A MIR17HG-derived long noncoding RNA provides an essential chromatin scaffold for protein interaction and myeloma growth.

Long noncoding RNAs (lncRNAs) can drive tumorigenesis and are susceptible to therapeutic intervention. Here, we used a large-scale CRISPR interference viability screen to interrogate cell-growth dependency to lncRNA genes in multiple myeloma (MM) and identified a prominent role for the miR-17-92 cluster host gene (MIR17HG). We show that an MIR17HG-derived lncRNA, named lnc-17-92, is the main mediator of cell-growth dependency acting in a microRNA- and DROSHA-independent manner. Lnc-17-92 provides a chromatin scaffold for the functional interaction between c-MYC and WDR82, thus promoting the expression of ACACA, which encodes the rate-limiting enzyme of de novo lipogenesis acetyl-coA carboxylase 1. Targeting MIR17HG pre-RNA with clinically applicable antisense molecules disrupts the transcriptional and functional activities of lnc-17-92, causing potent antitumor effects both in vitro and in vivo in 3 preclinical animal models, including a clinically relevant patient-derived xenograft NSG mouse model. This study establishes a novel oncogenic function of MIR17HG and provides potent inhibitors for translation to clinical trials.

A Cross-Species Analysis Reveals Dysthyroidism of the Ovaries as a Common Trait of Premature Ovarian Aging.

Although the imbalance of circulating levels of Thyroid Hormones (THs) affects female fertility in vertebrates, its involvement in the promotion of Premature Ovarian Aging (POA) is debated. Therefore, altered synthesis of THs in both thyroid and ovary can be a trait of POA. We investigated the relationship between abnormal TH signaling, dysthyroidism, and POA in evolutionary distant vertebrates: from zebrafish to humans. Ovarian T3 signaling/metabolism was evaluated by measuring T3 levels, T3 responsive transcript, and protein levels along with transcripts governing T3 availability (deiodinases) and signaling (TH receptors) in distinct models of POA depending on genetic background and environmental exposures (e.g., diets, pesticides). Expression levels of well-known (Amh, Gdf9, and Inhibins) and novel (miR143/145 and Gas5) biomarkers of POA were assessed. Ovarian dysthyroidism was slightly influenced by genetics since very few differences were found between C57BL/6J and FVB/NJ females. However, diets exacerbated it in a strain-dependent manner. Similar findings were observed in zebrafish and mouse models of POA induced by developmental and long-life exposure to low-dose chlorpyrifos (CPF). Lastly, the T3 decrease in follicular fluids from women affected by diminished ovarian reserve, as well as of the transcripts modulating T3 signaling/availability in the cumulus cells, confirmed ovarian dysthyroidism as a common and evolutionary conserved trait of POA.

Insight into Nephrocan Function in Mouse Endoderm Patterning.

Endoderm-derived organs as liver and pancreas are potential targets for regenerative therapies, and thus, there is great interest in understanding the pathways that regulate the induction and specification of this germ layer. Currently, the knowledge of molecular mechanisms that guide the in vivo endoderm specification is restricted by the lack of early endoderm specific markers. Nephrocan (Nepn) is a gene whose expression characterizes the early stages of murine endoderm specification (E7.5-11.5) and encodes a secreted N-glycosylated protein. In the present study, we report the identification of a new transcript variant that is generated through alternative splicing. The new variant was found to have differential and tissue specific expression in the adult mouse. In order to better understand Nepn role during endoderm specification, we generated Nepn knock-out (KO) mice. Nepn-/- mice were born at Mendelian ratios and displayed no evident phenotype compared to WT mice. In addition, we produced nullizygous mouse embryonic stem cell (mESC) line lacking Nepn by applying (CRISPR)/CRISPR-associated systems 9 (Cas9) and employed a differentiation protocol toward endoderm lineage. Our in vitro results revealed that Nepn loss affects the endoderm differentiation impairing the expression of posterior foregut-associated markers.

Early versus delayed hormonal treatment in locally advanced or asymptomatic metastatic prostatic cancer patient dilemma.

