Structure-function studies of DELE1-mediated activation of the integrated stress response

ABSTRACT Protein homeostasis (proteostasis) is tightly regulated by an intricate network of finely tuned cellular pathways. Among the pathways required for responding to internal or external cellular insults is the Integrated Stress Response, which halts general protein production while specifically increasing production of a select subset of

“pro-survival” proteins. As we age, our cells decline in their ability to maintain proteostasis, serving as a hallmark for a range of age-related diseases such as Alzheimer’s, Parkinson’s, Huntington’s, and amyotrophic lateral sclerosis. This pathological loss of proteostasis correlates with increases in ISR activity, establishing the

importance of gaining a detailed understanding of the mechanisms involved in ISR activation. Four kinases serve as upstream triggers of the ISR. Each kinase is activated by distinct forms of cellular stress, but all convergently phosphorylate the eIF2α translation complex to activate the ISR pathway. One of these kinases, Heme-

Regulated Inhibitor (HRI), was recently shown to signal mitochondrial stress to the ISR via a protein called DELE1. The HRI kinase is the principal mediator of ISR activation in neurons with perturbed proteostasis, and is thus of particular relevance to age-related brain diseases. However, the detailed mechanism through which

DELE1 activates this kinase to relay mitochondrial stress to the ISR is unknown. Recent structural and cellular studies on the DELE1 protein from our group have shown that that ISR activation is dependent on oligomerization of DELE1 in the cytosol. Our high-resolution single particle cryo-EM structure of DELE1, combined with

biochemical and cellular studies, indicate that the higher-order assembly likely serves as a structural scaffold for HRI binding and activation. We plan to elucidate how interactions between DELE1, HRI, and eIF2α transduce mitochondrial stress to ISR activity using single-particle cryo-EM, crystallographic, and functional studies.

Atomic-level descriptions of the interacting elements of this pathway could enable us to precisely counter human mitochondrial pathologies without impacting the capacity of cells to respond to other stresses, such as ER stress or viral infections. We will use a range of complementary methodologies to probe the mechanistic underpinnings

of the DELE1-HRI-eIF2α pathway to gain much-needed insights into this branch of ISR activation. We will combine biophysical, biochemical, structural, and cellular studies in three Aims that: 1) tests our hypothesis that DELE1 oligomerization leads to auto- or trans-phosphorylation of bound HRI kinases; 2) defines the role of

cleavage in DELE1 oligomerization and ISR activation; and 3) examine the structural details of eIF2α recruitment to HRI and the mechanism of its activation. These studies will provide a comprehensive, mechanistic description of the key interactions that relay mitochondrial stress to the ISR, and will provide novel avenues to specifically

target the DELE1-HRI-eIF2α pathway to tune both the mitochondrial signaling and the adaptive ISR signaling for therapeutic interventions.