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Ubiquitination of the actin polymerase VASP

ABSTRACT: Filopodia are dynamic, actin-rich structures that extend outward from the cell to explore and respond to cues in the local environment. The actin polymerase VASP is a component of the filopodial tip complex, where it regulates actin polymerization and filopodial dynamics. Previously, we showed that VASP transiently co-localizes with the brain-enriched E3 ubiquitin ligase TRIM9 at the tips of neuronal filopodia. TRIM9 was required for the reversible, non-degradative ubiquitination of VASP and this modification was associated with decreased filopodia number and stability. Furthermore, the axon guidance cue netrin promoted deubiquitination of VASP. We hypothesize mono or multi-monoubiquitination of VASP is a mechanism to negatively regulate actin dynamics by blocking VASP and actin interactions. To determine the lysine residues that are ubiquitinated in VASP, we immunoprecipitated exogenously expressed human VASP from HEK293 cells. The immunopurified protein was analyzed by mass spectrometry to identify post-translational modifications. We identified numerous lysine residues that are ubiquitinated in VASP, including a high prevalence at K240 and K286.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Line Cell

SUBMITTER: Aurora Cabrera

LAB HEAD: Stephanie Gupton

PROVIDER: PXD041196 | Pride | 2024-01-17

REPOSITORIES: Pride

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			Action	DRS
	20210327_PHCOQEHF_PC823-Gupton_1_R1.raw	Raw
	20210327_PHCOQEHF_PC823-Gupton_1_R2.raw	Raw
	20210327_PHCOQEHF_PC823-Gupton_2_R1.raw	Raw
	20210327_PHCOQEHF_PC823-Gupton_2_R2.raw	Raw
	20210327_PHCOQEHF_PC823-Gupton_4_R1.raw	Raw

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Project description:Huntington’s disease is caused by a polyglutamine repeat expansion at the N-terminus of the huntingtin protein which affects the function and folding of the protein, and results in intracellular protein aggregates. Here, we examined whether this mutation leads to altered ubiquitination of huntingtin and other proteins in both soluble and insoluble fractions of brain lysates of the Q175 knock-in Huntington disease mouse model and the Q20 wild-type mouse model. Ubiquitination sites are detected by identification of Gly-Gly (diGly) remnant motifs that remain on modified lysine residues after digestion. We identified K6, K9, K132, K804 and K837 as endogenous ubiquitination sites of soluble huntingtin, with wild-type huntingtin being mainly ubiquitinated at K132, K804 and K837. Mutant huntingtin protein levels were strongly reduced in the soluble fraction while K6 and K9 were mainly ubiquitinated. In the insoluble fraction increased levels of huntingtin K6 and K9 diGly sites were observed for mutant huntingtin as compared to wild type. Besides huntingtin, proteins with various roles, including membrane organization, transport, mRNA processing, gene transcription, translation, catabolic processes and oxidative phosphorylation, were differently expressed or ubiquitinated in wild-type and mutant huntingtin brain tissues. Correlating protein and diGly site fold-changes in the soluble fraction revealed that diGly site abundances of the majority of the proteins were not related to protein fold-changes, indicating that these proteins were differentially ubiquitinated in the Q175 mice. In contrast, both the fold-change of the protein level and diGly site level were increased for several proteins in the insoluble fraction, including ubiquitin, ubiquilin-2, sequestosome-1/p62 and myo5a. Our data sheds light on putative novel proteins involved in different cellular processes as well as their ubiquitination status in Huntington’s disease, which forms the basis for further mechanistic studies to understand the role of differential ubiquitination of huntingtin as well as ubiquitin-regulated processes in Huntington’s disease.

Project description:During development, neurons undergo expansive growth and shape change to achieve their mature morphology and physiological function. As cellular remodeling occurs, proteins are synthesized, transported, degraded, and recycled. As such, protein levels and localization are tightly regulated. E3 ubiquitin ligases play a key role in proteostasis by altering protein localization, intracellular trafficking, and protein lifetime via the addition of ubiquitin modifiers. TRIM9 and TRIM67 are brain-enriched E3 ubiquitin ligases implicated in numerous stages of neuronal morphogenesis. We previously demonstrated both proteins are required for axon pathfinding and growth cone shape changes in developing neurons. In particular, TRIM9 and TRIM67 regulate filopodia number and stability in early stages of neuronal development. Our published in vivo studies suggest TRIM9 and TRIM67 may function at the synapse as well. We observed distinct deficits in spatial learning and memory of Trim9-/- and Trim67-/- mice in the Morris Water Maze test, compared to their littermate controls. Furthermore, adult-born neurons in the dentate gyrus in Trim9-/- mice also displayed a decreased number of dendritic spines. Here we demonstrate TRIM9 and TRIM67 localize to the post-synaptic density (PSD), a structure attached to the post-synaptic membrane in dendritic spines. We identified 148 proteins that were significantly changed (p < 0.05) in the Trim67-/- mice compared to their Trim67+/+ littermates. Gene Ontology analysis of these proteins demonstrated enrichment of several cellular pathways, including peptidyl-amino acid modification, microtubule dynamics, and Ras signal transduction. Likewise, we identified 109 proteins that were significantly changed (p < 0.05) in the Trim9-/- mice compared to their Trim9+/+ littermates. Following Gene Ontology analysis, we observed the prominent enrichment of one pathway in our significantly different proteins: the actin cytoskeleton. Changes in these actin cytoskeleton proteins were bidirectional, suggesting the presence of altered cytoskeletal architecture and organization in the Trim9-/- PSD.

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Ubiquitination of the actin polymerase VASP

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