INTRODUCTION: The objective of this work is to compare the effectiveness of hormonal treatment (luteinizing hormone-releasing hormone agonists and/or antiandrogens) as an early or as a deferred intervention for patients with locally advanced prostate cancer (LAPC) and/or asymptomatic metastasis. MATERIALS AND METHODS: Systematic review of trials published in 1950-2007. Sources included MEDLINE and bibliographies of retrieved articles. Eligible trials included adults with a history of LAPC who are not suitable for curative local treatment of prostate cancer. We retrieved 22 articles for detailed review, of which 8 met inclusion criteria. The Veterans Administration Cooperative Urological Research Group suggested that delaying hormonal therapy did not compromise overall survival and that many of the patients died of causes other than prostate cancer. In European Organisation for Research and Treatment of Cancer (EORTC) 30846 trial, the median survival for delayed endocrine treatment was 6.1 year, and for immediate treatment 7.6 year, the HR for survival on delayed versus immediate treatment was 1.23 (95 % CI 0.88-1.71), indicating a 23 % nonsignificant trend in favour of early treatment. In EORTC 30891, the immediate androgen deprivation resulted in a modest but statistically significant increase in overall survival. The protocol SAKK 08/88 showed the lack of any major advantage of immediate compared with deferred hormonal treatment regarding quality of life or overall survival. CONCLUSIONS: The early intervention with hormonal treatment for patients with LAPC provides important reductions in all-cause mortality, prostate cancer-specific mortality, overall progression, and distant progression compared with deferring their use until standard care has failed to halt the disease.

Exercise performance of chronic heart failure patients in the early period of support by an axial-flow left ventricular assist device as destination therapy.

Axial-flow left ventricular assist devices (LVADs) are increasingly used as destination therapy in end-stage chronic heart failure (CHF), as they improve survival and quality of life. Their effect on exercise tolerance in the early phase after implantation is still unclear. The aim of this study was to evaluate the effect of LVADs on the exercise capacity of a group of CHF patients within 2 months after initiation of circulatory support. Cardiopulmonary exercise test data were collected for 26 consecutive LVAD-implanted CHF patients within 2 months of initiation of assistance; the reference group consisted of 30 CHF patients not supported by LVAD who were evaluated after an episode of acute heart failure. Both LVAD and reference groups showed poor physical performance; LVAD patients achieved lower workload (LVAD: 36.3 ± 9.0 W, reference: 56.6 ± 18.2 W, P < 0.001) but reached a similar peak oxygen uptake (peak VO2 ; LVAD: 12.5 ± 3.0 mL/kg/min, reference: 13.6 ± 2.9 mL/kg/min, P = ns) and similar percentages of predicted peak VO2 (LVAD: 48.8 ± 13.9%, reference: 54.2 ± 15.3%, P = ns). While the values of the O2 uptake efficiency slope were 12% poorer in LVAD patients than in reference patients (1124.2 ± 226.3 vs. 1280.2 ± 391.1; P = ns), the kinetics of VO2 recovery after exercise were slightly better in LVAD patients (LVAD: 212.5 ± 62.5, reference: 261.1 ± 80.2 sec, P < 0.05). In the first 2 months after initiation of circulatory support, axial-flow LVAD patients are able to sustain a low-intensity workload; though some cardiopulmonary exercise test parameters suggest persistence of a marked physical deconditioning, their cardiorespiratory performance is similar to that of less compromised CHF patients, possibly due to positive hemodynamic effects beginning to be produced by the assist device.

A role for variable region-containing chitin-binding proteins (VCBPs) in host gut-bacteria interactions.

A number of different classes of molecules function as structural matrices for effecting innate and adaptive immunity. The most extensively characterized mediators of adaptive immunity are the immunoglobulins and T-cell antigen receptors found in jawed vertebrates. In both classes of molecules, unique receptor specificity is effected through somatic variation in the variable (V) structural domain. V region-containing chitin-binding proteins (VCBPs) consist of two tandem Ig V domains as well as a chitin-binding domain. VCBPs are encoded at four loci (i.e., VCBPA-VCBPD) in Ciona, a urochordate, and are expressed by distinct epithelial cells of the stomach and intestine, as well as by granular amoebocytes present in the lamina propria of the gut and in circulating blood. VCBPs are secreted into the gut lumen, and direct binding to bacterial surfaces can be detected by immunogold analysis. Affinity-purified native and recombinant VCBP-C, as well as a construct consisting only of the tandem V domains, enhance bacterial phagocytosis by granular amoebocytes in vitro. Various aspects of VCBP expression and function suggest an early origin for the key elements that are central to the dialogue between the immune system of the host and gut microflora.

Evaluation of penile cavernosal artery intima-media thickness in patients with erectile dysfunction. A new parameter in the diagnosis of vascular erectile dysfunction. Our experience on 59 cases.

