A Protein Kinase C Phosphorylation Motif in GLUT1 Affects Glucose Transport and is Mutated in GLUT1 Deficiency Syndrome.

Protein kinase C has been implicated in the phosphorylation of the erythrocyte/brain glucose transporter, GLUT1, without a clear understanding of the site(s) of phosphorylation and the possible effects on glucose transport. Through in vitro kinase assays, mass spectrometry, and phosphospecific antibodies, we identify serine 226 in GLUT1 as a PKC phosphorylation site. Phosphorylation of S226 is required for the rapid increase in glucose uptake and enhanced cell surface localization of GLUT1 induced by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Endogenous GLUT1 is phosphorylated on S226 in primary endothelial cells in response to TPA or VEGF. Several naturally occurring, pathogenic mutations that cause GLUT1 deficiency syndrome disrupt this PKC phosphomotif, impair the phosphorylation of S226 in vitro, and block TPA-mediated increases in glucose uptake. We demonstrate that the phosphorylation of GLUT1 on S226 regulates glucose transport and propose that this modification is important in the physiological regulation of glucose transport.

Vascular Actions of Angiotensin 1-7 in the Human Microcirculation: Novel Role for Telomerase.

OBJECTIVE: This study examined vascular actions of angiotensin 1-7 (ANG 1-7) in human atrial and adipose arterioles. APPROACH AND RESULTS: The endothelium-derived hyperpolarizing factor of flow-mediated dilation (FMD) switches from antiproliferative nitric oxide (NO) to proatherosclerotic hydrogen peroxide in arterioles from humans with coronary artery disease (CAD). Given the known vasoprotective properties of ANG 1-7, we tested the hypothesis that overnight ANG 1-7 treatment restores the NO component of FMD in arterioles from patients with CAD. Endothelial telomerase activity is essential for preserving the NO component of vasodilation in the human microcirculation; thus, we also tested whether telomerase activity was necessary for ANG 1-7-mediated vasoprotection by treating separate arterioles with ANG 1-7±the telomerase inhibitor 2-[[(2E)-3-(2-naphthalenyl)-1-oxo-2-butenyl1-yl]amino]benzoic acid. ANG 1-7 dilated arterioles from patients without CAD, whereas dilation was significantly reduced in arterioles from patients with CAD. In atrial arterioles from patients with CAD incubated with ANG 1-7 overnight, the NO synthase inhibitor NG-nitro-l-arginine methyl ester abolished FMD, whereas the hydrogen peroxide scavenger polyethylene glycol catalase had no effect. Conversely, in vessels incubated with ANG 1-7+2-[[(2E)-3-(2-naphthalenyl)-1-oxo-2-butenyl1-yl]amino]benzoic acid, NG-nitro-l-arginine methyl ester had no effect on FMD, but polyethylene glycol catalase abolished dilation. In cultured human coronary artery endothelial cells, ANG 1-7 significantly increased telomerase activity. These results indicate that ANG 1-7 dilates human microvessels, and dilation is abrogated in the presence of CAD. Furthermore, ANG 1-7 treatment is sufficient to restore the NO component of FMD in arterioles from patients with CAD in a telomerase-dependent manner. CONCLUSIONS: ANG 1-7 exerts vasoprotection in the human microvasculature via modulation of telomerase activity.

An exonic splicing enhancer within a bidirectional coding sequence regulates alternative splicing of an antisense mRNA.

The discovery of increasing numbers of genes with overlapping sequences highlights the problem of expression in the context of constraining regulatory elements from more than one gene. This study identifies regulatory sequences encompassed within two genes that overlap in an antisense orientation at their 3' ends. The genes encode the alpha-thyroid hormone receptor gene (TRalpha or NR1A1) and Rev-erbalpha (NR1D1). In mammals TRalpha pre-mRNAs are alternatively spliced to yield mRNAs encoding functionally antagonistic proteins: TRalpha1, an authentic thyroid hormone receptor; and TRalpha2, a non-hormone-binding variant that acts as a repressor. TRalpha2-specific splicing requires two regulatory elements that overlap with Rev-erbalpha sequences. Functional mapping of these elements reveals minimal splicing enhancer elements that have evolved within the constraints of the overlapping Rev-erbalpha sequence. These results provide insight into the evolution of regulatory elements within the context of bidirectional coding sequences. They also demonstrate the ability of the genetic code to accommodate multiple layers of information within a given sequence, an important property of the code recently suggested on theoretical grounds.

