Biology subjects

Dekkers, D. H. W.

Publications and source records attributed to Dekkers, D. H. W..

13 recordsLinked to original sources

bioRxiv2026

A role for tubulin in cellular quality control and proteostasis

Microtubules, stiff rods built up from tubulin dimers, form a cytoskeletal network whose structure, behaviour, and function have been extensively investigated, mainly from a mechanical perspective. Here, we describe a role for tubulin in the cellular stress response. We overexpressed tubulin dimers in a controlled fashion in 293F cells. Despite the engagement of autoregulation, a mechanism that degrades tubulin-encoding mRNAs when tubulin levels are high, a surplus of tubulin and microtubules is detected in overexpressing cells. This leads to altered microtubule behaviour, mitotic problems, deregulation of the cell cycle, and replication stress. Surprisingly, we also observe proteostasis defects in tubulin overexpressing cells, which we attribute to mitochondrial stress-related translation attenuation. Conversely, tubulin and microtubules are downregulated as part of the response to oxygen or glutamine deprivation. Together, our data link tubulin levels, and hence autoregulation, to cellular quality control and proteostasis. We propose that competitive interactions with key partners, including the mitochondrial protein import and general translation machinery, underlie the tubulin-mediated control of cellular homeostasis.

cell biology↗

bioRxiv2026

SHANK3-CAMSAP2 interaction links synapses to dendritic microtubule organisation in PV neurons

The microtubule cytoskeleton plays an essential role in establishing and maintaining neuronal polarity. In neurons, microtubules are initially generated at the centrosome which gradually loses this role in development. In mature neurons, microtubules are stabilised by the microtubule minus end-binding protein CAMSAP2. How and where microtubule minus ends are anchored within dendrites of mammalian neurons remains an open question. Here, we show that microtubules are directly attached to the postsynaptic density of excitatory synapses through an interaction of CAMSAP2 with the scaffolding protein SHANK3. This process is particularly relevant in parvalbumin-positive GABAergic neurons, in which excitatory synapses are predominantly located directly on the dendritic shaft in direct proximity to microtubules. Notably, this association is strongly enhanced by the Autism Spectrum Disorder (ASD)-associated SHANK3L68P mutation, leading to an increase of synaptic levels of CAMSAP2. This promotes an increased microtubule association with the synapse and alters microtubule dynamics. Downregulation of CAMSAP2 in SHANK3L68P parvalbumin neurons tips the balance between site-specific microtubule stabilisation and dynamics, reshaping dendritic architecture, connecting SHANK3-CAMSAP2 dependent microtubule regulation to synaptic ASD pathology. TeaserInteraction between synapses and microtubules is enhanced in parvalbumin neurons bearing the ASD-associated SHANK3 L68P mutation.

neuroscience↗

bioRxiv2025

Proteome-wide ubiquitinome profiling reveals substrate-specific dynamics within the USP7 network

USP7 is a pleiotropic deubiquitylating enzyme that is involved in tumor suppression, (neuro)development, chromatin regulation and the DNA damage response. How USP7 regulates these diverse pathways is still unclear. Here, we report data-independent acquisition and label free quantitation mass spectrometry (DIA-LFQ-MS) to profile the proteome-wide impact of USP7 on substrate de-ubiquitylation and overall protein abundance. First, we identified proteins associated with endogenous USP7 by immunopurification followed by DIA-LFQ-MS. Integration of our new results with earlier interactomes of epitope-tagged USP7 yielded a consensus set of high-confidence protein targets. Domain mapping analysis revealed that, in addition to the TRAF domain, the ubiquitin-like domains of USP7 play a key role in substrate selection. Using specific enrichment of tryptic K-{varepsilon}-GG peptides, we mapped proteome-wide changes in ubiquitinome dynamics following inhibition of USP7. Combining unbiased proteome-wide and targeted quantitative mass spectrometry revealed that deubiquitylation by USP7 can have different effects on the stability of distinct substrates, and suggests that USP7s activity profile is substrate-dependent rather than an intrinsic enzymatic property. Thus, in addition to providing a proteome-wide map of USP7 target sites, our multi-angle proteomics approach reveals that the effects of USP7-mediated deubiquitylation on its targets are remarkably variable and substrate-specific. Finally, based on these detailed molecular insights we show how USP7 connects various neurodevelopmental syndromes and tumor suppression pathways.

biochemistry↗

bioRxiv2025

Effect of mosquito saliva from distinct species on human dermal endothelial cell function in vitro and West Nile virus pathogenesis in vivo

