Revisiting PC1/3 Mutants: Dominant-Negative Effect of Endoplasmic Reticulum-Retained Mutants

Abstract

Prohormone convertase 1/3 (PC1/3), encoded by the gene PCSK1, is critical for peptide hormone synthesis. An increasing number of studies have shown that inactivating mutations in PCSK1 are correlated with endocrine pathologies ranging from intestinal dysfunction to morbid obesity, whereas the common nonsynonymous polymorphisms rs6232 (N221D) and rs6234-rs6235 (Q665E-S690T) are highly associated with obesity risk. In this report, we revisited the biochemical and cellular properties of PC1/3 variants in the context of a wild-type PC1/3 background instead of the S357G hypermorph background used for all previous studies. In the wild-type background the PC1/3 N221D variant exhibited 30% lower enzymatic activity in a fluorogenic assay than wild-type PC1/3; this inhibition was greater than that detected in an equivalent experiment using the PC1/3 S357G background. A PC1/3 variant with the linked carboxyl-terminal polymorphisms Q665E-S690T did not show this difference. We also analyzed the biochemical properties of 2 PC1/3 mutants, G209R and G593R, which are retained in the endoplasmic reticulum (ER), and studied their effects on wild-type PC1/3. The expression of ER-retained mutants induced ER stress markers and also resulted in dominant-negative blockade of wild-type PC1/3 prodomain cleavage and decreased expression of wild-type PC1/3, suggesting facilitation of the entry of wild-type protein to a degradative proteasomal pathway. Dominant-negative effects of PC1/3 mutations on the expression and maturation of wild-type protein, with consequential effects on PC1/3 availability, add a new element which must be considered in population and clinical studies of this gene.

PCSK1 Mutations and Human Endocrinopathies: From Obesity to Gastrointestinal Disorders

“…Creemers et al proposed that mutations in close proximity of this calcium-binding site could alter enzyme stability which could lead to protein misfolding and thereby decreasing the amount of active enzyme (73). Recent evidence from our group and the Lindberg group confirmed that some PCSK1 mutations lead to protein instability (89,205). In particular, our results indicate that several of the identified heterozygous mutations caused a delayed exit of the mutant protein from the cell (205).…”

“…As described above, the inhibitory function of PC1/3 prodomain was already established in vitro (54,56,57). The earlier described PCSK1-p.G209R mutant which was shown to retain the PCSK1-p.P258T hypomorph in the ER, could have a similar limited dominant negative effect (89). Aside from a clear relation with increased BMI in the reported patients, it remains unclear whether heterozygosity can lead to other endocrinopathies which are reported for null patients.…”

“…The dimers and oligomers are constituted of 87 kDa PC1/3 and incubation with substrate stabilizes PC1/3 activity. Interestingly, Blanco et al recently demonstrated that some ER-retained PC1/3 mutants inhibited PC1/3-WT secretion and activity (89). The proposed mechanism to explain this effect was that ER-retained mutants can oligomerize with PC1/3-WT in the ER lumen, and the resulting accumulated heterocomplex can be targeted to degradation via ERAD (Figure 3).…”

“…However, the patient with the PCSK1-p.P258T mutation was compound homozygous for both the deleterious PCSK1-p.G209R mutation and the PCSK1-p.P258T mutation. Coexpression of the two mutant proteins in vitro showed that the catalytically inactive PCSK1-p.G209R mutant retains the PCSK1-p.P258T in the ER and therefore renders it inactive against substrates in trans (89). The expression of the different symptoms of the syndrome varies between patients.…”

“…However rs6232 is extremely rare in nonCaucasian ethnicities (ϳ5% in Caucasian). In vitro assays showed that the PCSK1-p.N221D variation causes a significant decrease in enzymatic activity (89,97). The PCSK1-p.Q665E-p.S690T variation was not shown to decrease enzymatic activity but could have an effect on protein sorting and stability as it is located in the C-terminal domain (98).…”