Carbon monoxide differentially inhibits TLR signaling pathways by regulating ROS-induced trafficking of TLRs to lipid rafts.

Carbon monoxide (CO), a byproduct of heme catabolism by heme oxygenase (HO), confers potent antiinflammatory effects. Here we demonstrate that CO derived from HO-1 inhibited Toll-like receptor (TLR) 2, 4, 5, and 9 signaling, but not TLR3-dependent signaling, in macrophages. Ligand-mediated receptor trafficking to lipid rafts represents an early event in signal initiation of immune cells. Trafficking of TLR4 to lipid rafts in response to LPS was reactive oxygen species (ROS) dependent because it was inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase, and in gp91(phox)-deficient macrophages. CO selectively inhibited ligand-induced recruitment of TLR4 to lipid rafts, which was also associated with the inhibition of ligand-induced ROS production in macrophages. TLR3 did not translocate to lipid rafts by polyinosine-polycytidylic acid (poly(I:C)). CO had no effect on poly(I:C)-induced ROS production and TLR3 signaling. The inhibitory effect of CO on TLR-induced cytokine production was abolished in gp91(phox)-deficient macrophages, also indicating a role for NADPH oxidase. CO attenuated LPS-induced NADPH oxidase activity in vitro, potentially by binding to gp91(phox). Thus, CO negatively controlled TLR signaling pathways by inhibiting translocation of TLR to lipid rafts through suppression of NADPH oxidase-dependent ROS generation.

Differential regulation and localization of carboxypeptidase D and carboxypeptidase E in human and mouse ß-cells.

Hyperglycemia can result from a relative or absolute lack of functional insulin secreted by the pancreatic ß-cells. Prohormone processing enzymes play an essential role in the secretion of mature and fully functional insulin. Defects in insulin processing enzymes including prohormone convertases 1/3 and 2, and carboxypeptidase E (CPE) can lead to ß-cell stress and hyperproinsulinemia, both of which are features of type 2 diabetes. Despite their importance, the regulation and role of this family of enzymes remain to be fully elucidated. Previously, we demonstrated that lipotoxicity led to the degradation of CPE, but did not affect its related enzyme, carboxypeptidase D (CPD). In this study, we found that CPD was significantly up-regulated by elevated glucose, while CPE was not. Low doses of insulin also increased CPD protein levels, consistent with a role for autocrine signaling. Glucose and insulin did not affect CPD or CPE expression in an α-cell line. Furthermore, insulin treatment altered the CPD sub-cellular localization, which was distinct from CPE. Somewhat surprisingly, the loss of CPE did not affect the levels of CPD. Knockdown of CPD exerted no effect on CPE protein levels. In addition, while our previous study demonstrated that even modest reduction of CPE was sufficient to induce ß-cell apoptosis, CPD knockdown did not affect cell viability. Taken together, our data demonstrate that CPE and CPD are differentially localized, differentially regulated and unlikely to have compensatory functions in pancreatic ß-cells.

Derlin-1 promotes the efficient degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR folding mutants.

A complex involving Derlin-1 and p97 mediates the retrotranslocation and endoplasmic reticulum (ER)-associated degradation of misfolded proteins in yeast and is used by certain viruses to promote host cell protein degradation (Romisch, K. (2005) Annu. Rev. Cell Dev. Biol. 21, 435-456; Lilley, B. N., and Ploegh, H. L. (2004) Nature 429, 834-840; Ye, Y., Shibata, Y., Yun, C., Ron, D., and Rapoport, T. A. (2004) Nature 429, 841-847). We asked whether the components of this pathway are involved in the endoplasmic reticulum-associated degradation of the mammalian integral membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), a substrate for the ubiquitin-proteasome system. We report that Derlin-1 and p97 formed complexes with CFTR in human airway epithelial cells. Derlin-1 interacted with nonubiquitylated CFTR, whereas p97 associated with ubiquitylated CFTR. Exogenous expression of Derlin-1 led to its co-localization with CFTR in the ER where it reduced wild type (WT) CFTR expression and efficiently degraded the disease-associated CFTR folding mutants, DeltaF508 and G85E (>90%). Consistent with this, Derlin-1 also reduced the amount of WT or DeltaF508 CFTR appearing in detergent-in-soluble aggregates. An approximately 70% knockdown of endogenous Derlin-1 by RNA interference increased the steady-state levels of WT and DeltaF508 CFTR by 10-15-fold, reflecting its significant role in CFTR degradation. Derlin-1 mediated the degradation of N-terminal CFTR fragments corresponding to the first transmembrane domain of CFTR, but CFTR fragments that incorporated additional domains were degraded less efficiently. These findings suggest that Derlin-1 recognizes misfolded, nonubiquitylated CFTR to initiate its dislocation and degradation early in the course of CFTR biogenesis, perhaps by detecting structural instability within the first transmembrane domain.

A functional link between glucokinase binding to insulin granules and conformational alterations in response to glucose and insulin.

Glucokinase (GK) activity is essential for the physiological regulation of insulin secretion by glucose. Because the enzyme exerts nearly total control over glucose metabolism in the beta-cell, even small changes in GK activity exert effects on glucose-stimulated insulin secretion and, consequently, the blood glucose concentration. Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the association of GK with insulin granules is regulated by glucose in the beta-cell. Glucose stimulation increased the rate of fluorescence recovery after photobleaching of GK to insulin granules, indicating that GK is released into the cytoplasm after glucose stimulation. Changes in fluorescence resonance energy transfer between two different fluorescent protein variants inserted on opposing ends of GK were observed after glucose stimulation and correlated with increased enzyme activity. Furthermore, glucose-stimulated changes in GK regulation were blocked by two inhibitors of insulin secretion. Insulin treatment restored GK regulation in inhibited cells and stimulated GK translocation and activation by itself. Together, these data support a model for post-translational regulation of GK whereby insulin regulates both the association of GK with secretory granules and the activity of the enzyme within the pancreatic beta-cell.