During probing and feeding, an infected mosquito injects both virus and saliva into the host skin. The presence of mosquito saliva in the skin increases arbovirus pathogenesis in the bitten host, however the exact mechanism behind this remains to be determined. It is hypothesized that disease enhancement is dependent on the function of the dermal endothelium, where an increased permeability aids in the influx of virus-susceptible cells to the bite site and therefore more cells for the virus to replicate in. Here, we investigate the effects of saliva from Culex and Aedes species on the human dermal endothelial cell function in vitro. Furthermore, we investigate the effect of Culex saliva on West Nile virus (WNV) pathogenesis in a mouse model. We found that salivary gland extract from anthropophilic mosquito species (Aedes and Cx. pipiens molestus) induce permeability of the human dermal endothelium, while an ornithophilic mosquito species (Cx. pip. pipiens) does not. We identified that this effect is likely due to the presence of protease(s) in Cx. pipiens molestus saliva that are absent in Cx. pipiens pipiens saliva. In addition, we show that the presence of Culex saliva at the WNV inoculation site in vivo leads to more consistent weight loss, increased permeability in the inoculation site, and increased mortality compared to inoculation of WNV alone. Identification and characterization of novel salivary proteins from similar but genetically distinct mosquito species will advance the development of intervention methods to combat potential transmission risks and disease severity of emerging mosquito-borne pathogens.

microbiology↗

bioRxiv2024

Hao-Fountain syndrome protein USP7 controls neuronal differentiation via BCOR-ncPRC1.1

Pathogenic variants in the ubiquitin-specific protease 7 (USP7) gene cause a neurodevelopmental disorder called Hao-Fountain syndrome. However, which of USP7s pleiotropic functions are relevant for neurodevelopment remains unclear. Here, we present a combination of quantitative proteomics, transcriptomics and epigenomics to define the USP7 regulatory circuitry during neuronal differentiation. USP7 activity is required for the transcriptional programs that direct both differentiation of embryonic stem cells into neural stem cells, and the neuronal differentiation of SH-SY5Y neuroblastoma cells. USP7 controls the dosage of the Polycomb H2AK119ub1 ubiquitin ligase complexes ncPRC1.1 and ncPRC1.6. Loss-of-function experiments revealed that BCOR-ncPRC1.1, but not ncPRC1.6, is a key effector of USP7 during neuronal differentiation. Indeed, BCOR-ncPRC1.1 mediates a major portion of USP7-dependent gene regulation during this process. Besides providing a detailed map of the USP7 regulome during neurodifferentiation, our results suggest that USP7 and ncPRC1.1-associated neurodevelopmental disorders involve dysregulation of a shared epigenetic network.

molecular biology↗

bioRxiv2024

STK19 drives Transcription-Coupled Repair by stimulating repair complex stability, Pol II ubiquitylation and TFIIH recruitment

DNA damage forms a major obstacle for gene transcription by RNA polymerase II (Pol II). Transcription-coupled nucleotide excision repair (TC-NER) efficiently eliminates transcription-blocking lesions (TBLs), thereby safeguarding accurate transcription, preserving correct cellular function and counteracting aging. TC-NER initiation involves the recognition of lesion-stalled Pol II by CSB, which recruits the CRL4CSA E3 ubiquitin ligase complex and UVSSA. TBL-induced ubiquitylation of Pol II at lysine 1268 of the RPB1 subunit by CRL4CSA serves as a critical TC-NER checkpoint, governing Pol II stability and initiating TBL excision by TFIIH recruitment. However, the precise regulatory mechanisms of the CRL4CSA E3 ligase activity and TFIIH recruitment remain elusive. Here, we reveal Inactive Serine/Threonine Kinase 19 (STK19) as a novel TC-NER factor, that is essential for correct TBL removal repair and subsequent transcription restart. Cryo-EM studies demonstrate that STK19 is an integral part of the Pol II-TC-NER complex, bridging CSA with UVSSA, RPB1 and downstream DNA. Live-cell imaging and interaction studies show that STK19 stimulates TC-NER complex stability and CRL4CSA activity, resulting in efficient Pol II ubiquitylation and correct UVSSA and TFIIH binding. These findings underscore the crucial role of STK19 as a core component of the TC-NER machinery and its key involvement in the cellular responses to DNA damage that interfere with transcription.

molecular biology↗

bioRxiv2024

KIF2A maintains cytokinesis in mouse embryonic stem cells by stabilising intercellular bridge microtubules

