Patterns and Tempo of PCSK9 Pseudogenizations Suggest an Ancient Divergence in Mammalian Cholesterol Homeostasis Mechanisms
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Genetica (2021) 149:1–19 https://doi.org/10.1007/s10709-021-00113-x ORIGINAL PAPER Patterns and tempo of PCSK9 pseudogenizations suggest an ancient divergence in mammalian cholesterol homeostasis mechanisms Barbara van Asch1 · Luís Filipe Teixeira da Costa2 Received: 8 September 2020 / Accepted: 4 January 2021 / Published online: 30 January 2021 © The Author(s) 2021 Abstract Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a central role in cholesterol homeostasis in humans as a major regulator of LDLR levels. PCSK9 is an intriguing protease in that it does not act by proteolysis but by preventing LDLR recirculation from endosomes to the plasma membrane. This, and the inexistence of any other proteolytic substrate but itself could suggest that PCSK9 is an exquisite example of evolutionary fne-tuning. However, the gene has been lost in several mammalian species, and null alleles are present (albeit at low frequencies) in some human populations without apparently deleterious health efects, raising the possibility that the PCSK9 may have become dispensable in the mammalian lineage. To address this issue, we systematically recovered, assembled, corrected, annotated and analysed publicly available PCSK9 sequences for 420 eutherian species to determine the distribution, frequencies, mechanisms and timing of PCSK9 pseudogeni- zation events, as well as the evolutionary pressures underlying the preservation or loss of the gene. We found a dramatic diference in the patterns of PCSK9 retention and loss between Euarchontoglires—where there is strong pressure for gene preservation—and Laurasiatheria, where multiple independent events have led to PCSK9 loss in most species. These results suggest that there is a fundamental diference in the regulation of cholesterol metabolism between Euarchontoglires and Laurasiatheria, which in turn has important implications for the use of Laurasiatheria species (e.g. pigs) as animal models of human cholesterol-related diseases. Keywords Cholesterol homeostasis · Molecular evolution · Gene loss · PCSK9 · Human disease · Animal models Introduction Elucidating the underlying mechanisms took several years and led to unexpected results. As a member of a protease A central role for Proprotein convertase subtilisin/kexin type family, PCSK9 was suspected to be involved in low-density 9 (PCSK9) in cholesterol homeostasis was frst revealed by lipoprotein receptor (LDLR) degradation, as confrmed by the discovery that mutations in the gene were responsible PCSK9 over-expression experiments (Maxwell and Breslow for cases of autosomal dominant hypercholesterolemia 2004; Maxwell et al. 2005). Surprisingly, however, further (Abifadel et al. 2003; Leren 2004; Timms et al. 2004). experiments revealed that PCSK9 promotes LDLR degra- dation by a mechanism independent of its catalytic abil- Supplementary information The online version of this article ity (which is only required for proper PCSK9 sub-cellular (doi:https ://doi.org/10.1007/s1070 9-021-00113 -x) contains localization) (Seidah et al. 2003; Maxwell et al. 2005; Cam- supplementary material, which is available to authorized users. eron et al. 2006; Lagace et al. 2006; McNutt et al. 2007; Li et al. 2007). In most proprotein convertases, autocatalytic * Luís Filipe Teixeira da Costa [email protected]; [email protected] cleavage between the prodomain and the mature protein is followed by a second cleavage within the prodomain, which Barbara van Asch [email protected] is necessary for its release and activation of the protease (Anderson et al. 2002). In contrast, only the initial cleavage 1 Department of Genetics, Stellenbosch University, Private (Naureckiene et al. 2003) occurs in PCSK9, and the pro- Bag X1, Matieland 7602, South Africa tein is secreted while still in a complex with the prodomain 2 Unit for Cardiac and Cardiovascular Genetics, Department (Seidah et al. 2003; Benjannet et al. 2004). Furthermore, of Medical Genetics, Oslo University Hospital, Nydalen, crystalographic studies provided an explanation for the tight P.O. Box 4956, 0424 Oslo, Norway Vol.:(0123456789)1 3 2 Genetica (2021) 149:1–19 association between PCSK9’s prodomain and the mature orders was analysed, including 346 species for which PCSK9 protein, and suggested that the topology of that association gene and pseudogene sequences were newly assembled or blocks access to the catalytic triad, thus inhibiting further annotated (Fig. 1). This comprehensive dataset provides a catalytic activity (Piper et al. 2007). general picture of the patterns and timing of pseudogeniza- But how could a proteolytically inactive PCSK9 promote tion events of PCSK9 in placental mammals and the selec- LDLR