The Genetics of Obesity Meets Basic Cell Biology Through Prohormone Convertase 1/3

Examination of individuals with “extreme phenotypes” has revealed some rare monogenic disorders that were previously unknown. Monogenic obesity is one example of a severe phenotype that associates with mutations from specific genes that play roles in the regulation of metabolism and energy expenditure (1). One of these genes is PCSK1, which encodes a prohormone-processing enzyme, prohormone convertase (PC)1/3, in tissues that produce metabolic hormones. The story of the link between PC1/3 and obesity illustrates the need for basic cell biological investigations to reveal mechanisms of how gene variants can lead to the development of a metabolic disorder. In their article in this issue of Endocrinology, Prabhu et al (2) have taken the cell biology approach in an elegant study to determine the link between a variant of PCSK1, known as N222D, and obesity. PCs are enzymes that process, or convert, prohormones and proproteins into bioactive peptides. Currently, 9 members of the PC family of endoproteolytic enzymes have been identified in mammals (reviewed in Reference 3). Three of them, PC1/3, PC2, and PC5, are found primarily in the secretory granules of endocrine and neuroendocrine tissues in which prohormone processing occurs, and they function optimally in the high-Ca2+, low-pH environment within secretory granules. Both PC1/3 and PC2 have been localized within the secretory granules of the anterior and neurointermediate lobes of the pituitary, pancreatic islets, the endocrine cells of the heart, and neuropeptide-rich regions of the intestine and brain. Both PC1/3 and PC2 act in a concerted manner to cleave prohormones, the peptide hormone products of which regulate several metabolic processes. For example, PC1/3 and PC2 cleave proopiomelanocortin in the anterior pituitary to yield ACTH and α-MSH, respectively (4), which are both key peptides in the regulation of energy expenditure. Proglucagon is processed to glucagon in the α-cells of pancreatic islets by PC2, and to glucagon-like peptide-1 and glucagon-like peptide-2 in intestinal L cells by PC1/3 (5, 6), and these hormones are responsible for nutrient homeostasis. Finally, proinsulin is cleaved in a sequential manner to yield insulin in the pancreatic β-cell. The B chain-C peptide junction is cleaved by PC1/3, followed by cleavage at the C peptide-A chain junction by PC2 (7, 8). These examples illustrate the importance of PC1/3 and PC2 in metabolic regulation and predict disturbances in nutrient homeostasis and energy expenditure upon disruption of their functions. Such disturbances would result in development of diseases such as diabetes, obesity, or metabolic syndrome. Interestingly, impairment of PC1/3 activity, in particular, is strongly linked to the development of early-onset obesity. The PCSK1 gene resides in a 5.6-MB interval within chromosome 5q that links with obesity-associated traits (9, 10). A number of recent genome-wide association studies have identified variants in PCSK1 that are associated with monogenic early-onset obesity (11–13). The elucidation of the role of PC1/3 in the development of obesity has been driven by the initial finding of PC1/3 deficiency in a subject who showed extreme childhood obesity, abnormal glucose homeostasis, hypogonadotrophic hypogonadism, hypocortisolism, and elevated plasma levels of unprocessed proinsulin and proopiomelanocortin (14). This report identified 2 mutations in the PCSK1 gene that caused a deficiency in PC1/3 activity: a heterozygous G483R mutation that prevents removal of the C-terminal tail of PC1/3 and causes its retention in the endoplasmic reticulum (ER); and a frameshift mutation that creates a premature stop codon within the catalytic domain (Figure 1). The recapitulation of the Gly483Arg mutation in a heterologous cell line demonstrated the importance of proper intracellular trafficking of PC1/3 to its biological activity. This key experiment illustrates how cell biological studies can determine the mechanism by which PCSK1 variants could result in the multiple metabolic disturbances. Events governing PC1/3 maturation during transit through the regulated secretory pathway. The structural domains of prepro-PC1/3 consist of the signal peptide (black), the prodomain (orange), catalytic domain (green) containing the catalytic motif DHS and the oxyanion residue N, the P domain (gray), and the C-terminal domain (yellow) containing the sorting domain within amino acids 711–753. Glycosylation sites are marked by ball-and-stick symbols. The signal peptide is removed during translocation into the ER. Within the ER, the prodomain is removed by cleavage at 80RSKR83 (arrow). The N222D mutant is processed at this site and is retained in the ER. Wild-type 87-kDa PC1/3 transits through the Golgi and is targeted to secretory granules by the sorting domain contained within amino acids 711–753. Within granules, 87-kDa PC1/3 is cleaved at 617RR618 (arrow) to yield the soluble 66-kDa form, which then cleaves its prohormone substrates to yield their bioactive forms. How does PC1/3 acquire its biological activity? Several studies, many of which were carried out by