Biology subjects

Vertegaal, A. C. O.

Publications and source records attributed to Vertegaal, A. C. O..

16 recordsLinked to original sources

bioRxiv2026

Survey of the human proteostasis network: the ubiquitin-proteasome system

Modification by ubiquitination governs the half-lives of thousands of proteins that are fated for elimination by either the proteasome or autophagy pathways, depending on the intricate architectures of ubiquitin modification. This system mediates quality control for individual proteins, protein complexes, and organelles, as well as myriad purely regulatory functions. Here we provide a comprehensive survey of the ubiquitin-proteasome system (UPS), the scope of which is at present poorly defined. The UPS, with the inclusion of pathways involving ubiquitin-like modifiers, comprises in our estimate over 1430 distinct proteins in humans, a vast set of activities whose collective impact on the biology of the cell is pervasive. The UPS is an integral component of the proteostasis network (PN), the remainder of which we have also surveyed in recent studies. With the addition of molecular chaperones, proteins from autophagy-lysosome pathway, and related activities, the PN includes in total over 3150 components by our estimates. Comprehensive and systematic definition of these pathways should support a range of ongoing investigations in the areas of genomics, proteomics, biochemistry, cell biology, and disease research.

bioinformatics↗

bioRxiv2025

FibrilPaints as a tool to bind and modulate Huntingtin amyloids

Huntingtons disease (HD) is caused by expansion of a polyglutamine tract in the huntingtin (Htt) protein, leading to aggregation of the exon 1 fragment (HttEx1) into amyloid fibrils. HttEx1 forms one of the lowest-complexity amyloid cores known, its fibril core consists of a single amino acid, glutamine. With emerging therapies improving patients prospects by silencing expression of HTT, tools to monitor HttEx1 aggregation become essential for timely intervention and next-generation therapeutics. Here, we show that the peptide FibrilPaint1 selectively binds HttEx1Q44 fibrils without interacting with monomeric protein, allowing to measure and trace HttEx1 amyloid fibrils. Using the FibrilRuler assay, we tracked fibril formation from early species to larger clustered assemblies. The non-fluorescent variant, FibrilPaint20, was used to recruit the E3 ubiquitin ligase CHIP to HttEx1 fibrils, enabling site-specific ubiquitin tagging. However, unlike Tau fibrils, ubiquitinated HttEx1 fibrils resisted proteasomal degradation. This reveals a fundamental difference in how amyloids with extremely low-complexity cores respond to cellular clearance machinery. Together, our findings establish the FibrilPaint peptide family as a toolset for the detection and molecular targeting of amyloids, providing new opportunities to study protein aggregation and act as building blocks for future diagnostic and therapeutic strategies in neurodegenerative diseases. HighlightsO_LIFibrilPaint1 selectively binds HttEx1Q44 amyloid fibrils and allows monitoring of fibril growth using the hydrodynamic radius (FibrilRuler). C_LIO_LIFibrilPaint20 recruits the E3 ligase CHIP to Htt fibrils, enabling ubiquitination. C_LIO_LIDespite successful ubiquitination, Htt fibrils resist proteasomal degradation in vitro, highlighting structural barriers. C_LIO_LIFibrilPaint provides a scaffold for functional targeting of amyloids with diagnostic and therapeutic potential. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=160 SRC="FIGDIR/small/695423v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@18acc7corg.highwire.dtl.DTLVardef@1771f3borg.highwire.dtl.DTLVardef@1a35e51org.highwire.dtl.DTLVardef@85270a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO The FibrilRuler Test: FibrilPaint enables measurement of Huntingtin fibril size during aggregation After a short lag-phase following removal of the protective MBP tag by Factor Xa, fibrillation proceeds rapidly. Subsequent fibril clustering further accelerates growth, leading to exponential increases in aggregate size. C_FIG

biochemistry↗

bioRxiv2025

THE NSL COMPLEX PROMOTES NEURAL DEVELOPMENT BY PREVENTING R-LOOP INDUCED REPLICATION STRESS

Early neuronal development relies on the proliferation of neural progenitor cells, making this developmental stage particularly vulnerable to DNA replication impediments. Here, we identify a non-canonical role for the non-specific lethal (NSL) complex in safeguarding DNA replication during neurodevelopment. The NSL complex, which acetylates histone 4 at gene promoters and is mutated in Koolen-de Vries syndrome (KdVS), prevents unscheduled R-loop accumulation at weakly transcribed promoters that are typically devoid of R-loops. Single-cell sequencing reveals that loss of NSL causes replisome stalling and delayed S-phase progression. Neural organoids derived from KdVS patients exhibit impaired DNA replication, developmental abnormalities, and reduced synapse formation, driven entirely by unscheduled R-loop accumulation. These findings reveal that faithful DNA replication is critical for early neurodevelopment.

