Biology subjects

Snijders, A. P.

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

23 records · Page 2Linked to original sources

bioRxiv2019

Proteasome-mediated protein degradation resets the cell division cycle and triggers ESCRT-III-mediated cytokinesis in an archaeon

The archaeon Sulfolobus acidocaldarius is a relative of eukaryotes known to progress orderly through its cell division cycle despite lacking obvious CDK/cyclin homologues. Here, in exploring the mechanisms underpinning archaeal cell division cycle control, we show that the proteasome of S. acidocaldarius, like its eukaryotic counterpart, regulates the transition from the end of one cell division cycle to the beginning of the next. Further, we identify the archaeal ESCRT-III homologue CdvB as a key target of the proteasome, and show that state-dependent degradation of CdvB triggers archaeal cell division by allowing constriction of a CdvB1:CdvB2 ESCRT-III division ring. These findings suggest an ancient role for proteasome-mediated degradation in resetting the cell division cycle in both archaea and eukaryotes.

cell biology↗

bioRxiv2019

Protein ubiquitylation is essential for the schizont to merozoite transition in Plasmodium falciparum blood-stage development

Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intracellular parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, indicating a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the late schizont stage. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites. The ubiquitylation of many merozoite proteins and their disappearance in ring stages are consistent with the idea that ubiquitylation leads to their destruction via the proteasome once their function is complete following invasion, which would allow amino acid recycling in the period prior to the parasites elaboration of a new food vacuole.

microbiology↗

bioRxiv2019

An Integrated Chemical Proteomics Approach for Quantitative Profiling of Intracellular ADP-Ribosylation

ADP-ribosylation is integral to a diverse range of cellular processes such as DNA repair, chromatin regulation and RNA processing. However, proteome-wide investigation of its cellular functions has been limited due to numerous technical challenges including the complexity of the poly(ADP-ribose) (PAR) chains, low abundance of the modification and lack of sensitive enrichment methods. To facilitate live-cell profiling of ADP-ribosylated proteins, we show that 2-alkyne-adenosine (2YnAd) is metabolically incorporated in mammalian cells and enables fluorescent detection and robust affinity enrichment of the modified proteins. We then present an integrated chemical biology approach that involves simultaneous metabolic incorporation of 2YnAd and the previously reported 6-alkyne-adenosine (6YnAd) in live cells followed by click chemistry with a capture reagent to facilitate highly sensitive and comprehensive enrichment of the modified proteins. By combining this dual metabolic labelling strategy with the tandem mass tag (TMT) isobaric mass spectrometry, we have quantified the responses of thousands of endogenous proteins to clinical PARP inhibitors Olaparib and Rucaparib. Our study provides insight into the wider scope of PARP targets and will help to advance further characterisation of their functional roles.

biochemistry↗

bioRxiv2018

Functional cross-talk between allosteric effects of activating and inhibiting ligands underlies PKM2 regulation

Allosteric regulation is central to the role of the glycolytic enzyme pyruvate kinase M2 (PKM2) in cellular metabolism. Multiple activating and inhibitory allosteric ligands regulate PKM2 activity by controlling the equilibrium between high activity tetramers and low activity dimers and monomers. However, it remains elusive how allosteric inputs upon simultaneous binding of different ligands are integrated to regulate PKM2 activity. Here, we show that, in the presence of the allosteric inhibitor L-phenylalanine (Phe), the activator fructose 1,6-bisphosphate (FBP) can induce PKM2 tetramerisation, but fails to maximally increase enzymatic activity. Guided by a new computational framework we developed to identify residues that mediate FBP-induced allostery, we generated two PKM2 mutants, A327S and C358A, in which activation by FBP remains intact but cannot be attenuated by Phe. Our findings demonstrate a role for residues involved in FBP-induced allostery in enabling the integration of allosteric input from Phe and reveal a mechanism that underlies the co-ordinate regulation of PKM2 activity by multiple allosteric ligands.

biochemistry↗

bioRxiv2018

Repression of divergent noncoding transcription by a sequence-specific transcription factor

Many active eukaryotic gene promoters exhibit divergent noncoding transcription, but the mechanisms restricting expression of these transcripts are not well understood. Here we demonstrate how a sequence-specific transcription factor represses divergent noncoding transcription at highly expressed genes in yeast. We find that depletion of the transcription factor Rap1 induces noncoding transcription in a large fraction of Rap1 regulated gene promoters. Specifically, Rap1 prevents transcription initiation at cryptic promoters near its binding sites, which is uncoupled from transcription regulation in the protein-coding direction. We further provide evidence that Rap1 acts independently of chromatin-based mechanisms to repress cryptic or divergent transcription. Finally, we show that divergent transcription in the absence of Rap1 is elicited by the RSC chromatin remodeller. We propose that a sequence-specific transcription factor limits access of basal transcription machinery to regulatory elements and adjacent sequences that act as divergent cryptic promoters, thereby providing directionality towards productive transcription.

molecular biology↗