Dynamics and Functional Interplay of Nonhistone Lysine Crotonylome and Ubiquitylome in Vascular Smooth Muscle Cell Phenotypic Remodeling

Abstract

BackgroundThe crotonylation of histones is discovered of late as one of the post-translational modifications (PTMs) that can regulate gene expression. However, the function of crotonylation on nonhistone proteins in vascular smooth muscle cells (VSMCs) is unclear. Here, we aim to find the cellular characteristics of crotonylated nonhistone proteins and the cross talk with ubiquitinated proteins in VSMC phenotypic remodeling using the modified omics and proteomic analysis.MethodsWe performed the modified omics and proteomic analysis of VSMCs before and after the stimulation with platelet-derived growth factor-BB (PDGF-BB). The crotonylated and ubiquitinated pan-antibody was used to enrich proteins and then subjected to a high-throughput mass spectrometry analysis. The enrichment analysis was performed within differentially modified proteins in regard to GO terms, KEGG, and protein domains.ResultsAs a result, there were 2,138 crotonylation sites in 534 proteins and 1,359 ubiquitination sites corresponding to 657 proteins. These crotonylated proteins detected after PDGF-BB stimulation might be involved in various vital cellular pathways and carry out important functions in VSMCs. Some of them closely took part in significant physiological processes of VSMC phenotypic remodeling, including glycolysis/gluconeogenesis, vascular smooth muscle contraction, and the PI3K-Akt signaling pathway. Furthermore, the KEGG pathway enrichment analysis showed the involvement of ubiquitinated proteins in the physiological processes of VSMC phenotypic remodeling, including glycolysis/gluconeogenesis, vascular smooth muscle contraction, RAS signaling pathway, or the PI3K-Akt signaling pathway. A cross talk analysis showed that there were 199 sites within the 177 proteins modified by crotonylation and ubiquitination simultaneously. Protein–protein interaction (PPI) network analysis indicated that crotonylated and ubiquitinated proteins play an important role in cellular bioprocess commonly and possibly have a synergistic effect.ConclusionIn summary, our bioinformatics analysis shows that the crotonylation and ubiquitination of nonhistone proteins play an essential role in VSMC phenotypic transformation induced by PDGF-BB stimulation. The cross talk between crotonylation and ubiquitination in glycolysis is possibly a novel mechanism during VSMC phenotypic remodeling.

MTMR7 suppresses the phenotypic switching of vascular smooth muscle cell and vascular intimal hyperplasia after injury via regulating p62/mTORC1-mediated glucose metabolism

“…3A). In has been con rmed that enhanced glycolysis is closely related to PDGF-BBmediated phenotypic change of VSMC (Cao et al, 2022;Heiss et al, 2016). Then we screened mRNAs via microarray in PDGF-BB-challenged VSMCs transfected with Len-Con or Len-Mtmr7, and found that glucose metabolism-related pathway was signi cant downregulated after MTMR7 overexpression, indicating the MTMR7-mediated regulation of VSMC may involve the stability of glycolysis (Fig.…”

Protein lysine crotonylation in cellular processions and disease associations

“… 78 Phenotypic changes in these VSMCs contribute to the remodeling of the vasculature. In one recent report, several non-histone proteins were identified as targets of Kcr within VSMCs, 79 with 2386 Kcr sites (570 proteins) and 2138 Kcr sites (534 proteins) in VSMCs that were or were not treated using PDGF-BB. Of these targets, the Kcr modification of 5 markers of VMSC contractility was detected at multiple sites, including 16 and 15 sites in caldesman1 and myosin9, respectively.…”

LDHA maintains the growth and migration of vascular smooth muscle cells and promotes neointima formation via crosstalk between K5 crotonylation and K76 mono-ubiquitination

“…We found that enzymes of the glucose metabolism pathways including glycolysis/gluconeogenesis, pentose phosphate pathway, pyruvate metabolism and TCA cycle, were modified extensively by crotonyl and ubiquitin. 20 The modification of the enzymes might modulate the pathways directly or indirectly. Among these enzymes, both the crotonylation and ubiquitination of LDHA were significantly increased in PDGF-BB-induced VSMCs, following that the expression of LDHA was also upregulated, thus we screen LDHA as an important enzyme that may regulate VSMC phenotypic switching.…”

“…In our recent study, the interplay of non-histone lysine crotonylome and ubiquitylome in VSMCs phenotypic remodeling were analyzed through bioinformatics. 20 Given the potential link between glucose metabolism reprogramming and phenotypic switching of VSMCs, here we focused on the key enzymes of glucose metabolism. We find that crotonylation and ubiquitination of LDHA were significantly increased in PDGF-BB-induced proliferative VSMCs.…”