genomics↗

bioRxiv2025

TRIM28 regulates pre-mRNA splicing via phosphorylation and SUMOylation networks

Pre-mRNA splicing is a highly regulated process orchestrated by splicing factors, cis-acting elements, and interconnected cellular processes such as transcription and chromatin remodeling. Here, we identify a novel regulatory axis involving phosphorylation and SUMOylation that governs the function of Tripartite motif-containing 28 protein (TRIM28) and its role in pre-mRNA splicing. We demonstrate that TRIM28 interacts with the spliceosomal protein USP39 in a phosphorylation-dependent manner, with non-phosphorylated TRIM28 promoting USP39 SUMOylation at defined lysine residues. This post-translational modification enhances USP39s role within the U4/U6.U5 tri-snRNP complex. Functionally, TRIM28 knockdown induces widespread alterations in alternative splicing patterns, underscoring its importance in splicing regulation. Together, our findings uncover a mechanistic link between TRIM28-mediated post-translational modifications and the modulation of spliceosomal activity, offering new insights into how splicing decisions are integrated with cellular signaling pathways.

cell biology↗

bioRxiv2024

Lrif1 modulates Trim28-mediated repression of the Dux locus in mouse embryonic stem cells

Germline mutations in SMCHD1, DNMT3B and LRIF1 can cause facioscapulohumeral muscular dystrophy type 2 (FSHD2). FSHD is an epigenetic skeletal muscle disorder in which partial failure in heterochromatinization of the D4Z4 macrosatellite repeat causes spurious expression of the repeat-embedded DUX4 gene in skeletal muscle, ultimately leading to muscle weakness and wasting. All three proteins play a role in chromatin organization and gene silencing; however, their functional relationship has not been fully elucidated. Here, we show that knockdown of Lrif1, but not of the other two FSHD2 genes, in mouse embryonic stem cells leads to modest upregulation of the 2-cell cleavage stage transcriptional program driven by the transcription factor Dux, which is the mouse functional homologue of human DUX4. Furthermore, we show that Lrif1 interacts with Trim28, a known Dux repressor and that this interaction is independent of Cbx proteins and Smchd1. We uncover that modest Dux upregulation in Lrif1 knockdown mESCs coincides with decreased Trim28 occupancy at the Dux locus. Together, our results provide evidence for a conserved function of Lrif1 in repressing an early zygotic genome activator in mice and humans.

molecular biology↗

bioRxiv2024

SUMOylation differentially regulates SMCHD1 complex formation and function in a genomic context-specific manner

Structural Maintenance of Chromosomes Hinge Domain Containing 1 (SMCHD1) is a chromatin repressor regulating gene expression and chromatin architecture of distinct autosomal and X-chromosomal loci. SMCHD1 mutations cause derepression of the D4Z4 macrosatellite repeat-embedded DUX4 gene in skeletal muscle of facioscapulohumeral muscular dystrophy (FSHD) patients Little is known about the regulation and post-translational modification of SMCHD1. Here we report the SUMOylation dynamics of SMCHD1 and its impact on autosomal single copy and repetitive loci, and the inactive X chromosome (Xi). We identify that SMCHD1 is SUMOylated primarily at lysine 1374, uncover factors regulating SMCHD1 SUMOylation, and demonstrate that SMCHD1 interacts with chromatin repressors TRIM28, HNRNPK and SETDB1 in a SUMO-dependent manner. We find that SUMOylation impacts Xi engagement of SMCHD1, maintenance of a repressive D4Z4 chromatin structure preventing DUX4 expression, and regulation of LRIF1 promoter activity. The rapid, SUMO-dependent upregulation of DUX4 could explain the bursts of DUX4 expression typical for FSHD muscle.

