Biology subjects

Bollen, M.

Publications and source records attributed to Bollen, M..

8 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

POLO kinase inhibits Protein Phosphatase 1 to promote the Spindle Assembly Checkpoint and prevent aneuploidy

Protein phosphatase 1 (PP1) is essential for spindle assembly checkpoint (SAC) silencing and mitotic exit, but its regulation during mitosis remains ill-defined. Here, we demonstrate in vitro and in Drosophila cells that the mitotic kinase POLO phosphorylates PP187B at a conserved residue (T286) within a pocket implicated in the recognition of RVxF-containing target proteins. Phosphorylation of T286 inhibits PP187B binding to the RVxF motif of the SAC kinase MPS1, dampening the dephosphorylation of MPS1 T-loop. Phosphorylation of T286 is dynamically regulated during mitosis. It occurs at unattached/tensionless kinetochores and decreases as chromosomes congress. Expression of phosphomimetic PP187BT286D prevents MPS1 inactivation in metaphase and causes a SAC-dependent delay of anaphase onset. Conversely, an unphosphorylatable PP187BT286A mutant impairs MPS1 activation at unattached kinetochores and weakens the SAC. In vivo, larval neuroblasts expressing PP187BT286 phosphomutants exhibit frequent chromosome mis-segregation and aneuploidy. Thus, our findings identify POLO-mediated phosphorylation of PP187B as a critical regulatory strategy that fine-tunes phosphatase activity to ensure a robust and timely SAC and prevent genome instability.

cell biology↗

bioRxiv2022

Simulation-based assessment of the performance of hierarchical abundance estimators for camera trap surveys

The need for knowledge about abundance to guide conservation and management strategies in combination with low detectability of many species has led to a widespread use in ecology and management of a range of hierarchical models (HMs) for abundance. These models also appear like good candidates for inference about local abundance in nature reserves studied by camera traps. However, the best choice among these models is unclear, particularly how they perform in the face of several complicating features of realistic populations that include: (i) movements relative to sites, (ii) multiple detections of unmarked individuals within a single survey, and (iii) low probabilities of detection. We conducted a simulation-based comparison of three HMs (Royle-Nichols, binomial N-mixture and Poisson N-mixture model) in the context of small populations of elusive animals in a single study area, where animals cannot be distinguished individually and hence double counting occurs. We generated count data by simulating camera traps monitoring individuals moving according to a Gaussian random walk. Under the simulated scenarios none of the three HMs yielded accurate abundance estimates. Moreover, the performance of each HM depended on the interpretation of abundance. By pooling abundance estimates for trend estimation, each models performance markedly improves. Overall, the Royle-Nichols and Poisson N-mixture models outperform a binomial N-mixture model. This emphasizes the importance of choosing the appropriate HM for the data problem.

ecology↗

bioRxiv2022

Sugar transporter Slc37a2 regulates bone metabolism via a dynamic tubular lysosomal network in osteoclasts

Osteoclasts are giant bone-digesting cells that harbour specialized lysosome-related organelles termed secretory lysosomes (SLs). SLs store cathepsin K and serve as a membrane precursor to the ruffled border, the osteoclasts resorptive apparatus. Yet, the molecular composition and spatiotemporal organization of SLs remains incompletely understood. Here, using organelle-resolution proteomics, we identify member a2 of the solute carrier 37 family (Slc37a2) as a SL sugar transporter. We demonstrate that Slc37a2 localizes to the SL limiting membrane and that these organelles adopt a hitherto unnoticed but dynamic tubular network in living osteoclasts that is required for bone digestion. Accordingly, mice lacking Slc37a2 accrue high bone mass owing to uncoupled bone metabolism and disturbances in SL export of monosaccharide sugars, a prerequisite for SL delivery to the ruffled border. Thus, Slc37a2 is a physiological component of the osteoclasts unique secretory organelle and a potential therapeutic target for metabolic bone diseases.

