Donald F. Steiner

The proprotein convertases

The major endoproteolytic processing enzymes of the secretory pathway are the subtilisin-like proprotein convertases (SPCs). Furin (SPC1) has emerged as one of the major processing enzymes of the constitutive secretory pathway and its localization in the trans-Golgi network and mechanism of autoactivation have been studied in considerable detail. Recent gene disruption experiments and the study of naturally-occurring mutations underscore the importance of PC2 (prohormones convertase 2, or SPC2) and PC1/PC3 (prohormone convertase 1/3, or SPC3) in the processing of a wide variety of hormone and neuropeptide precursors. The role of Carboxypeptidase E (CPE) in the removal of carboxy-terminal basic residues exposed by the endoproteases was shown to be necessary for efficient endoproteolytic processing of proinsulin and several other protein precursors. Many biologically active peptides are also amidated after their proteolytic processing by peptidylglycine alpha-amidating monooxygenase (PAM) and recent X-ray studies of the peptidyl alpha-hydroxylating monooxygenase component of PAM have shed new light on the role of copper in the mechanism of this reaction.

Hyperproinsulinaemia in obese fat/fat mice associated with a carboxypeptidase E mutation which reduces enzyme activity.

Mice homozygous for the fat mutation develop obesity and hyperglycaemia that can be suppressed by treatment with exogenous insulin. The fat mutation maps to mouse chromosome 8, very close to the gene for carboxypeptidase E (Cpe), which encodes an enzyme (CPE) that processes prohormone intermediates such as proinsulfn. We now demonstrate a defect in proinsulin processing associated with the virtual absence of CPE activity in extracts of fat/fat pancreatic islets and pituitaries. A single Ser202Pro mutation distinguishes the mutant Cpe allele, and abolishes enzymatic activity in vitro. Thus, the fat mutation represents the first demonstration of an obesity–diabetes syndrome elicited by a genetic defect in a prohormone processing pathway.

Determination of free and total insulin and C-peptide in insulin-treated diabetics.

Serum levels of free and total insulin as well as total C-peptide immunoreactivity (C-peptide and proinsulin) and C-peptide were measured in insulin-treated diabetics with circulating insulin antibodies by the addition of polyethylene glycol (PEG) before and after acidification. PEG resulted in complete precipitation of insulin antibodies from serum and made it possible to measure free insulin in the supernatant. Incubation of serum at 37δ C. for two hours before addition of PEG resulted in values for free insulin that probably resembled the in-vivo levels most closely. The same method could also be used to remove proinsulin bound to circulating insulin antibodies and permitted the measurement of C-peptide in the supernatant. Clinical studies using this approach indicate that combined measurements of serum free and total insulin and C-peptide provide information that is helpful in understanding the contribution of endogenous and exogenous insulin to the course and metabolic control of insulin-requiring diabetic patients.

Proteolytic processing in the secretory pathway.

Many cellular processes, including embryogenesis (1–4), gene expression (5), cell cycle (6), programmed cell death (7), intracellular protein targeting (8) and endocrine/neural functions (9–13) are regulated by limited proteolysis of precursor proteins (14, 15). These functions are carried out by proteolytic enzyme families that are strategically localized within cells or on cell surfaces (3, 5–7, 9, 10). This review focuses on the serine proteases that process protein precursors (proproteins) traversing the secretory pathway (for recent reviews, see Refs. 9, 11–13, 16–19, 62). The early development of this field is reviewed in Ref. 14.

Insulin gene mutations as a cause of permanent neonatal diabetes

We report 10 heterozygous mutations in the human insulin gene in 16 probands with neonatal diabetes. A combination of linkage and a candidate gene approach in a family with four diabetic members led to the identification of the initial INS gene mutation. The mutations are inherited in an autosomal dominant manner in this and two other small families whereas the mutations in the other 13 patients are de novo. Diabetes presented in probands at a median age of 9 weeks, usually with diabetic ketoacidosis or marked hyperglycemia, was not associated with β cell autoantibodies, and was treated from diagnosis with insulin. The mutations are in critical regions of the preproinsulin molecule, and we predict that they prevent normal folding and progression of proinsulin in the insulin secretory pathway. The abnormally folded proinsulin molecule may induce the unfolded protein response and undergo degradation in the endoplasmic reticulum, leading to severe endoplasmic reticulum stress and potentially β cell death by apoptosis. This process has been described in both the Akita and Munich mouse models that have dominant-acting missense mutations in the Ins2 gene, leading to loss of β cell function and mass. One of the human mutations we report here is identical to that in the Akita mouse. The identification of insulin mutations as a cause of neonatal diabetes will facilitate the diagnosis and possibly, in time, treatment of this disorder.

