Biology subjects

Cobbold, S. A.

Publications and source records attributed to Cobbold, S. A..

4 recordsLinked to original sources

bioRxiv2025

Differentiation of Toxoplasma into latent forms is linked to central carbon metabolism and requires a GID/CTLH-type E3 ubiquitin ligase

Toxoplasma and other Apicomplexan parasites, switch between different developmental stages to persist in and transmit between hosts. Toxoplasma can alternate between systemic tachyzoites and encysted bradyzoite forms found in the CNS and muscle tissues. How parasites sense these tissue types and trigger differentiation remains largely unknown. We show that Toxoplasma differentiation is induced under glucose-limiting conditions and using a CRISPR screen identify parasite genes required for growth under these conditions. From [~]25 identified genes important for differentiation we show that lactate and glutamine metabolism is linked to differentiation and demonstrate the importance of an E3 ubiquitin ligase complex, orthologous to glucose induced degradation deficient (GID) complex in yeast and CTLH complex in humans. We show that TgGID likely regulates translational repression of a key transcription factor required for differentiation, BFD1, through its 3 utr. Overall, this work provides important new insight into how these divergent parasites sense different host cell niches and trigger stage conversion through a ubiquitination-dependent program.

microbiology↗

bioRxiv2025

Global analysis of cancer cell responses to USP9X inhibition

The ubiquitin specific protease (USP) enzyme USP9X is amongst the best studied human deubiquitinases (DUBs), with a myriad of described targets and cellular roles. In cancer, USP9X has been touted as both an oncogene and a tumour suppressor in different contexts, which has confounded the field and questioned its viability as a cancer target. We here describe WEHI-092, a novel piperazine-based USP9X specific small molecule inhibitor and map its binding site to a unique region in the USP9X fingers subdomain, distinct from known DUB inhibitor binding sites. Using proteomics and ubiquitinomics, we show that USP9X has a distinct set of substrates compared to USP7 indicating remarkable DUB target specificity, yet the substrate profile of USP9X varies significantly across cancer cell lines. Interestingly, we reveal a core set of 17 proteins commonly regulated by USP9X in most or all cell lines, which we consider as proximal biomarkers for USP9X inhibition. Consistent with our proteomic analyses, we show that WEHI-092 treatment arrests cells in metaphase without inducing cell death, which may account for growth suppression seen in long-term clonogenic assays in most cancer cell lines, and positions USP9X inhibitors as a new potential class of selective mitotic poisons.

cell biology↗

bioRxiv2025

Quantification and differential analysis of mass spectrometry proteomics data with probabilistic recovery of information from missing values

Mass spectrometry (MS) is the technology standard for expression proteomics, but statistical analysis of the resulting data is complicated by the occurrence of missing values. Missing values remain ubiquitous in MS-based proteomics data and are especially frequent in the emerging fields of single-cell and spatial proteomics. The limpa package implements new methods for quantification and differential expression analysis of MS proteomics data, including probabilistic information recovery from missing values. limpa summarises peptide-level data to estimate an expression value for every protein in every sample. Expression values that are supported by fewer detected peptides or involve more missing values are treated as less precise and are downweighted in the differential expression analysis, maximising statistical power while avoiding false discoveries. limpa produces a linear model object suitable for downstream analysis with the limma package, allowing complex experimental designs and other downstream tasks such as the gene ontology or pathway analysis.

bioinformatics↗

bioRxiv2024

The RBR E3 ubiquitin ligase HOIL-1 can ubiquitinate diverse non-proteinaceous substrates in vitro

HOIL-1 is a RING-between-RING (RBR)-family E3 ubiquitin ligase and component of the linear ubiquitin chain assembly complex (LUBAC). While most E3 ubiquitin ligases conjugate ubiquitin to protein lysine sidechains, HOIL-1 has been reported to ubiquitinate hydroxyl groups in protein serine and threonine sidechains and glucosaccharides, such as glycogen and its building block maltose, in vitro. However, HOIL-1 substrate specificity is currently poorly defined. Here we show that HOIL-1 is unable to ubiquitinate lysine but can efficiently ubiquitinate serine as well as a variety of model and physiologically relevant di- and monosaccharides in vitro. We identify a critical catalytic histidine residue, His510, in the flexible catalytic site of HOIL-1 that enables this O-linked ubiquitination and prohibits ubiquitin discharge onto lysine sidechains. Finally, we utilise HOIL-1s in vitro non-proteinaceous ubiquitination activity and an engineered, constitutively active HOIL-1 variant to produce preparative amounts of different ubiquitinated saccharides that can be used as tool compounds and standards in the rapidly emerging field of non-proteinaceous ubiquitination.

biochemistry↗