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Ubiquitinated Protein Validation by PRM Analysis in SMG vs SO of E. tenella

ABSTRACT: Eimeria tenella is highly host-specific and presents a monoxenous life cycle with multiple developmental stages. Each stage of E. tenella has different morphology, pathogenesis, gene and protein expressions which may change through gene regulatory and post-translational modifications (PTMs). Ubiquitination modulates a wide range of cellular functions and plays an important role in diverse cellular activities. Hitherto, there is no information on ubiquitination in E. tenella except inexact report on the enzymes related to ubiquitination. Dissecting the regulatory mechanism of E. tenella stage-conversion of ubiquitin is essential to understand parasite development. In this study, We used the ubiquitinated protein of SMG and SO stages to validate the proteomic data using PRM analysis. Skyline (v. 21.1) software was used for the alignment of the retention times, the peak detection of the peptide fragments and their quantification. We selected 14 ubiquitinated protein sites in 13 proteins for PRM validation, including L-dopachrome isomerase, glyceraldehyde-3-phosphate dehydrogenase, putative GRA9 protein, elongation factor 1-alpha, sag13, histone H2B, putative L-lactate dehydrogenase, putative polyubiquitin, putative myosin light chain TgMLC1, DNA-directed RNA polymerase II, putative myosin A, putative UV excision repair protein rhp23, and putative DNA damage inducible protein. We found that there is consistency in the SMG/SO trend by using the previous 4D label free method and PRM technique.

ORGANISM(S): Eimeria Tenella

SUBMITTER: Zigang Qu

PROVIDER: PXD067512 | iProX | Wed Aug 20 00:00:00 BST 2025

REPOSITORIES: iProX

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Project description:NguyenLK2011 - Ubiquitination dynamics in Ring1B-Bmi1 system This theoretical model investigates the dynamics of Ring1B/Bmi1 ubiquitination to identify bistable switch-like and oscillatory behaviour in the system. Michaelis-Menten (MM) equations are used to formulate the model. However, the authors show that the dynamics persist even for Mass-Action kinetics. This SBML file is the MM version of the model. This model is described in the article: Switches, excitable responses and oscillations in the Ring1B/Bmi1 ubiquitination system. Nguyen LK, Muñoz-García J, Maccario H, Ciechanover A, Kolch W, Kholodenko BN. PLoS Comput. Biol. 2011 Dec; 7(12): e1002317 Abstract: In an active, self-ubiquitinated state, the Ring1B ligase monoubiquitinates histone H2A playing a critical role in Polycomb-mediated gene silencing. Following ubiquitination by external ligases, Ring1B is targeted for proteosomal degradation. Using biochemical data and computational modeling, we show that the Ring1B ligase can exhibit abrupt switches, overshoot transitions and self-perpetuating oscillations between its distinct ubiquitination and activity states. These different Ring1B states display canonical or multiply branched, atypical polyubiquitin chains and involve association with the Polycomb-group protein Bmi1. Bistable switches and oscillations may lead to all-or-none histone H2A monoubiquitination rates and result in discrete periods of gene (in)activity. Switches, overshoots and oscillations in Ring1B catalytic activity and proteosomal degradation are controlled by the abundances of Bmi1 and Ring1B, and the activities and abundances of external ligases and deubiquitinases, such as E6-AP and USP7. This model is hosted on BioModels Database and identified by: BIOMD0000000622. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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.

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Ubiquitinated Protein Validation by PRM Analysis in SMG vs SO of E. tenella

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