degradation? The puzzle was eventually solved with tive pressures acting upon the gene in diferent evolutionary the realization that PCSK9 binds LDLR (particularly at lineages. The results suggest that the usefulness of mammal its EGF-A domain) more tightly at low pH than at neutral species as models of cholesterol metabolism-related diseases pH, and the observation that the addition of PCSK9 to cell has more limitations than previously thought. medium leads to a redistribution of LDLR from the plasma membrane to the lysosomes. These results implied that the binding of PCSK9 and its tightening at acidic pH prevent Materials and methods a conformational change in the LDLR that is necessary for its recycling from the endosomes to the plasma membrane, Species selection thus re-directing the LDLR to the lysosomes and leading to its degradation (Cunningham et al. 2007; Zhang et al. 2007; This study aimed at obtaining the broadest possible view of Fisher et al. 2007). the PCSK9 gene status and evolution in eutherian mammals, A protease which has been fne-tuned into having only as provided by publicly available sequence data. All spe- itself as a substrate and acting as something of a chaper- cies for which sequences could be recovered from NCBI’s one in directing a protein for degradation could be seen, RefSeq, Gene and whole-genome shotgun contig (wgs) data- from frst principles, as an exquisite example of evolution- bases, as well as a subset of sequences from the Sequence ary refnement. However, non-functional alleles of PCSK9 Read Archive (SRA) database, as of January 31st, 2019, have been found in some human populations without appar- were included. SRA-recovered data were selected so as to ent negative health efects, even in individuals carrying two have a fair chance of recovering full or nearly full sequences, null alleles (Cohen et al. 2005; Zhao et al. 2006; Hooper and included: (a) whole-exome exon capture data; (b) whole et al. 2007). Moreover, some studies suggested that PCSK9 genome sequencing projects with at least 10× coverage; (c) had undergone pseudogenization in other mammalian spe- RNA-seq data from mRNA isolated from liver or kidney (or cies (Ding et al. 2007; Cameron et al. 2008). Could it be that "mixed tissues" including at least one of these). RNA-seq PCSK9 had become evolutionarily unimportant in mammals, data searches excluded species belonging to clades where and on the path to pseudogenization? Taking advantage of gene loss was thought to be the ancestral state, based on the conserved synteny around the PCSK9 locus from sharks knowledge gathered from genomic data. When sequences to placental mammals, a preliminary survey was taken were available for wild and domesticated individuals of the by counting the number of placental mammal entries in same species, the former were used. Some species were NCBI’s Gene database for PCSK9 and its fanking genes— later excluded from the study due to poor coverage (most BSND and USP24. At the time when this study was initi- frequently observed in exon capture SRA data) or reiter- ated there were 67 entries for PCSK9, 93 for BSND, and ated unavailability of SRA sequences. In the three mem- 94 for USP24, suggesting that a large percentage of species bers of the family Heteromyidae for which data was avail- had lost PCSK9. Interestingly, most potential losses were able, genomic data strongly suggested that a multiplication found among Laurasiatheria, one of the four major clades of the PCSK9 gene had occurred. In every case, it was not of placental mammals. A similar—and rigorously sup- clear how many PCSK9-like genes were present, or if the ported—picture, based on a general analysis of gene losses assembled contigs available were correct and/or represented in a panel of 62 mammals, was recently reported (Sharma the full complement of gene family members. Since it was and Hiller 2020). These results suggested that PCSK9 may beyond the scope of the present work to deal with those have become unimportant, at least in some mammalian line- issues, Heteromyidae was also excluded. Finally, there was ages, but left many unanswered questions about the patterns an instance in which searches against two supposedly difer- of conservation and loss of PCSK9 in placental mammals, ent species (the hoary bamboo rat—Rhyzomys pruinosus— particularly regarding the distribution, frequency, mecha- and the chinese zokor—Eospalax fontanierii) gave the exact nisms and timing of pseudogenization events. Some of these same number of hits which displayed diferent run numbers questions were addressed in the present study by exhaus- but identical spot numbers. Since both samples had been run tively recovering and performing comparative analyses of in the same lab at the same time, and comparison of a few the eutherian PCSK9 sequences available in the NCBI data- sequences