Biology subjects

Chong, P. A.

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

3 recordsLinked to original sources

bioRxiv2023

Pseudomonas aeruginosa AlgF is a protein-protein interaction mediator required for acetylation of the alginate exopolysaccharide

Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, acetylation of alginate reduces opsonic killing by phagocytes and improves reactive oxygen species scavenging. Although it is well-known that alginate acetylation in P. aeruginosa requires AlgI, AlgJ, AlgF, and AlgX, how these proteins coordinate polymer modification at a molecular level remains unclear. Here, we describe the structural characterization of AlgF and its protein interaction network. We characterize direct interactions between AlgF and both AlgJ and AlgX in vitro, and demonstrate an association between AlgF and AlgX, as well as AlgJ and AlgI, in P. aeruginosa. We determine that AlgF does not exhibit acetylesterase activity and is unable to bind to polymannuronate in vitro. Therefore, we propose that AlgF functions to mediate protein-protein interactions between alginate acetylation enzymes, forming the periplasmic AlgJFXK (AlgJ-AlgF-AlgX-AlgK) acetylation and export complex required for robust biofilm formation.

microbiology↗

bioRxiv2022

Exploration of O-GlcNAc-transferase (OGT) glycosylation sites reveals a target sequence compositional bias

O-GlcNAc transferase (OGT) is an essential glycosylating enzyme that catalyzes the addition of N-acetylglucosamine to serine or threonine residues of nuclear and cytoplasmic proteins. The enzyme glycosylates a broad range of peptide sequences and prediction of glycosylation sites has proven challenging. The lack of an experimentally verified set of polypeptide sequences that are not glycosylated by OGT has made prediction of legitimate glycosylation sites more difficult. Here, we tested a number of intrinsically disordered protein regions as substrates of OGT to establish a set of sequences that are not glycosylated by OGT. The negative data set suggests an amino acid compositional bias for OGT targets. This compositional bias was validated by modifying the amino acid composition of the protein Fused in sarcoma (FUS) to enhance glycosylation. NMR experiments demonstrate that the tetratricopeptide repeat (TPR) region of OGT can bind FUS and that glycosylation-promoting mutations enhance binding. These results provide evidence that the TPR recognizes disordered segments of substrates with particular compositions to promote glycosylation, providing insight into the broad specificity of OGT.

biochemistry↗

bioRxiv2020

FUS-ALS mutants alter FMRP phase separation equilibrium and impair protein translation

Mutations in the RNA binding protein (RBP) FUS cause amyotrophic lateral sclerosis (ALS) and result in its nuclear depletion and cytoplasmic mislocalisation, with cytoplasmic gain of function thought to be crucial in pathogenesis. Here, we show that expression of mutant FUS at physiological levels drives translation inhibition in both mouse and human motor neurons. Rather than acting directly on the translation machinery, we find that mutant FUS forms cytoplasmic condensates that promote the phase separation of FMRP, another RBP associated with neurodegeneration and robustly involved in translation regulation. FUS and FMRP co-partition and repress translation in vitro. In our in vivo model, FMRP RNA targets are depleted from ribosomes. Our results identify a novel paradigm by which FUS mutations favour the condensed state of other RBPs, impacting on crucial biological functions, such as protein translation.

neuroscience↗