Biology subjects

Muqit, M. M. K.

Publications and source records attributed to Muqit, M. M. K..

4 recordsLinked to original sources

bioRxiv2026

Structural Basis for Activation of HRI by the DELE1 C-terminal domain

Haem-Regulated Inhibitor (HRI, aka EIF2AK1) is one of four stress-sensing kinases that phosphorylate eIF2 as part of the integrated stress response (ISR). HRI was first characterized as a haem-sensing kinase where it is inhibited when bound to haem. Recent studies have revealed another role for HRI in sensing mitochondrial stress via the mitochondrial protein DELE1. Upon stress, DELE1 is cleaved, and its C-terminal fragment (DELE1CTD) is released from the mitochondria to the cytoplasm, where it interacts with HRI, to trigger the ISR. This pathway is critical for mitochondrial quality control and neuronal health. Here, we perform biophysical analysis to demonstrate that purified recombinant DELE1CTD binds with high affinity to the N-terminal region of HRI, competing with and displacing haem to activate HRI and pointing to a shared binding site. Using Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS), we map the binding footprint of DELE1CTD on HRI and confirm that it overlaps with the haem-binding site. These findings support a simple activation mechanism: DELE1CTD activates HRI by excluding inhibitory haem from its binding site.

biochemistry↗

bioRxiv2024

Heme binding causes structural rearrangements in HRI to inhibit activation via autophosphorylation

Heme-Regulated Inhibitor (HRI) is one of the four mammalian kinases which phosphorylates eIF2 to facilitate a cellular response to stress through the regulation of mRNA translation. Originally identified for its role as a heme sensor in erythroid progenitor cells, it has since materialised as a potential therapeutic target in both cancer and neurodegeneration. Here we characterise two modes of HRI inhibition of using structural mass spectrometry, biochemical and biophysical techniques. We demonstrate that several ATP-mimetic compounds, including BRAF inhibitors and a compound, GCN2iB, thought to be specific to GCN2, are capable of potently inhibiting HRI. We demonstrate that hemin, a haem-like molecule, inactivates HRI structurally using hydrogen-deuterium exchange mass spectrometry (HDX-MS), and this results in wide-spread structural rearrangement of the protein and how that impacts on the kinase domain through a series of allosteric interactions. This inhibition mainly impacts autophosphorylation, which includes tyrosine phosphorylation, not observed before in the eIF2 kinases.

biochemistry↗

bioRxiv2023

Genetic screening identifies integrated stress response kinase HRI (EIF2AK1) as a negative regulator of PINK1 and mitophagy signalling

Loss-of-function mutations of the PINK1 kinase cause familial early-onset Parkinsons disease (PD). PINK1 is activated upon mitochondrial damage to phosphorylate Ubiquitin and Parkin to trigger removal of damaged mitochondria by autophagy (mitophagy). PINK1 also indirectly phosphorylates a subset of Rab GTPases including Rab8A. We have performed an siRNA screen targeting all human Ser/Thr kinases in HeLa cells and discovered that knockdown of the eukaryotic translation initiation factor 2-alpha kinase 1 (EIF2AK1), also known as heme-regulated inhibitor (HRI) kinase, a branch of the integrated stress response (ISR), selectively enhances mitochondrial depolarization-induced stabilization of PINK1 and increased phosphorylation of ubiquitin and Rab8A. We confirm our findings in multiple human cell lines, including SK-OV-3, U2OS and ARPE-19 cells. Knockdown of the upstream mitochondrial-cytosol relay component, DELE1, enhanced PINK1 stabilisation and activation similar to EIF2AK1 knockdown. Strikingly, we demonstrate that the small molecule ISR inhibitor, ISRIB, also enhances PINK1 activation and signaling under conditions of mitochondrial damage. Using the mito-QC mitophagy reporter in human cells, we observe that EIF2AK1 knockdown or ISRIB treatment significantly enhances PINK1-dependent mitophagy but does not alter deferiprone-induced mitophagy. Our findings indicate that the DELE1-EIF2AK1 ISR signaling relay is a negative regulator of PINK1-dependent mitophagy and suggest that inhibitors of DELE1-EIF2AK1 and/or ISRIB analogues could have therapeutic benefits in PD and related disorders.

biochemistry↗

bioRxiv2022

Structure-based design and characterization of Parkin activating mutations

Human autosomal recessive mutations in the Parkin gene are causal for Parkinsons disease (PD). Parkin encodes a ubiquitin E3 ligase that functions together with the PD associated kinase, PINK1, in a mitochondrial quality control pathway. Structural studies reveal that Parkin exists in an inactive conformation mediated by multiple autoinhibitory domain interfaces. Here we have performed comprehensive mutational analysis of both human and rat Parkin to unbiasedly determine Parkin activating mutations across all major autoinhibitory interfaces. Out of 31 mutations tested, we identify 11 activating mutations clustered near the RING0:RING2 or REP:RING1 interfaces, which reduce the thermal stability of Parkin. Of these, we demonstrate that three mutations, V393D, A401D, and W403A located at the REP:RING1 interface were able to completely rescue a Parkin S65A mutant, defective in mitophagy, in cell-based studies. Overall our data extends previous analysis of Parkin activation mutants and suggests that small molecules that mimic REP:RING1 destabilisation offer therapeutic potential for PD patients harbouring select Parkin mutations. Summary blurbParkin, an E3 ubiquitin ligase involved in Parkinsons disease, is inactive in the basal state and is activated by PINK1 to mediate mitophagy. Here we characterized 31 mutations and discovered three that activate Parkin and rescue loss of PINK1 phosphorylation.

biochemistry↗