Biology subjects

Snijders, A. P.

Publications and source records attributed to Snijders, A. P..

At least 19 recordsLinked to original sources

bioRxiv2023

A malaria parasite phospholipase facilitates efficient asexual blood stage egress

Malaria parasite release (egress) from host red blood cells involves parasite-mediated membrane poration and rupture, thought to involve membrane-lytic effector molecules such as perforin-like proteins and/or phospholipases. With the aim of identifying these effectors, we disrupted the expression of two Plasmodium falciparum perforin-like proteins simultaneously and showed that they have no essential roles during blood stage egress. Proteomic profiling of parasite proteins discharged into the parasitophorous vacuole (PV) just prior to egress detected the presence in the PV of a lecithin:cholesterol acyltransferase (LCAT; PF3D7_0629300). Conditional ablation of LCAT resulted in abnormal egress and a reduced replication rate. Lipidomic profiles showed drastic changes in several phosphatidylserine and acylphosphatidylglycerol species during egress. We thus show that, in addition to its previously demonstrated role in liver stage merozoite egress, LCAT is required to facilitate efficient egress in asexual blood stage malaria parasites. Author SummaryMalaria kills over half a million people every year worldwide. It is caused by a single-celled parasite called Plasmodium falciparum that grows and multiplies within a bounding vacuole, inside red blood cells of the infected individuals. Following each round of multiplication, the infected cell is ruptured in a process known as egress to release a new generation of parasites. Egress is required for the disease to progress and is orchestrated by the parasite. The parasite sends out various molecules to puncture and destroy the membranes of the vacuole and the red blood cell. However, little is known about these molecules. In this work, we set out to identify these molecules by using genetic and proteomics approaches. We screened the molecules the parasite sends out during egress and identified a parasite enzyme called LCAT present in the vacuole. Our experiments found that mutant parasites that were unable to make LCAT clumped together and could not escape the infected cell properly. As a result, we saw a reduction in the rate at which these parasites spread through the red blood cells. Taken together, our findings suggest that P. falciparum needs LCAT to efficiently break out of red blood cells.

microbiology↗

bioRxiv2023

The yeast RNA methylation complex consists of conserved yet reconfigured components with m6A-dependent and independent roles

N6-methyladenosine (m6A), the most abundant mRNA modification, is deposited in mammals/insects/plants by m6A methyltransferase complexes (MTC) comprising a catalytic subunit and at least five additional proteins. The yeast MTC is critical for meiosis and was known to comprise three proteins, of which two were conserved. We uncover three novel MTC components (Kar4/Ygl036w-Vir1/Dyn2). All MTC subunits, except for Dyn2, are essential for m6A deposition and have corresponding mammalian MTC orthologs. Unlike the mammalian bipartite MTC, the yeast MTC is unipartite, yet multifunctional. The mRNA interacting module, comprising Ime4, Mum2, Vir1, and Kar4, exerts the MTCs m6A-independent function, while Slz1 enables the MTC catalytic function in m6A deposition. Both functions are critical for meiotic progression. Kar4 also has a mechanistically separate role from the MTC during mating. The yeast MTC constituents play distinguishable m6A-dependent, MTC-dependent and MTC-independent functions, highlighting their complexity and paving the path towards dissecting multi-layered MTC functions in mammals.

molecular biology↗

bioRxiv2023

A chemoproteomic platform for reactive fragment profiling against the deubiquitinases

Chemoproteomics is a powerful method capable of detecting interactions between small molecules and the proteome, however its use as a high-throughput screening method for chemical libraries has so far been limited. To address this need, we have further developed a chemoproteomics workflow to screen cysteine reactive covalent fragments in cell lysates against the deubiquitinating (DUB) enzymes using activity-based protein profiling. By using targeted ubiquitin probes, we have addressed sensitivity and affinity limitations, enabling target identification and covalent fragment library profiling in a 96-well plate format. The use of data independent acquisition (DIA) methods for MS analysis combined with automated Evosep liquid chromatography (LC) reduced instrument runtimes to 21 minutes per sample and simplified the workflow. In this proof-of-concept study, we have profiled 138 covalent fragments against 57 DUB proteins and validated four hit fragments against OTUD7B and UCHL3 through site identification experiments and orthogonal biochemical activity assays. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/526632v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@2e036borg.highwire.dtl.DTLVardef@e27962org.highwire.dtl.DTLVardef@8f1caaorg.highwire.dtl.DTLVardef@1a3159a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

m6A reader Pho92 is recruited co-transcriptionally and couples translation efficacy to mRNA decay to promote meiotic fitness in yeast

