Firefly luciferase mutants as sensors of proteome stress

Kasturi, Prasad; Bracher, A.; Loew, C.; Zheng, Min; Villella, Adriana; Garza, Dan; Hartl, F. Ulrich; Raychaudhuri, Santwana

doi:10.1038/nmeth.1697

Cited by 208 publications

(267 citation statements)

References 408 publications

(556 reference statements)

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“…This model is supported by studies showing that expression of mutant htt causes the manifestation of multiple mutant phenotypes at permissive temperatures in Caenorhabditis elegans harboring temperature-sensitive alleles of unrelated genes (Gidalevitz et al, 2006) and induces the misfolding of engineered, metastable luciferase-based "proteostasis sensors" in cultured mammalian cells (Gupta et al, 2011). The cumulative deficit caused by the challenge of mutant htt to global protein folding capacity could interfere with the folding of otherwise normal proteins, diverting misfolded proteins to the UPS and eventually exceeding the capacity of the 26S proteasome (Hipp et al, 2012).…”

Section: Introductionmentioning

confidence: 90%

Protein misfolding specifies recruitment to cytoplasmic inclusion bodies

Bersuker¹,

Brandeis²,

Kopito³

2016

J Exp Med

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Section: Introductionmentioning

confidence: 90%

Protein misfolding specifies recruitment to cytoplasmic inclusion bodies

Bersuker¹,

Brandeis²,

Kopito³

2016

J Exp Med

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“…In human cells, we used a destabilized mutant firefly luciferase (FlucDM-GFP) that clusters in stressed cells and loses its luciferase activity (22). We cotransfected HeLa cells with HYPE constructs and FlucDM-GFP and monitored reporter clustering as well as luciferase activity.…”

Section: Fic-1 (E274g) Is Present In the Cytosolmentioning

confidence: 99%

Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock response

Truttmann

Hanke

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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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.AMPylation | FIC protein | chaperones | proteostasis | HSP70

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“…5F). To further challenge the folding environment, we expressed a destabilized luciferase construct (DM Fluc), which contains two point mutations that impair the ability of the protein to fold (49). DM Fluc had 5% of the activity of WT Fluc in control cells (Fig.…”

Section: Insulin-regulated Proteostasismentioning

confidence: 99%

“…Luciferase Plasmids, Transfection, and Luciferase AssaypCIneo-WT-Fluc and pCIneo-DM-Fluc were kindly provided by Dr. Ulrich Hartl and Dr. Swasti Raychaudhuri (Max Planck Institute) (49). pEGFP-N1 was from Clontech.…”

Section: Statistical Comparison Of Insulin and Control M Syn And M Degmentioning

confidence: 99%

Hyperactivation of the Insulin Signaling Pathway Improves Intracellular Proteostasis by Coordinately Up-regulating the Proteostatic Machinery in Adipocytes

Minard

Wong

Chaudhuri

et al. 2016

Journal of Biological Chemistry

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Edited by Jeffrey PessinHyperinsulinemia, which is associated with aging and metabolic disease, may lead to defective protein homeostasis (proteostasis) due to hyperactivation of insulin-sensitive pathways such as protein synthesis. We investigated the effect of chronic hyperinsulinemia on proteostasis by generating a time-resolved map of insulin-regulated protein turnover in adipocytes using metabolic pulse-chase labeling and high resolution mass spectrometry. Hyperinsulinemia increased the synthesis of nearly half of all detected proteins and did not affect protein degradation despite suppressing autophagy. Unexpectedly, this marked elevation in protein synthesis was accompanied by enhanced protein stability and folding and not by markers of proteostasis stress such as protein carbonylation and aggregation. The improvement in proteostasis was attributed to a coordinate upregulation of proteins in the global proteostasis network, including ribosomal, proteasomal, chaperone, and endoplasmic reticulum/mitochondrial unfolded protein response proteins. We conclude that defects associated with hyperactivation of the insulin signaling pathway are unlikely attributed to defective proteostasis because up-regulation of protein synthesis by insulin is accompanied by up-regulation of proteostatic machinery. The insulin signaling pathway (ISP)4 is a master regulator of protein metabolism. Many of its effects, such as increased protein synthesis and reduced autophagy, are orchestrated via mTORC1 (1, 2). The ISP is hyperactivated by chronic hyperinsulinemia, a common consequence of insulin resistance and aging (3). This in turn exacerbates insulin resistance, obesity (4 -6), and aging (7-10). Chronic ISP activity may partially cause these detrimental effects by dysregulating protein homeostasis (proteostasis) (11,12). Supporting this view, genetic manipulations that lead to either endoplasmic reticulum (ER) stress, reduced proteasomal activity, or reduced autophagy are associated with insulin resistance in mice (13-15). Moreover, the mTORC1 inhibitor rapamycin extends lifespan in a range of animals, and in Drosophila these effects are at least in part due to reduced protein synthesis and enhanced autophagy (16).Proteins are made via a carefully orchestrated process that minimizes misfolding. This involves chaperones that ensure efficient folding of newly synthesized proteins, the ubiquitinproteasome system that degrades misfolded proteins, and autophagy for removal of protein aggregates (17, 18). It has been proposed that chronic insulin signaling impairs protein folding and promotes aggregation because it simultaneously activates protein synthesis, suppresses autophagy, and inhibits the activity of FOXO, a transcription factor that regulates chaperone gene expression (11, 12, 19 -21).The effect of loss of function of daf-2/ISP activity on global proteostasis was recently studied in Caenorhabditis elegans (22), but few studies have examined the effects of hyperactivation of the ISP on proteostasis, and none have examined the...

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Firefly luciferase mutants as sensors of proteome stress

Cited by 208 publications

References 408 publications

Protein misfolding specifies recruitment to cytoplasmic inclusion bodies

Protein misfolding specifies recruitment to cytoplasmic inclusion bodies

Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock response

Hyperactivation of the Insulin Signaling Pathway Improves Intracellular Proteostasis by Coordinately Up-regulating the Proteostatic Machinery in Adipocytes

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