scholarly journals Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock response

2016 ◽  
Vol 114 (2) ◽  
pp. E152-E160 ◽  
Author(s):  
Matthias C. Truttmann ◽  
Xu Zheng ◽  
Leo Hanke ◽  
Jadyn R. Damon ◽  
Monique Grootveld ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Heat Shock ◽  
Protein Aggregation ◽  
Heat Shock Response ◽  
Protein Translation ◽  
Cellular Protein ◽  
Interacting Protein ◽  
Endogenous Protein ◽  
Shock Response ◽  
Influenza Virus Replication

Protein AMPylation is a conserved posttranslational modification with emerging roles in endoplasmic reticulum homeostasis. However, the range of substrates and cell biological consequences of AMPylation remain poorly defined. We expressed human and Caenorhabditis elegans AMPylation enzymes—huntingtin yeast-interacting protein E (HYPE) and filamentation-induced by cyclic AMP (FIC)-1, respectively—in Saccharomyces cerevisiae, a eukaryote that lacks endogenous protein AMPylation. Expression of HYPE and FIC-1 in yeast induced a strong cytoplasmic Hsf1-mediated heat shock response, accompanied by attenuation of protein translation, massive protein aggregation, growth arrest, and lethality. Overexpression of Ssa2, a cytosolic heat shock protein (Hsp)70, was sufficient to partially rescue growth. In human cell lines, overexpression of active HYPE similarly induced protein aggregation and the HSF1-dependent heat shock response. Excessive AMPylation also abolished HSP70-dependent influenza virus replication. Our findings suggest a mode of Hsp70 inactivation by AMPylation and point toward a role for protein AMPylation in the regulation of cellular protein homeostasis beyond the endoplasmic reticulum.

scholarly journals Management of the endoplasmic reticulum stress by activation of the heat shock response in yeast

2013 ◽  
Vol 14 (3) ◽  
pp. 481-494 ◽  
Author(s):  
Jin Hou ◽  
Hongting Tang ◽  
Zihe Liu ◽  
Tobias Österlund ◽  
Jens Nielsen ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Heat Shock ◽  
Endoplasmic Reticulum Stress ◽  
Heat Shock Response ◽  
Shock Response

scholarly journals Neurodegenerative disease-associated protein aggregates are poor inducers of the heat shock response in neuronal-like cells

2020 ◽  
Author(s):  
R. San Gil ◽  
D. Cox ◽  
L. McAlary ◽  
T. Berg ◽  
A. K. Walker ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Neurodegenerative Diseases ◽  
Protein Aggregation ◽  
Neurodegenerative Disease ◽  
Heat Shock Response ◽  
Flow Cytometric Analysis ◽  
Protein Aggregates ◽  
Shock Response ◽  
Shock Proteins

AbstractProtein aggregation that results in the formation of inclusions is strongly correlated with neuronal death and is a pathological hallmark common to many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Huntington’s disease. Cells are thought to dramatically up-regulate the levels of heat shock proteins during periods of cellular stress via induction of the heat shock response (HSR). Heat shock proteins are well-characterised molecular chaperones that interact with aggregation-prone proteins to either stabilise, refold, or traffic protein for degradation. The reason why heat shock proteins are unable to maintain the solubility of particular proteins in neurodegenerative disease is unknown. We sought to determine whether neurodegenerative disease-associated protein aggregates can induce the HSR. Here, we generated a neuroblastoma cell line that expresses a fluorescent reporter under conditions of HSR induction, for example heat shock. Using these cells, we show that the HSR is not induced by exogenous treatment with aggregated forms of Parkinson’s disease-associated α-synuclein or the ALS-associated G93A mutant of superoxide dismutase-1 (SOD1G93A). Furthermore, flow cytometric analysis revealed that intracellular expression of SOD1G93A or a pathogenic form of polyQ-expanded huntingtin (Htt72Q), similarly, results in no or low induction of the HSR. In contrast, expression of a non-pathogenic but aggregation-prone form of firefly luciferase (Fluc) did induce an HSR in a significantly greater proportion of cells. Finally, we show that HSR induction is dependent on the intracellular levels of the aggregation-prone proteins, but the pathogenic proteins (SOD1G93A and Htt72Q) elicit a significantly lower HSR compared to the non-pathogenic proteins (Fluc). These results suggest that pathogenic proteins either evade detection or impair induction of the HSR in neuronal-like cells. Therefore, defective HSR induction may facilitate the initiation of protein aggregation leading to inclusion formation in neurodegenerative diseases.