N6-methyladenosine (m6A) RNA modification impacts mRNA fate primarily via reader proteins, which dictate processes in development, stress, and disease. Yet little is known about m6A function in Saccharomyces cerevisiae, which occurs solely during early meiosis. Here we perform a multifaceted analysis of the m6A reader protein Pho92/Mrb1. Cross-linking immunoprecipitation analysis reveals that Pho92 associates with the 3end of meiotic mRNAs in both an m6A-dependent and independent manner. Within cells, Pho92 transitions from the nucleus to the cytoplasm, and associates with translating ribosomes. In the nucleus Pho92 associates with target loci through its interaction with transcriptional elongator Paf1C. Functionally, we show that Pho92 promotes and links protein synthesis to mRNA decay. As such, the Pho92-mediated m6A-mRNA decay is contingent on active translation and the CCR4-NOT complex. We propose that the m6A reader Pho92 is loaded co-transcriptionally to facilitate protein synthesis and subsequent decay of m6A modified transcripts, and thereby promotes meiosis.

biochemistry↗

bioRxiv2021

RNA polymerase II dynamics and mRNA stability feedback determine mRNA scaling with cell size

A fundamental feature of cellular growth is that protein and RNA amounts scale with cell size so that concentrations remain constant. A key component to this is that global transcription rates increase in larger cells, but the underlying mechanism has remained unknown. Here, we identify RNAPII as the major limiting factor increasing transcription with cell size in budding yeast as transcription is highly sensitive to the dosage of RNAPII but not to other components of the general transcriptional machinery. Our experiments support a dynamic equilibrium model where global transcription at a given size is set by the mass-action recruitment kinetics of unengaged nucleoplasmic RNAPII, and DNA content. This drives a sub-linear increase in transcription with size, which is precisely compensated for by a decrease in mRNA decay rates as cells enlarge. Thus, limiting RNAPII and feedback on mRNA stability work in concert to ensure mRNA concentration homeostasis in growing cells.

molecular biology↗

bioRxiv2021

The master energy homeostasis regulator PGC-1alpha couples transcriptional co-activation and mRNA nuclear export

PGC-1 plays a central role in maintaining the mitochondrial and energy metabolism homeostasis, linking external stimuli to the transcriptional co-activation of genes involved in adaptive and age-related pathways. The carboxyl-terminus encodes a serine/arginine-rich (RS) region and a putative RNA recognition motif, however potential RNA-processing role(s) have remained elusive for the past 20 years. Here, we show that the RS domain of human PGC-1 directly interacts with RNA and the nuclear RNA export factor NXF1. Inducible depletion of endogenous PGC-1 and expression of RNAi-resistant RS-deleted PGC-1 further demonstrate that the RNA-binding activity is required for nuclear export of co-activated transcripts and mitochondrial homeostasis. Moreover, a quantitative proteomics approach confirmed PGC-1-dependent RNA transport and mitochondrial-related functions, identifying also novel mRNA nuclear export targets in age-related telomere maintenance. Discovering a novel function for a major cellular homeostasis regulator provides new directions to further elucidate the roles of PGC-1 in gene expression, metabolic disorders, ageing and neurodegenerative diseases.

molecular biology↗

bioRxiv2021

Cell-specific Bioorthogonal Tagging of Glycoproteins in Co-culture

Altered glycosylation is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function.

synthetic biology↗

bioRxiv2021

Exploitation of the Secretory Pathway by SARS-CoV-2 Envelope

The {beta}-coronavirus SARS-CoV-2 is the causative agent of the global Covid-19 pandemic. Coronaviral Envelope (E) proteins are pentameric viroporins that play essential roles in assembly, release and pathogenesis. We developed an inert tagging strategy for SARS-CoV-2 E and find that it localises to the Golgi and to lysosomes. We identify sequences in E, conserved across Coronaviridae, responsible for ER-to-Golgi export, and relate this activity to interaction with COP-II via SEC24. Using proximity biotinylation, we identify host-cell factors that interact with E and identify an ARFRP1/AP-1 dependent pathway allowing Golgi-to-lysosome trafficking of E. We identify sequences in E that bind AP-1, are conserved across {beta}-coronaviruses and allow E to be trafficked from Golgi to lysosomes. We show that E acts to deacidify lysosomes and by developing a trans-complementation assay, we show that both lysosomal trafficking of E and its viroporin activity are necessary for efficient viral replication and release.