molecular biology↗

bioRxiv2023

Comparative phosphoproteome analysis of primary and metastatic uveal melanoma cell lines

Uveal melanoma (UM) is an ocular tumor that often develops asymptomatically. Statistically, every second patient eventually develops metastases that drastically worsen the prognosis by several months of overall survival. While isolated liver perfusion with melphalan and more recently immunotherapy (Tebentafusp) are the few treatment options available for metastatic UM patients, their application is complex or expensive. There is an urgent need to understand drug response and identify potential avenues for therapy. Hence, we focused on uncovering altered phosphorylation signaling events in metastatic UM using proteomics as an approach to identify potential drug targets. We analyzed the phosphoproteomes of the primary UM cell line Mel270 and two cell lines OMM2.3 and OMM2.5, derived from metastatic lesions of the same patient. We found 177 phosphosites to be altered significantly between primary and metastatic cell lines. Pathway analysis of up-regulated phosphosites in metastatic lines suggests that Rho signaling and mitotic cell cycle to be significantly altered uncovering potential routes of signaling for metastasis. Clinical data from LUMC and TCGA datasets uncovered MARK3 expression (which links to Rho signaling) correlation with chromosome 3 status, a prognostic marker in UM, suggesting that MARK3 kinase might be involved in metastatic UM signaling.

cancer biology↗

bioRxiv2023

BioE3 enables the identification of bona fide targets of E3 ligases

The post-translational modification of proteins by ubiquitination is a highly regulated process that involves a dynamic, three-step enzymatic cascade, where more than 600 E3 ligases play a critical role in recognizing specific substrates for modification. Separating bona fide targets of E3s from E3-interacting proteins remains a major challenge in the field. In this study, we present BioE3, a novel approach for identifying substrates of ubiquitin-like (UbL) E3 ligases of interest. Using BirA-E3 ligase fusion proteins and bioUbLs, the method facilitates site-specific biotinylation of UbL-modified substrates of particular E3s for proteomic identification. We demonstrate that the BioE3 system can identify both known and novel targets of two RING-type ubiquitin E3 ligases: RNF4, known to be involved in DNA damage response and the regulation of PML nuclear bodies, and MIB1, implicated in endocytosis, autophagy, and centrosomal protein homeostasis. We further show the versatility of BioE3 by identifying targets of an organelle-specific E3 (MARCH5) and a relatively uncharacterized E3 (RNF214). Furthermore, we show that BioE3 works with HECT-type E3 ligases and identify novel targets of NEDD4 involved in vesicular trafficking. BioE3 is a powerful tool that enables identification of bona fide substrates of UbL E3 ligases and how they change with chemical perturbations. BioE3 may also be applicable for UbLs beyond Ub and SUMO, as well as other E3 ligase classes. The resulting knowledge can shed light on the regulation of cellular processes by the complex UbL network and provide information useful for strategies such as targeted protein degradation (TPD), advancing our understanding of fundamental biological mechanisms and their applications.

cell biology↗

bioRxiv2023

BRCA1/BARD1 ubiquitinates PCNA in unperturbed conditions to promote replication fork stability and continuous DNA synthesis.

Deficiencies in the BRCA1 tumor suppressor gene are the main cause of hereditary breast and ovarian cancer. BRCA1 is involved in the Homologous Recombination DNA repair pathway, and, together with BARD1, forms a heterodimer with ubiquitin E3 activity. The relevance of the BRCA1/BARD1 ubiquitin E3 activity for tumor suppression and DNA repair remains controversial and most efforts aimed to identify BRCA1/BARD1 ubiquitination substrates rely on indirect evidence. Here, we observed that the BRCA1/BARD1 ubiquitin E3 activity was not required for Homologous Recombination or resistance to Olaparib. Using TULIP2 methodology, which enables the direct identification of E3-specific ubiquitination substrates, we identified substrates for BRCA1/BARD1. We found that PCNA is ubiquitinated by BRCA1/BARD1 in unperturbed conditions independently of RAD18. PCNA ubiquitination by BRCA1/BARD1 avoids the formation of ssDNA gaps during DNA replication and promotes replication fork stability epistatically to BRCA1 S114 phosphorylation, addressing the controversy about the function of BRCA1/BARD1 E3 activity in Homologous Recombination.

molecular biology↗

bioRxiv2022

SUMO Activated Target Traps (SATTs) enable the identification of a comprehensive E3-specific SUMO proteome.

