In pancreatic islets, formation of beta-secretory granule cores involves early proinsulin homohexamerization and subsequent insulin condensation. We examined proinsulin conformational maturation by monitoring accessibility of protein disulfide bonds. Proinsulin disulfides are intact immediately upon synthesis, but are > or = 90% sensitive to in vivo reduction with 2 mM dithiothreitol; wash out of dithiothreitol leads to reoxidation, proinsulin transport, and conversion to insulin. With t1/2 approximately 10 min, newly synthesized proinsulin becomes resistant to disulfide reduction, correlating with endoplasmic reticulum (ER) export. However, inhibition of ER export with brefeldin A blocks acquisition of resistance to reduction, and once proinsulin arrives in the Golgi, it resists reduction despite brefeldin treatment. Moreover, in vivo, resistance of proinsulin disulfides is overcome after increasing [dithiothreitol] > 10-fold, or in vitro, in islets lysed in a zinc-free, but not a zinc-containing, medium. Employing 30 mM dithiothreitol in vivo, a further decrease in disulfide accessibility is observed following proinsulin conversion to insulin. Incubation of islets with chloroquine or zinc enhances and diminishes accessibility of insulin disulfides, respectively. We hypothesize that two major conformational changes culminating in granule core formation, proinsulin hexamerization and insulin condensation, are sensitive to zinc and occur upon ER exit and arrival in immature secretory granules, respectively.

For several secretory proteins, it has been hypothesized that disulfide-bonded loop structures are required for sorting to secretory granules. To explore this hypothesis, we employed dithiothreitol (DTT) treatment in live pancreatic islets, as well as in PC-12 and GH4C1cells. In islets, disulfide reduction in the distal secretory pathway did not increase constitutive or constitutive-like secretion of proinsulin (or insulin). In PC-12 cells, DTT treatment caused a dramatic increase in unstimulated secretion of newly synthesized chromogranin B (CgB), presumably as a consequence of reducing the single conserved chromogranin disulfide bond (E. Chanat, U. Weiss, W. B. Huttner, and S. A. Tooze. EMBO J.12: 2159–2168, 1993). However, in GH4C1cells that also synthesize CgB endogenously, DTT treatment reduced newly synthesized prolactin and blocked its export, whereas newly synthesized CgB was routed normally to secretory granules. Moreover, on transient expression in GH4C1cells, CgA and a CgA mutant lacking the conserved disulfide bond showed comparable multimeric aggregation properties and targeting to secretory granules, as measured by stimulated secretion assays. Thus the conformational perturbation of regulated secretory proteins caused by disulfide disruption leads to consequences in protein trafficking that are both protein and cell type dependent.

No abstract

To examine the possibility of independent cytoplasmic/transmembrane domain-based apical sorting, we have investigated paramyxovirus SV5 hemagglutininneuraminidase (HN), a type II membrane protein with a small N-terminal signal/anchor region. In SV5-infected Madin-Darby canine kidney (MDCK) cells, >90% of HN is found on the apical surface. We have expressed chimeric proteins in which the N terminus of HN, including its signal/anchor region, is attached to a (normally cytosolic) reporter pyruvate kinase (PK). PK itself expressed immediately downstream from a cleavable signal peptide was converted to a 58-kDa N-linked glycosylated form, which was secreted predominantly (80%) to the basolateral surface of MDCK cells. By contrast, stably expressed PK chimeras, now anchored as type II membrane proteins with either the first 48 or 72 amino acids of HN, received similar N-linked glycosylation, yet exhibited polarized transport with a preferentially (75%) apical distribution. These results suggest that the Nterminal signal/anchor region of HN contains independent sorting information for apical specific targeting in MDCK cells.

We developed a microplate-based enhanced chemiluminescence system for the direct detection of circulating miRNAs. The system exhibited a high target sensitivity and specificity, with a detection limit of 3.02 fM.