Authors:

Shaye Ross

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About Paper:

The metazoan unfolded protein response (UPR) is an integrated signaling network initiated within the endoplasmic reticulum (ER) that regulates proteostasis in response to misfolded proteins. Previously, the Mattoo lab established the sole human FIC (filamentation induced by cyclic AMP) protein HYPE (Huntingtin- yeast Partner E) as a key regulator of UPR. HYPE modifies the HSP70 chaperone BiP/GRP78 via reversible adenylylation/AMPylation (adds AMP moiety), which affects BiP's ATPase activity and subsequent ability to fold misfolded proteins. Several insults, including infection by the bacterial pathogen Brucella abortus, are known to induce the mammalian UPR. B. abortus secretes an effector protein, VceC (VirB Co-regulated Effector C), via its Type IV secretion system that induces UPR, a process integral to Brucella's ability to colonize the mammalian ER lumen. VceC is a membrane-tethered protein facing the ER lumen and has been suggested to interact with BiP based on pulldown experiments using cellular lysates. However, the exact mechanism and the role of UPR activation during Brucella infection is not understood. To address our gap in knowledge, we have used different biochemical and biophysical techniques to study VceC's structure, function, and potential interactions with BiP and HYPE. Using bacterially expressed and affinity purified VceC, we find that its expression in E. coli induces toxicity. Further, size exclusion chromatography and mass photometry indicate that VceC exists in multimeric states, none of which bind directly to BiP. Instead, we find a strong interaction with HYPE. Collectively, our data challenge the idea of a direct VceC-BiP interaction and provide new insights into VceC's toxicity and stoichiometry, which may influence its role in manipulating ER stress. Keywords: VceC; HYPE; Unfolded Protein Response; Endoplasmic Reticulum; Brucella Abortus

Source:

Purdue University / 2024

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Co-authors:

Shaye Ross