Unrestrained AMPylation targets cytosolic chaperones and activates the heat shock response

Abstract

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

rAMPing Up Stress Signaling: Protein AMPylation in Metazoans

“…This leads to the formation of protein aggregates. If expressed in human cells, HYPE (E234G)-mediated target AMPylation likewise triggers a heat-shock response, attenuates translational activities and interferes with HSP70’s ability to cycle between the cytoplasm and the nucleus, recapitulating the findings made in S. cerevisiae (Truttmann et al, 2017). HYPE-mediated target AMPylation outside of the ER has obvious implications for regulation of cellular signaling cascades (Fig.…”

“…HYPE modifies a series of targets in vitro , including Rho family GTPases, core histones, heat shock proteins, nuclear envelope proteins, tubulins, components of the ATP synthase machinery as well as factors that regulate transcription and translation (Broncel et al, 2015, Engel et al, 2012, Ham et al, 2014, Mattoo et al, 2011, Preissler et al, 2015, Sanyal et al, 2015, Truttmann et al, 2016, Truttmann et al, 2015, Truttmann et al, 2017, Worby et al, 2009). The ability of recombinant HYPE and HYPE (E234G) to AMPylate purified proteins or putative targets offered in the context of cell lysates must be viewed critically, not unlike other enzyme-substrate combinations, such as protein kinases, when examined under artificial in vitro conditions.…”

“…Despite the concern that in vitro AMPylation assays might be prone to produce false positives, there is supporting evidence for HYPE-mediated modification of non-ER proteins: In addition to Grp78/BiP, ATP synthase subunits α and β, tubulin and translation elongation factor 1 directly interact with wild-type HYPE in vivo (Broncel et al, 2015). Expression of HYPE (E234G) in S. cerevisiae – an organism that lacks an endogenous Fic-type AMPylase – triggers a cytosolic heat shock response and evokes cytotoxic effects that limit growth (Truttmann et al, 2017). The primary target for HYPE (E234G) in S.cerevisiae is Ssa2, a major cytoplasmic HSP70 family-type chaperone critical for maintenance of proteostasis.…”

“…Fic-1 (E274G) AMPylates core histones, eEF1A-type translation elongation factors as well as heat shock (HSP) 40 and 70 family proteins in vitro (Truttmann et al, 2016, Truttmann et al, 2017). Among the endogenous HSP70 proteins modified are the two C. elegans BiP orthologs (HSP-3, HSP-4) as well as the major cytosolic HSP70 representative, HSP-1.…”

“…Fic-1’s enzymatic activity is not restricted to nematode targets, and can also AMPylate human (HSPA1A) and yeast (SSA2) cytosolic HSP70 proteins. When expressed in Saccharomyces cerevisiae , Fic-1 (E274G) reliably modifies Ssa2 and interferes with its function (Truttmann et al, 2017). …”