Here, we have described a novel LDLR-CD81 interaction that may contribute to HCV entry. or hinge-cysteine-histidine-rich domain domains of PCSK9 were able to reduce CD81 and LDLR levels. These data confirm that PCSK9 reduces CD81 levels via an intracellular pathway as reported for LDLR. Using immunocytochemistry, a proximity ligation assay, and co-immunoprecipitation, we found that the cell surface level of PCSK9 was enhanced upon overexpression of CD81 and that both PCSK9 and LDLR interact with this tetraspanin protein. Interestingly, using CHO-A7 cells lacking LDLR expression, we revealed that LDLR was not required for the degradation of CD81 by PCSK9, but its presence strengthened the PCSK9 effect. and denote amino acid positions at boundaries between domains. The N-terminal CAT construct and the HR-CHRD were individually subcloned for further study. Full-length PCSK9 and the truncated constructs (CAT and HR-CHRD) were fused with the TM-CT of LAMP1 or ACE2 to drive the proteins to the lysosomal compartment or to the cell surface, respectively. PCSK9 has been extensively studied for TCN238 its ability to bind cell surface receptor proteins such as the LDLR (4, 5), very low density SMAX1 lipoprotein (VLDL) receptor, and apolipoprotein E receptor 2 (8, 9) and sort them to endosomes/lysosomes for degradation (5, 6). In that context, the PCSK9-LDLR interaction has been shown to occur between the catalytic domain of PCSK9 (10) and the N-terminal part of epidermal growth factor-like repeat A (EGF-A) domain of the LDLR (10, 11), although the CHRD and the prodomain seem to possess some additional binding affinity for the -barrel ligand-binding domain of the LDLR (12,C15). There are two pathways by which PCSK9 leads to the degradation of the LDLR. In the extracellular pathway, following secretion from cells, extracellular PCSK9 binds to the LDLR at the cell surface, triggering PCSK9-LDLR complex internalization and its traffic to acidic endosomes/lysosomes where degradation occurs. In the intracellular pathway, mature PCSK9 traffics from the ER to the Golgi apparatus where it binds to the LDLR and sorts the receptor directly to lysosomes for degradation (16). In liver, PCSK9 seems to enhance the degradation of the LDLR mostly by the extracellular pathway (1, 6). Interestingly, several natural mutations in the human gene have been reported. They confer gain-of-function (GOF) or loss-of-function (LOF) activity on the LDLR. Therefore, individuals with GOF mutations will have reduced LDLR levels in hepatocytes and thus suffer from hypercholesterolemia. Various LOF and GOF mutants were TCN238 found to be associated with hypocholesterolemia and hypercholesterolemia, respectively (17). For example, the LOF mutations G236S and N354I are due to failure to exit the ER or to undergo autocatalytic cleavage, respectively (18). In contrast, typical GOF mutations include D129N, D374H, and D374Y, which enhance the binding of PCSK9 to the LDLR (19, 20). TCN238 The various strategic mutants used in this study are described in Table 1. TABLE 1 List of PCSK9 mutants used in this study to produce secreted forms (18).17R357HGOFFound related to FH patients (30).18D374HGOFFound in Portuguese FH patients (31).19D374YGOFFound in Norwegian patients with FH (32). Has partial resistance to furin (29) and higher affinity for LDLR by 25 times compared with wild type (33). Degrades LDLR 10-fold higher than wild type (23).20F379ALOFBased on data. Recently, we identified CD81 as a new cell surface protein that could be targeted by PCSK9 (21). Because PCSK9 reduces the protein levels of the LDLR and hepatitis C virus (HCV) uses both LDLR and CD81 as entry factors, we showed that PCSK9 could prevent Huh7 cells from infection with the virus. Surprisingly, we also found that stable PCSK9 overexpression in HuH7 cells could also decrease CD81 levels. In this study, we investigated further how PCSK9, CD81, and LDLR interact with each other and probed the effect of the expression of various PCSK9 mutants on CD81 levels. Experimental Procedures cDNA Constructs Human full-length PCSK9 and its truncated constructs catalytic domain (CAT), HR, and CHRD with or without the transmembrane-cytosolic tail (TM-CT) of lysosomal-associated membrane protein 1 (LAMP1) or angiotensin I-converting enzyme 2 (ACE2) proteins (hereby abbreviated as PCSK9-LAMP1, PCSK9-ACE2, CAT-ACE2, and HR-CHRD-ACE2) were cloned into pIRES2-eGFP (Clontech) and phCMV3 vectors as described (12). Plasmids coding for human PCSK9 mutants were produced from phCMV3-hPCSK9-ACE2 using the QuikChange Lightning kit (Agilent Technologies). The primer sequence information will be given if required. The integrity of all mutants was verified by sequencing. Cell Culture and Transfection Huh7 cells were cultured in.