OBJECTIVE: A precise characterization of erectile dysfunction (ED) of vascular origin has not yet been achieved, although cavernous peak systolic velocity (PSV) is generally considered a major parameter. Nevertheless the penile dynamic color Doppler is invasive and linked to several complications. The intima-media thicknesses (IMT) of cavernosal artery would add to the predictive value of vasculogenic ED risk and outcomes. We also hypothesized the existence of a correlation between IMT cavernosal artery and IMT carotid arteries. This study seeks to evaluate these hypotheses with our experience, investigating the predictive accuracy of carotid and cavernosal Doppler ultrasound findings for discriminating patients with vasculogenic ED. MATERIAL AND METHODS: A total of 59 subjects (32 vasculogenic ED patients - group A - and 27 no vasculogenic ED patients - group B) were evaluated in our andrological center from September 2012 to June 2013 and enrolled in the study. All subjects underwent medical history, erectile function domain of the International Index of Erectile Function, physical examination, routine and sex hormone blood tests, and high resolution dynamic color Doppler ultrasound evaluation of carotid and penile districts and valuation of IMT in both districts. RESULTS: The values of cavernosal artery IMT in group A were higher than in group B (0,28 ± 0,06 mm vs 0,17 ± 0,07 mm). Even the values of carotid artery IMT in vasculogenic ED group were higher than in no vasculogenic ED group (0,74 ± 0,14 mm vs 0,59 ± 0,11 mm). The cavernosal IMT showed a moderate (r = 0.61) positive linear correlation (p < 0.001) with the carotid artery IMT. CONCLUSIONS: An increased cavernous IMT might predict ED of vascular origin with more accuracy than PSV and could be a sensitive predictor also for systemic atherosclerosis at an earlier phase.

Downregulation of praja2 restrains endocytosis and boosts tyrosine kinase receptors in kidney cancer.

Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer in the adult population. Late diagnosis, resistance to therapeutics and recurrence of metastatic lesions account for the highest mortality rate among kidney cancer patients. Identifying novel biomarkers for early cancer detection and elucidating the mechanisms underlying ccRCC will provide clues to treat this aggressive malignant tumor. Here, we report that the ubiquitin ligase praja2 forms a complex with-and ubiquitylates the AP2 adapter complex, contributing to receptor endocytosis and clearance. In human RCC tissues and cells, downregulation of praja2 by oncogenic miRNAs (oncomiRs) and the proteasome markedly impairs endocytosis and clearance of the epidermal growth factor receptor (EGFR), and amplifies downstream mitogenic and proliferative signaling. Restoring praja2 levels in RCC cells downregulates EGFR, rewires cancer cell metabolism and ultimately inhibits tumor cell growth and metastasis. Accordingly, genetic ablation of praja2 in mice upregulates RTKs (i.e. EGFR and VEGFR) and induces epithelial and vascular alterations in the kidney tissue.In summary, our findings identify a regulatory loop between oncomiRs and the ubiquitin proteasome system that finely controls RTKs endocytosis and clearance, positively impacting mitogenic signaling and kidney cancer growth.

Stress urinary incontinence: long-term results of laparoscopic Burch colposuspension.

BACKGROUND: The open Burch colposuspension, first described in 1961 had been widely employed for the surgical treatment of women with stress urinary incontinence (SUI) caused by urethral hypermobility. We evaluated the long-term efficacy of laparoscopic Burch colposuspension (LBC) for SUI in women. METHODS: A randomized prospective trial was conducted from September 2010 to January 2013. The extraperitoneal laparoscopic Burch colposuspension was performed by an operator on 96 women, mean age was 54,3 ± 3,7 years all of whom suffered from SUI or mixed urinary incontinence. Patients completed a self-administered the Short Form-36 (SF-36), the Physical Component Summary (PCS) and Mental Component Summary (MCS), the Short Urinary Distress Inventory (SUDI) and Short Incontinence Impact Questionnaire (SIIQ). at both baseline and follow up(6 weeks, 6 months, 18 months postoperatively). The Genito-Urinary Treatment Satisfaction Scale (GUTSS) was used to assess satisfaction with surgery. RESULTS: After follow up was recorded an improvement of questionnaries scores. The general health score is improved after surgery (2,60 ± 1.02 versus 2,76 ± 1.06) with p = 0.09. The PCS baseline score is 46.29 ± 10.95 versus 49.54 ± 10.41 after treatment with p = 0.01, so there was a significant baseline to follow up improvement. The MCS improved also, infact baseline score is 42.19 ± 12.57 versus 42.70 ± 13.03 with p = 0.87. The SUDI baseline score is 50.22 ± 20.73 versus 23.92 ± 17.90, while SIIQ score is 49.98 ± 23.90 versus 31.40 ± 23.83 with p < 0.01. In both questionnaires there is an improvement. Satisfaction with treatment outcomes from the GUTSS at 6-month follow up is 29.5 ± 6.3 with p = 0.46. CONCLUSION: The LBC has significant advantages, without any apparent compromise in short-term and long term outcomes.