Ubiquitin and ubiquitin-like conjugation cascades consist of dedicated E1, E2 and E3 enzymes with E3s providing substrate specificity. Mass spectrometry-based approaches have enabled the identification of more than 60,000 acceptor sites for ubiquitin and 40,000 acceptor sites for SUMO2/3. However, E3-to-target wiring is poorly understood. The limited number of SUMO E3s provides the unique opportunity to systematically study E3-substrate wiring. We developed SUMO Activated Target Traps (SATTs) and systematically identified substrates for eight different SUMO E3s, PIAS1, PIAS2, PIAS3, PIAS4, NSMCE2, ZNF451, LAZSUL(ZNF451-3) and ZMIZ2. SATTs enabled us to identify 590 SUMO1 and 1195 SUMO2/3 targets in an E3-specific manner. We found pronounced E3 substrate preference, even at the substrate isoform level. Quantitative proteomics enabled us to measure substrate specificity of E3s, quantified using the SATT index. Furthermore, we developed the Polar SATTs web-based tool (https://amsterdamstudygroup.shinyapps.io/PolaRVolcaNoseR/) to browse the dataset in an interactive manner, increasing the accessibility of this resource for the community. Overall, we uncover E3-to-target wiring of 1681 SUMO substrates, highlighting unique and overlapping sets of substrates for eight different SUMO E3 ligases.

molecular biology↗

bioRxiv2022

Multi-level monitoring of EME1-MUS81 in CPT induced nucleolar rDNA repair

We identified EME1, the regulatory subunit of the structure-specific endonuclease complex EME1-MUS81, as substrate for the sumoylated UBC9 and demonstrated synergistic functions in promoting Camptothecin (CPT)-induced nucleolar ribosomal DNA (rDNA) repair (Nagamalleswari et al, co-submitted). Sumoylation of EME1 appears complex involving mono- and poly-sumoylation. Hence, we addressed here whether these modifications differentially regulate EME1 functions by analyzing EME1-variant expressing cell lines including mono-sumo(1)ylation and poly-sumo(2)ylation mimetic fusions. We complemented our analysis with the regulated endogenous EME1 interactome and our observation that Trichostatin A (TSA) induced EME1 and UBC9 sumoylation. Our findings are substantiated by identifying several regulatory proteins and by detecting CPT-induced endogenous di- and poly-sumoylated EME1 in different cell fractions. Together, our data suggest that Histone H4 acetylation, two mono-sumoylation events, poly-sumoylation, ISG15 and ubiquitination sequentially and in mutual dependence tightly control the intracellular localization, recruitment to DNA lesions, enzymatic activity, withdrawal from DNA lesions and the stability of EME1.

molecular biology↗

bioRxiv2021

Unbiased in vivo exploration of nuclear bodies-enhanced sumoylation reveals that PML orchestrates embryonic stem cell fate

Membrane-less organelles are condensates formed by phase separation whose functions often remain enigmatic. Upon oxidative stress, PML scaffolds Nuclear Bodies (NBs) to regulate senescence or metabolic adaptation, but their role in pluripotency remains elusive. Here we establish that PML is required for basal SUMO2/3 conjugation in mESCs and oxidative stress-driven sumoylation in mESCs or in vivo. PML NBs create an oxidation-protective environment for UBC9-driven SUMO2/3 conjugation of PML partners, often followed by their poly-ubiquitination and degradation. Differential in vivo proteomics identified several members of the KAP1 complex as PML NB-dependent SUMO2-targets. The latter drives functional activation of this key epigenetic repressor. Accordingly, Pml-/- mESCs re-express transposable elements and display features of totipotent-like cells, a process further enforced by PML-controlled SUMO2-conjugation of DPPA2. Finally, PML is required for adaptive stress responses in mESCs. Collectively, PML orchestrates mESC fate through SUMO2-conjugation of key transcriptional or epigenetic regulators, raising new mechanistic hypotheses about PML roles in normal or cancer stem cells.