cell biology↗

bioRxiv2021

Neural stem cells alter nucleocytoplasmic partitioning and accumulate nuclear polyadenylated transcripts during quiescence

Quiescence is a cellular state characterised by reversible cell-cycle arrest and diminished biosynthetic activity that protects against environmental insults, replicative exhaustion and proliferation-induced mutations1. Entry into and exit from this state controls development, maintenance and repair of tissues plus, in the adult central nervous system, generation of new neurons and thus cognition and mood2-4. Cancer stem cells too can undergo quiescence, which confers them resistance to current therapies5, 6. Despite clinical relevance, quiescence is poorly understood and is defined functionally given lack of molecular markers. Decrease of the most resource-intensive cellular process of protein synthesis is a feature of quiescence, controlled across species and cell types by inhibition of the Target of Rapamycin (TOR) pathway1, 7. Here, we combine Drosophila genetics and a mammalian model to show that altered nucleocytoplasmic partitioning and nuclear accumulation of polyadenylated RNAs are novel evolutionarily conserved hallmarks of quiescence regulation. Furthermore, nuclear accumulation of messenger RNA (mRNA) in quiescent NSCs (qNSCs) largely predicts protein downregulation, accounting for uncoupling between transcriptome and proteome in quiescence. These mechanisms provide a previously unappreciated regulatory layer to reducing protein synthesis in quiescent cells, whilst priming them for reactivation in response to appropriate cues.

developmental biology↗

bioRxiv2020

Inhibition of protein N-myristoylation blocks Plasmodium falciparum intraerythrocytic development, egress, and invasion

We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last ten to fifteen hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified sixteen NMT substrates for which myristoylation was significantly reduced by NMT inhibitor treatment, and of these, six proteins were substantially reduced in abundance. In a viability screen, we showed that for four of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least three mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of pseudoschizonts, which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition.

microbiology↗

bioRxiv2020

A multipass membrane protein interacts with the cGMP-dependent protein kinase to regulate critical calcium signals in malaria parasites

In malaria parasites, all cGMP-dependent signalling is mediated through a single cGMP-dependent protein kinase (PKG), a major function of which is to control essential calcium signals. However, how PKG transmits these signals in the absence of known second messenger-dependent calcium channels or scaffolding proteins is unknown. Here we identify a polytopic membrane protein, ICM1, with homology to transporters and calcium channels that is tightly-associated with PKG in both Plasmodium falciparum asexual blood stages and P. berghei gametocytes. Phosphoproteomic analyses in both Plasmodium species reveal multiple ICM1 phosphorylation events dependent upon PKG activity. Stage-specific depletion of P. berghei ICM1 blocks gametogenesis due to the inability of mutant parasites to mobilise intracellular calcium upon PKG activation, whilst conditional loss of P. falciparum ICM1 results in reduced calcium mobilisation, defective egress and lack of invasion. Our findings provide new insights into atypical calcium homeostasis in malaria parasites essential for pathology and disease transmission.

microbiology↗

bioRxiv2020

The malaria parasite sheddase SUB2 governs host red blood cell membrane sealing at invasion