Classification of Speaking and Singing Voices Using Bioimpedance Measurements and Deep Learning.

The acts of speaking and singing are different phenomena displaying distinct characteristics. The classification and distinction of these voice acts is vastly approached utilizing voice audio recordings and microphones. The use of audio recordings, however, can become challenging and computationally expensive due to the complexity of the voice signal. The research presented in this paper seeks to address this issue by implementing a deep learning classifier of speaking and singing voices based on bioimpedance measurement in replacement of audio recordings. In addition, the proposed research aims to develop a real-time voice act classification for the integration with voice-to-MIDI conversion. For such purposes, a system was designed, implemented, and tested using electroglottographic signals, Mel Frequency Cepstral Coefficients, and a deep neural network. The lack of datasets for the training of the model was tackled by creating a dedicated dataset 7200 bioimpedance measurement of both singing and speaking. The use of bioimpedance measurements allows to deliver high classification accuracy whilst keeping low computational needs for both preprocessing and classification. These characteristics, in turn, allows a fast deployment of the system for near-real-time applications. After the training, the system was broadly tested achieving a testing accuracy of 92% to 94%.

Myogenesis in C2C12 Cells Requires Phosphorylation of ATF6α by p38 MAPK.

Activating transcription factor 6α (ATF6α) is an endoplasmic reticulum protein known to participate in unfolded protein response (UPR) during ER stress in mammals. Herein, we show that in mouse C2C12 myoblasts induced to differentiate, ATF6α is the only pathway of the UPR activated. ATF6α stimulation is p38 MAPK-dependent, as revealed by the use of the inhibitor SB203580, which halts myotube formation and, at the same time, impairs trafficking of ATF6α, which accumulates at the cis-Golgi without being processed in the p50 transcriptional active form. To further evaluate the role of ATF6α, we knocked out the ATF6α gene, thus inhibiting the C2C12 myoblast from undergoing myogenesis, and this occurred independently from p38 MAPK activity. The expression of exogenous ATF6α in knocked-out ATF6α cells recover myogenesis, whereas the expression of an ATF6α mutant in the p38 MAPK phosphorylation site (T166) was not able to regain myogenesis. Genetic ablation of ATF6α also prevents the exit from the cell cycle, which is essential for muscle differentiation. Furthermore, when we inhibited differentiation by the use of dexamethasone in C2C12 cells, we found inactivation of p38 MAPK and, consequently, loss of ATF6α activity. All these findings suggest that the p-p38 MAPK/ATF6α axis, in pathophysiological conditions, regulates myogenesis by promoting the exit from the cell cycle, an essential step to start myoblasts differentiation.

Peripheral adaptation mechanisms in physical training and cardiac rehabilitation: the case of a patient supported by a CardioWest total artificial heart.

BACKGROUND: The benefits of exercise training in patients with chronic heart failure (CHF) are due to a combination of cardiac and peripheral adaptations. Separating these 2 components is normally impossible, except for patients implanted with total artificial heart (TAH), where cardiac adaptation cannot occur. METHODS AND RESULTS: We report the case of a patient implanted with a CardioWest-TAH who underwent a comprehensive strength and endurance training program and was evaluated by repeated peak cardiopulmonary exercise tests. The patient experienced a 24% increase of peak oxygen consumption and an improvement in recovery kinetics during the training period of 29 months. CONCLUSION: This unique situation of a patient with a TAH, and therefore a fixed peak cardiac output, allows us to isolate training-induced changes in the periphery, that suggest greater oxygen extraction and more efficient metabolic gas kinetics during the exercise and recovery phases.

Anemia does not preclude increments in cardiac performance during a short period of intensive, exercise-based cardiac rehabilitation.

