To identify novel regulators of G␣ o , the most abundant G-protein in brain, we used yeast two-hybrid screening with constitutively active G␣ o as bait and identified a new regulator of G-protein signaling (RGS) protein, RGS17 (RGSZ2), as a novel human member of the RZ (or A) subfamily of RGS proteins. RGS17 contains an aminoterminal cysteine-rich motif and a carboxyl-terminal RGS domain with highest homology to hRGSZ1-and hRGS-G␣-interacting protein. RGS17 RNA was strongly expressed as multiple species in cerebellum and other brain regions. The interactions between hRGS17 and active forms of G␣ i1-3 , G␣ o , G␣ z , or G␣ q but not G␣ s were detected by yeast two-hybrid assay, in vitro pull-down assay, and co-immunoprecipitation studies. Recombinant RGS17 acted as a GTPase-activating protein (GAP) on free G␣ i2 and G␣ o under pre-steady-state conditions, and on M2-muscarinic receptor-activated G␣ i1 , G␣ i2 , G␣ i3 , G␣ z , and G␣ in steady-state GTPase assays in vitro. Unlike RGSZ1, which is highly selective for G z , RGS17 exhibited limited selectivity for G o among G i /G o proteins. All RZ family members reduced dopamine-D2/ G␣ i -mediated inhibition of cAMP formation and abolished thyrotropin-releasing hormone receptor/G␣ q -mediated calcium mobilization. RGS17 is a new RZ member that preferentially inhibits receptor signaling via G i/o , G z , and G q over G s to enhance cAMP-dependent signaling and inhibit calcium signaling. Differences observed between in vitro GAP assays and whole-cell signaling suggest additional determinants of the G-protein specificity of RGS GAP effects that could include receptors and effectors. Regulators of G-protein signaling (RGS)1 proteins accelerate the intrinsic GTPase activity of heterotrimeric G-protein G␣ subunits. All RGS proteins contain a conserved RGS core domain, which is an interaction site for the G␣ subunits (1-3).There are more than 30 human RGS or RGS-like proteins that are classified into six subfamilies based upon their sequence homology and that have conserved functional and targeting domains outside of the RGS domain. For instance, a membrane-targeting domain immediately proximal to the RGS core domain directs small RGS proteins such as RGS1-5 and -16 to the cell membrane (4). Putative nuclear localization signals have been found within (5) and also outside of the RGS core domain (6), and this may direct certain RGS subtypes to the nucleus (7,8). RZ family members, such as RGSZ1 and RGS-G␣-interacting protein (GAIP), contain a cysteine-rich motif that may serve as a palmitoylation site for membrane association (9, 10). A more recent study showed that RZ family members also serve as adapter proteins for G␣ subunit degradation (11). The cysteine-rich motif interacts with the leucinerich region of GAIP-interacting protein N terminus, an E3 ubiquitin ligase responsible for G␣ i3 degradation. Recently RGSZ1 and RGS6 have been shown to associate with SCG-10, a protein involved in neuronal differentiation (12, 13), and the G z GTPase-activating protein (GAP) effect...

The transcriptional coactivator p/CIP(SRC-3/AIB1/ACTR/RAC3) binds liganded nuclear hormone receptors and facilitates transcription by directly recruiting accessory factors such as acetyltransferase CBP/p300 and the coactivator arginine methyltransferase CARM1. In the present study, we have established that recombinant p/CIP (p300/CBP interacting protein) is robustly methylated by CARM1 in vitro but not by other protein arginine methyltransferase family members. Metabolic labeling of MCF-7 breast cancer cells with S-adenosyl-L-[methyl-3 H]methionine and immunoblotting using dimethyl arginine-specific antibodies demonstrated that p/CIP is specifically methylated in intact cells. In addition, methylation of full-length p/CIP is not supported by extracts derived from CARM1 ؊/؊ mouse embryo fibroblasts, indicating that CARM1 is required for p/CIP methylation. Using mass spectrometry, we have identified three CARM1-dependent methylation sites located in a glutamine-rich region within the carboxy terminus of p/CIP which are conserved among all steroid receptor coactivator proteins. These results were confirmed by in vitro methylation of p/CIP using carboxyterminal truncation mutants and synthetic peptides as substrates for CARM1. Analysis of methylation site mutants revealed that arginine methylation causes an increase in full-length p/CIP turnover as a result of enhanced degradation. Additionally, methylation negatively impacts transcription via a second mechanism by impairing the ability of p/CIP to associate with CBP. Collectively, our data highlight coactivator methylation as an important regulatory mechanism in hormonal signaling.

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Duchenne muscular dystrophy (DMD) is an incurable neuromuscular degenerative disease, caused by a mutation in the dystrophin gene. Mdx mice recapitulate DMD features. Here we show that injection of wild-type (WT) embryonic stem cells (ESCs) into mdx blastocysts produces mice with improved pathology and function. A small fraction of WT ESCs incorporates into the mdx mouse nonuniformly to upregulate protein levels of dystrophin in the skeletal muscle. The chimeric muscle shows reduced regeneration and restores dystrobrevin, a dystrophin-related protein, in areas with high and with low dystrophin content. WT ESC injection increases the amount of fat in the chimeras to reach WT levels. ESC injection without dystrophin does not prevent the appearance of phenotypes in the skeletal muscle or in the fat. Thus, dystrophin supplied by the ESCs reverses disease in mdx mice globally in a dose-dependent manner.

Background and Purpose: The benefit of endovascular treatment (EVT) for large vessel occlusion in clinical practice in developing countries like China needs to be confirmed. The aim of the study was to determine whether the benefit of EVT for acute ischemic stroke in randomized trials could be generalized to clinical practice in Chinese population. Methods: We conducted a prospective registry of EVT at 111 centers in China. Patients with acute ischemic stroke caused by imaging-confirmed intracranial large vessel occlusion and receiving EVT were included. The primary outcome was functional independence at 90 days defined as a modified Rankin Scale score of 0 to 2. Outcomes of specific subgroups in the anterior circulation were reported and logistic regression was performed to predict the primary outcome. Results: Among the 1793 enrolled patients, 1396 (77.9%) had anterior circulation large vessel occlusion (median age, 66 [56–73] years) and 397 (22.1%) had posterior circulation large vessel occlusion (median age, 64 [55–72] years). Functional independence at 90 days was reached in 45% and 44% in anterior and posterior circulation groups, respectively. For anterior circulation population, underlying intracranial atherosclerotic disease was identified in 29% of patients, with higher functional independence at 90 days (52% versus 44%; P =0.0122) than patients without intracranial atherosclerotic disease. In the anterior circulation population, after adjusting for baseline characteristics, procedure details, and early outcomes, the independent predictors for functional independence at 90 days were age <66 years (odds ratio [OR], 1.733 [95% CI, 1.213–2.476]), time from onset to puncture >6 hours (OR, 1.536 [95% CI, 1.065–2.216]), local anesthesia (OR, 2.194 [95% CI, 1.325–3.633]), final modified Thrombolysis in Cerebral Infarction 2b/3 (OR, 2.052 [95% CI, 1.085–3.878]), puncture-to-reperfusion time ≤1.5 hours (OR, 1.628 [95% CI, 1.098–2.413]), and National Institutes of Health Stroke Scale score 24 hours after the procedure <11 (OR, 9.126 [95% CI, 6.222–13.385]). Conclusions: Despite distinct characteristics in the Chinese population, favorable outcome of EVT can be achieved in clinical practice in China. Registration: URL: https://www.clinicaltrials.gov . Unique identifier: NCT03370939.