Biology subjects

Wakarchuk, W. W.

Publications and source records attributed to Wakarchuk, W. W..

4 recordsLinked to original sources

bioRxiv2025

Differences in intrinsic cellular O-GlcNAcylation impact response to metabolic stress

The cellular response to metabolic stress is complex and involves various signals and pathways leading to cellular adaptation. AMP-activated Protein Kinase (AMPK) senses nutrient insufficiency reflected in the ratio of ATP to AMP/ADP and/or glucose availability. In addition, the dynamic modification of proteins with O-linked {beta}-N-Acetylglucosamine (O-GlcNAc) by O-GlcNAc transferase (OGT) is also nutrient sensitive, given that the substrate for this modification is the product of the hexosamine biosynthetic pathway. AMPK can also regulate the hexosamine biosynthetic pathway, and O-GlcNAc may reciprocally regulate AMPK activity under some contexts. However, it remains unclear which parameters establish the extent of reciprocal regulation of AMPK and protein O-GlcNAc modification and whether these signals can be orthogonal under some circumstances. Here, we used ARPE-19 and MDA-MB-231 cells to examine how nutrient limitation or pharmacological activation combined with inhibition of AMPK and O-GlcNAcylation impacts the activation of the reciprocal pathway. We found that the intrinsic level of global protein O-GlcNAc modification may impact how AMPK and metabolic stress regulate global O-GlcNAcylation, and that both signaling systems could be orthogonal in some contexts. These results contribute to understanding the complexity of cellular signaling in response to nutrient availability.

cell biology↗

bioRxiv2023

PROTEIN-O-MANNOSYLATION BY NON-SEC/TAT SECRETION TRANSLOCONS IN ACTINOBACTERIA

Protein-O-mannosylation (POM) is a form of O-glycosylation that is ubiquitous throughout all domains of life and has been extensively characterized in eukaryotic systems. However, in prokaryotes this process has only been investigated in terms of pathogenicity (in Mycobacterium tuberculosis) even though there are many non-pathogenic bacteria that are known to regularly carry out POM. To date, there is no consensus on what benefit POM imparts to the non-pathogenic bacteria that can perform it. Though the generation of a POM deficient mutant of Corynebacterium glutamicum - a widely utilized and known mannosylating actinobacteria - this work shows that even closely related actinobacterial GT-39s (the enzymes responsible for the initiation of POM) can potentially have different activities and substrate specificities for targets of POM. Moreover, presented here is evidence that POM does not only occur in a SEC-dependent manner; POM also occurs with TAT and non-SEC secreted substrates in a specific and likely tightly regulated manner. Together these results highlight the need for further biochemical characterization of POM in these and other bacterial species to help elucidate the true nature of its biological functions. ImportanceBoth the mechanism and overall cellular function of protein-O-mannosylation, a ubiquitous subset of O-glycosylation, is poorly understood in bacterial systems. In Mycobacterium tuberculosis and other pathogenic actinobacteria, numerous secreted virulence factors were identified as mannoproteins, with protein-O-mannosylation deficient mutants displaying a less virulent phenotype due to these proteins lacking the modification. However, these findings do not offer any explanations as to why non-pathogenic strains of actinobacteria also perform this modification as in these organisms it is seemingly dispensable. Corynebacterium glutamicum is a widely utilized, industrially relevant actinobacteria that also performs protein-O-mannosylation. This manuscript describes the utilization of C. glutamicum as a Gram-positive recombinant host for the in vivo study of actinobacterial protein-O-mannosylation and demonstrates the distinct lack of first-hand biochemical data of the process in prokaryotes.

microbiology↗

bioRxiv2022

O-GlcNAc transferase modulates formation of clathrin-coated pits

Clathrin-mediated endocytosis (CME) controls the internalization and function of a wide range of cell surface proteins. CME occurs by the assembly of clathrin and many other proteins on the inner leaflet of the plasma membrane into clathrin-coated pits (CCPs). These structures recruit specific membrane protein cargo destined for internalization and trigger the generation of membrane curvature that precedes eventual scission of CCPs from the plasma membrane to yield intracellular vesicles. The diversity of cell surface protein cargo thus controlled by CME indicates that CCP formation is regulated to allow cellular adaptation under different contexts. Of interest is how cues derived from cellular metabolism may regulate CME, given the reciprocal role of CME in controlling cellular metabolism. The modification of proteins with O-linked {beta}-N-acetylglucosamine (O-GlcNAc) is sensitive to nutrient availability and may allow cellular adaptation to different metabolic conditions. We examined how the modification of proteins with O-GlcNAc may control CCP formation and thus CME. We used perturbation of key enzymes responsible for protein O-GlcNAc modification, as well as specific mutants of the endocytic regulator AAK1 predicted to be impaired for O-GlcNAc modification. We identify that CCP initiation and the assembly of clathrin and other proteins within CCPs is controlled by O-GlcNAc protein modification. This reveals a new dimension of regulation of CME and highlights the important reciprocal regulation of cellular metabolism and endocytosis.

cell biology↗

bioRxiv2018

Genetics behind the biosynthesis of nonulosonic acid containing lipooligosaccharides in Campylobacter coli

Campylobacter jejuni and Campylobacter coli are the most common cause of bacterial gastroenteritis in the world. Ganglioside mimicry by C. jejuni lipooligosaccharide (LOS) is the triggering factor of Guillain-Barre syndrome (GBS), an acute polyneuropathy. Sialyltransferases from the glycosyltransferase (GT) family 42 are essential for the expression of ganglioside mimics in C. jejuni. Recently, two novel GT-42 genes, cstIV and cstV, have been identified in C. coli. Despite being present in [~]11% of currently available C. coli genomes, the biological role of cstIV and cstV is unknown. In the present study, mutation studies in two strains expressing either cstIV or cstV were performed and mass spectrometry was used to investigate differences in the chemical composition of LOS. Attempts were made to identify donor and acceptor molecules using in vitro activity tests with recombinant GT-42 enzymes. Here, we show that CstIV and CstV are involved in C. coli LOS biosynthesis. In particular, cstV is associated with LOS sialylation, while cstIV is linked to the addition of a diacetylated nonulosonic acid residue.\n\nIMPORTANCEDespite being a major foodborne pathogen, Campylobacter coli glycobiology has been largely neglected. The genetic makeup of the C. coli lipooligosaccharide biosynthesis locus was largely unknown until recently. C. coli harbour a large set of genes associated to lipooligosaccharide biosynthesis, including several putative glycosyltransferases involved in the synthesis of sialylated lipooligosaccharide in Campylobacter jejuni. In the present study, C. coli was found to express lipooligosaccharide structures containing sialic acid and other nonulosonate acids. These findings have a strong impact in understanding C. coli ecology, host-pathogen interaction, and pathogenesis.

microbiology↗