scholarly journals Heterogeneity in heat shock response dynamics caused by translation fidelity decline and proteostasis collapse

2019 ◽  
Author(s):  
Nadia Vertti-Quintero ◽  
Simon Berger ◽  
Xavier Casadevall i Solvas ◽  
Cyril Statzer ◽  
Jillian Annis ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Response ◽  
Early Adulthood ◽  
Surrogate Marker ◽  
Protein Translation ◽  
Single Individual ◽  
Response Dynamics ◽  
C Elegans ◽  
Translation Fidelity ◽  
Shock Response

AbstractGenetics, environment, and stochasticity influence the rate of ageing in living organisms. Individual Caenorhabditis elegans that are genetically identical and cultured in the same environment have different lifespans, suggesting a significant role of stochasticity in ageing. We have developed a novel microfluidic methodology to measure heat-shock response as a surrogate marker for heterogeneity associated with lifespan and have quantified the heat-shock response of C. elegans at the population, single individual, and tissue levels. We have further mathematically modelled our data to identify the major drivers determining such heterogeneity. This approach demonstrates that protein translation and degradation rate constants explain the individuality of the heat-shock time-course dynamic. We observed a decline of protein turnover capacity in early adulthood, co-incidentally occurring as the predicted proteostasis collapse. We identified a decline of intestinal response as the tissue that underlies the individual heterogeneity. Additionally, we verified that individuals with enhanced translation fidelity in early adulthood live longer. Altogether, our results reveal that the stochastic onset of proteostasis collapse of somatic tissues during early adulthood reflects individual protein translation capacity underlying heterogenic ageing of isogenic C. elegans.

Altered Cellular Protein Levels of Tumor Suppressor Genes and Heat Shock Elements (TRAP1) Indicate Sensitivity to the Proteasome Inhibitor Bortezomib (Velcade®) in Mantle Cell Lymphoma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2424-2424
Author(s):  
Marc Weinkauf ◽  
Malte Rieken ◽  
Alessandro Pastore ◽  
Elena Hartmann ◽  
Andreas Rosenwald ◽  
...  
Keyword(s):  
Heat Shock ◽  
Cell Lines ◽  
Heat Shock Response ◽  
Tumor Suppressor Genes ◽  
Proteasome Inhibition ◽  
Cellular Protein ◽  
Response Elements ◽  
Protein Levels ◽  
Bortezomib Treatment ◽  
Shock Response

