Biology subjects

Spruijt, C. G.

Publications and source records attributed to Spruijt, C. G..

8 recordsLinked to original sources

bioRxiv2026

Proteomics of human cancer-associated T cells identifies regulators of T cell functionality

CD8+ T cells in solid cancers progressively lose anti-tumor activity, yet the cell-intrinsic mechanisms driving this loss of function remain incompletely defined. Here, we performed matched proteomic and transcriptomic profiling of dysfunctional and bystander CD8+ tumor-infiltrating T cells isolated from primary tumors of treatment-naive non-small cell lung cancer patients. Proteomic analysis revealed widespread discordance with mRNA expression, with 8% of all quantified proteins displaying differential expression exclusively at the protein level. Genetic perturbation of such differentially expressed proteins identified the chromatin remodeler CHD4 and fatty acid synthase (FASN) as cell-intrinsic regulators of T cell function. CHD4 deletion resulted in altered gene-regulatory networks that promoted effector differentiation and enhanced cytokine production. In contrast, FASN deletion preserved mitochondrial fitness and sustained T cell functionality under chronic T cell receptor stimulation. Together, these findings demonstrate that proteomic profiling uncovers regulators of T cell functionality that are not apparent from transcriptomic analyses alone, highlighting an additional layer of regulatory control.

immunology↗

bioRxiv2026

Prolyl hydroxylation is required for maturation and secretion of Zika virus non-structural protein 1

Mosquito-borne orthoflaviviruses such as Zika virus (ZIKV) and dengue virus (DENV) have a high epidemic potential and are associated with a wide range of possible outcomes, from asymptomatic infections to severe complications. The orthoflavivirus non-structural protein 1 (NS1), which can be membrane-bound as well as secreted from the cell, plays important roles in viral replication, immune evasion and pathogenesis. To describe the interactome of ZIKV NS1, we infected Huh7 and neural progenitor cells with a recombinant ZIKV expressing FLAG-tagged NS1 and identified interacting proteins by quantitative mass spectrometry. In both cell types, multiple prolyl hydroxylases, glycosylation enzymes, ER-associated degradation proteins, and proteins involved in immunity were identified as NS1 interactors. Mass spectrometry data showed that NS1 contains potential hydroxyproline residues, suggesting that prolyl hydroxylase enzymes are involved in post-translational modification of NS1. Chemical inhibition of prolyl hydroxylases impaired NS1 intracellular trafficking and plasma membrane localization and reduced ZIKV RNA levels and titers. Mutagenesis of the proline residue at position 281 impaired NS1 secretion, although viral replication was unaffected. Moreover, substituting proline at position 267 strongly dysregulated NS1 expression and reverted to the wild-type sequence already after a single passage, suggesting that this mutation leads to severe replication defects. Our results identify prolyl hydroxylation as an essential post-translational modification of ZIKV NS1.

microbiology↗

bioRxiv2026

Plasmodium falciparum stomatin-like protein forms a putative complex with a metalloprotease in distinct mitochondrial loci

Withdrawal statementThis manuscript has been withdrawn owing to a duplicate posting of manuscript number BIORXIV/2024/604071. This was a technical error for which the authors are not responsible, and this DOI for the work should not be cited as reference. The record is retained here for purposes of transparency. If you have any questions, please contact the corresponding author. The correct preprint can be found at doi: 10.1101/2024.07.18.604071

microbiology↗

bioRxiv2024

Pfs230 Domain 12 is a potent malaria transmission-blocking vaccine candidate

Malaria transmission-blocking vaccines (TBV) target sexual stage parasites that are transmitted to mosquitoes and critical for spread of the pathogen. The clinically most advanced TBV candidate contains part of the Pro-domain (Pro) and Domain 1 (D1) of Plasmodium falciparum surface protein Pfs230. Subunit vaccines that contain other domains of Pfs230 have so far failed to induce functional antibodies. Here, we produced eight single domain fragments of Pfs230 in Drosophila melanogaster S2 cells and assessed their immunogenicity in mouse immunizations. In addition to D1-specific antibodies, antibodies raised against Domain 12 (D12) showed strong recognition of Pfs230 on the surface of female parasites. Importantly, D12-specific antibodies showed strong functional transmission-reducing activity in membrane feeding assays with cultured parasites, an activity that was complement-dependent. Murine D12-specific antibodies further reduced mosquito transmission of parasites acquired from naturally infected parasite carriers. The D12 antigen was recognized by sera from an all-age cohort of individuals who had been naturally exposed to Plasmodium falciparum with antibody levels increasing with age. In conclusion, we identified Pfs230D12 as a promising new TBV candidate. Impact statementPfs230 Domain 12 induces antibodies in mice that strongly reduce transmission of lab-cultured and naturally circulating malaria parasites to mosquitoes.

