ICAM-1 in acute myocardial infarction: a potential therapeutic target

Current treatments for AMI centre on prompt restoration of epicardial coronary blood flow. Despite improvements, AMI is still associated with significant morbidity and mortality. Novel approaches are therefore keenly sought. Intercellular adhesion molecule-1 (ICAM-1, CD54) is a member of the immunoglobulin superfamily. It is implicated in neutrophil and monocyte-endothelial cell adhesion, processes contributing to myocardial neutrophil infiltration and microvascular coronary slow flow, both viewed as important to the pathophysiologic responses in AMI. ICAM-1 would therefore appear an important potential therapeutic target in this context, and is the subject of this review.     

Cosupplementation with a synthetic, lipid-soluble polyphenol and vitamin C inhibits oxidative damage and improves vascular function yet does not inhibit acute renal injury in an animal model of rhabdomyolysis

We investigated whether cosupplementation with synthetic tetra-tert-butyl bisphenol (BP) and vitamin C (Vit C) ameliorated oxidative stress and acute kidney injury (AKI) in an animal model of acute rhabdomyolysis (RM). Rats were divided into groups: Sham and Control (normal chow), and BP (receiving 0.12% w/w BP in the diet; 4 weeks) with or without Vit C (100mg/kg ascorbate in PBS ip at 72, 48, and 24h before RM induction). All animals (except the Sham) were treated with 50% v/v glycerol/PBS (6 mL/kg injected into the hind leg) to induce RM. After 24h, urine, plasma, kidneys, and aortae were harvested. Lipid oxidation (assessed as cholesteryl ester hydroperoxides and hydroxides and F(2)-isoprostanes accumulation) increased in the kidney and plasma and this was coupled with decreased aortic levels of cyclic guanylylmonophosphate (cGMP). In renal tissues, RM stimulated glutathione peroxidase (GPx)-4, superoxide dismutase (SOD)-1/2 and nuclear factor kappa-beta (NFκβ) gene expression and promoted AKI as judged by formation of tubular casts, damaged epithelia, and increased urinary levels of total protein, kidney-injury molecule-1 (KIM-1), and clusterin. Supplementation with BP±Vit C inhibited the two indices of lipid oxidation, down-regulated GPx-4, SOD1/2, and NF-κβ gene responses and restored aortic cGMP, yet renal dysfunction and altered kidney morphology persisted. By contrast, supplementation with Vit C alone inhibited oxidative stress and diminished cast formation and proteinuria, while other plasma and urinary markers of AKI remained elevated. These data indicate that lipid- and water-soluble antioxidants may differ in terms of their therapeutic impact on RM-induced renal dysfunction.     

Clathrin-mediated endocytosis of the epithelial sodium channel. Role of epsin

Here we present evidence that the epithelial sodium channel (ENaC), a heteromeric membrane protein whose surface expression is regulated by ubiquitination, is present in clathrin-coated vesicles in epithelial cells that natively express ENaC. The channel subunits are ubiquitinated and co-immunoprecipitate with both epsin and clathrin adaptor proteins, and epsin, as expected, co-immunoprecipitates with clathrin adaptor proteins. The functional significance of these interactions was evaluated in a Xenopus oocyte expression system where co-expression of epsin and ENaC resulted in a down-regulation of ENaC activity; conversely, co-expression of epsin sub-domains acted as dominant-negative effectors and stimulated ENaC activity. These results identify epsin as an accessory protein linking ENaC to the clathrin-based endocytic machinery thereby regulating the activity of this ion channel at the cell surface.     

Cysteine string protein monitors late steps in cystic fibrosis transmembrane conductance regulator biogenesis

