Abstract
Protein palmitoylation is a reversible post-translational modification by fatty acids (FA), mainly a palmitate (C16:0). Palmitoylation allows protein shuttling between the plasma membrane and cytosol to regulate protein stability, sorting and signaling activity and its deficiency leads to diseases. We aimed to characterize the palmitoyl-proteome of ovarian follicular cells and molecular machinery regulating protein palmitoylation within the follicle. For the first time, 84 palmitoylated proteins were identified from bovine granulosa cells (GC), cumulus cells (CC) and oocytes by acyl-biotin exchange proteomics. Of these, 32 were transmembrane proteins and 27 proteins were detected in bovine follicular fluid extracellular vesicles (ffEVs). Expression of palmitoylation and depalmitoylation enzymes as palmitoyltransferases (ZDHHCs), acylthioesterases (LYPLA1 and LYPLA2) and palmitoylthioesterases (PPT1 and PPT2) were analysed using transcriptome and proteome data in oocytes, CC and GC. By immunofluorescence, ZDHHC16, PPT1, PPT2 and LYPLA2 proteins were localized in GC, CC and oocyte. In oocyte and CC, abundance of palmitoylation-related enzymes significantly varied during oocyte maturation. These variations and the involvement of identified palmitoyl-proteins in oxidation-reduction processes, energy metabolism, protein localization, vesicle-mediated transport, response to stress, G-protein mediated and other signaling pathways suggests that protein palmitoylation may play important roles in oocyte maturation and ffEV-mediated communications within the follicle.
Highlights
Palmitic acid (C16:0) is the most abundant fatty acid (FA) produced in mammalian cells, which represents 32% of total fatty acids (FA) content in bovine oocyte [1]
In order to identify palmitoylated proteins in bovine follicular cells, we performed purification of the proteins with thioester-bonded palmitate, using an acyl-biotinyl exchange (ABE) protocol from bovine granulosa cells (GC) and COCs
We demonstrated that genes of acyl-protein thioesterases (APTs) LYPLA1 and LYPLA2, palmitoyl-thioesterases PPT1 and PPT2 (PPT2), as well as two alpha-beta hydrolase-domain (ABHD) enzymes strongly and cell- expressed in follicular somatic cells and enclosed oocyte
Summary
Palmitic acid (C16:0) is the most abundant fatty acid (FA) produced in mammalian cells, which represents 32% of total FA content in bovine oocyte [1]. About 10–20% of proteins are modified by thioester linkage of deprotonated palmitate C16:0 to cysteine (Cys) in a process known as protein palmitoylation [2,3]. Protein palmitoylation is one of the major post-translational modifications (PTM) by lipids, which mediates the association of soluble proteins with membrane, their subcellular trafficking between membrane compartments and involved in protein-protein interactions, signaling and other intracellular processes determining protein localization, activity, and cell differentiation in different tissues [4,5,6]. Palmitoylation regulates functional activity of many integral and peripheral membrane proteins involved in cell signaling, including G-proteins and their receptors (GPCRs) [8,9,10,11]. Analyses of palmitoylomes in human diseases indicated a pivotal role of protein palmitoylation in nervous system disorders and cancers [14]
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