Regulation of the stability and transcriptional activity of NFATc4 by ubiquitination

AbstractNuclear factor of activated T cells (NFATc4) has been implicated as a critical regulator of the cardiac development and hypertrophy. However, the mechanisms for regulating NFATc4 stability and transactivation remain unclear. We showed that NFATc4 protein was predominantly ubiquitinated through the formation of Lysine 48-linked polyubiquitin chains, and this modification decreased NFATc4 protein levels and its transcriptional activity. Furthermore, activation of GSK3β markedly enhanced NFATc4 ubiquitination and decreased its transactivation, whereas inhibition of GSK3β had opposite effects. Importantly, ubiquitination and phosphorylation induced by GSK3β repressed NFATc4-dependent cardiac-specific gene expression. These results demonstrate that the ubiquitin–proteasome system plays an important role in regulating NFATc4 stability and transactivation.Structured summaryMINT-6798349:NFATc4 (uniprotkb:Q14934) physically interacts (MI:0218) with Ubiquitin (uniprotkb:P62988) by anti bait coimmunoprecipitation (MI:0006)MINT-6798334:NFATc4 (uniprotkb:Q14934) physically interacts (MI:0218) with Ubiquitin (uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)MINT-6798321:Ubiquitin (uniprotkb:P62988) physically interacts (MI:0218) with NFATc4 (uniprotkb:Q14934) by pull down (MI:0096

Structure of the Protein Phosphatase 2A Holoenzyme

SummaryProtein Phosphatase 2A (PP2A) plays an essential role in many aspects of cellular physiology. The PP2A holoenzyme consists of a heterodimeric core enzyme, which comprises a scaffolding subunit and a catalytic subunit, and a variable regulatory subunit. Here we report the crystal structure of the heterotrimeric PP2A holoenzyme involving the regulatory subunit B′/B56/PR61. Surprisingly, the B′/PR61 subunit has a HEAT-like (huntingtin-elongation-A subunit-TOR-like) repeat structure, similar to that of the scaffolding subunit. The regulatory B′/B56/PR61 subunit simultaneously interacts with the catalytic subunit as well as the conserved ridge of the scaffolding subunit. The carboxyterminus of the catalytic subunit recognizes a surface groove at the interface between the B′/B56/PR61 subunit and the scaffolding subunit. Compared to the scaffolding subunit in the PP2A core enzyme, formation of the holoenzyme forces the scaffolding subunit to undergo pronounced conformational rearrangements. This structure reveals significant ramifications for understanding the function and regulation of PP2A

C Terminus of Hsc70-interacting Protein Promotes Smooth Muscle Cell Proliferation and Survival through Ubiquitin-mediated Degradation of FoxO1

Forkhead transcription factors (FoxOs) play a pivotal role in controlling cellular proliferation and survival. The cellular level of these factors is tightly regulated through the phosphoinositide 3-kinase/Akt and ubiquitin-mediated degradation. However, the ubiquitin ligases responsible for the degradation of FoxO1 and the relevance of this regulation to smooth muscle cell (SMC) proliferation and survival have not been fully identified. Here we showed that overexpression of C terminus of Hsc70-interacting protein (CHIP) promoted ubiquitination and degradation of FoxO1 in SMCs in response to tumor necrosis factor-α. Both the U-box (containing ubiquitin ligase activity) and the charged (essential for FoxO1 binding) domains within CHIP were required for CHIP-mediated FoxO1 down-regulation. Moreover, interaction and ubiquitination of FoxO1 by CHIP depended on phos pho ryl a tion of FoxO1 at Ser-256. Furthermore, overexpression of CHIP repressed FoxO1-mediated transactivation and its proapo pto tic function following tumor necrosis factor-α treatment. In contrast, knockdown of CHIP by small interfering RNA enhanced FoxO1-mediated transactivation and its effect on SMC proliferation and survival. Taken together, our data indicate that CHIP is a negative regulator of FoxO1 activity through ubiquitin-mediated degradation, and inhibition of CHIP may serve as a potential therapeutic target for reducing proliferative arterial diseases

CHIP Represses Myocardin-Induced Smooth Muscle Cell Differentiation via Ubiquitin-Mediated Proteasomal Degradation▿

Myocardin, a coactivator of serum response factor (SRF), plays a critical role in the differentiation of vascular smooth muscle cells (SMCs). However, the molecular mechanisms regulating myocardin stability and activity are not well defined. Here we show that the E3 ligase C terminus of Hsc70-interacting protein (CHIP) represses myocardin-dependent SMC gene expression and transcriptional activity. CHIP interacts with and promotes myocardin ubiquitin-mediated degradation by the proteasome in vivo and in vitro. Furthermore, myocardin ubiquitination by CHIP requires its phosphorylation. Importantly, CHIP overexpression reduces the level of myocardin-dependent SMC contractile gene expression and diminishes arterial contractility ex vivo. These findings for the first time, to our knowledge, demonstrate that CHIP-promoted proteolysis of myocardin plays a key role in the physiological control of SMC phenotype and vessel tone, which may have an important implication for pathophysiological conditions such as atherosclerosis, hypertension, and Alzheimer's disease

ECAS score: a web-based risk model to predict moderate and severe extracranial carotid artery stenosis

<p>To develop and validate a risk model (Extracranial Carotid Artery Stenosis score, ECAS score) to predict moderate and severe ECAS. Furthermore, we compared discrimination of the ECAS score and three existing models with regard to both moderate and severe ECAS.</p> <p>The ECAS score was developed based on the Renqiu Stroke Screening Study (RSSS), in which eligible patients were randomly divided into derivation (60%) and validation (40%) cohorts. ECAS was diagnosed by carotid duplex ultrasound according to the published criteria. Independent predictors of moderate (≥50%) and severe (≥70%) ECAS were obtained using multivariable logistic regression. The area under the receiver operating characteristic curve (AUROC) and the Hosmer–Lemeshow test were used to assess model discrimination and calibration.</p> <p>A total of 5010 participants were included and the mean age was 64.3. The proportion of ECAS of < 50%, 50–69%, 70–99% and occlusion was 4.4, 0.5, 0.4, and 0.4%, respectively. The ECAS score was developed from sets of predictors of moderate and severe ECAS. The ECAS score demonstrated good discrimination in the derivation and validation cohorts (AUROC range: 0.785–0.846). The Hosmer–Lemeshow tests of ECAS score for moderate and severe ECAS were not significant in the derivation and validation cohorts (all <i>P</i> > 0.05). When compared to the three existing models, the ECAS score showed significantly better discrimination for both moderate and severe ECAS (all <i>P</i> < 0.001).</p> <p>The ECAS score is a valid model for predicting moderate and severe ECAS. Further validation of the ECAS score in different populations and larger samples is warranted.</p