Biology subjects

Croteau, N.

Publications and source records attributed to Croteau, N..

4 recordsLinked to original sources

bioRxiv2023

Identification and structural characterization of small-molecule inhibitors of PINK1

Mutations in PTEN-induced putative kinase 1 (PINK1) cause early-onset autosomal recessive Parkinsons Disease (PD). PINK1 is a Ser/Thr protein kinase which functions as a mitochondrial damage sensor and initiates mitochondrial quality control by accumulating on the damaged organelle. There, it will autophosphorylate and then phosphorylate ubiquitin chains, which in turn will recruit and activate Parkin, and E3 ubiquitin ligase also implicated in PD. Ubiquitylation of mitochondrial proteins leads to the autophagic degradation of the damaged organelle. Pharmacological modulation of PINK1 constitutes an appealing avenue to study its physiological function and develop PD therapeutics. In this study, we used a thermal shift assay to identify small-molecule inhibitors of PINK1. In vitro kinase activity assays demonstrate that these molecules are ATP competitive inhibitors that block ubiquitin phosphorylation. PRT062607 (a SYK inhibitor) is the most potent inhibitor of PINK1 in our screen and has an IC50 of 2 M against insect PINK1 and 1 M in HeLa cells expressing human PINK1. The crystal structures of PINK1 in complex with PRT062607 or CYC116 reveal how the compounds interact with the ATP-binding pocket. PRT062607 notably engages with the catalytic aspartate (type-1 inhibition) and causes a destabilization of insert-2 at the autophosphorylation dimer interface. Our findings provide a scaffold for the development of more selective and potent inhibitors of PINK1 that can be used as chemical probes.

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↗

bioRxiv2021

Selective localization of Mfn2 near PINK1 enable its preferential ubiquitination by Parkin on mitochondria

Mutations in Parkin and PINK1 cause an early-onset familial Parkinsons disease. Parkin is a RING-In-Between-RING (RBR) E3 ligase that transfers ubiquitin from an E2 enzyme to a substrate in two steps: 1) thioester intermediate formation on Parkin, and 2) acyl transfer to a substrate lysine. The process is triggered by PINK1, which phosphorylates ubiquitin on damaged mitochondria, which in turn recruits and activates Parkin. This leads to the ubiquitination of outer mitochondrial membrane proteins and clearance of the organelle. While the targets of Parkin on mitochondria are known, the factors determining substrate selectivity remain unclear. To investigate this, we examined how Parkin catalyzes ubiquitin transfer to substrates. We found that His433 in the RING2 domain catalyzes acyl transfer. In cells, mutation of His433 impairs mitophagy. In vitro ubiquitination assays with isolated mitochondria show that Mfn2 is a kinetically preferred substrate. Using proximity-ligation assays, we show that Mfn2 specifically co-localizes with PINK1 and phospho-ubiquitin in U2OS cells upon mitochondrial depolarization. We propose a model whereby ubiquitination of Mfn2 is efficient by virtue of its localization near PINK1, which leads to the recruitment and activation of Parkin via phospho-ubiquitin at these sites.

biochemistry↗

bioRxiv2019

Crystal structure of human PACRG in complex with MEIG1

In human, the Parkin Co-Regulated Gene (PACRG) shares a bidirectional promoter with Parkin, a gene involved in Parkinsons disease, mitochondrial quality control and inflammation. The PACRG protein is essential to the formation of the inner junction between doublet microtubules of the axoneme, a structure found in flagella and cilia. PACRG interacts with tubulin as well as the meiosis expressed gene 1 (MEIG1) protein, which is essential for spermiogenesis in mice. However, the 3D structure of human PACRG is unknown. Here, we report the crystal structure of the C-terminal domain of human PACRG in complex with MEIG1 at 2.1 [A] resolution. PACRG adopts an -helical structure with a loop insertion that mediates a conserved network of interactions with MEIG1. Using the cryo-electron tomography structure of the axonemal doublet microtubule from the flagellated protozoan Chlamydomonas reinhardtii, we generate a model of a mammalian microtubule doublet inner junction, which reveals how PACRG interacts with tubulin subunits in both the A- and B-tubules. Furthermore, the model shows that MEIG1 interacts with {beta}-tubulin on the outer surface of the B-tubule, facing towards the central pair of the axoneme. We also model the PACRG-like protein (PACRGL), a homolog of PACRG with potential roles in microtubule remodelling and axonemal inner junction formation. Finally, we explore the evolution of the PACRG and Parkin head-to-head gene structure and analyze the tissue distribution of their transcripts. Our work establishes a framework to assess the function of the PACRG family of proteins and its adaptor proteins in the function of motile and non-motile cilia.

cell biology↗