Biology subjects

Wilhelm, L. P.

Publications and source records attributed to Wilhelm, L. P..

5 recordsLinked to original sources

bioRxiv2026

NLRX1 is an essential, druggable regulator of mitochondrial permeability transition

The molecular composition of the mitochondrial permeability transition pore (mPTP) remains contested, and several efficacious mPTP inhibitors act through undefined, cyclophilin D (CypD)-independent targets. Using two structurally distinct chemotypes of optimised, brain-penetrant mPTP inhibitors as chemical probes, we applied affinity-based chemoproteomics to identify the mitochondrial NOD-like receptor NLRX1 as their shared target. Both chemotypes bind NLRX1, and binding potency across a compound series tracks mPTP-inhibitory activity. Using CRISPR-Cas9-edited human cells and Nlrx1-/- mouse tissues, we show that NLRX1 is required for normal calcium-induced mPTP opening: its loss raises the calcium threshold for pore opening and its overexpression lowers it, independently of CypD. NLRX1 associates with postulated mPTP components, including ATP synthase and the adenine nucleotide translocase, in a compound-sensitive manner, and sustains mitochondrial protein homeostasis over longer timescales. The lead compound, GSK900, is orally bioavailable, brain-penetrant, and active in an mPTP-sensitive neurological injury model. These findings, converging with recent genetic studies, establish NLRX1 as an essential, CypD-independent regulator of mitochondrial permeability transition and provide brain-penetrant chemical tools to interrogate this biology.

cell biology↗

bioRxiv2026

Exploration of targeted electrophilic kinase probes identifies a covalent ULK1 degrader

Kinases have proven to be one of the most fertile target classes for new drug approvals. However, classical reversible inhibitors may not be capable of the levels of specificity or target modulation required across a broad spectrum of disease areas. Approaches that chemically modify kinase inhibitors in solvent exposed regions are unveiling a swathe of mechanisms to address kinase function in new ways. For example, by either covalently recruiting nucleophilic residues outside of the ATP-binding pocket to inhibit, or by recruiting secondary effector proteins to degrade. Here, we systematically assessed the impact of minimal electrophilic modifications to ATP-site binding scaffolds, leading us to identify molecules that can control the activity and abundance of the master autophagy regulator, Unc-51-like autophagy activating kinase 1 (ULK1).

biochemistry↗

bioRxiv2025

Reconstitution of multistep recruitment of ULK1 to membranes in autophagy

The Human ULK1 autophagy-initiating complex consists of ULK1, FIP200, and the HORMA domain heterodimer ATG13:ATG101. PI3P is essential to recruit ULK1C to membranes for ULK1, but ULK1C subunits do not contain PI3P-binding domains. Here we show that the ATG13:ATG101 dimer forms a complex with the PI3P-binding protein WIPI3, as well as WIPI2. Bound to WIPI2-3, ATG13:ATG101 inserts its Trp-Phe (WF) finger into the membrane. MD simulations show that WIPIs and the WF finger cooperatively stabilizes the complex on membranes. Biochemical reconstitution and cell-based assays show that WIPI3:ATG13 engagement promotes ATG16L1 phosphorylation, autophagy and mitophagy. A kinase domain (KD)-proximal PVP motif in the ULK1 IDR docks onto the ATG13:ATG101 HORMA dimer brings the ULK1 KD close to the membrane. The PVP motif is essential for in vitro ULK1 phosphorylation of ATG16L1 and important for autophagy and mitophagy. These data establish a stepwise pathway for recruitment of the ULK1 KD to the vicinity of the membrane surface.

cell biology↗

bioRxiv2023

PINK1 regulated basal mitophagy is evident in skeletal muscles

PINK1, mutated in familial forms of Parkinsons disease, initiates mitophagy following mitochondrial depolarization. However, it is difficult to monitor this pathway physiologically in mice as loss of PINK1 does not alter basal mitophagy levels in most tissues. To further characterize this pathway in vivo, we used mito-QC mice in which loss of PINK1 was combined with the mitochondrial-associated POLGD257A mutation. We focused on skeletal muscle as gene expression data indicates that this tissue has the highest PINK1 levels. We found that loss of PINK1 in oxidative hindlimb muscle significantly reduced mitophagy. Of interest, the presence of the POLGD257A mutation, while having a minor effect in most tissues, restored levels of muscle mitophagy caused by the loss of PINK1. Although our observations highlight that multiple mitophagy pathways operate within a single tissue, we identify skeletal muscle as a tissue of choice for the study of PINK1-dependant mitophagy under basal conditions.

cell biology↗

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↗