Functional characterization of the p97/valosin-containing protein (VCP) interacting network in Leishmania Functional characterization of the p97/valosin-containing protein (VCP) interacting network in Leishmania

ABSTRACT: Valosin‐containing protein (VCP)/p97/Cdc48 is an essential AAA+ ATPase associated with many ubiquitin-dependent cellular pathways that plays a central role in protein quality control in all eukaryotes. Its functional diversity relies on its ability to cooperate with a large number of protein cofactors that provide pathway selectivity and fine-tune substrate processing. Here, we identified the main partners of the recently characterized Leishmania infantum VCP homolog (LiVCP) and we provide the first insights into the biology of the LiVCP network in Trypanosomatidae. Among the LiVCP core partners detected by immunoprecipitation (IP) and LC-MS/MS mass spectra acquisition, we determined p47, FAF1, UFD1 and PUB1 as the major cofactors of the Leishmania VCP. Furthermore, we provide in silico analyses demonstrating the conserved regions of these cofactors that potentially interact with LiVCP. We employed ‘‘network proteomics’’ using IP and LC-MS/MS studies for each cofactor to confirm their close partnership with LiVCP and to identify the cofactor-specific interacting partners as well as the partners that are shared among multiple cofactors and LiVCP complexes, such as the important heterotrimer VCP-NPL4-UFD1 complex in Leishmania. Our results suggest a glycosome/peroxisome association of LiFAF1. Gene Ontology analysis of each proteome coupled with digitonin fractionation and immunofluorescence studies indicated nuclear association for Lip47, cytoplasmic and vacuolar association for LiUFD1, glycosome association for LiFAF1, and endoplasmic reticulum interaction for LiPUB1 protein. All together, these data provide the first VCP protein network in Trypanosomatidae. Further functional and structural analysis of the essential LiVCP quality control protein network may lead to the development of new anti-parasitic drugs.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Leishmania Infantum Jpcm5

SUBMITTER: Bruno Aguiar  

LAB HEAD: Barbara Papadopoulou

PROVIDER: PXD013731 | Pride | 2020-08-06

REPOSITORIES: Pride