Biology subjects

van Veelen, P. A.

Publications and source records attributed to van Veelen, P. A..

25 records · Page 2Linked to original sources

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

WWP2 osteoarthritis risk allele rs1052429-A confers risk by affecting cartilage matrix deposition via hypoxia associated genes

ObjectiveTo explore the co-expression network of the osteoarthritis (OA) risk gene WWP2 in articular cartilage and study cartilage characteristics when mimicking the effect of OA risk allele rs1052429-A on WWP2 expression in a human 3D in vitro model of cartilage. MethodsCo-expression behavior of WWP2 with genes expressed in lesioned OA articular cartilage (N=35 samples) was explored. By applying lentiviral particle mediated WWP2 upregulation in 3D in vitro pellet cultures of human primary chondrocytes (N=8 donors) the effects of upregulation on cartilage matrix deposition was evaluated. Finally, we transfected primary chondrocytes with miR-140 mimics to evaluate whether miR-140 and WWP2 are involved in similar pathways. ResultsUpon performing Spearman correlations in lesioned OA cartilage, 98 highly correlating genes (|{rho}|>0.7) were identified. Among these genes, we identified GJA1, GDF10, STC2, WDR1, and WNK4. Subsequent upregulation of WWP2 on 3D chondrocyte pellet cultures resulted in a decreased expression of COL2A1 and ACAN and an increase in EPAS1 expression. Additionally, we observed a decreased expression of GDF10, STC2, and GJA1. Proteomics analysis identified 42 proteins being differentially expressed with WWP2 upregulation, which were enriched for ubiquitin conjugating enzyme activity. Finally, upregulation of miR-140 in 2D chondrocytes resulted in significant upregulation of WWP2 and WDR1. ConclusionsMimicking the effect of OA risk allele rs1052429-A on WWP2 expression initiates detrimental processes in the cartilage shown by a response in hypoxia associated genes EPAS1,GDF10, and GJA1 and a decrease in anabolic markers, COL2A1 and ACAN.

cell biology↗

bioRxiv2021

Clostridioides difficile phosphoproteomics shows an expansion of phosphorylated proteins in stationary growth phase

Phosphorylation is a post-translational modification that can affect both house-keeping functions and virulence characteristics in bacterial pathogens. In the Gram-positive enteropathogen Clostridioides difficile the extent and nature of phosphorylation events is poorly characterized, though a protein-kinase mutant strain demonstrates pleiotropic phenotypes. Here, we used an immobilized metal affinity chromatography strategy to characterize serine, threonine and tyrosine phosphorylation in C. difficile. We find limited protein phosphorylation in the exponential growth phase but a sharp increase in the number of phosphopeptides after the onset of stationary growth phase. Among the overall more than 1500 phosphosites, our approach identifies expected targets and phosphorylation sites, including the protein kinase PrkC, the anti-sigma-F factor antagonist (SpoIIAA), the anti-sigma-B factor antagonist (RsbV) and HPr kinase/phosphorylase (HprK). Analysis of high-confidence phosphosites shows that phosphorylation on serine residues is most common, followed by threonine and tyrosine phosphorylation. This work forms the basis for a further investigation into the contributions of individual kinases to the overall phosphoproteome of C. difficile and the role of phosphorylation in C. difficile physiology and pathogenesis. ImportanceIn this manuscript, we present a comprehensive analysis of protein phosphorylation in the Gram-positive enteropathogen Clostridioides difficile. To date, only limited evidence on the role of phosphorylation in regulation in this organism has been published; the current study is expected to form the basis for research on this post-translational modification in C. difficile.

microbiology↗

bioRxiv2020

Healthy cells functionally present TAP-independent SSR1 peptides: implications for selection of clinically relevant antigens