cell biology↗

bioRxiv2020

Identification of proximal SUMO-dependent interactors using SUMO-ID

The fast dynamics and reversibility of posttranslational modifications by the ubiquitin family pose significant challenges for research. Here we present SUMO-ID, a technology that merges proximity biotinylation by TurboID and protein-fragment complementation to find SUMO-dependent interactors of proteins of interest. We developed an optimized split-TurboID version and show SUMO interaction-dependent labelling of proteins proximal to PML and RANGAP1. SUMO-dependent interactors of PML are involved in transcription, DNA damage, stress response and SUMO modification and are highly enriched in SUMO Interacting Motifs, but may only represent a subset of the total PML proximal proteome. Likewise, SUMO-ID also allowed us to identify novel interactors of SUMOylated SALL1, a less characterized SUMO substrate. Thus, SUMO-ID is a powerful tool that allows to study the consequences of SUMO-dependent interactions, and may further unravel the complexity of the ubiquitin code.

biochemistry↗

bioRxiv2020

ER-embedded UBE2J1/RNF26 ubiquitylation complex in spatio-temporal control of the endolysosomal pathway

The endolysosomal system fulfills a wide variety of cellular functions, many of which are modulated through interactions with other organelles. In particular, the ER exerts spatiotemporal constraints on the organization and motility of endosomes and lysosomes. We have recently described the ER transmembrane E3 ubiquitin ligase RNF26 to control perinuclear positioning and transport dynamics of the endolysosomal vesicular network. We now report that the ubiquitin conjugating enzyme UBE2J1, also anchored in the ER membrane, collaborates with RNF26 in this context, and that the cellular activity of this E2/E3 pair, localized in a perinuclear ER subdomain, is underpinned by transmembrane interactions. Through modification of its substrate SQSTM1/p62, the ER-embedded UBE2J1/RNF26 ubiquitylation complex recruits endosomal adaptors to immobilize their cognate vesicles in the perinuclear region. The resulting spatiotemporal compartmentalization of the endolysosomal system between the perinuclear vesicle cloud and the cell periphery facilitates timely downregulation of endocytosed cargoes, such as EGFR.

cell biology↗

bioRxiv2020

A CSB-PAF1C axis restores processive transcription elongation after DNA damage repair

The coordinated transcription of genes involves the regulated release of RNA polymerase II (RNAPII) from promoter-proximal sites into active elongation. DNA lesions in transcribed strands block elongation and induce a strong transcriptional arrest. The transcription-coupled repair (TCR) pathway efficiently removes transcription-blocking DNA lesions, but this is not sufficient to resume transcription. Through proteomics screens, we find that the TCR-specific CSB protein loads the evolutionary conserved PAF1 complex (PAF1C) onto RNAPII in promoter-proximal regions in response to DNA damage. PAF1C is dispensable for TCR-mediated repair, but is essential for recovery of RNA synthesis after UV irradiation, suggesting an uncoupling between DNA repair and transcription recovery. Moreover, we find that PAF1C promotes RNAPII pause release in promoter-proximal regions and subsequently acts as a processivity factor that stimulates transcription elongation throughout genes. Our findings expose the molecular basis for a non-canonical PAF1C-dependent pathway that restores transcription throughout the human genome after genotoxic stress.

cell biology↗

bioRxiv2019

The sequential and cooperative action of CSB, CSA and UVSSA targets the TFIIH complex to DNA damage-stalled RNA polymerase II

The response to DNA damage-stalled RNA polymerase II (RNAPIIo) involves the assembly of the transcription-coupled repair (TCR) complex on actively transcribed strands. The function of the TCR proteins CSB, CSA and UVSSA and the manner in which the core DNA repair complex, including transcription factor IIH (TFIIH), is recruited are largely unknown. Here, we define the assembly mechanism of the TCR complex in human isogenic knockout cells. We show that TCR is initiated by RNAPIIo-bound CSB, which recruits CSA through a newly identified CSA-interaction motif (CIM). Once recruited, CSA facilitates the association of UVSSA with stalled RNAPIIo. Importantly, we find that UVSSA is the key factor that recruits the TFIIH complex in a manner that is stimulated by CSB and CSA. Together these findings reveal a sequential and highly cooperative assembly mechanism of TCR proteins and reveal the mechanism for TFIIH recruitment to DNA damage-stalled RNAPIIo to initiate repair.

molecular biology↗