cell biology↗

bioRxiv2022

Nuclear inhibitor of protein phosphatase 1 (NIPP1) regulates CNS tau phosphorylation and myelination during development

Nuclear inhibitor of protein phosphatase 1 (NIPP1) is a known regulator of gene expression and has been shown to play roles in many physiological or pathological processes such as stem cell proliferation and skin inflammation. While NIPP1 has many regulatory roles in proliferating cells, its function in the central nervous system (CNS) has not been directly investigated. In the present study, we examined NIPP1 CNS function using a conditional knockout (cKO) mouse model, in which the nipp1 gene is excised from neural precursor cells. These mice demonstrate severe developmental impairments that lead to premature lethality within the first few postnatal weeks. To delineate some of the neurological changes occurring in these animals, we first assessed microtubule associated protein tau, a known target of NIPP1 activity. Furthermore, observed tremors prompted exploration of myelin integrity, an integral structure for CNS function, whose disruption is associated with various neurological disorders and neurodegenerative diseases. First, immunoblotting demonstrated increased phospho-tau and altered AKT and PP1 activity in NIPP1 cKO mice, suggesting increased tau phosphorylation likely results from a shift in kinase/phosphatase activity. Second, immunoblots, electron microscopy, and electrophysiology demonstrated a myelin deficit within the brain and optic nerve. Our study suggests that NIPP1 in neural precursors regulates phosphorylation of tau and CNS myelination and may represent a novel therapeutic target for neurodegenerative diseases.

neuroscience↗

bioRxiv2021

Managing African Swine Fever: Assessing the Potential of Camera Traps in Monitoring Wild Boar Occupancy Trends in Infected and Noninfected Zones, Using Spatio-temporal Statistical Models

The recent spreading of African swine fever (ASF) over the Eurasian continent has been acknowledged as a serious economic threat for the pork industry. Consequently, an extensive body of research focuses on the epidemiology and control of ASF. Nevertheless, little information is available on the combined effect of ASF and ASF-related control measures on wild boar (Sus scrofa) population abundances. This is crucial information given the role of the remaining wild boar that act as an important reservoir of the disease. Given the high potential of camera traps as a non-invasive method for ungulate trend estimation, we assess the effectiveness of ASF control measures using a camera trap network. In this study, we focus on a major ASF outbreak in 2018-2020 in the South of Belgium. This outbreak elicited a strong management response, both in terms of fencing off a large infected zone as well as an intensive culling regime. We apply a Bayesian multi-season site-occupancy model to wild boar detection-nondetection data. Our results show that (1) occupancy rates at the onset of our monitoring period reflect the ASF infection status; (2) ASF-induced mortality and culling efforts jointly lead to decreased occupancy over time; and (3) the estimated mean total extinction rate ranges between 22.44% and 91.35%, depending on the ASF infection status. Together, these results confirm the effectiveness of ASF-control measures implemented in Wallonia (Belgium), which has regained its disease-free status in December 2020, as well as the usefulness of a camera trap network to monitor these effects.

ecology↗

bioRxiv2021

Protein Phosphatase 1 regulates atypical chromosome segregation and cell polarity during mitotic and meiotic division in Plasmodium sexual stages

PP1 is a conserved eukaryotic serine/threonine phosphatase that regulates many aspects of mitosis and meiosis, often working in concert with other phosphatases, such as CDC14 and CDC25. The proliferative stages of the malaria parasite life cycle include sexual development within the mosquito vector, with male gamete formation characterized by an atypical rapid mitosis, consisting of three rounds of DNA synthesis, successive spindle formation with clustered kinetochores, and a meiotic stage during zygote to ookinete development following fertilization. It is unclear how PP1 is involved in these unusual processes. Using real-time live-cell and ultrastructural imaging, conditional gene knockdown, RNA-seq and proteomic approaches, we show that Plasmodium PP1 is implicated in both mitotic exit and, potentially, establishing cell polarity during zygote development in the mosquito midgut, suggesting that small molecule inhibitors of PP1 should be explored for blocking parasite transmission.

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↗