IgG anti-laminin-332 autoantibodies are present in a subset of patients with mucous membrane, but not bullous, pemphigoid.

BACKGROUND: Antiepiligrin cicatricial pemphigoid is a mucosal-predominant subepidermal blistering disease associated with an increased relative risk of cancer. In contrast to prior reports showing that anti-laminin (L)-332 autoantibodies are a reliable marker for patients with antiepiligrin cicatricial pemphigoid, a recent report suggested that as many as 40% of patients with bullous pemphigoid (BP) have IgG reactive with this laminin isoform. OBJECTIVE: We sought to determine whether patients with BP possess circulating IgG anti-L-332 autoantibodies. METHODS: Sera from 100 adults with BP were analyzed by indirect immunofluorescence testing of intact skin, immunoblot studies of human keratinocyte (HK) extracts, and a new L-332 enzyme-linked immunosorbent assay. Sera showing reactivity suggestive of anti-L-332 autoantibodies in these assays were further analyzed in immunoblot studies of HK extracellular matrix and immunoprecipitation studies of biosynthetically radiolabeled HK extracts. RESULTS: IgG from all patients with BP bound intact epidermal basement membrane by indirect immunofluorescence microscopy and immunoblotted bullous pemphigoid antigen-1, -2, or both in HK extracts. None of these sera immunoblotted L-332 in HK extracts, although 13 did score above the cut point of a new IgG(4) L-332 enzyme-linked immunosorbent assay (sensitivity = 0.91, specificity = 0.98, Youden index = 0.89). Further analysis of sera from these 13 patients found: (1) all had IgG that bound the epidermal side of 1 mol/L NaCl split skin by indirect immunofluorescence microscopy; (2) none immunoblotted L-332 purified from HK extracellular matrix; and (3) none immunoprecipitated L-332 from biosynthetically radiolabeled HK extracts. LIMITATIONS: The basis of false-positive enzyme-linked immunosorbent assay determinations for anti-L-332 IgG among patients with BP is unknown. CONCLUSION: Anti-L-332 autoantibodies remain a reliable marker for patients with antiepiligrin cicatricial pemphigoid.

Role of intramolecular epitope spreading in pemphigus vulgaris.

Pemphigus vulgaris (PV) is an acquired immunobullous disorder. At the early stage of the disease (mucosal PV), patients display only autoimmunity to desmoglein (Dsg) 3 and develop mucosal blisters; while at the later stage of the disease (mucocutaneous PV), patients exhibit non-cross-reactive autoimmunity to both Dsg3 and Dsg1 and acquire cutaneous as well as mucosal blisters. At these two disease stages, Dsg3 autoantibodies exhibit different tissue-binding patterns and pathogenic activities, suggesting that they may recognize distinct epitopes. To test this hypothesis and to investigate the mechanism underlying the disease transition, we studied Dsg3 autoantibody epitopes from mucosal PV patients and patients exhibiting disease transition to mucocutaneous PV. We demonstrated that autoantibodies from the majority of mucosal PV patients target epitopes at the COOH-terminal portion of the Dsg3 ectodomain. Interestingly, only autoantibodies against the Dsg3 NH2-terminal epitope(s) are able to bind human skin. Moreover, we discovered that the intramolecular epitope spreading from Dsg3(87-566) to Dsg3(1-88) is a critical step that precedes the intermolecular epitope spreading from Dsg3 to Dsg1. During disease transition, this mechanism dictates the development of Dsg3 autoantibodies that recognize human skin and lead to expression of cutaneous PV lesions.