The vascular endothelium is a highly dynamic
The vascular endothelium is a highly dynamic multifunctional organ that is critically involved in the regulation of vessel integrity, vascular growth and remodeling, tissue growth and metabolism, immune responses, cell adhesion, angiogenesis, hemostasis and vascular permeability . Therefore, the proper functioning of the endothelium is important for the homeostasis of the body, and endothelial dysfunction is associated with several pathophysiological conditions, including atherosclerosis, hypertension, and diabetes . Previous studies have reported that endothelial jw products sale could internalize eFABP4 and that eFABP4 co-localized with CK1 in the membrane of endothelial cells forming specific protein complexes . Considering this, the present study analyzes whether eFABP4 cellular uptake is dependent on CK1 interaction or if eFABP4 can pass through the membrane and be internalized in endothelial cells in a CK1-independent mechanism. The results of the current study showed that FABP4 may interact with a CK1 derived peptide physically. Currently, the specific form of CK1 folding into a transmembrane protein in endothelial cells remains unclear. Therefore, to characterize the FABP4-CK1 binding, we based our work on previously determined structural characteristics. CK1 as an intermediate filament protein consists of 3 domains; a long central α-helical region, known as the rod domain (residues 180–489), flanked by two end domains, the head (amino-terminal, residues 2–179) and tail (carboxy-terminal, residues 490–644) [25,26]. The rod region has extensive regions of the coiled-coil structure; besides the amino- and carboxyl-end domains are highly flexible and characteristically have glycine-rich sequences forming glycine-loops through cell membranes leading to the interaction with other membrane proteins. Thus, in order to map the putative FABP4 binding domain in the CK1 protein, we selected the H1 subdomain, which is the region that separates the rod region from the glycine-rich region in the head which terminates the penetration of the N-terminal region into cell membranes [17,27]. H1 is a highly conserved sequence in keratins, and previous studies reported this region as the binding domain of CK1 to HK, one of its best-characterized ligands in endothelial cells. Three peptides covering the selected region were designed, however, the synthesis of the peptide covering the entire region (named as P1) was not feasible, leading to determine the binding affinity between FABP4 and the peptides P2 and P3. Using this approach, we reported for the first time, that FABP4 and CK1 physically bind and that the FABP4 binding domain would be located among the 151GIQEVTINQSLLQPLNVEID170 sequence corresponding to the P3 peptide. The specificity of this interaction was corroborated because no binding affinity was observed between FABP4 and the peptide P2. According to the primary structure analysis of Badowski et al. on the CK1 amino acid sequence , while the P2 peptide is covering a regulatory region containing several aromatic residues, such as phenylalanine (F) and tyrosine (Y) interspersed among glycine (G), cysteine (C), valine (V) and proline (P), forming a hydrophobic pocket, the P3 peptide is covering an aliphatic-rich region containing 50% of the residues with polar; glutamine (Q), threonine (T), asparagine (N) and serine (S), or charged characteristics; glutamate (E) and aspartate (D), properties. These differences would explain the different affinity shown by FABP4 towards one or the other peptide. The role of CK1 as a receptor-like protein in the membrane of endothelial cells was previously described. Astern et al.  proposed that CK1 is part of a possible endothelial receptor complex formed together with the uPAR and the receptor for the globular head of complement 1q protein (gC1qR) and that this multiprotein receptor complex was the platform for the assembly and the activation of the vasoregulatory plasma kallikrein-kinin system. CK1 and uPAR, but not gC1qR, co-localize on the membrane of endothelial cells. Although uPAR binds to cell membrane constituents by a phosphatidylinositol linkage, CK1 has neither a phosphatidylinositol bond nor the structure of a transmembrane protein . These results let us to hypothesize that CK1 and uPAR would form a receptor complex and their cell surface expression would be regulated by endocytosis and recycling. Therefore, the molecular mechanism through which CK1 and FABP4 interacts with the cell membrane of endothelial cells would be similar to the mechanism previously proposed for uPAR-mediated endocytosis; urokinase-plasminogen activator inhibitor 1 (uPA-PAI1) complexes bound to uPAR associated with low-density lipoprotein receptor-related protein 1 (LRP1) leading to clathrin-dependent endocytosis, lysosomal degradation of uPA andPAI1 and recycling of UPAR and LRP1 to the plasma membrane [29,30].