Chris West Archives - Center for Tropical and Emerging Global Diseases

Synergy between a cytoplasmic vWFA/VIT protein and a WD40-repeat F-box protein controls development in Dictyostelium

Donna Huber on September 14, 2023

Like most eukaryotes, the pre-metazoan social amoeba Dictyostelium depends on the SCF (Skp1/cullin-1/F-box protein) family of E3 ubiquitin ligases to regulate its proteome. In Dictyostelium, starvation induces a transition from unicellular feeding to a multicellular slug that responds to external signals to culminate into a fruiting body containing terminally differentiated stalk and spore cells. These transitions are subject to regulation by F-box proteins and O₂-dependent posttranslational modifications of Skp1. Here we examine in greater depth the essential role of FbxwD and Vwa1, an intracellular vault protein inter-alpha-trypsin (VIT) and von Willebrand factor-A (vWFA) domain containing protein that was found in the FbxwD interactome by co-immunoprecipitation. Reciprocal co-IPs using gene-tagged strains confirmed the interaction and similar changes in protein levels during multicellular development suggested co-functioning. FbxwD overexpression and proteasome inhibitors did not affect Vwa1 levels suggesting a non-substrate relationship. Forced FbxwD overexpression in slug tip cells where it is normally enriched interfered with terminal cell differentiation by a mechanism that depended on its F-box and RING domains, and on Vwa1 expression itself. Whereas vwa1-disruption alone did not affect development, overexpression of either of its three conserved domains arrested development but the effect depended on Vwa1 expression. Based on structure predictions, we propose that the Vwa1 domains exert their negative effect by artificially activating Vwa1 from an autoinhibited state, which in turn imbalances its synergistic function with FbxwD. Autoinhibition or homodimerization might be relevant to the poorly understood tumor suppressor role of the evolutionarily related VWA5A/BCSC-1 in humans.

Andrew W Boland, Elisabet Gas-Pascual, Hanke van der Wel, Hyun W Kim, Christopher M West. Front Cell Dev Biol. 2023 Sep 14;11:1259844. doi: 10.3389/fcell.2023.1259844. eCollection 2023.

Glycomics, Glycoproteomics and Glycogenomics: an Inter-Taxa Evolutionary Perspective

Donna Huber on September 29, 2020

Glycosylation is a highly diverse set of co- and post-translational modification of proteins. For mammalian glycoproteins, glycosylation is often site-, tissue- and species-specific, and diversified by microheterogeneity. Multitudinous biochemical, cellular, physiological and organismic effects of their glycans have been revealed, either intrinsic to the carrier proteins or mediated by endogenous reader proteins with carbohydrate recognition domains. Furthermore, glycans frequently form the first line of access by or defense from foreign invaders, and new roles for nucleocytoplasmic glycosylation are blossoming. We now know enough to conclude that the same general principles apply in invertebrate animals and unicellular eukaryotes – different branches of which spawned the plants or fungi and animals. The two major driving forces for exploring the glycomes of invertebrates and protists are (i) to understand the biochemical basis of glycan-driven biology in these organisms, especially of pathogens, and (ii) to uncover the evolutionary relationships between glycans, their biosynthetic enzyme genes, and biological functions for new glycobiological insights. With an emphasis on emerging areas of protist glycobiology, here we offer an overview of glycan diversity and evolution, to promote future access to this treasure trove of glycobiological processes.

Christopher M West, Daniel Malzl, Alba Hykollari, Iain B H Wilson. Mol Cell Proteomics. 2020 Sep 29;mcp.R120.002263. doi: 10.1074/mcp.R120.002263.