immunology↗

bioRxiv2024

The role of stomatin-like protein (STOML) in Plasmodium falciparum

Members of the Stomatin, Prohibitin, Flotillin and HflK/C (SPFH) protein family form large membrane anchored or spanning complexes and are involved in various functions in different organelles. The human malaria causing parasite Plasmodium falciparum harbors four SPFH proteins, including prohibitin 1 and 2, prohibitin-like protein (PHBL), and stomatin-like protein (STOML), which all localize to the parasite mitochondrion. In the murine model parasite Plasmodium berghei, STOML appears essential for asexual blood-stage (ABS) development and is localized to puncta on mitochondrial branching points in oocyst stages. In this study, we show that deletion of STOML causes a significant growth defect and slower ABS development, while sexual-stage development remains unaffected. Parasites lacking STOML were not more sensitive to respiratory chain targeting drugs, rendering a function of STOML in respiratory chain assembly unlikely. Epitope tagging of endogenous STOML revealed a distinct punctate localization on branching points and endings of the ABS mitochondrial network. STOML resides in a large protein complex and pulldown experiments identified a zinc dependent metalloprotease, FtsH, as a likely interaction partner. The predicted AlphaFold2 structure of STOML shows high similarity with the bacterial HflK/C, which has been shown to form a large vault-like structure around bacterial FtsH hexamers. Combined, our results suggest that a similar STOML-FtsH complex localized to specific loci of P. falciparum mitochondria facilitate the parasites ABS development.

microbiology↗

bioRxiv2022

Galectin-9 interacts with Vamp-3 to regulate cytokine secretion in dendritic cells

Intracellular vesicle transport is essential for cellular homeostasis and is partially mediated by SNARE proteins. Endosomal trafficking to the plasma membrane ensures cytokine secretion in dendritic cells (DCs) and the initiation of immune responses. Despite its critical importance, the specific molecular agents that regulate DC cytokine secretion are poorly characterised. Galectin-9, a {beta}-galactoside-binding protein, has emerged as a novel cellular modulator although its exact intracellular roles in regulating (immune) cell homeostasis and vesicle transport are virtually unknown. We investigated galectin-9 function in primary human DCs and report that galectin-9 is essential for intracellular cytokine trafficking to the cell surface. Galectin-9-depleted DCs accumulate cytokine-containing vesicles in the Golgi complex that eventually undergo lysosomal degradation. We observed galectin-9 to molecularly interact with Vamp-3 using immunoprecipitation-mass-spectrometry and identified galectin-9 was required for rerouting Vamp-3-containing endosomes upon DC activation as the underlying mechanism. Overall, this study identifies galectin-9 as a necessary mechanistic component for intracellular trafficking. This may impact our general understanding of vesicle transport and shed new light into the multiple roles galectins play in governing cell function.

cell biology↗

bioRxiv2022

Arbovirus-vector protein interactomics identifies Loquacious as a co-factor for dengue virus replication in Aedes mosquitoes

Efficient virus replication in Aedes vector mosquitoes is essential for the transmission of arboviral diseases such as dengue virus (DENV) in human populations. Like in vertebrates, virus-host protein-protein interactions are essential for viral replication and immune evasion in the mosquito vector. Here, 79 mosquito host proteins interacting with DENV non-structural proteins NS1 and NS5 were identified by label-free mass spectrometry, followed by a functional screening. We confirmed interactions with host factors previously observed in mammals, such as the oligosaccharyltransferase complex, and we identified protein-protein interactions that seem to be specific for mosquitoes. Among the interactors, the double-stranded RNA (dsRNA) binding protein Loquacious (Loqs), an RNA interference (RNAi) cofactor, was found to be essential for efficient replication of DENV and Zika virus (ZIKV) in mosquito cells. Loqs did not affect viral RNA stability or translation of a DENV replicon and its proviral activity was independent of its RNAi regulatory activity. Interestingly, Loqs colocalized with DENV dsRNA in viral replication organelles in infected cells and directly interacted with high affinity with DENV RNA in the 3 untranslated region in vitro (KD = 100-200 nM). Our study provides an interactome for DENV NS1 and NS5 and identifies Loqs as a key proviral host factor in mosquitoes. We propose that DENV hijacks a factor of the RNAi mechanism for replication of its own RNA. AUTHOR SUMMARYDengue virus is a mosquito-transmitted virus endemic to the tropics and subtropics, affecting an estimated 390 million people yearly. While the mechanisms of infection, pathogenesis and immune evasion have been extensively studied in humans, replication in Aedes mosquitoes has received much less attention, despite being a critical step in the arbovirus transmission cycle. Here, we used a proteomic approach to identify Aedes mosquito proteins recruited by dengue virus non- structural proteins NS1 and NS5. In addition to previously established host proteins that interact with DENV in mammals, we identified Loquacious, a double-stranded RNA binding protein involved in the antiviral RNAi immune response of mosquitoes. Unexpectedly, our data showed Loquacious functions as a proviral factor that is recruited to replication organelles to facilitate viral RNA replication. We propose that DENV exploits host immune components, such as Loquacious, for its own benefit.

microbiology↗

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↗