We examined the role of the cysteine string protein (Csp) in cystic fibrosis transmembrane conductance regulator (CFTR) biogenesis in relation to another J-domain protein, Hdj-2, a recognized CFTR cochaperone. Increased expression of Csp produced a dose-dependent reduction in mature (band C) CFTR and an increase in immature (band B) CFTR. Exogenous expression of Hdj-2 also increased CFTR band B, but unlike Csp, Hdj-2 increased band C as well. The Csp-induced block of CFTR maturation required Hsp70, because a J-domain mutant (H43Q) that interferes with the ability of Csp to stimulate Hsp70 ATPase activity relieved the Csp-induced block of CFTR maturation. Nevertheless, Csp H43Q still increased immature CFTR. Csp-induced band B CFTR was found adjacent to the nucleus, co-localizing with calnexin, and it remained detergent-soluble. These data indicate that Csp did not block CFTR maturation by promoting the aggregation or degradation of immature CFTR. Csp knockdown by RNA interference produced a 5-fold increase in mature CFTR and augmented cAMP-stimulated CFTR currents. Thus, the production of mature CFTR is inversely related to the expression level of Csp. Both Csp and Hdj-2 associated with the CFTR R-domain in vitro, and Hdj-2 binding was displaced by Csp, suggesting common interaction sites. Combined expression of Csp and Hdj-2 mimicked the effect of Csp alone, a block of CFTR maturation. But together, Csp and Hdj-2 produced additive increases in CFTR band B, and this did not depend on their interactions with Hsp70, consistent with direct chaperone actions of these proteins. Like Hdj-2, Csp reduced the aggregation of NBD1 in vitro in the absence of Hsp70. Our data suggest that both Csp and Hdj-2 facilitate the biosynthesis of immature CFTR, acting as direct CFTR chaperones, but in addition, Csp is positioned later in the CFTR biogenesis cascade where it regulates the production of mature CFTR by limiting its exit from the endoplasmic reticulum.     

Cysteine String Protein Promotes Proteasomal Degradation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Increasing Its Interaction with the C Terminus of Hsp70-interacting Protein and Promoting CFTR Ubiquitylation

