Endoplasmic reticulum quality control of LDLR variants associated with familial hypercholesterolemia

Abstract

Loss‐of‐function mutations in the low‐density lipoprotein receptor (LDLR) gene can cause familial hypercholesterolemia (FH), but detailed functional evidence for pathogenicity is limited to a few reported mutations. Here, we investigated the cellular pathogenic mechanisms of three mutations in LDLR causing FH, which are structurally identical to pathogenic mutations in the very low‐density lipoprotein receptor (VLDLR). Similar to the VLDLR mutants, LDLR mutants D482H and C667F were found to be localized to the ER, while D445E, which is a conserved amino acid change, did not affect the trafficking of the receptor to the plasma membrane, as confirmed by the N‐glycosylation profile. Although the ER‐retained mutant proteins were soluble, induction of ER stress was observed as indicated by spliced X‐box binding protein‐1 (XBP‐1) mRNA levels. The mutants were found to associate with ER quality control components, and their stability was enhanced by inhibitors of proteasome. Our results contribute to the growing list of transport‐deficient class II LDLR variants leading to FH and provide evidence for the involvement of endoplasmic reticulum‐associated degradation in their stability.

Proteostasis Regulation in the Endoplasmic Reticulum: An Emerging Theme in the Molecular Pathology and Therapeutic Management of Familial Hypercholesterolemia

“…Other chaperones GRP94, ERP72 (PDIA4), and CNX have also been found to associate with class II mutants (G544V) but not with wild type LDLR ( Sørensen et al, 2006 ). Recently we have also reported that three LDLR class II variants were found to be associated with ER chaperones: GRP78 (BiP), GRP94, the lectin chaperone CNX ( Kizhakkedath et al, 2019 ). In cells overexpressing the G544V mutant, the ER-retention of the mutant was shown to induce ER-stress and activation of UPR as evidenced by the upregulation of mRNAs for GRP78, GRP94, ERP72, attributed to the activity of ER sensors IRE1 and PERK ( Sørensen et al, 2006 ).…”

“…Many membrane and secretory proteins that fail to conform to the ER quality control (ERQC) are dislocated into the cytosol and degraded by the proteasome by a process termed as ER-associated degradation (ERAD) ( Vembar and Brodsky, 2008 ; Ruggiano et al, 2014 ; Sun and Brodsky, 2019 ). Misfolded proteins can still retain their function and premature ERAD of mutant misfolded proteins is accounted for the cellular pathogenesis of several congenital disorders ( Ward et al, 1995 ; Hume et al, 2009 ; Ali et al, 2011 ; Al-Kindi et al, 2014 ; Kizhakkedath et al, 2014 , 2019 ; John et al, 2015 ). Sometimes the quality control mechanisms fail to recognize folding-incompetent forms which leads to the accumulation of folding-intermediates in the ER, causing ER stress.…”

“…Very little information is available on the ERAD components involved in the substrate recognition, retrotranslocation and degradation of LDLR mutants. Recent results from our lab indicate that the LDLR mutants interact with HRD1 and its partners SEL1L and OS9 ( Kizhakkedath et al, 2019 ). Our results also demonstrated that proteasomal inhibition leads to stabilization of the ER-retained mutants, but had no effect on their folding.…”

“…Our results also demonstrated that proteasomal inhibition leads to stabilization of the ER-retained mutants, but had no effect on their folding. Further, inhibitors of ER mannosidase 1 also had a stabilizing effect on the mutants ( Kizhakkedath et al, 2019 ). ER-retained variants of VLDLR, another LDLR family member, were also found to be degraded by the HRD1-SEL1L mediated proteasomal degradation ( Kizhakkedath et al, 2018 ).…”

“…ER-retained variants of VLDLR, another LDLR family member, were also found to be degraded by the HRD1-SEL1L mediated proteasomal degradation ( Kizhakkedath et al, 2018 ). Unlike VLDLR mutants, the ER retained LDLR mutants were not observed to be aggregation-prone, though overexpression of mutants caused ER stress ( Kizhakkedath et al, 2019 ). The ERAD adaptor protein SEL1L is reported to also play an ERAD independent role in the maturation and processing of lipoprotein lipase (LPL) and hepatic lipid metabolism ( Sha et al, 2014 ).…”