Islet-Cell-Surface Antibodies in Juvenile Diabetes Mellitus

Using an indirect immunofluorescence test on suspensions of viable, insulin-producing islet cells from rats, we found that 32 per cent (28/88) of insulin-treated patients with juvenile diabetes have islet-cell-surface antibodies in their circulation. These antibodies also occurred in four of nine children with glucose intolerance, in one of 24 healthy children and in nondiabetic children with thyroid disorders. In the diabetic children, the immunofluorescent reaction was inhibited by preadsorption of serum to islet cells but was little affected by preadsorption to rat hepatocytes or erythrocytes or to acetone powders of various rat tissues, including pancreas. These results show that organ-specific, nonspecies-specific antibodies reactive with the cell surface of the islet cells can be present in serum from diabetic children, and provide an approach to investigation of immunopathological aspects of diabetes mellitus.

Proteolytic Processing Mechanisms in the Biosynthesis of Neuroendocrine Peptides: The Subtilisin-like Proprotein Convertases

The recent discovery of a novel family of precursor processing endoproteases has greatly accelerated progress in understanding the complex mechanisms underlying the maturation of prohormones, neuropeptides, and many other precursor-derived proteins. At least six members of this family have been found thus far in mammalian species, several having alternatively spliced isoforms, and related enzymes have been identified in many invertebrates, including molluscs, insects, nematodes, and coelenterates. The proprotein convertases are all dependent on calcium for activity and all possess highly conserved subtilisin-like domains with the characteristic catalytic triad of this serine protease (ordered Asp, His, and Ser along the polypeptide chain). Two members of this family, PC2 (SPC2) and PC1/PC3 (SPC3), appear to play a preeminent role in neuroendocrine precursor processing. Both convertases are expressed only in the brain and in the extended neuroendocrine system, while another important family member--furin/PACE (SPC1)--is expressed more ubiquitously, in almost all tissues, and at high levels in liver. SPC2 and SPC3 exhibit acidic pH optima and other properties which enhance their activity in the acidic, calcium-enriched environment of the dense-core secretory granules of the regulated pathway in neuroendocrine cells, while furin has a neutral pH optimum and is localized predominantly to the trans Golgi network where it is retained by a C-terminal transmembrane domain. Furin processes a wide variety of precursors in the constitutive pathway, such as those of growth factors, receptors, coagulation factors, and viral glycoproteins. Recent findings on the processing of proopiomelanocortin, proinsulin, proglucagon, and several other neuroendocrine precursors by SPC2 and SPC3 are discussed, along with information on the structure, properties, evolution, developmental expression, and regulation of the convertases. An inherited defect in the fat/fat mouse which affects the processing of proinsulin, and probably also many other prohormones, due to a point mutation in carboxypeptidase E has recently been identified and has begun to provide new insights into the functional integration of the individual processing steps.

PROCESSING MECHANISMS IN THE BIOSYNTHESIS OF PROTEINS

Limited proteolysis is a widely occurring mechanism in protein biosynthesis. Protein precursors can be classified according to their functions, localization within cell compartments, and enzymic cleavage mechanisms. The presecretory proteins represent an important class of very rapidly turning over precursors which play an early role in the sequestration of secretory products and whose cleavage appears to be intimately associated with structures formed at the ribosome-membrane junction during protein synthesis. A model is proposed which predicts that the prepeptide forms a beta-pleated sheet structure with other components of the membrane which results in the transfer of a loop of peptide across the microsomal membrane. Proinsulin is representative of the general class of proproteins that are processed post-translationally within their secretory cells either during the formation and maturation of secretory granules (peptides hormones and neurotransmitters, serum albumins) or during the assembly of macromolecular structures (virus capsules, membrane-associated enzyme complexes). The former group are cleaved by Golgi-associated proteases having tryptic and carboxypeptidase B-like specificity. Some precursors are secreted as such and processed extracellularly either in the circulation or at special sites (procollagens, zymogens, provenoms, vitellogenins).