Cysteine string protein (Csp) is a J-domain-containing protein whose overexpression blocks the exit of cystic fibrosis transmembrane conductance regulator (CFTR) from the endoplasmic reticulum (ER). Another method of blocking ER exit, the overexpression of Sar1-GTP, however, yielded twice as much immature CFTR compared with Csp overexpression. This finding suggested that Csp not only inhibits CFTR ER exit but also facilitates the degradation of immature CFTR. This was confirmed by treatment with a proteasome inhibitor, which returned the level of immature CFTR to that found in cells expressing Sar1-GTP only. CspH43Q, which does not interact with Hsc70/Hsp70 efficiently, did not promote CFTR degradation, suggesting that the pro-degradative effect of Csp requires Hsc70/Hsp70 binding/activation. In agreement with this, Csp overexpression increased the amount of Hsc70/Hsp70 co-immunoprecipitated with CFTR, whereas overexpression of CspH43Q did not. The Hsc70/Hsp70 binding partner C terminus of Hsp70-interacting protein (CHIP) can target CFTR for proteasome-mediated degradation. Csp overexpression also increased the amount of CHIP co-immunoprecipitated with CFTR. In addition, CHIP interacted directly with Csp, which was confirmed by in vitro binding experiments. Csp overexpression also increased CFTR ubiquitylation and reduced the half-life of immature CFTR. These findings indicate that Csp not only regulates the exit of CFTR from the ER, but that this action is accompanied by Hsc70/Hsp70 and CHIP-mediated CFTR degradation.Cysteine string protein (Csp, DnaJC5)² is a member of the DnaJ/Hsp40 protein chaperone family (1, 2). Csp contains a short N-terminal sequence, followed by the J-domain, a linker region, and the central cysteine-rich region, which gives these proteins their name. The cysteine residues in this region are post-translationally modified by the palmitate groups that serve for the membrane attachment of Csp (3). This structure is followed by a C-terminal domain, which is the most variable region among the three Csp paralogs in the human genome.Csp was originally discovered as an abundant protein in presynaptic junctions of Drosophila neurons (4). Csps are expressed at high levels in neurons and in cell types involved in regulated exocytosis (5–7), where they have been shown to govern exocytic secretory functions. For example, depolarization-induced synaptic vesicle exocytosis is impaired in neurons from Csp-deficient Drosophila (8), and Csp overexpression produced decreases in stimulated insulin release from β-cells and stimulated catecholamine release from chromaffin cells (9–11). Deletion of the Csp gene proved to be lethal both in Drosophila and in mice causing a progressive, fatal sensorimotor disorder characterized by developing neurodegenerative changes (12, 13).As for other Hsp40 homologs, the J-domain is responsible for the ability of Csp to bind Hsc70/Hsp70 and stimulate its ATPase activity (14). Similar to other chaperone proteins, Csp can be found in many different protein complexes in the cell, and depending on the composition of these complexes, Csp has been linked to many different cellular processes, ranging from membrane fusion to protein folding and G-protein-mediated signaling.Based on its direct interaction with the SNARE proteins syntaxin 1A and vesicle-associated membrane protein (synaptobrevin) Csp has been proposed to be a direct regulator of SNARE function (15, 16), a role further supported by Csp''s phosphorylation-dependent, direct interaction with synaptotagmin, a Ca²⁺-sensing, SNARE-binding protein (17). As part of a chaperone complex with Hsp90, Hsp70, and α-guanine nucleotide dissociation inhibitor, Csp coordinates Ca²⁺-induced neurotransmitter release by regulating the retrieval of Rab3b from presynaptic membranes (18).Csp forms a ternary complex with Hsc70/Hsp70 and small glutamine-rich tetratricopeptide repeat-containing protein. This complex is present on synaptic vesicles, and it can re-fold luciferase, leading to the proposal that Csp assists with the reactivation of unfolded proteins at the synapse (19). This same Csp-Hsc70-small glutamine-rich tetratricopeptide repeat-containing protein complex also interacts with heterotrimeric G-proteins. In this context Csp functions as a guanine-nucleotide exchange factor for Gα_S, which leads to stimulation of G-protein-dependent signaling and to G-protein-mediated inhibition of N-type Ca²⁺ channels (20, 21).Our laboratory previously established a novel role for Csp at endoplasmic reticulum (ER) membranes in modulating the trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR). The overexpression of Csp blocked the maturation of CFTR (22, 23), producing a dose-dependent reduction in mature (band C) CFTR and an increase in immature (band B) CFTR, which was localized to the ER. The Csp-induced block of CFTR maturation required Hsc70/Hsp70, because expression of a J-domain mutant of Csp (CspH43Q) that cannot stimulate Hsc70/Hsp70 ATPase activity allowed CFTR maturation. Conversely, the knock down of Csp promoted increased formation of mature CFTR. Together, these findings indicated that Csp negatively regulates CFTR progression to post-ER compartments.In the present study, we provide evidence for another ER-based function of Csp: its involvement in proteasome-mediated CFTR degradation. Overexpression of Csp increased the association of CFTR with Hsp70/Hsc70 and with the E3 ubiquitin ligase, CHIP (C terminus of Hsp70-interacting protein). Using co-immunoprecipitation and in vitro binding assays, we demonstrated a direct interaction between Csp and CHIP. The overexpression of Csp also increased the ubiquitylation of CFTR, in agreement with its ability to reduce CFTR steadystate levels and its increased association with CHIP. Pairwise interactions between Csp, Hsp70/Hsc70, and CHIP suggest a model in which Csp coordinates the formation of a complex that facilitates the degradation of CFTR as it blocks CFTR exit from the ER.     

IRAK1 and IRAK4 Promote Phosphorylation,Ubiquitination, and Degradation of MyD88 Adaptor-like (Mal)

Signal transduction by Toll-like receptor 2 (TLR2) and TLR4 requires the adaptors MyD88 and Mal (MyD88 adaptor-like) and serine/threonine kinases, interleukin-1 receptor-associated kinases IRAK1 and IRAK4. We have found that both IRAK1 and IRAK4 can directly phosphorylate Mal. In addition, co-expression of Mal with either IRAK resulted in depletion of Mal from cell lysates. This is likely to be due to Mal phosphorylation by the IRAKs because kinase-inactive forms of either IRAK had no effect. Furthermore, lipopolysaccharide stimulation resulted in ubiquitination and degradation of Mal, which was inhibited using an IRAK1/4 inhibitor or by knocking down expression of IRAK1 and IRAK4. MyD88 is not a substrate for either IRAK and did not undergo degradation. We therefore conclude that Mal is a substrate for IRAK1 and IRAK4 with phosphorylation promoting ubiquitination and degradation of Mal. This process may serve to negatively regulate signaling by TLR2 and TLR4.