Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secretory protein that promotes low-density lipoprotein receptor (LDLR) degradation and thereby regulating plasma levels of LDL cholesterol. Previous studies have revealed the role of the C-terminal domain (CTD) of PCSK9 in its secretion, however, how CTD regulates PCSK9 secretion is not completely understood. Additionally, SEC24A, the cargo adaptor protein of the coat protein complex II, has been implicated in the secretion of mouse PCSK9. Here, we investigated how CTD and SEC24 regulated PCSK9 secretion in humans. We found that mutant PCSK91-528, in which amino acids from 529 to the end (amino acid 692) were deleted, was maturated and secreted from cells as effectively as the wild-type protein. On the other hand, lacking amino acids 454 to 692 in mutant PCSK91-453 significantly reduced its maturation and secretion, but to a lesser extent when compared to mutants PCSK91-446, PCSK91-445 and PCSK91-444, that all markedly impaired PCSK9 maturation. However, mutant PCSK91-444 virtually eliminated PCSK9 secretion while PCSK91-446 and PCSK91-445 could still be adequately detected in culture medium. Interestingly, mutation of Pro445 to other amino acid residues considerably impaired the secretion of mutant PCSK91-445 but not the full-length protein. We also found that natural variants in CTD including S462P, S465L, E482G, R495Q and A522T impaired PCSK9 secretion. Further, the knockdown of SEC24A, SEC24B, SEC24C but not SEC24D reduced secretion of the full-length PCSK9 but not mutant PCSK91-446. Therefore, SEC24A, SEC24B, and SEC24C facilitate endogenous PCSK9 secretion from cultured human hepatocytes, that are most likely mediated by the CTD of PCSK9. Our studies also indicate that the CTD of PCSK9 may allosterically and independently modulate the stability of the hinge region. Collectively, these data revealed that the CTD of PCSK9 and the hinge region play a critical role in PCSK9 maturation and secretion.

中文翻译：

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PCSK9和SEC24亚型的C末端结构域在PCSK9分泌中的作用。

前蛋白转化酶枯草杆菌蛋白酶/ kexin 9型（PCSK9）是一种分泌蛋白，可促进低密度脂蛋白受体（LDLR）降解，从而调节LDL胆固醇的血浆水平。先前的研究揭示了PCSK9的C末端结构域（CTD）在其分泌中的作用，但是，CTD如何调节PCSK9的分泌尚不完全清楚。此外，SEC24A，外壳蛋白复合物II的货物衔接蛋白，已与小鼠PCSK9的分泌有关。在这里，我们研究了CTD和SEC24如何调节人类PCSK9的分泌。我们发现，突变PCSK91-528（其中从529到末端的氨基酸（第692个氨基酸）被删除）已成熟并从细胞中像野生型蛋白一样有效地分泌出来。另一方面，在突变体PCSK91-453中缺少454至692位氨基酸的氨基酸显着降低了其成熟和分泌，但与突变体PCSK91-446，PCSK91-445和PCSK91-444相比，它们的程度都明显降低，它们均明显损害了PCSK9的成熟。然而，突变体PCSK91-444实际上消除了PCSK9的分泌，而PCSK91-446和PCSK91-445仍能在培养基中被充分检测到。有趣的是，Pro445向其他氨基酸残基的突变大大损害了突变体PCSK91-445的分泌，但没有损害全长蛋白的分泌。我们还发现CTD中的天然变体，包括S462P，S465L，E482G，R495Q和A522T会损害PCSK9的分泌。此外，敲除SEC24A，SEC24B，SEC24C而不是SEC24D可以减少全长PCSK9的分泌，但不能减少突变型PCSK91-446的分泌。因此，SEC24A，SEC24B，SEC24C和SEC24C促进培养的人肝细胞分泌内源性PCSK9，这很可能是由PCSK9的CTD介导的。我们的研究还表明，PCSK9的CTD可能会变构且独立地调节铰链区的稳定性。这些数据共同表明，PCSK9的CTD和铰链区在PCSK9的成熟和分泌中起关键作用。