Cytokinesis, the final stage of cell division, serves to physically separate daughter cells while ensuring correct segregation of cellular components. In cultured naive mouse embryonic stem cells cytokinesis lasts unusually long but the underlying mechanisms are not well understood. Here, using cellular and in vitro approaches, we describe a novel function for the kinesin-13 member KIF2A in this process. In genome-engineered mouse embryonic stem cells we find that KIF2A mainly localises to spindle poles during metaphase and regulates spindle length in a manner consistent with its known role as microtubule minus-end depolymerase. By contrast, during cytokinesis we observe tight binding of KIF2A on the lattices of intercellular bridge microtubules. At this stage KIF2A maintains microtubule length and number, and controls microtubule acetylation. Based on in vitro experiments we propose that the conversion of KIF2A from a depolymerase to a stabiliser is driven both by the inhibition of its ATPase activity, which increases affinity for the lattice, and by a preference of KIF2A for compacted lattices. We propose that during cytokinesis KIF2A maintains the compacted microtubule state, thereby dampening acetylation. As KIF2A depletion causes pluripotency problems and affects mRNA homeostasis our results furthermore indicate that KIF2A-mediated microtubule stabilisation prolongs cytokinesis to maintain pluripotency.

cell biology↗

bioRxiv2023

Interactome mapping reveals a role for LRP10 in autophagy and NDFIP1-mediated alpha-synuclein secretion

Variants in the LRP10 gene have been found in a spectrum of neurodegenerative disorders, including Lewy body diseases (LBDs). In brains of LBD patients, LRP10 is found in neuronal -synuclein-containing Lewy bodies, astrocytes, and vasculature, but not in inclusion-free neurons. Furthermore, recent work suggests that LRP10 is involved in -synuclein processing and transmission, which is disrupted by the LBD-associated LRP10:c.1424+5G>A variant (LRP10-Splice). In spite of the cumulating genetic and functional evidence for a role of LRP10 in neurodegenerative disorders, our knowledge about the biological processes in which LRP10 is involved is incomplete. In this work, we provide a list of LRP10 interactors identified via LRP10 co-immunoprecipitation and mass spectrometry in LRP10-overexpressing cells and induced pluripotent stem cells (iPSC)-derived astrocytes. In addition to interactors and biological processes previously associated with LRP10, we identified novel interactors and pathways that may provide new insights into LRP10 function. Based on these findings, we focused on the involvement of LRP10 in the autophagy and unconventional secretion pathways via its interaction with the autophagy receptor SQSTM1/p62 and the ubiquitin-proteasome adaptor protein NDFIP1, respectively. We demonstrate that changes in LRP10 levels, either via knock-out or overexpression, affect p62 levels and autophagy in HuTu-80 cells and iPSC-derived astrocytes. Furthermore, we found that both LRP10 and NDFIP1 stimulate -synuclein secretion and synergistically affect intracellular -synuclein levels. Next, we studied the LRP10 interactome and related biological processes in iPSC-derived astrocytes carrying the LRP10-Splice variant. Although various interactors and biological processes were shared between wild-type LRP10 (LRP10-WT) and LRP10-Splice, others were only found in either LRP10-WT or LRP10-Splice. Interestingly, we found that LRP10-Splice responded differently to autophagy-modulating drugs in comparison to LRP10-WT. Furthermore, we show that LRP10-Splice interferes with the LRP10-WT:NDFIP1 interaction and NDFIP1-mediated -synuclein secretion. Finally, we investigated the interactome of a secreted LRP10 species only found in conditioned media from LRP10-Splice carrier cells, and identify biological processes that might be impacted by the secreted LRP10-Splice specific protein. In summary, this study enhances our understanding of LRP10 biology, describes LRP10 functions in autophagy and NDFIP1-mediated -synuclein secretion, and reveals potentially interesting differences between LRP10-WT and LRP10-Splice carrier cells that might be relevant to better understand the role of LRP10 in LBDs pathogenesis.

neuroscience↗

bioRxiv2021

In vivo Analysis Reveals that ATP-hydrolysis Couples Remodeling to SWI/SNF Release from Chromatin

ATP-dependent chromatin remodelers control the accessibility of genomic DNA through nucleosome mobilization. However, the dynamics of genome exploration by remodelers, and the role of ATP hydrolysis in this process remain unclear. We used live-cell imaging of Drosophila polytene nuclei to monitor Brahma (BRM) remodeler interactions with its chromosomal targets. In parallel, we measured local chromatin condensation and its effect on BRM association. Surprisingly, only a small portion of BRM is bound to chromatin at any given time. BRM binds decondensed chromatin but is excluded from condensed chromatin, limiting its genomic search space. BRM-chromatin interactions are highly dynamic, whereas histone-exchange is limited and much slower. Intriguingly, loss of ATP hydrolysis enhanced chromatin retention and clustering of BRM, which was associated with reduced histone turnover. Thus, ATP hydrolysis couples nucleosome remodeling to remodeler release, driving a continuous transient probing of the genome.