BACKGROUND AND AIMS: Anemia seems to be rather common in cardiac rehabilitation patients but it is not known whether it could influence cardiovascular performance indexes and prognosis immediately after an acute cardiac event. The purposes of this study were to define its prevalence and to investigate the safety and efficacy of an intensive exercise-based cardiac rehabilitation in patients with and without anemia. METHODS: 436 participants (77% males; mean age 64 ± 13 years) were submitted to a two-week cardiac rehabilitation program consisting of low to medium intensity, individualized training with respiratory, aerobic and calisthenic exercises (three sessions daily, six times per week). A six-minute walking test was performed at enrolment and repeated at discharge together with a cardiopulmonary test. RESULTS: Anemia, as defined according to World Health Organization criteria, was detected in 328 patients (75.2% of the entire population). The distance walked increased from 381 ± 117 m at baseline to 457 ± 110 m (p < 0.001) after a mean period of 12.4 ± 4 days. A direct correlation was found between hemoglobin concentrations and both the absolute distance walked (r = 0.48; p < 0.001) and peak VO(2) (r = 0.39; p < 0.001). Anemic patients walked a significantly shorter distance at baseline and at discharge (p < 0.001); however, both groups showed the same increment in the distance walked: 76.0 ± 61 m vs 76.0 ± 60 m (p = 0.99). CONCLUSIONS: Our data indicate: 1) a high prevalence of anemia in the study population and 2) that, in spite of a clear reduction in exercise capacity, a moderate anemia does not preclude increments in cardiac performance during a short period of intensive, exercise-based cardiac rehabilitation.

Multi Species Analyses Reveal Testicular T3 Metabolism and Signalling as a Target of Environmental Pesticides.

Thyroid hormones (THs) regulate many biological processes in vertebrates, including reproduction. Testicular somatic and germ cells are equipped with the arrays of enzymes (deiodinases), transporters, and receptors necessary to locally maintain the optimal level of THs and their signalling, needed for their functions and spermatogenesis. Pesticides, as chlorpyrifos (CPF) and ethylene thiourea (ETU), impair the function of thyroid and testis, affecting male fertility. However, their ability to disarrange testicular T3 (t-T3) metabolism and signalling is poorly considered. Here, a multi-species analysis involving zebrafish and mouse suggests the damage of t-T3 metabolism and signalling as a mechanism of gonadic toxicity of low-doses CPF and ETU. Indeed, the developmental exposure to both compounds reduces Dio2 transcript in both models, as well as in ex-vivo cultures of murine seminiferous tubules, and it is linked to alteration of steroidogenesis and germ cell differentiation. A major impact on spermatogonia was confirmed molecularly by the expression of their markers and morphologically evidenced in zebrafish. The results reveal that in the adopted models, exposure to both pesticides alters the t-T3 metabolism and signalling, affecting the reproductive capability. Our data, together with previous reports suggest zebrafish as an evaluable model in assessing the action of compounds impairing locally T3 signalling.

Utility of Preoperative Neutrophil/Lymphocyte Ratio as a New Objective Prognostic Tool in Endoscopically Treated Upper Tract Urothelial Carcinoma: A Retrospective Evaluation.

Introduction: This exploratory retrospective analysis examined any potential prognostic role of preoperative neutrophil lymphocyte ratio (NLR) for progression-free survival (PFS) and time to endoscopically verified upper tract or bladder recurrence-free survival (RFS) in upper tract urothelial cancer (UTUC) patients selected for endoscopic treatment with subsequent endosurveillance. Patients and Methods: Eligibility criteria were natural orifice endoscopically retrogradely treated low-risk and imperative UTUC patients treated between 2005 and 2019, with biopsy confirmed diagnosis and 12 months minimum follow-up. For PFS, optimal NLR cutoff value was derived by log-rank test. Subsequently, both PFS and RFS were assessed for differences using Kaplan-Meier survival curves and log-rank test. Multivariate proportional Cox regression analysis adjusted for clinicopathologic variables was performed to examine end points for NLR-independent prognostic significance. Results: There were 100 eligible patients (63 truly low risk and 37 imperative cases). The optimal PFS log-rank test NLR cutoff value was 2.7. NLR ≥2.7 was significantly associated with shorter PFS (p = 0.01), and shorter upper tract RFS (p = 0.03), but not with bladder RFS (p = 0.90). Only positive high-grade cytology (hazard ratio [HR] 5.92, 95% confidence interval [CI] 2.140-16.35, p = 0.002) and NLR ≥2.7 (HR 4.28, 95% CI 1.34-13.72, p = 0.014) independently predicted PFS in multivariate analysis. Recurrence and progression were not significantly linked in the low-risk subset. Conclusions: This exploratory analysis showed that baseline NLR evaluation before first endoscopic UTUC treatment may be a valuable predictor and prognosticator of defined disease progression and of upper tract recurrence risk. In conjunction with high-grade urine cytology, NLR may improve risk stratification to optimize future individualized management.