Abstract Mantle cell lymphoma (MCL) is a distinct lymphatic neoplasia characterized by a poor clinical outcome with only transient response to conventional chemotherapy. In various phase II studies the proteasome inhibitor Bortezomib (Velcade®) has demonstrated a high clinical efficacy with up to 60% remission rates in relapsed MCL, however, the target pathways of proteasome inhibition remain largely unknown. Additionally, as the mode of action involves proteasome mediated protein degradation, only analysis of the protein level may reliably identify the cellular signal pathways involved. Four MCL cell lines (HBL-2, Granta 519, Jeko-1, NCEB-1) were exposed to Bortezomib at the minimal cytotoxic concentration (25nmol) and harvested for analysis at 0, 1, 4 and 8 hours of exposure. To assess the early alterations of cellular protein levels induced by Bortezomib-effected proteasome inhibition, the whole cellular proteome was screened applying two-dimensional gel electrophoresis. A high percentage of proteins were initially affected by proteasome inhibition, increasing their cellular content. Analyzing triplicate experiments, approximately 20 % of all cellular proteins detected on the 2D-gels (201/1013) exhibited an alteration of cellular protein content by a factor of >5, while 14 % (148) of the proteins were altered by >10 x. As expected about 79 % (158/201) of these proteins showed an increase of cellular protein, and only 21 % (44/201) of the proteins were downregulated. Approximately 65 % (133/201) of these proteins were already dysregulated after 1 hour whereas the 68 remaining proteins reached the threshold level after 4 hours. Cross cell line comparison yielded 41 reproducibly altered proteins in 3 responder cell lines. Among these proteins are (putative) tumor suppressors (ENO-1, fumarate hydratase), heat shock response elements (TRAP1, HSPA8, HSP72) and structural proteins (centrosomal protein CEP290). In the non-responder cell line (NCEB-1), expression of only 22 of these 41 proteins was altered after exposure to Bortezomib. These unspecific proteins involved some heat shock response elements (HSPA8, HSP72), the respiratory chain (NADH-ubiquinon oxidoreductase 24kDa mitochondrial precursor) and signal transduction (LSP1). In contrast, the cellular protein levels of the putative tumor suppressor genes, as well as the actin cross-linking protein alpha-fodrin were altered only in the responder cell lines. RNA-expression analysis detected highly diverging expression patterns after Bortezomib treatment in all four cell lines. No down-regulation of proliferation associated genes was detected, and the expression of heat shock response related genes were increased. As predicted, due to its mode of action, there was no correlation between the detected alterations of cellular protein levels and RNA-expression after Bortezomib treatment. In summary, Bortezomib treatment specifically affected the cellular protein levels of tumor suppressor genes and distinct heat shock response elements (TRAP1) in sensitive cell lines. Currently primary patient samples are being analyzed to confirm the predictive value of these molecular markers.

scholarly journals Impaired proteostasis in senescent vascular endothelial cells: a perspective on estrogen and oxidative stress in the aging vasculature

2019 ◽  
Vol 316 (2) ◽  
pp. H421-H429 ◽  
Author(s):  
HyunTae V. Hwang ◽  
Yun Lin ◽  
Michelle N. Rebuffatti ◽  
Darlene T. Tran ◽  
Lily Lee ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Heat Shock ◽  
Protein Aggregation ◽  
Cellular Senescence ◽  
Heat Shock Response ◽  
Human Endothelial Cells ◽  
Early Passage ◽  
Shock Response ◽  
17Β Estradiol

The heat shock response is an important cytoprotective mechanism for protein homeostasis and is an essential protective response to cellular stress and injury. Studies on changes in the heat shock response with aging have been mixed with regard to whether it is inhibited, and this, at least in part, reflects different tissues and different models. Cellular senescence is a key feature in aging, but work on the heat shock response in cultured senescent (SEN) cells has largely been limited to fibroblasts. Given the prevalence of oxidative injury in the aging cardiovascular system, we investigated whether SEN primary human coronary artery endothelial cells have a diminished heat shock response and impaired proteostasis. In addition, we tested whether this downregulation of heat shock response can be mitigated by 17β-estradiol (E2), which has a critical cardioprotective role in women, as we have previously reported that E2 improves the heat shock response in endothelial cells (Hamilton KL, Mbai FN, Gupta S, Knowlton AA. Arterioscler Thromb Vasc Biol 24: 1628–1633, 2004). We found that SEN endothelial cells, despite their unexpectedly increased proteasome activity, had a diminished heat shock response and had more protein aggregation than early passage cells. SEN cells had increased oxidative stress, which promoted protein aggregation. E2 treatment did not decrease protein aggregation or improve the heat shock response in either early passage or SEN cells. In summary, cellular senescence in adult human endothelial cells is accompanied by increased oxidative stress and a blunting of proteostasis, and E2 did not mitigate these changes. NEW & NOTEWORTHY Senescent human endothelial cells have a diminished heat shock response and increased protein aggregates. Senescent human endothelial cells have increased basal oxidative stress, which increases protein aggregates. Physiological level of 17β-estradiol did not improve proteostasis in endothelial cells. Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/proteostasis-in-senescent-endothelial-cells/ .

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