the Lindberg group in the 1990s (15, 16), have shown that PC1/3 must undergo a maturation process that is strictly dependent upon proper subcellular trafficking, as illustrated in Figure 1. PC1/3 is initially synthesized as a 91-kDa proenzyme in the ER. ProPC1/3 is quickly converted to an 87-kDa PC1/3 by removal of its prodomain at an 80RSKR83 cleavage site in an autocatalytic, intramolecular manner in the ER. The 87-kDa full-length PC1/3, which has enzymatic activity, then transits through the Golgi. Upon reaching the trans-Golgi network, PC1/3 is targeted to the secretory granules of the regulated secretory pathway through sorting information contained within its C-terminal domain. Specifically, residues 711–753 contain 2 α-helices that anchor PC1/3 to the trans-Golgi network membrane through a hydrophobic patch and aggregate the C-terminal domain in a Ca2+-dependent manner (17). Once in the immature granule, PC1/3 is processed at a 617RR618 site at its C-terminal end in an autocatalytic manner to yield a soluble 64- to 66-kDa form. The catalytic activity of 64- to 66-kDa PC1/3 is optimal at pH 5.0–5.5, the pH of the mature and immature secretory granules, respectively. Whereas the 64- to 66-kDa form of PC1/3 is secreted as soluble cargo, a significant proportion of PC1/3 remains membrane associated within the immature and mature secretory granule. Mutations that interfere with this maturation process may be linked with obesity-associated metabolic disorders. The N222D mutation was found in an obese mouse that phenocopies the PC1/3 deficiency in human obesity (18), in contrast to mice haplodeficient in PC1/3, which do not develop obesity (19). The site of the mutation is right next to a known human variant, N221D (rs6232), that associates with increased risk of obesity (11, 13), making this mouse the only known obese mouse model harboring a mutation in PC1/3 that is similar to a human gene variant. Prabhu et al first carried out experiments in islets from these mice and showed that proinsulin processing was defective at the B chain-C peptide junction, which was predicted, demonstrating that the N222D mutation severely diminished the activity of PC1/3. They then expressed the N222D mutation in cell lines to more closely examine the mechanism behind the diminished activity. A previous study had attributed the reduced activity of N222D solely to an impaired maturation process (18). To circumvent this process, Prabhu et al generated the N222D mutation within an “uncleavable” PC1/3 by also mutating 617RR618 and 654RR655 in the C-terminal end. This additional manipulation ensured that any trafficking defects would be due solely to the N222D mutation and not to differential cleavages related to maturation of the enzyme. With this strategy, Prabhu et al showed that the N222D mutation indeed accumulated in the ER in both an insulin-secreting cell line and Neuro2a cells. However, they also found that processing of PC1/3 at 80RSKR83 to remove the prodomain remained intact. This finding demonstrates that the impairment in cellular trafficking is due solely to the N222D mutation and not to effects on maturation, and that this behavior is not cell type specific. What happens when N222D PC1/3 is retained in the ER? One would assume that the protein is misfolded and targeted for disposal by ER-associated degradation. However, the story with N222D PC1/3 is not that simple. Although a substantial proportion of the mutant enzyme is indeed ubiquitinated and targeted for proteosomal degradation, Prabhu et al showed that N222D could also associate with wild-type PC1/3 in the ER and exert a small dominant-negative effect on its intracellular transport. In addition, they found that a proportion of the N222D mutant is secreted but does not contain any enzymatic activity. This finding is interesting in light of the fact that the 87-kDa form of PC1/3 is enzymatically active in the distal compartments of the Golgi and secretory granules (20). That the mutation occurs in a conserved Ca2+-binding site may explain this apparent discrepancy, because the activities of both the 87-kDa and 66-kDa forms of PC1/3 are Ca2+ dependent, as previously shown by Lindberg’s group (16). Therefore, the picture that emerges is one in which a mutation in PC1/3 renders it enzymatically inactive and prevents its normal transit though the secretory pathway independently of the maturation process. Such dramatic aberrations in the normal trafficking and activation patterns of PC1/3 result in impaired proinsulin processing, which is characteristic of metabolic disorders such as diabetes and obesity. The study by Prabhu et al highlights the need for mechanistic cell biological studies that extend beyond the usual analysis of PC1/3 maturation events. High-throughput next-generation sequencing is now identifying many more mutations in genes associated with obesity (21, 22), and future cell biology studies will address how other identified variants in the PCSK1 gene are linked to the development of early-onset obesity and other metabolic disorders. Disclosure Summary: The author has nothing to disclose. endoplasmic reticulum prohormone convertase.

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