Reversible protein O-GlcNAc modification has emerged as an essential intracellular signaling system in several tissues, including cardiovascular pathophysiology related to diabetes and acute ischemic stress. We tested the hypothesis that cardiac O-GlcNAc signaling is altered in chronic cardiac hypertrophy and failure of different etiologies. Global protein O-GlcNAcylation and the main enzymes regulating O-GlcNAc, O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), and glutamine-fructose-6-phosphate amidotransferase (GFAT) were measured by immunoblot and/or real-time RT-PCR analyses of left ventricular tissue from aortic stenosis (AS) patients and rat models of hypertension, myocardial infarction (MI), and aortic banding (AB), with and without failure. We show here that global O-GlcNAcylation was increased by 65% in AS patients, by 47% in hypertensive rats, by 81 and 58% post-AB, and 37 and 60% post-MI in hypertrophic and failing hearts, respectively (P < 0.05). Noticeably, protein O-GlcNAcylation patterns varied in hypertrophic vs. failing hearts, and the most extensive O-GlcNAcylation was observed on proteins of 20-100 kDa in size. OGT, OGA, and GFAT2 protein and/or mRNA levels were increased by pressure overload, while neither was regulated by myocardial infarction. Pharmacological inhibition of OGA decreased cardiac contractility in post-MI failing hearts, demonstrating a possible role of O-GlcNAcylation in development of chronic cardiac dysfunction. Our data support the novel concept that O-GlcNAc signaling is altered in various etiologies of cardiac hypertrophy and failure, including human aortic stenosis. This not only provides an exciting basis for discovery of new mechanisms underlying pathological cardiac remodeling but also implies protein O-GlcNAcylation as a possible new therapeutic target in heart failure.

Emerin is a conserved membrane component of nuclear lamina structure. Here, we report an advance in understanding the molecular basis of emerin function: intermolecular emerin-emerin association. There were two modes: one mediated by association of residues 170-220 in one emerin molecule to residues 170-220 in another, and the second involving residues 170-220 and 1-132. Deletion analysis showed residues 187-220 contain a positive element essential for intermolecular association in cells. By contrast, deletion of residues 168-186 inactivated a proposed negative element, required to limit or control association. Association of GFP-emerin with nuclear BAF in cells required the LEM domain (residues 1-47) and the positive element. Emerin peptide arrays revealed direct binding of residues 170-220 to residues 206-225 (the proposed positive element), residues 147-174 (particularly P 153 MYGRDSAYQSITHYRP 169 ) and the LEM domain. Emerin residues 1-132 and 159-220 were each sufficient to bind lamin A or B1 tails in vitro, identifying two independent regions of molecular contact with lamins. These results, and predicted emerin intrinsic disorder, support the hypothesis that there are multiple 'backbone' and LEM-domain configurations in a proposed intermolecular emerin network at the nuclear envelope.

Liver X receptor (LXR)4 ␣ (NR1H3) and LXR␤ (NR1H2) are ligand-activated transcription factors belonging to the nuclear receptor family and work as heterodimers with the retinoic X receptor (RXR). LXR␣ is expressed primarily in liver, macrophages, adipose tissue, and the interstitial epithelium, whereas

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Members of the poly-ADP-ribose polymerase (PARP) family catalyse the ADP-ribosylation of target proteins and are known to play important roles in many cellular processes, including DNA repair, differentiation and transcription. The majority of PARPs exhibit mono-ADP-ribosyltransferase activity rather than PARP activity; however, little is known about their biological activity. In the present study, we report that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly-ADP-ribose polymerase (TIPARP), mono-ADP-ribosylates and positively regulates liver X receptor α (LXRα) and LXRβ activity. Overexpression of TIPARP enhanced LXR-reporter gene activity. TIPARP knockdown or deletion reduced LXR regulated target gene expression levels in HepG2 cells and in Tiparp(-/-)mouse embryonic fibroblasts (MEFs) respectively. Deletion and mutagenesis studies showed that TIPARP's zinc-finger and catalytic domains were required to enhance LXR activity. Protein interaction studies using TIPARP and LXRα/β peptide arrays revealed that LXRs interacted with an N-terminal sequence (a.a. 209-236) of TIPARP, which also overlapped with a putative co-activator domain of TIPARP (a.a. 200-225). Immunofluorescence studies showed that TIPARP and LXRα or LXRβ co-localized in the nucleus.In vitroribosylation assays provided evidence that TIPARP mono-ADP-ribosylated both LXRα and LXRβ. Co-immunoprecipitation (co-IP) studies revealed that ADP-ribosylase macrodomain 1 (MACROD1), but not MACROD2, interacted with LXRs in a TIPARP-dependent manner. This was complemented by reporter gene studies showing that MACROD1, but not MACROD2, prevented the TIPARP-dependent increase in LXR activity. GW3965-dependent increases in hepatic Srebp1 mRNA and protein expression levels were reduced in Tiparp(-/-)mice compared with Tiparp(+/+)mice. Taken together, these data identify a new mechanism of LXR regulation that involves TIPARP, ADP-ribosylation and MACROD1.