Red blood cell (RBC) invasion by malaria merozoites involves formation of a parasitophorous vacuole into which the parasite moves. The vacuole membrane seals and pinches off behind the parasite through an unknown mechanism, enclosing the parasite within the RBC. During invasion, several parasite surface proteins are shed by a membrane-bound protease called SUB2. Here we show that genetic depletion of SUB2 abolishes shedding of a range of parasite proteins, identifying previously unrecognized SUB2 substrates. Interaction of SUB2-null merozoites with RBCs leads to either abortive invasion with rapid RBC lysis, or successful entry but developmental arrest. Selective failure to shed the most abundant SUB2 substrate, MSP1, reduces intracellular replication, whilst conditional ablation of the substrate AMA1 produces host RBC lysis. We conclude that SUB2 activity is critical for host RBC membrane sealing following parasite internalisation and for correct functioning of merozoite surface proteins. Key highlightsO_LIMany malaria parasite surface proteins are shed by SUB2 during RBC invasion C_LIO_LISUB2-null merozoites either induce rapid host RBC lysis, or invade then die C_LIO_LIMerozoite surface protein shedding is crucial for host RBC membrane sealing C_LI

microbiology↗

bioRxiv2020

Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

ABSTRACTPPP-family phosphatases such as PP1 have little intrinsic specificity. Cofactors can target PP1 to substrates or subcellular locations, but it remains unclear how they might confer sequence-specificity on PP1. The cytoskeletal regulator Phactr1 is a neuronally-enriched PP1 cofactor that is controlled by G-actin. Structural analysis showed that Phactr1 binding remodels PP1’s hydrophobic groove, creating a new composite surface adjacent to the catalytic site. Using phosphoproteomics, we identified numerous fibroblast and neuronal Phactr1/PP1 substrates, which include cytoskeletal components and regulators. We determined high-resolution structures of Phactr1/PP1 bound to the dephosphorylated forms of its substrates IRSp53 and spectrin αII. Inversion of the phosphate in these holoenzyme-product complexes supports the proposed PPP-family catalytic mechanism. Substrate sequences C-terminal to the dephosphorylation site make intimate contacts with the composite Phactr1/PP1 surface, which are required for efficient dephosphorylation. Sequence specificity explains why Phactr1/PP1 exhibits orders-of-magnitude enhanced reactivity towards its substrates, compared to apo-PP1 or other PP1 holoenzymes.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2020

Structure of the cysteine-rich domain of Plasmodium falciparum P113 identifies the location of the RH5 binding site

Plasmodium falciparum RH5 is a secreted parasite ligand that is essential for erythrocyte invasion through direct interaction with the host erythrocyte receptor basigin. RH5 forms a tripartite complex with two other secreted parasite proteins: CyRPA and RIPR, and is tethered to the surface of the parasite through membrane-anchored P113. Antibodies against RH5, CyRPA and RIPR inhibit parasite invasion, suggesting that vaccines containing these three components have the potential to prevent blood-stage malaria. To further explore the role of the P113-RH5 interaction, we selected monoclonal antibodies against P113 that were either inhibitory or non-inhibitory for RH5 binding. Using a Fab fragment as a crystallisation chaperone, we determined the crystal structure of the RH5-binding region of P113 and showed that it is composed of two domains with structural similarities to rhamnose-binding lectins. We identified the RH5 binding site on P113 by using a combination of hydrogen-deuterium exchange mass spectrometry and site directed mutagenesis. We found that a monoclonal antibody to P113 that bound to this interface and inhibited the RH5-P113 interaction did not inhibit parasite blood-stage growth. These findings provide further structural information on the protein interactions of RH5 and will be helpful in the development of blood-stage malaria vaccines that target RH5. ImportanceMalaria is a deadly infectious disease primarily caused by the parasite Plasmodium falciparum. It remains a major global health problem and there is no highly effective vaccine. A parasite protein called RH5 is centrally involved in the invasion of host red blood cells, making it - and the other parasite proteins it interacts with - promising vaccine targets. We recently identified a protein called P113 that binds RH5 suggesting that it anchors RH5 to the parasite surface. In this paper, we use structural biology to locate and characterize the RH5 binding region on P113. These findings will be important to guide the development of new anti-malarial vaccines to ultimately prevent this disease which affects some of the poorest people on the planet.

microbiology↗

bioRxiv2020

Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe GalNAzMe that is specific for cancer-relevant Ser/Thr-N-acetylgalactosamine (O-GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn (N)-linked glycans. We equip cells with the capacity to biosynthesize the nucleotide-sugar donor UDP-GalNAzMe from a caged precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR knock-out (KO) screen, and imaging of intestinal organoids. GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation. Significance statementA large portion of all secreted and cell surface proteins in humans are modified by Ser/Thr(O)-linked glycosylation with N-acetylgalactosamine (GalNAc). While of fundamental importance in health and disease, O-GalNAc glycosylation is technically challenging to study because of a lack of specific tools to be used in biological assays. Here, we design an O-GalNAc specific reporter molecule termed GalNAzMe to selectively label O-GalNAc glycoproteins in living human cells. GalNAzMe is compatible with a range of experiments in quantitative biology to broaden our understanding of glycosylation. We further demonstrate that labeling is genetically programmable by expression of a mutant glycosyltransferase, allowing application even to experiments with low inherent sensitivity.

