Biology subjects

Vandewinkel, B.

Publications and source records attributed to Vandewinkel, B..

3 recordsLinked to original sources

bioRxiv2025

The adeno-associated virus Rep proteins target PP4:SMEK1 by preventing substrate-recruitment

Adeno-associated virus (AAV) vectors are widely used for in vivo gene therapy, but their clinical effectiveness and scalable production remain limited by gaps in our understanding of AAV biology. In particular, the host-virus interactions that regulate AAV gene expression and replication are not fully defined, yet they hold key insights for engineering improved recombinant AAV (rAAV) vectors. Here, we identify the PP4:SMEK1/2 phosphatase complex as a host restriction factor that suppresses wildtype AAV replication. Active replication and Rep expression correlates with hyperphosphorylation of KAP1S824 and RPA2S4/8/33, known PP4 substrates. Rep proteins directly bind the HEAT/Arm domain of the SMEK1 regulatory subunit of PP4. We further reveal that the KAP1:SMEK1 interaction is mediated by a MAPP short linear motif (SLiM) that interacts with the substrate recruitment groove of the SMEK1 EVH1 domain. Rep68 disrupts this interaction and interferes with substrate recruitment by the PP4:SMEK1/2 complex, leading to substrate hyperphosphorylation. We also uncover a multifunctional complex containing PP4:SMEK1 and PP1:NIPP1, both implicated in KAP1S824 dephosphorylation. Together, these findings reveal a substrate-recruitment interference mechanism by which AAV Rep proteins inhibit host phosphatase activity. This mechanistic insight provides a blueprint for enhancing rAAV vector potency and production in gene therapy applications.

cell biology↗

bioRxiv2024

PEX14 acts as the interface linking optineurin to cell type-selective pexophagy

Pexophagy, the selective degradation of peroxisomes, is essential for removing excess or dysfunctional peroxisomes, and its dysregulation is linked to various diseases. Previous research has shown that optineurin (OPTN), an autophagy receptor involved in mitophagy, aggrephagy, and xenophagy, can induce pexophagy in HEK-293 cells. However, the underlying mechanism remains unclear. In this study, we used proximity labeling to identify PEX14, a peroxisomal membrane protein, as a neighboring partner of OPTN. Biochemical analyses revealed that PEX14 and OPTN interact through their respective coiled-coil and ubiquitin-binding domains. Further analyses demonstrated that the C-terminal half of overexpressed OPTN triggers pexophagy, likely by forming oligomers with endogenous OPTN. The co-localization of PEX14-OPTN complexes with LC3, combined with the suppression of OPTN-mediated peroxisome degradation by bafilomycin A1, supports a model in which PEX14 acts as a docking site for OPTN on the peroxisomal membrane, enabling the recruitment of the autophagic machinery for OPTN-mediated pexophagy. SummaryThis study uncovers and defines the peroxisomal membrane protein PEX14 as a key player in optineurin-driven pexophagy, advancing our mechanistic understanding of this cellular process. These findings open new avenues for developing therapeutic strategies targeting diseases associated with defective pexophagy.

cell biology↗

bioRxiv2020

The Cryo-EM structure of AAV2 Rep68 in complex with ssDNA reveals a malleable AAA+ machine that can switch between oligomeric states.

The adeno-associated virus (AAV) Rep proteins use a unique AAA+ domain to catalyze DNA replication, transcription regulation, and genome packaging. Also, they mediate site-specific integration during a latent phase. To understand the mechanisms underlying AAV Rep function, we investigated the cryo-EM and X-ray structures of Rep68-ssDNA complexes. Surprisingly, Rep68 generates hybrid ring structures where the Origin-Binding-Domain (OBD) forms octameric rings while the helicase domain (HD) forms heptamers. Moreover, the binding to ATP{gamma}S promotes a large conformational change in the entire AAA+ domain that leads the HD to form both heptamer and hexamers. The HD oligomerization is driven by an interdomain linker region that acts as a latch to catch the neighboring HD subunit and is flexible enough to permit the formation of different stoichiometric ring structures. Overall, our studies show the structural basis of AAV Reps structural flexibility required to fulfill its multifunctional role during the AAV life cycle.

biochemistry↗