Disruption of PC1/3 expression in mice causes dwarfism and multiple neuroendocrine peptide processing defects

The subtilisin-like proprotein convertases PC1/3 (SPC3) and PC2 (SPC2) are believed to be the major endoproteolytic processing enzymes of the regulated secretory pathway. They are expressed together or separately in neuroendocrine cells throughout the brain and dispersed endocrine system in both vertebrates and invertebrates. Disruption of the gene-encoding mouse PC1/3 has now been accomplished and results in a syndrome of severe postnatal growth impairment and multiple defects in processing many hormone precursors, including hypothalamic growth hormone-releasing hormone (GHRH), pituitary proopiomelanocortin to adrenocorticotropic hormone, islet proinsulin to insulin and intestinal proglucagon to glucagon-like peptide-1 and -2. Mice lacking PC1/3 are normal at birth, but fail to grow normally and are about 60% of normal size at 10 weeks. They lack mature GHRH, have low pituitary growth hormone (GH) and hepatic insulin-like growth factor-1 mRNA levels and resemble phenotypically the “little” mouse (Gaylinn, B. D., Dealmeida, V. I., Lyons, C. E., Jr., Wu, K. C., Mayo, K. E. & Thorner, M. O. (1999) Endocrinology 140, 5066–5074) that has a mutant GHRH receptor. Despite a severe defect in pituitary proopiomelanocortin processing to mature adrenocorticotropic hormone, blood corticosterone levels are essentially normal. There is marked hyperproinsulinemia but without impairment of glucose tolerance. In contrast, PC2-null mice lack mature glucagon and are chronically hypoglycemic (Furuta, M., Yano, H., Zhou, A., Rouille, Y., Holst, J., Carroll, R., Ravazzola, M., Orci, L., Furuta, H. & Steiner, D. (1997) Proc. Natl. Acad. Sci. USA 94, 6646–6651). The PC1/3-null mice differ from a human subject reported with compound heterozygosity for defects in this gene, who was of normal stature but markedly obese from early life. The PC1/3-null mice are not obese. The basis for these phenotypic differences is an interesting topic for further study. These findings prove the importance of PC1/3 as a key neuroendocrine convertase.

Insulin Mutation Screening in 1,044 Patients With Diabetes: Mutations in the INS Gene Are a Common Cause of Neonatal Diabetes but a Rare Cause of Diabetes Diagnosed in Childhood or Adulthood

OBJECTIVE— Insulin gene (INS) mutations have recently been described as a cause of permanent neonatal diabetes (PND). We aimed to determine the prevalence, genetics, and clinical phenotype of INS mutations in large cohorts of patients with neonatal diabetes and permanent diabetes diagnosed in infancy, childhood, or adulthood. RESEARCH DESIGN AND METHODS— The INS gene was sequenced in 285 patients with diabetes diagnosed before 2 years of age, 296 probands with maturity-onset diabetes of the young (MODY), and 463 patients with young-onset type 2 diabetes (nonobese, diagnosed <45 years). None had a molecular genetic diagnosis of monogenic diabetes. RESULTS— We identified heterozygous INS mutations in 33 of 141 probands diagnosed at <6 months, 2 of 86 between 6 and 12 months, and none of 58 between 12 and 24 months of age. Three known mutations (A24D, F48C, and R89C) account for 46% of cases. There were six novel mutations: H29D, L35P, G84R, C96S, S101C, and Y103C. INS mutation carriers were all insulin treated from diagnosis and were diagnosed later than ATP-sensitive K+ channel mutation carriers (11 vs. 8 weeks, P < 0.01). In 279 patients with PND, the frequency of KCNJ11, ABCC8, and INS gene mutations was 31, 10, and 12%, respectively. A heterozygous R6C mutation cosegregated with diabetes in a MODY family and is probably pathogenic, but the L68M substitution identified in a patient with young-onset type 2 diabetes may be a rare nonfunctional variant. CONCLUSIONS— We conclude that INS mutations are the second most common cause of PND and a rare cause of MODY. Insulin gene mutation screening is recommended for all diabetic patients diagnosed before 1 year of age.