synthetic biology↗

bioRxiv2020

A Structure-Based Mechanism for DNA Entry into the Cohesin Ring

Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here, we combine biophysical approaches and cryo-EM to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design biochemical experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against a shut ATPase gate. ATP hydrolysis leads to ATPase gate opening to complete DNA entry. Whether DNA loading is successful, or rather results in loop extrusion, might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.

molecular biology↗

bioRxiv2020

Differential protein phosphorylation affects the localisation of two secreted Toxoplasma proteins and is widespread during stage conversion

The intracellular parasite Toxoplasma gondii resides within a membrane bound parasitophorous vacuole (PV) and secretes an array of proteins to establish this replicative niche. It has been shown previously that Toxoplasma both secretes kinases and that numerous proteins are phosphorylated after secretion. Here we assess the role of phosphorylation of SFP1 and the related GRA29, two secreted proteins with unknown function. We show that both proteins form stranded structures in the PV that are independent of the previously described intravacuolar network or actin. GRA29 likely acts as a seed for SFP1 strand formation, and these structures can form independently of other Toxoplasma secreted proteins. We show that an unstructured region at the C-terminus of SFP1 and GRA29 is required for the formation of strands and that mimicking phosphorylation of this domain negatively regulates strand development. When tachyzoites convert to chronic stage bradyzoites, both proteins show a dispersed localisation throughout the cyst matrix. Many secreted proteins are reported to dynamically redistribute as the cyst forms and secreted kinases are known to play a role in cyst formation. Using quantitative phosphoproteome and proteome analysis comparing tachyzoite and early bradyzoite stages, we reveal widespread differential phosphorylation of secreted proteins. These data support a model in which secreted kinases and phosphatases are important to dynamically regulate parasite secreted proteins during stage conversion. IMPORTANCEToxoplasma gondii is a common parasite that infects up to one third of the human population. Initially the parasite grows rapidly, infecting and destroying cells of the host, but subsequently switches to a slow-growing form and establishes chronic infection. In both stages the parasite lives within a membrane bound vacuole within the host cell, but in the chronic stage a durable cyst wall is synthesized that provides protection to the parasite during transmission to a new host. Toxoplasma secretes proteins into the vacuole to build its replicative niche and previous studies identified many of these proteins as phosphorylated. We investigate two secreted proteins and show that phosphorylation plays an important role in their regulation. We also observed widespread phosphorylation of secreted proteins when parasites convert from acute to chronic stages, providing new insight into how the cyst wall may be dynamically regulated.

microbiology↗

bioRxiv2019

Engineering transplantable jejunal mucosal grafts using primary patient-derived organoids from children with intestinal failure

Intestinal failure (IF), following extensive anatomical or functional loss of small intestine (SI), has debilitating long-term effects on infants and children with this condition. Priority of care is to increase the childs length of functional intestine, jejunum in particular, to improve nutritional independence. Here we report a robust protocol for reconstruction of autologous intestinal mucosal grafts using primary IF patient materials. Human jejunal intestinal organoids derived from paediatric IF patients can be expanded efficiently in vitro with region-specific markers preserved after long-term culture. Decellularized human intestinal matrix with intact ultrastructure is used as biological scaffolds. Proteomic and Raman spectroscopic analyses reveal highly analogous biochemical composition of decellularized human SI and colon matrix, implying that they can both be utilised as scaffolds for jejunal graft reconstruction. Indeed, seeding of primary human jejunal organoids to either SI or colonic scaffolds in vitro can efficiently reconstruct functional jejunal grafts with persistent disaccharidase activity as early as 4 days after seeding, which can further survive and mature after transplantation in vivo. Our findings pave the way towards regenerative medicine for IF patients.

bioengineering↗