3.1 CD36 ubiquitination
Protein ubiquitination is the coupling of protein and ubiquitin, mediated by ubiquitin ligase (E3)(Popovic et al., 2014). CD36 is the target of E3 ligase Parkin(Abumrad and Moore, 2011) (Figure 1). Differing from the degradation effect of ubiquitination, monoubiquitylation of CD36 by Parkin enhances its stability and increase its plasma membrane level. In the presence of parkin(Kim et al., 2011), LCFA-induces polyubiquitination and degradation of CD36 is significantly down-regulated, probably because one of the ubiquitination sites (Lys469 and Lys472) of LCFA is also the target of Parkin. A recent study confirmed that USP14 is a deubiquitinating enzyme that mediates CD36 deubiquitylation on macrophages, USP14 cleaves ubiquitin chains from ubiquitinated CD36 proteins, thus avoiding the fate of CD36 being transported into the proteasome for degradation(Zhang et al., 2020)(Figure 1). However, whether Parkin and USP14 respectively act as E3 ligase and deubiquitinating enzyme in cardiomyocytes remains unclear.
LCFA, a ligand of CD36, significantly enhances the ubiquitination of CD36(Tran et al., 2011) and promotes its degradation after a long-term interaction(Smith et al., 2008) (Figure 1). This is a negative feedback for the cellular uptake of fatty acids. Recent studies have shown that ubiquitinated CD36 in myocytes stabilizes the structure of insulin receptor substrates 1(Sun et al., 2018), and thus maintains insulin signaling. Giving that insulin reduces the ubiquitination of it(Smith et al., 2008) (Figure 1), CD36 may be involved in self-regulation of insulin signaling pathway. Moreover, CD36 ubiquitination in macrophages inhibits the formation of atherosclerosis by decreasing fatty acids uptake(Srikanthan et al., 2014). But the role of ubiquitinated CD36 in heart have not yet been elucidated.
3.2 CD36 glycosylation
The glycosylation of CD36 is N-linked at asparagine residues (Asn) mediated by glycosyltransferase(Hoosdally et al., 2009; McDonald et al., 2018). There are 10 glycosylation modification sites of CD36 in human, located in the extracellular segment of CD36(Vinals et al., 2003).
Apart from increasing the molecular weight of CD36, glycosylation could also stabilize the tertiary folding of polypeptides and is therefore essential for forming the spatial structure of CD36(Mitra et al., 2006). And it affects the folding of CD36 theoretically thus influences the correct translocation of it to the cell membrane. It has been proved that carboxyl-terminal sites Asn247, Asn321, and Asn417 are indispensable for CD36 trafficking(Hoosdally et al., 2009). And mutations in Asn108 and Asn173 sites result in the abnormal distribution of CD36 on the COS m6 cells membrane(Vinals et al., 2003). Mutations in Asn 102 of CD36 has been found in spontaneously hypertensive rats (SHR)(Lauzier et al., 2011). Since Asn102 is located in the fatty acid binding pocket, mutations at this glycosylation site may have a greater possibility to affect fatty acid docking in the CD36 pocket then affect fatty acid transport. CD36 protein levels in SHR are significantly downregulated and fatty acid intake is reduced, which may be related to the glycosylation mutation of Asn102. However, the role of Asn102 in SHR has not yet been confirmed by experiments. Further researches are required to clarify the role of Asn102 and other glycosylation sites in cardiovascular diseases.
3.3 CD36 phosphorylation
CD36 has two phosphorylation sites, Thr92 and Ser237, phosphorylated by PKC(Chu and Silverstein, 2012) and PKA(Hatmi et al., 1996), respectively(Figure 1). CD36 in small intestinal epithelial cells is also dephosphorylated by intestinal alkaline phosphatase (IAP)(Lynes et al., 2011) (Figure 1). In platelets, phosphorylation of Thr92 mediated the binding of CD36 and thrombospondin(Asch et al., 1993). Phosphorylation of CD36 at Thr92 is also necessary for the adhesion of plasmodium falciparum-infected erythrocytes to human dermal microvascular endothelial cells under flow condition(Ho et al., 2005). CD36 phosphorylated at Ser237 downregulates the fatty acid uptake rate of platelets and enterocytes(Guthmann et al., 2002; Lynes et al., 2011).
3.4 CD36 palmitoylation
Most members of the DHHC(Asp–His–His–Cys)family of proteins have palmitoyl transferase activity, and are confirmed to be the main PATs(De and Sadhukhan, 2018). DHHC4/5 has been proved to be the palmitoylase of CD36 in adipocytes (Figure 1). The absence of either DHHC4 or DHHC5 prevents the palmitoylation and the insertion of CD36 on the adipose membrane, thereby destroying the CD36-dependent fatty acid uptake(Wang et al., 2019). Besides, SelK(Meiler et al., 2013), which belongs to neither PATs nor PPT, is also required for palmitoylation of CD36 in macrophages, suggesting that other proteins may also be involved in the palmitoylation of CD36.
CD36 has four palmitoylation sites Cys 3, Cys7, Cys464, and Cys466 (Figure 1). And the palmitoylation is strengthened by insulin stimulation. Combined mutations of these four palmitoylation sites hinders CD36 translocate to the cell membrane for fatty acid uptake even in the presence of insulin and AMPK(van Oort et al., 2014). With the inhibition of CD36 palmitoylation by ceruloplasmin(Thorne et al., 2010), a kind of palmitoylation specific inhibitor, the processing of CD36 at the endoplasmic reticulum and transport through the secretory pathway extends from 90 minutes to 4 hours in melanoma cells, indicating that palmitoylation is essential for the transport and translocation of CD36.
Increased palmitoylation of CD36 is found in liver steatosis and fibrosis(Zhao et al., 2018) .The decrease of CD36 palmitoylation down-regulates the uptake of fatty acids and helps to balance the fatty acid metabolism in the liver cells, thus eliminates the liver steatosis and fibrosis(Zhao et al., 2018). However, no research has revealed the effects of the palmitoylation of CD36 in cardiovascular system.