacterized. Richardson et al previously demonstrated a role for the UPR in innate immunity in C elegans. Here, in an effort to understand this role, we demonstrate that intestinal expression of XBP-1 is sufficient to overcome PMK-1 dependent larval lethality on a lawn of pathogenic Pseudomonas aeruginosa. Further, we demonstrate that XBP-1 deficiency results in constitutive ER stress even in the absence of pathogenic infection. This elevated ER stress is reflected by increased activities of both IRE- 1 and PEK-1 under physiological conditions. Our data suggest that negative feedback loops involving the activation of IRE-1-XBP-1 and PEK-1 pathways serve essential roles not only at the extremes of ER stress but also in the maintenance of ER homeostasis under physiological conditions.by Stephanie A. Kinkel.S.M

mTOR Complex 2 Is Required for the Development of Prostate Cancer Induced by Pten Loss in Mice

mTOR complex 2 (mTORC2) contains the mammalian target of rapamycin (mTOR) kinase and the Rictor regulatory protein and phosphorylates Akt. Whether this function of mTORC2 is critical for cancer progression is unknown. Here, we show that transformed human prostate epithelial cells lacking PTEN require mTORC2 to form tumors when injected into nude mice. Furthermore, we find that Rictor is a haploinsufficient gene and that deleting one copy protects Pten heterozygous mice from prostate cancer. Finally, we show that the development of prostate cancer caused by Pten deletion specifically in prostate epithelium requires mTORC2, but that for normal prostate epithelial cells, mTORC2 activity is nonessential. The selective requirement for mTORC2 in tumor development suggests that mTORC2 inhibitors may be of substantial clinical utility.W. M. Keck FoundationDamon Runyon Cancer Research Foundation (Research Fellowship)Leukemia & Lymphoma Society of America (Career Development Award)Howard Hughes Medical Institute (Investigator)National Institutes of Health (U.S.) (K99 CA1296613-01A1)National Institutes of Health (U.S.) (R01 CA107166)National Institutes of Health (U.S.) (R01 AI04389)National Institutes of Health (U.S.) (R01 CA103866

Physiological IRE-1-XBP-1 and PEK-1 Signaling in Caenorhabditis elegans Larval Development and Immunity

Endoplasmic reticulum (ER) stress activates the Unfolded Protein Response, a compensatory signaling response that is mediated by the IRE-1, PERK/PEK-1, and ATF-6 pathways in metazoans. Genetic studies have implicated roles for UPR signaling in animal development and disease, but the function of the UPR under physiological conditions, in the absence of chemical agents administered to induce ER stress, is not well understood. Here, we show that in Caenorhabditis elegans XBP-1 deficiency results in constitutive ER stress, reflected by increased basal levels of IRE-1 and PEK-1 activity under physiological conditions. We define a dynamic, temperature-dependent requirement for XBP-1 and PEK-1 activities that increases with immune activation and at elevated physiological temperatures in C. elegans. Our data suggest that the negative feedback loops involving the activation of IRE-1-XBP-1 and PEK-1 pathways serve essential roles, not only at the extremes of ER stress, but also in the maintenance of ER homeostasis under physiological conditions.National Institutes of Health (U.S.) (grant R01-GM084477

Challenging Varieties of Capitalism's Account of Business Interests: The New Social Market Initiative and German Employers' Quest for Liberalization, 2000-2014

