Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Role of Partitioning-defective 1/Microtubule Affinity-regulating Kinases in the Morphogenetic Activity of Helicobacter pylori CagA

Helicobacter pylori CagA plays a key role in gastric carcinogenesis. Upon delivery into gastric epithelial cells, CagA binds and deregulates SHP-2 phosphatase, a bona fide oncoprotein, thereby causing sustained ERK activation and impaired focal adhesions. CagA also binds and inhibits PAR1b/MARK2, one of the four members of the PAR1 family of kinases, to elicit epithelial polarity defect. In nonpolarized gastric epithelial cells, CagA induces the hummingbird phenotype, an extremely elongated cell shape characterized by a rear retraction defect. This morphological change is dependent on CagA-deregulated SHP-2 and is thus thought to reflect the oncogenic potential of CagA. In this study, we investigated the role of the PAR1 family of kinases in the hummingbird phenotype. We found that CagA binds not only PAR1b but also other PAR1 isoforms, with order of strength as follows: PAR1b > PAR1d ≥ PAR1a > PAR1c. Binding of CagA with PAR1 isoforms inhibits the kinase activity. This abolishes the ability of PAR1 to destabilize microtubules and thereby promotes disassembly of focal adhesions, which contributes to the hummingbird phenotype. Consistently, PAR1 knockdown potentiates induction of the hummingbird phenotype by CagA. The morphogenetic activity of CagA was also found to be augmented through inhibition of non-muscle myosin II. Because myosin II is functionally associated with PAR1, perturbation of PAR1-regulated myosin II by CagA may underlie the defect of rear retraction in the hummingbird phenotype. Our findings reveal that CagA systemically inhibits PAR1 family kinases and indicate that malfunctioning of microtubules and myosin II by CagA-mediated PAR1 inhibition cooperates with deregulated SHP-2 in the morphogenetic activity of CagA.Infection with Helicobacter pylori strains bearing cagA (cytotoxin-associated gene A)-positive strains is the strongest risk factor for the development of gastric carcinoma, the second leading cause of cancer-related death worldwide (1–3). The cagA gene is located within a 40-kb DNA fragment, termed the cag pathogenicity island, which is specifically present in the genome of cagA-positive H. pylori strains (4–6). In addition to cagA, there are ∼30 genes in the cag pathogenicity island, many of which encode a bacterial type IV secretion system that delivers the cagA-encoded CagA protein into gastric epithelial cells (7–10). Upon delivery into gastric epithelial cells, CagA is localized to the plasma membrane, where it undergoes tyrosine phosphorylation at the C-terminal Glu-Pro-Ile-Tyr-Ala motifs by Src family kinases or c-Abl kinase (11–14). The C-terminal Glu-Pro-Ile-Tyr-Ala-containing region of CagA is noted for the structural diversity among distinct H. pylori isolates. Oncogenic potential of CagA has recently been confirmed by a study showing that systemic expression of CagA in mice induces gastrointestinal and hematological malignancies (15).When expressed in gastric epithelial cells, CagA induces morphological transformation termed the hummingbird phenotype, which is characterized by the development of one or two long and thin protrusions resembling the beak of the hummingbird. It has been thought that the hummingbird phenotype is related to the oncogenic action of CagA (7, 16–19). Pathophysiological relevance for the hummingbird phenotype in gastric carcinogenesis has recently been provided by the observation that infection with H. pylori carrying CagA with greater ability to induce the hummingbird phenotype is more closely associated with gastric carcinoma (20–23). Elevated motility of hummingbird cells (cells showing the hummingbird phenotype) may also contribute to invasion and metastasis of gastric carcinoma.In host cells, CagA interacts with the SHP-2 phosphatase, C-terminal Src kinase, and Crk adaptor in a tyrosine phosphorylation-dependent manner (16, 24, 25) and also associates with Grb2 adaptor and c-Met in a phosphorylation-independent manner (26, 27). Among these CagA targets, much attention has been focused on SHP-2 because the phosphatase has been recognized as a bona fide oncoprotein, gain-of-function mutations of which are found in various human malignancies (17, 18, 28). Stable interaction of CagA with SHP-2 requires CagA dimerization, which is mediated by a 16-amino acid CagA-multimerization (CM)² sequence present in the C-terminal region of CagA (29). Upon complex formation, CagA aberrantly activates SHP-2 and thereby elicits sustained ERK MAP kinase activation that promotes mitogenesis (30). Also, CagA-activated SHP-2 dephosphorylates and inhibits focal adhesion kinase (FAK), causing impaired focal adhesions. It has been shown previously that both aberrant ERK activation and FAK inhibition by CagA-deregulated SHP-2 are involved in induction of the hummingbird phenotype (31).Partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) is an evolutionally conserved serine/threonine kinase originally isolated in C. elegans (32–34). Mammalian cells possess four structurally related PAR1 isoforms, PAR1a/MARK3, PAR1b/MARK2, PAR1c/MARK1, and PAR1d/MARK4 (35–37). Among these, PAR1a, PAR1b, and PAR1c are expressed in a variety of cells, whereas PAR1d is predominantly expressed in neural cells (35, 37). These PAR1 isoforms phosphorylate microtubule-associated proteins (MAPs) and thereby destabilize microtubules (35, 38), allowing asymmetric distribution of molecules that are involved in the establishment and maintenance of cell polarity.In polarized epithelial cells, CagA disrupts the tight junctions and causes loss of apical-basolateral polarity (39, 40). This CagA activity involves the interaction of CagA with PAR1b/MARK2 (19, 41). CagA directly binds to the kinase domain of PAR1b in a tyrosine phosphorylation-independent manner and inhibits the kinase activity. Notably, CagA binds to PAR1b via the CM sequence (19). Because PAR1b is present as a dimer in cells (42), CagA may passively homodimerize upon complex formation with the PAR1 dimer via the CM sequence, and this PAR1-directed CagA dimer would form a stable complex with SHP-2 through its two SH2 domains.Because of the critical role of CagA in gastric carcinogenesis (7, 16–19), it is important to elucidate the molecular basis underlying the morphogenetic activity of CagA. In this study, we investigated the role of PAR1 isoforms in induction of the hummingbird phenotype by CagA, and we obtained evidence that CagA-mediated inhibition of PAR1 kinases contributes to the development of the morphological change by perturbing microtubules and non-muscle myosin II.     