Tumors with an impaired transporter associated with antigen processing (TAP) present several ER-derived self-antigens on HLA class I (HLA-I) which are absent on healthy cells. Selection of such TAP-independent antigens for T cell-based immunotherapy should include analysis of their expression on healthy cells to prevent therapy-induced adverse toxicities. However, it is unknown how the absence of clinically relevant antigens on healthy cells needs to be validated. Here we monitored TAP-independent antigen presentation on various healthy cells using a new toolbox consisting of a T cell clone recognizing a TAP-independent SSR1-derived antigen. We found that most but not all healthy cells present this antigen under normal and inflammatory conditions, indicating that TAP-independent antigen presentation is a variable phenomenon. Our data emphasize the necessity of extensive testing of a wide variety of healthy cell types to define clinically relevant TAP-independent antigens that can be safely targeted by immunotherapy.

immunology↗

bioRxiv2020

Site-specific glycosylation mapping of Fc gamma receptor IIIb from neutrophils of health donors

Fc gamma receptors (Fc{gamma}R) translate antigen-recognition by immunoglobulin G (IgG) into various immune responses. A better understanding of this key element of immunity promises novel insights into mechanisms of (auto-/allo-)immune diseases and more rationally designed antibody-based drugs. Glycosylation on both IgG and Fc{gamma}R impacts their interaction dramatically. In this study, we developed a straightforward and comprehensive analytical methodology to map Fc{gamma}RIIIb glycosylation from primary human material. In contrast to recently published alternatives, we assessed all glycosylation sites in a single LC-MS/MS run and simultaneously determined the donor allotype. Studying Fc{gamma}RIIIb derived from healthy donor neutrophils, we observed profound differences as compared to the soluble variant and the homologous Fc{gamma}RIIIa on natural killer cells. This method will allow assessment of Fc{gamma}RIII glycosylation differences between individuals, cell types, subcellular locations and pathophysiological conditions.

immunology↗

bioRxiv2020

PAKC: A novel Panel of HLA class I Antigen presentation machinery Knockout Cells from the same genetic origin

With the emergence of immunotherapies, the understanding of functional HLA class I antigen presentation to T cells is more relevant than ever. Current knowledge on antigen presentation is based on decades of research in a wide variety of cell types with varying antigen presentation machinery (APM) expression patterns, proteomes and HLA haplotypes. This diversity complicates the establishment of individual APM contributions to antigen generation, selection and presentation. Therefore, we generated a novel Panel of APM Knockout Cell lines (PAKC) from the same genetic origin. After CRISPR/Cas9 genome-editing of ten individual APM components in a human cell line, we derived clonal cell lines and confirmed their knockout status and phenotype. We then show how PAKC will accelerate research on the functional interplay between APM components and their role in antigen generation and presentation. This will lead to improved understanding of peptide-specific T cell responses in infection, cancer and autoimmunity. Key pointsO_LIWe generated a panel of cell lines to study HLA class I antigen presentation C_LIO_LIWe show how this will spark research in infection, tumor biology and autoimmunity C_LI

immunology↗

bioRxiv2019

A Small-Molecule Activity-Based Probe for Monitoring Ubiquitin C-terminal Hydrolase L1 (UCHL1) Activity in Live Cells and Zebrafish Embryos

Many reagents have been emerged to study the function of specific enzymes in vitro. On the other hand, target specific reagents are scarce or need improvement allowing investigations of the function of individual enzymes in a cellular context. We here report the development of a target-selective fluorescent small-molecule activity-based DUB probe that is active in live cells and whole animals. The probe labels active Ubiquitin Carboxy-terminal Hydrolase L1 (UCHL1), also known as neuron-specific protein PGP9.5 (PGP9.5) and parkinson disease 5 (PARK5), a DUB active in neurons that constitutes 1-2% of total brain protein. UCHL1 variants have been linked with the neurodegenerative disorders Parkinsons and Alzheimers disease. In addition, high levels of UCHL1 also correlate often with cancer and especially metastasis. The function of UCHL1 or its role in cancer and neurodegenerative disease is poorly understood and few UCHL1 specific research tools exist. We show that the reagents reported here are specific for UCHL1 over all other DUBs detectable by competitive activity-based protein profiling and by mass spectrometry. Our probe, which contains a cyanimide reactive moiety, binds to the active-site cysteine residue of UCHL1 irreversibly in an activity-dependent manner. Its use is demonstrated by labelling of UCHL1 both in vitro and in cells. We furthermore show that this probe can report UCHL1 activity during the development of zebrafish embryos.

biochemistry↗