Do employers in coordinated market economies (CME's) actively defend the non-liberal, market- constraining institutions upon which their strategic coordination and competitive success depends? This paper revisits the debate over firms' employer preferences with an in-depth examination of employers in Germany - a paradigmatic CME and crucial "test case" for Varieties of Capitalism. It is based on interviews with key officials and an in-depth examination of a large-scale campaign - the New Social Market Initiative or INMS - founded and funded by German metalworking employers to shape public opinion. The paper argues that German employers have a strong preference for liberalization: they have pushed hard for the liberalization of labor markets, the reduction of government expenditures, the expansion of market-oriented freedoms, and cuts to social protection, employment protection and benefit entitlements. I find no empirical support for the claim that the INSM is an attempt to appease discontented firms within employers' associations. On the contrary: for many employers, the Agenda 2010 reforms did not go far enough. Following the discrediting of the Anglo-American model in the financial crisis, far-reaching concessions by employees, and the unexpected revitalization of the German economy, employers have moderated their demands - but liberalization remains their default preference. This paper also addresses the role of ideas and the conditions under which employer campaigns can influence policy.Verteidigen Arbeitgeber in koordinierten Marktwirtschaften aktiv die nichtliberalen, marktbeschränkenden Institutionen, von denen ihre Möglichkeiten zur strategischen Koordination und ihr Erfolg im Wettbewerb abhängen? Mit einer umfassenden Untersuchung der Präferenzen von Arbeitgebern in Deutschland, das als typisches Beispiel einer koordinierten Marktwirtschaft und wegweisender "Testfall" für die Theorie über Spielarten des Kapitalismus gilt, greift dieses Discussion Paper die Debatte über die Präferenzen von Unternehmen in ihrer Eigenschaft als Arbeitgeber auf. Es basiert auf Interviews mit führenden Arbeitgeberfunktionären sowie einer detaillierten Untersuchung der Initiative Neue Soziale Marktwirtschaft (INSM): einer groß angelegten, von deutschen Metallarbeitgebern initiierten und finanzierten Kampagne zur öffentlichen Meinungsbildung. Der Beitrag belegt eine deutliche Präferenz deutscher Arbeitgeber für die Liberalisierung. Mit Nachdruck haben sie sich für eine Liberalisierung der Arbeitsmärkte, eine Senkung der Staatsausgaben und eine Ausweitung marktorientierter Gestaltungsfreiheiten ebenso eingesetzt wie für Einschnitte bei der sozialen Sicherung, dem Kündigungsschutz und den Versorgungsansprüchen. Die Behauptung, die INSM sei ein Versuch, unzufriedene Unternehmen innerhalb der Arbeitgeberverbände zu beschwichtigen, lässt sich durch die empirischen Befunde nicht stützen. Im Gegenteil: Vielen Arbeitgebern gingen die Reformen im Zuge der Agenda 2010 nicht weit genug. Zwar haben die deutschen Arbeitgeber nach der Diskreditierung des angloamerikanischen Modells während der Finanzkrise, weitreichenden Zugeständnissen seitens der Arbeitnehmer sowie der unerwarteten Wiederbelebung der deutschen Wirtschaft ihre Forderungen gemäßigt - doch bleibt ihre grundlegende Präferenz für die Liberalisierung bestehen. Dieser Beitrag befasst sich außerdem mit der Rolle von Ideen sowie den Bedingungen, unter denen Arbeitgeberkampagnen politische Maßnahmen beeinflussen können

The Catabolite Control Protein CcpA Binds to Pmga and Influences Expression of the Virulence Regulator Mga in the Group A Streptococcus▿ †

Carbon catabolite repression (CCR) allows bacteria to alter metabolism in response to the availability of specific sugar sources, and increasing evidence suggests that CCR is involved in regulating virulence gene expression in many pathogens. A scan of the M1 SF370 group A streptococcus (GAS) genome using a Bacillus subtilis consensus identified a number of potential catabolite-responsive elements (cre) important for binding by the catabolite control protein A (CcpA), a mediator of CCR in gram-positive bacteria. Intriguingly, a putative cre was identified in the promoter region of mga upstream of its distal P1 start of transcription. Electrophoretic mobility shift assays showed that a His-CcpA fusion protein was capable of binding specifically to the cre in Pmga in vitro. Deletion analysis of Pmga using single-copy Pmga-gusA reporter strains found that Pmga P1 and its upstream cre were not required for normal autoregulated mga expression from Pmga P2 as long as Mga was produced from its native locus. In fact, the Pmga P1 region appeared to show a negative influence on Pmga P2 in these studies. However, deletion of the cre at the native Pmga resulted in a reduction of total mga transcripts as determined by real-time reverse transcription-PCR, supporting a role for CcpA in initial expression. Furthermore, normal transcriptional initiation from the Pmga P1 start site alone was dependent on the presence of the cre. Importantly, inactivation of ccpA in the M6 GAS strain JRS4 resulted in a reduction in Pmga expression and Mga protein levels in late-logarithmic-phase cell growth. These data support a role for CcpA in the early activation of the mga promoter and establish a link between CCR and Mga regulation in the GAS