Functional analysis of natural PCSK9 mutants in modern and archaic humans

Abstract

PCSK9 is the last member of the proprotein convertases (PCs) family and its gene is mutated in ~ 2% to 3% of individuals with familial hypercholesterolemia (FH). This protein enhances the degradation of the low‐density lipoprotein receptor (LDLR) and hence increases the levels of circulating LDL‐cholesterol (LDLc). Studies of the underlying mechanism(s) regulating the activity of different mutations in the PCSK9 gene are ongoing as they enhance our understanding of the biology and clinical relevance of PCSK9 and its partners. In an attempt to unravel the regulation of PCSK9 transcription and possibly identify mutation ‘hot spot’ regions with alterations in CpG methylation, we present for the first time the complete methylome profile of the PCSK9 gene in modern and archaic humanoids. Our data showed that the genomes of modern humans and archaic PCSK9 exhibit a similar methylation pattern. Next, we defined the mechanistic consequences of three PCSK9 natural mutations (PCSK9‐R96L, ‐R105W, and ‐P174S) and one archaic Denisovan mutation (PCSK9‐H449L) using various complementary cellular and in vitro binding assays. Our results showed that the PCSK9‐H449L is a loss‐of‐function (LOF) mutation, likely due to its lower binding affinity to the LDLR. Similarly, PCSK9‐R96L and ‐R105W are LOF mutations, even though they have been identified in FH patients. The PCSK9‐R105W mutation leads to a significantly lower autocatalytic processing of proPCSK9. PCSK9‐P174S resulted in a LOF in both extracellular and intracellular pathways. In conclusion, our extensive analyses revealed that all studied mutations result in PCSK9 LOF, via various mechanisms, leading to lower levels of LDLc.

The Multifaceted Biology of PCSK9

“…The reason why the lack of PCSK9 activity has not yet been associated with defined negative outcomes is surprising and begs the question of why PCSK9 was kept during mammalian evolution, but lost in some species, and very rarely in human. PCSK9 LOF variations reducing circulating LDLc levels might have been problematic for our ancestors, such as the hunter-gatherer Denisovans ( 285 ), who probably had a cholesterol-poor diet, but can now be particularly advantageous for people on cholesterol-rich diets, such as those living in industrialized countries. In fact, it has been suggested that in some placental mammalian species PCSK9 underwent “pseudogenization” leading to the loss of active full length PCSK9 protein ( 286 , 287 ).…”

The PCSK9 discovery, an inactive protease with varied functions in hypercholesterolemia, viral infections, and cancer

“…Tissue-specific PCSK9 KO in mouse small intestine ( 146 ), pancreatic β-cells ( 47 ), and lung epithelia ( 128 ) allowed some dissection of the relationship between the autocrine and endocrine functions of PCSK9 but clearly emphasized the dominant role of circulating PCSK9 originating from liver ( 48 , 80 ). Furthermore, the intracellular activity of PCSK9 found in cell lines ( 73 ) seems distinct from that of circulating PCSK9 ( 148 , 149 ) and may play important roles during embryonic development ( 41 ) and in placenta ( 85 , 150 ), and possibly explain the GOF of some PCSK9 variants.…”

Is It Ever Wise to Edit Wild-Type Alleles? Engineered CRISPR Alleles Versus Millions of Years of Human Evolution

“…Ancient humans were hunter-gatherers likely living on hunted fowl and berries. 26 Denisovan and Neanderthal sequencing revealed unique PCSK9 mutations predicted to result in loss-of-function alleles. 26 Loss-of-function variants of PCSK9 may have been tolerated if dietary lipid was in abundance but might have been disadvantageous to human ancestors who had to survive long periods of lipid-poor meals.…”

“…26 Denisovan and Neanderthal sequencing revealed unique PCSK9 mutations predicted to result in loss-of-function alleles. 26 Loss-of-function variants of PCSK9 may have been tolerated if dietary lipid was in abundance but might have been disadvantageous to human ancestors who had to survive long periods of lipid-poor meals. 13,26 Our fundamental understanding of genetic selection stipulates that while a few rare families have loss-of-function alleles of PCSK9, which serve to protect them from cardiovascular disease, the wild-type protein has important functions precisely because it has remained functional under selective pressure for over 350 million years throughout vertebrate evolution from fishes to humans.…”

“…26 Loss-of-function variants of PCSK9 may have been tolerated if dietary lipid was in abundance but might have been disadvantageous to human ancestors who had to survive long periods of lipid-poor meals. 13,26 Our fundamental understanding of genetic selection stipulates that while a few rare families have loss-of-function alleles of PCSK9, which serve to protect them from cardiovascular disease, the wild-type protein has important functions precisely because it has remained functional under selective pressure for over 350 million years throughout vertebrate evolution from fishes to humans. As we will describe below, there is evidence that PCSK9 influences immunity either directly through its effects on the surface localization of the LDL receptor and by its ability to modulate plasma lipids.…”