��-Defensins in Enteric Innate Immunity: FUNCTIONAL PANETH CELL ��-DEFENSINS IN MOUSE COLONIC LUMEN*

Paneth cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric α-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric α-defensins of Paneth cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative α-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as α-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1–4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth cell α-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts.Antimicrobial peptides (AMPs)² are released by epithelial cells onto mucosal surfaces as effectors of innate immunity (1–5). In mammals, most AMPs derive from two major families, the cathelicidins and defensins (6). The defensins comprise the α-, β-, and θ-defensin subfamilies, which are defined by the presence of six cysteine residues paired in characteristic tridisulfide arrays (7). α-Defensins are highly abundant in two primary cell lineages: phagocytic leukocytes, primarily neutrophils, of myeloid origin and Paneth cells, which are secretory epithelial cells located at the base of the crypts of Lieberkühn in the small intestine (8–10). Neutrophil α-defensins are stored in azurophilic granules and contribute to non-oxidative microbial cell killing in phagolysosomes (11, 12), except in mice whose neutrophils lack defensins (13). In the small bowel, α-defensins and other host defense proteins (14–18) are released apically as components of Paneth cell secretory granules in response to cholinergic stimulation and after exposure to bacterial antigens (19). Therefore, the release of Paneth cell products into the crypt lumen is inferred to protect mitotically active crypt cells from colonization by potential pathogens and confer protection against enteric infection (7, 20, 21).Under normal, homeostatic conditions, Paneth cells are not found outside the small bowel, although they may appear ectopically in response to local inflammation throughout the gastrointestinal tract (22, 23). Paneth cell numbers increase progressively throughout the small intestine, occurring at highest numbers in the distal ileum (24). Mouse Paneth cells express numerous α-defensin isoforms, termed cryptdins (Crps) (25), that have broad spectrum antimicrobial activities (6, 26). Collectively, α-defensins constitute approximately seventy percent of the bactericidal peptide activity in mouse Paneth cell secretions (19), selectively killing bacteria by membrane-disruptive mechanisms (27–30). The role of Paneth cell α-defensins in gastrointestinal mucosal immunity is evident from studies of mice transgenic for human enteric α-defensin-5, HD-5, which are immune to infection by orally administered Salmonella enterica sv. typhimurium (S. typhimurium) (31).The biosynthesis of mature, bactericidal α-defensins from their inactive precursors requires activation by lineage-specific proteolytic convertases. In mouse Paneth cells, inactive ∼8.4-kDa Crp precursors are processed intracellularly into microbicidal ∼4-kDa Crps by specific cleavage events mediated by matrix metalloproteinase-7 (MMP-7) (32, 33). MMP-7 null mice exhibit increased susceptibility to systemic S. typhimurium infection and decreased clearance of orally administered non-invasive Escherichia coli (19, 32). Although the α-defensin proregions are sensitive to proteolysis, the mature, disulfide-stabilized peptides resist digestion by their converting enzymes in vitro, whether the convertase is MMP-7 (32), trypsin (34), or neutrophil serine proteinases (35). Because α-defensins resist proteolysis in vitro, we hypothesized that Paneth cell α-defensins resist degradation and remain in a functional state in the large bowel, a complex, hostile environment containing varied proteases of both host and microbial origin.Here, we report on the isolation and characterization of a population of enteric α-defensins from the mouse colonic lumen. Full-length and N-terminally truncated Paneth cell α-defensins were identified and are abundant in the distal large bowel lumen.