Biology subjects

Veyron, S.

Publications and source records attributed to Veyron, S..

6 recordsLinked to original sources

bioRxiv2026

Evolutionary analysis of PINK1 reveals key roles of the N- and C-terminal extensions and TOM20 in folding its kinase domain

PTEN-induced kinase 1 (PINK1) is a mitochondrial serine/threonine kinase that initiates ubiquitin-dependent mitophagy and is mutated in early-onset Parkinson's disease. Despite extensive characterization of insect PINK1 orthologues, obtaining soluble and catalytically active recombinant Homo sapiens PINK1 (HsPINK1) has remained challenging, limiting its biochemical and structural investigation. Here, we identify an evolutionary divergence in the mechanisms supporting PINK1 stability and activity. Comparative analysis of 48 metazoan PINK1 orthologues revealed that predicted binding to the mitochondrial import receptor TOM20 is predominantly a vertebrate feature and coincides with divergence of the PINK1 N- and C-terminal extensions (NTE and CTE). Consistent with this distinction, invertebrate PINK1 orthologues were purified as active kinases, whereas vertebrate orthologues lacked detectable ubiquitin kinase activity. NMR spectroscopy demonstrated that humanization of ten residues within the Tribolium castaneum (Tc)PINK1 NTE-CTE interface is sufficient to confer direct binding to human TOM20, while co-expression with TOM20 increased the activity of recombinant HsPINK1. Guided by these evolutionary differences, we engineered a chimeric PINK1 containing a predominantly human kinase domain supported by Tc-derived NTE-CTE elements. The resulting kinase was catalytically active and required compatible interactions between the NTE-CTE and kinase C-lobe for activity. The chimera enabled characterization of PINK1 variants and revealed R152W as a likely pathogenic variant. Together, our findings support a model in which the NTE and CTE act as intramolecular determinants of PINK1 kinase stability and suggest that vertebrate PINK1 has evolved an increased dependence on the mitochondrial import machinery to maintain a functional state.

biochemistry↗

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↗

bioRxiv2021

Structure of PINK1 reveals autophosphorylation dimer and provides insights into binding to the TOM complex

Mutations in PINK1 causes autosomal-recessive Parkinsons disease. Mitochondrial damage results in PINK1 import arrest on the Translocase of the Outer Mitochondrial Membrane (TOM) complex, resulting in the activation of its ubiquitin kinase activity by autophosphorylation and initiation of Parkin-dependent mitochondrial clearance. Herein we report crystal structures of the entire cytosolic domain of insect PINK1. Our structures reveal a dimeric autophosphorylation complex targeting phosphorylation at the invariant Ser205 (human Ser228). The dimer interface requires insert 2, which is unique to PINK1. The structures also reveal how an N-terminal helix binds to the C-terminal extension and provide insights into stabilization of PINK1 on the core TOM complex.

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↗

bioRxiv2019

The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications

Microtubules are cytoskeletal structures involved in structural support, microtubule-based transport and the organization of organelles in the cells. The building blocks of the microtubule, the - and {beta}-tubulin heterodimers, polymerize into protofilaments, that associate laterally to form the hollow microtubule. There exists a specific type of microtubule structures in the cilia, termed doublet microtubules, where high stability is required for ciliary beating and function. The doublet microtubule, consisting of a complete A-tubule and a partial B-tubule maintains its stability through unique interactions at its outer and inner junctions, where the A- and B-tubules meet.\n\nUsing cryo-electron microscopy, we present the answer to the long-standing question regarding the identities, localizations and structures of the Chlamydomonas doublet microtubule inner junction proteins. Using a combination of sequence bioinformatics and mass spectrometry, we identified two new inner junction proteins, FAP276 and FAP106, and an inner junction associated protein FAP126. We show that inner junction proteins PACRG and FAP20, together with FAP52, previously unidentified FAP276, FAP106 and FAP126, form an interaction hub at the inner junction, which involves tubulin sites for post-translational modifications. We further compare the Chlamydomonas and Tetrahymena doublet microtubule structures to understand the common and species-specific features of the inner junction.

biophysics↗

bioRxiv2018

A metal-regulated AMPylation/deAMPylation switch in FIC proteins

FIC proteins regulate molecular processes in bacteria and animals by carrying out various post-translational modifications (PTM) of proteins by phosphate-containing compounds. The most frequent FIC-catalyzed PTM is the addition of AMP using ATP as a cofactor, a reaction coined AMPylation. In a large subgroup of FIC proteins, AMPylation is inhibited by a structurally conserved glutamate, but a diffusible signal able to relieve autoinhibition has not been identified. Here, we addressed this issue by studying two members of this subgroup, a single-domain FIC protein from the bacterial pathogen Enterococcus faecalis (EfFIC) and human HYPE/FicD, which is involved in the unfolded protein response in the endoplasmic reticulum. By combining structural and biochemical analysis, we find that EfFIC catalyzes both AMPylation and deAMPylation, and that both enzymatic activities are borne by the same active site. Remarkably, the conserved glutamate implements a multi-position metal switch, whereby different metals support or inhibit each of these reactions. As a result, the balance between the AMPylation and deAMPylation activities of EfFIC is controlled by the Mg2+/Ca2+ ratio, with Ca2+ favoring deAMPylation. Furthermore, we show that deAMPylation of the endoplasmic reticulum BIP chaperone by human FicD/HYPE is dependent on the Mg2+/Ca2+ ratio, with high Ca2+ concentration impairing deAMPylation. Our findings suggest that the conserved glutamate is a signature of AMPylation/deAMPylation bifunctionnality in FIC proteins. They also identify for the first time a diffusible signal that can rapidly modulate these opposing activities, which opens important perspectives for their functions in bacterial stress and human ER homeostasis.

biochemistry↗