Biology subjects

Claes, Z.

Publications and source records attributed to Claes, Z..

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↗

bioRxiv2017

Choice of PP1 catalytic subunit affects neither the requirement for G-actin nor the insensitivity to Sephin1 of PPP1R15A-regulated eIF2αP dephosphorylation

The integrated stress response (ISR) is regulated by kinases that phosphorylate the subunit of translation initiation factor 2 and phosphatases that dephosphorylate it. Genetic and biochemical observations indicate that the eIF2P-directed holophosphatase - a therapeutic target in diseases of protein misfolding - is comprised of a regulatory, PPP1R15, and a catalytic, Protein Phosphatase 1 (PP1) subunit. In mammals, there are two isoforms of the regulatory subunit, PPP1R15A and PPP1R15B, with overlapping roles in promoting the essential function of eIF2P dephosphorylation. However, conflicting reports have appeared regarding the requirement for an additional co-factor, G-actin, in enabling substrate-specific de-phosphorylation by PPP1R15-containing PP1 holoenzymes. An additional concern relates to the sensitivity of the PPP1R15A-containing PP1 holoenzyme to the [(ochlorobenzylidene)amino]guanidines (Sephin1 or Guanabenz), small molecule proteostasis modulators. It has been suggested that the source and method of purification of the PP1 catalytic subunit and the presence or absence of an N-terminal repeat-containing region in the PPP1R15A regulatory subunit might influence both the requirement for G-actin by the eIF2P-directed holophosphatase and its sensitivity to inhibitors. Here we report that in the absence of G-actin, PPP1R15A regulatory subunits were unable to accelerate eIF2P dephosphorylation beyond that affected by a catalytic subunit alone, whether PPP1R15A regulatory subunit had or lacked the N-terminal repeat-containing region and whether paired with native PP1 purified from rabbit muscle, or recombinant PP1 expressed in and purified from bacteria. Furthermore, none of the PPP1R15A-containing PP1c holophosphatases were inhibited by Sephin1 or Guanabenz.

biochemistry↗