molecular biology↗

bioRxiv2021

dCas9 targeted chromatin and histone enrichmentfor mass spectrometry (Catchet-MS) identifies IKZF1as a novel drug-able target for HIV-1 latency reversal

A major pharmacological strategy toward HIV cure aims to reverse latency in infected cells as a first step leading to their elimination. While the unbiased identification of molecular targets physically associated with the latent HIV-1 provirus would be highly valuable to unravel the molecular determinants of HIV-1 transcriptional repression and latency reversal, due to technical limitations, this has not been possible. Here we use a dCas9 targeted chromatin and histone enrichment strategy coupled to mass spectrometry (Catchet-MS) to describe the protein composition of the latent and activated HIV-1 5LTR. Catchet-MS identified known and novel latent 5LTR-associated host factors. Among these, IKZF1 is a novel HIV-1 transcriptional repressor, required for Polycomb Repressive Complex 2 recruitment to the LTR. We find the clinically advanced thalidomide analogue iberdomide, and the FDA approved analogues lenalidomide and pomalidomide, to be novel LRAs that, by targeting IKZF1 for degradation, reverse HIV-1 latency in CD4+T-cells isolated from virally suppressed people living with HIV-1. One Sentence SummarydCas9 targeted chromatin and histone enrichment for mass spectrometry (Catchet-MS) led to the identification of IKZF1-targeting thalidomide analogues as novel HIV-1 latency reversal agents

molecular biology↗

bioRxiv2020

Synaptic anchoring of the endoplasmic reticulum depends on myosin V and caldendrin activity

Excitatory synapses of principal hippocampal neurons are frequently located on dendritic spines. The dynamic strengthening or weakening of individual inputs results in a great structural and molecular diversity of dendritic spines. Active spines with large Ca2+ transients are frequently invaded by a single protrusion from the endoplasmic reticulum (ER), which is dynamically transported into and out of spines by the actin-based motor myosin V. An increase in synaptic strength often correlates with stable anchoring of the ER, followed by the formation of the spine apparatus organelle. Here we show that synaptic ER stabilization depends on the interplay of two Ca2+-binding proteins: calmodulin serves as a light chain of myosin V and activates the motor function, whereas caldendrin acts as an inhibitor which transforms myosin into a stationary F-actin tether. Together, they provide a Ca2+-sensing module for fine-tuning myosin V activity and thereby regulate the formation of the spine apparatus in a subset of active dendritic spines.

neuroscience↗

bioRxiv2019

Characterization of neuronal synaptopodin reveals a myosin V-dependent mechanism of synaptopodin clustering at the post-synaptic sites

The spine apparatus (SA) is an endoplasmic reticulum-related organelle which is present in a subset of dendritic spines in cortical and pyramidal neurons. The synaptopodin protein localizes between the stacks of the spine apparatus and is essential for the formation of this unique organelle. Although several studies have demonstrated the significance of the SA and synaptopodin in calcium homeostasis and plasticity of dendritic spines, it is still unclear what factors contribute to its stability at the synapse and whether the SA is locally formed or it is actively delivered to the spines. In this study we show that synaptopodin clusters are stable at their locations. We found no evidence of active microtubule-based transport for synaptopodin. Instead new clusters were emerging in the spines, which we interpret as the SA being assembled on-site. Furthermore, using super-resolution microscopy we show a tight association of synaptopodin with actin filaments. We identify the actin-based motor proteins myosin V and VI as novel interaction partners of synaptopodin and demonstrate that myosin V is important for the formation and/or maintenance of the SA.

neuroscience↗

bioRxiv2018

HSF2BP Negatively Regulates Homologous Recombination in DNA Interstrand Crosslink Repair in Human Cells by Direct Interaction With BRCA2

The tumor suppressor BRCA2 is essential for homologous recombination, replication fork stability and DNA interstrand crosslink (ICL) repair in vertebrates. We show that a functionally uncharacterized protein, HSF2BP, is involved in a novel, direct and highly evolutionarily conserved interaction with BRCA2. Although HSF2BP was previously described as testis-specific, we find it is expressed in mouse ES cells, in human cancer cell lines, and in tumor samples. Elevated levels of HSF2BP sensitize human cells to ICL-inducing agents (mitomycin C and cisplatin) and PARP inhibitors, resulting in a phenotype characteristic of cells from Fanconi anemia (FA) patients. We biochemically recapitulate the suppression of ICL repair and establish that excess HSF2BP specifically compromises homologous recombination by preventing BRCA2 and RAD51 loading at the ICL. As increased ectopic expression of HSF2BP occurs naturally, we suggest that it can be considered as a causative agent in FA and a source of cancer-promoting genomic instability.

molecular biology↗