Peripheral T3 signaling is the target of pesticides in zebrafish larvae and adult liver.

The intra-tissue levels of thyroid hormones (THs) regulate organ functions. Environmental factors can impair these levels by damaging the thyroid gland and/or peripheral TH metabolism. We investigated the effects of embryonic and/or long-life exposure to low-dose pesticides, ethylene thiourea (ETU), chlorpyrifos (CPF) and both combined on intra-tissue T4/T3 metabolism/signaling in zebrafish at different life stages. Hypothyroidism was evident in exposed larvae that showed reduced number of follicles and induced tshb mRNAs. Despite that, we found an increase in free T4 (fT4) and free T3 (fT3) levels/signaling that was confirmed by transcriptional regulation of TH metabolic enzymes (deiodinases) and T3-regulated mRNAs (cpt1, igfbp1a). Second-generation larvae showed that thyroid and TH signaling was affected even when not directly exposed, suggesting the role of parental exposure. In adult zebrafish, we found that sex-dependent damage of hepatic T3 level/signaling was associated with liver steatosis, which was more pronounced in females, with sex-dependent alteration of transcripts codifying the key enzymes involved in 'de novo lipogenesis' and ß-oxidation. We found impaired activation of liver T3 and PPARα/Foxo3a pathways whose deregulation was already involved in mammalian liver steatosis. The data emphasizes that the intra-tissue imbalance of the T3 level is due to thyroid endocrine disruptors (THDC) and suggests that the effect of a slight modification in T3 signaling might be amplified by its direct regulation or crosstalk with PPARα/Foxo3a pathways. Because T3 levels define the hypothyroid/hyperthyroid status of each organ, our findings might explain the pleiotropic and site-dependent effects of pesticides.

A novel member of Prame family, Gm12794c, counteracts retinoic acid differentiation through the methyltransferase activity of PRC2.

Embryonic stem cells (ESCs) fluctuate among different levels of pluripotency defined as metastates. Sporadically, metastable cellular populations convert to a highly pluripotent metastate that resembles the preimplantation two-cell embryos stage (defined as 2C stage) in terms of transcriptome, DNA methylation, and chromatin structure. Recently, we found that the retinoic acid (RA) signaling leads to a robust increase of cells specifically expressing 2C genes, such as members of the Prame family. Here, we show that Gm12794c, one of the most highly upregulated Prame members, and previously identified as a key player for the maintenance of pluripotency, has a functional role in conferring ESCs resistance to RA signaling. In particular, RA-dependent expression of Gm12794c induces a ground state-like metastate, as evaluated by activation of 2C-specific genes, global DNA hypomethylation and rearrangement of chromatin similar to that observed in naive totipotent preimplantation epiblast cells and 2C-like cells. Mechanistically, we demonstrated that Gm12794c inhibits Cdkn1A gene expression through the polycomb repressive complex 2 (PRC2) histone methyltransferase activity. Collectively, our data highlight a molecular mechanism employed by ESCs to counteract retinoic acid differentiation stimuli and contribute to shed light on the molecular mechanisms at grounds of ESCs naive pluripotency-state maintenance.

Feedback inhibition of cAMP effector signaling by a chaperone-assisted ubiquitin system.

Activation of G-protein coupled receptors elevates cAMP levels promoting dissociation of protein kinase A (PKA) holoenzymes and release of catalytic subunits (PKAc). This results in PKAc-mediated phosphorylation of compartmentalized substrates that control central aspects of cell physiology. The mechanism of PKAc activation and signaling have been largely characterized. However, the modes of PKAc inactivation by regulated proteolysis were unknown. Here, we identify a regulatory mechanism that precisely tunes PKAc stability and downstream signaling. Following agonist stimulation, the recruitment of the chaperone-bound E3 ligase CHIP promotes ubiquitylation and proteolysis of PKAc, thus attenuating cAMP signaling. Genetic inactivation of CHIP or pharmacological inhibition of HSP70 enhances PKAc signaling and sustains hippocampal long-term potentiation. Interestingly, primary fibroblasts from autosomal recessive spinocerebellar ataxia 16 (SCAR16) patients carrying germline inactivating mutations of CHIP show a dramatic dysregulation of PKA signaling. This suggests the existence of a negative feedback mechanism for restricting hormonally controlled PKA activities.