A terminal α3-galactose modification regulates an E3 ubiquitin ligase subunit in Toxoplasma gondii

Donna Huber on May 15, 2020

Skp1, a subunit of E3 Skp1/Cullin-1/F-box protein ubiquitin ligases, is modified by a prolyl hydroxylase that mediates O₂-regulation of the social amoeba Dictyostelium and the parasite Toxoplasma gondii. The full effect of hydroxylation requires modification of the hydroxyproline by a pentasaccharide that, in Dictyostelium, influences Skp1 structure to favor assembly of Skp1/F-box protein subcomplexes. In Toxoplasma, the presence of a contrasting penultimate sugar assembled by a different glycosyltransferase enables testing of the conformational control model. To define the final sugar and its linkage, here we identified the glycosyltransferase that completes the glycan and found that it is closely related to glycogenin, an enzyme that may prime glycogen synthesis in yeast and animals. However, the Toxoplasma enzyme catalyzes formation of a Galα1,3Glcα- rather than the Glcα1,4Glcα- linkage formed by glycogenin. Kinetic and crystallographic experiments showed that the glycosyltransferase Gat1 is specific for Skp1 in Toxoplasma and also in another protist, the crop pathogen Pythium ultimum. The fifth sugar is important for glycan function as indicated by the slow-growth phenotype of gat1Δ parasites. Computational analyses indicated that, despite the sequence difference, the Toxoplasma glycan still assumes an ordered conformation that controls Skp1 structure and revealed the importance of non-polar packing interactions of the fifth sugar. The substitution of glycosyltransferases in Toxoplasma and Pythium by an unrelated bifunctional enzyme that assembles a distinct but structurally compatible glycan in Dictyostelium is a remarkable case of convergent evolution, that emphasizes the importance of the terminal α-galactose and establishes the phylogenetic breadth of Skp1 glycoregulation.

Msano Mandalasi, Hyun W. Kim, David Thieker, M. Osman Sheikh, Elisabet Gas-Pascual, Kazi Rahman, Peng Zhao, Nitin G. Daniel, Hanke van der Wel, H. Travis Ichikawa, John N. Glushka, Lance Wells, Robert J. Woods, Zachary A. Wood, and Christopher M. West. J Biol Chem. 2020 May 15. pii: jbc.RA120.013792. doi: 10.1074/jbc.RA120.013792.

Trainee Spotlight: Msano Mandalasi

Donna Huber on October 31, 2017

trainee Msano Mandalasi
Msano Mandalasi, a post-doctoral trainee in Chris West‘s laboratory, is originally from Malawi, (located in southeastern Africa) and obtained her bachelor’s degree in Chemistry from the University of Malawi. After graduation, she worked briefly for the University of Malawi and then came to the US to obtain a Master’s degree in Chemistry. Later, she enrolled in a doctoral graduate program at the University of Maryland Eastern Shore where she graduated in 2012. She spent two years teaching undergraduate Chemistry before deciding to get back into research. She joined Dr. West’s group while he was at the University of Oklahoma and moved with the lab to the University of Georgia.

Msano’s research focus

The focus of Msano’s project is on the role of prolyl hydroxylation and glycosylation of E3 Ubiquitin ligase on Toxoplasma growth.

With a research background mostly in chemistry and biochemistry, her graduate research introduced her to some aspect of parasitology working on Schistosome glycobiology. However, she did not have a strong background in molecular biology prior to joining the West lab. This current project merges glycobiology and molecular biology and also extends some parasitology studies, thus giving her the opportunity to learn molecular biology and parasitology to complement her chemistry background. A combination of this expert knowledge will benefit her to address the research objectives on her Toxoplasma project.

Capstone experience

Each T32 trainee is provided with the opportunity to complete a capstone experience at the end of their fellowship. This experience allows for an extended visit to a collaborator’s laboratory or travel to a scientific meeting where they present their research and interact with colleagues. Msano plans to use her capstone experience to give oral presentations at scientific meetings, to publish some of the studies conducted within this time period, and interact with other trainees in the program.

T32 fellowship helps trainees achieve their goals

“Through the funding provided by the T32 Training Grant, I will be able to address research questions that should lead to launching my own area of research,” said Msano.

Msano hopes to run her own independent research program in academia one day.

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