Quality control and fate determination of Hsp90 client proteins

AbstractQuality control processes regulate the proteome by determining whether a protein is to be folded or degraded. Hsp90 is a hub in the network of molecular chaperones that maintain this process because it promotes both folding and degradation, in addition to regulating expression of other quality control components. The significance of Hsp90's role in quality control is enhanced by the function of its clients, which include protein kinases and transcription factors, in cellular signaling. The inhibition of Hsp90 with small molecules results in the rapid degradation of such clients via the ubiquitin/proteasome pathway, and also in the induction of the Hsp70 molecular chaperone. These two events result in markedly different outcomes depending on cell type. For tumor cells there is a profound loss of signaling in growth promoting pathways. By contrast, increased amounts of Hsp70 in neuronal cells ameliorate the toxicity that is associated with the formation of aggregates observed in neurodegenerative conditions. In this review we discuss the mechanisms underlying these differential effects of Hsp90 inhibition on the quality control of distinct client proteins. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90)

Specificity in the actions of the UBR1 ubiquitin ligase in the degradation of nuclear receptors☆

The UBR1 ubiquitin ligase promotes degradation of proteins via the N-end rule and by another mechanism that detects a misfolded conformation. Although UBR1 was shown recently to act on protein kinases whose misfolding was promoted by inhibition of Hsp90, it was unknown whether this ubiquitin ligase targeted other client types of the chaperone. We analyzed the role of UBR1 in the degradation of nuclear receptors that are classical clients of Hsp90. Our results showed that UBR1 deletion results in impaired degradation of the glucocorticoid receptor and the androgen receptor but not the estrogen receptor α. These findings demonstrate specificity in the actions of the UBR1 ubiquitin ligase in the degradation of Hsp90 clients in the presence of small molecule inhibitors that promote client misfolding

Epidemiology, practice of ventilation and outcome for patients at increased risk of postoperative pulmonary complications: LAS VEGAS - An observational study in 29 countries

BACKGROUND Limited information exists about the epidemiology and outcome of surgical patients at increased risk of postoperative pulmonary complications (PPCs), and how intraoperative ventilation was managed in these patients. OBJECTIVES To determine the incidence of surgical patients at increased risk of PPCs, and to compare the intraoperative ventilation management and postoperative outcomes with patients at low risk of PPCs. DESIGN This was a prospective international 1-week observational study using the ‘Assess Respiratory Risk in Surgical Patients in Catalonia risk score’ (ARISCAT score) for PPC for risk stratification. PATIENTS AND SETTING Adult patients requiring intraoperative ventilation during general anaesthesia for surgery in 146 hospitals across 29 countries. MAIN OUTCOME MEASURES The primary outcome was the incidence of patients at increased risk of PPCs based on the ARISCAT score. Secondary outcomes included intraoperative ventilatory management and clinical outcomes. RESULTS A total of 9864 patients fulfilled the inclusion criteria. The incidence of patients at increased risk was 28.4%. The most frequently chosen tidal volume (V T) size was 500 ml, or 7 to 9 ml kg−1 predicted body weight, slightly lower in patients at increased risk of PPCs. Levels of positive end-expiratory pressure (PEEP) were slightly higher in patients at increased risk of PPCs, with 14.3% receiving more than 5 cmH2O PEEP compared with 7.6% in patients at low risk of PPCs (P ˂ 0.001). Patients with a predicted preoperative increased risk of PPCs developed PPCs more frequently: 19 versus 7%, relative risk (RR) 3.16 (95% confidence interval 2.76 to 3.61), P ˂ 0.001) and had longer hospital stays. The only ventilatory factor associated with the occurrence of PPCs was the peak pressure. CONCLUSION The incidence of patients with a predicted increased risk of PPCs is high. A large proportion of patients receive high V T and low PEEP levels. PPCs occur frequently in patients at increased risk, with worse clinical outcome.</p

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Spontaneously-forming spheroids as an in vitro cancer cell model for anticancer drug screening.

The limited translational value in clinic of analyses performed on 2-D cell cultures has prompted a shift toward the generation of 3-dimensional (3-D) multicellular systems. Here we present a spontaneously-forming in vitro cancer spheroid model, referred to as spheroids(MARY-X), that precisely reflects the pathophysiological features commonly found in tumor tissues and the lymphovascular embolus. In addition, we have developed a rapid, inexpensive means to evaluate response following drug treatment where spheroid dissolution indices from brightfield image analyses are used to construct dose-response curves resulting in relevant IC50 values. Using the spheroids(MARY-X) model, we demonstrate the unique ability of a new class of molecules, containing the caged Garcinia xanthone (CGX) motif, to induce spheroidal dissolution and apoptosis at IC50 values of 0.42 +/-0.02 μM for gambogic acid and 0.66 +/-0.02 μM for MAD28. On the other hand, treatment of spheroids(MARY-X) with various currently approved chemotherapeutics of solid and blood-borne cancer types failed to induce any response as indicated by high dissolution indices and subsequent poor IC50 values, such as 7.8 +/-3.1 μM for paclitaxel. Our studies highlight the significance of the spheroids(MARY-X) model in drug screening and underscore the potential of the CGX motif as a promising anticancer pharmacophore

Fluorescent molecular rotors as versatile in situ sensors for protein quantitation

Abstract Accurate protein quantitation is essential for many cellular mechanistic studies. Existing technology relies on extrinsic sample evaluation that requires significant volumes of sample as well as addition of assay-specific reagents and importantly, is a terminal analysis. This study exploits the unique chemical features of a fluorescent molecular rotor that fluctuates between twisted-to-untwisted states, with a subsequent intensity increase in fluorescence depending on environmental conditions (e.g., viscosity). Here we report the development of a rapid, sensitive in situ protein quantitation method using ARCAM-1, a representative fluorescent molecular rotor that can be employed in both non-terminal and terminal assays

Hsp110 Chaperones Control Client Fate Determination in the Hsp70–Hsp90 Chaperone System

The Hsp110 family of protein chaperones was known to promote maturation of Hsp90 client proteins. The yeast Hsp110 ortholog Sse1 is now shown to influence the decision to fold or degrade substrates of the Hsp70–Hsp90 chaperone system when maturation is compromised

Spontaneously-forming spheroids as an in vitro cancer cell model for anticancer drug screening.

The limited translational value in clinic of analyses performed on 2-D cell cultures has prompted a shift toward the generation of 3-dimensional (3-D) multicellular systems. Here we present a spontaneously-forming in vitro cancer spheroid model, referred to as spheroids(MARY-X), that precisely reflects the pathophysiological features commonly found in tumor tissues and the lymphovascular embolus. In addition, we have developed a rapid, inexpensive means to evaluate response following drug treatment where spheroid dissolution indices from brightfield image analyses are used to construct dose-response curves resulting in relevant IC50 values. Using the spheroids(MARY-X) model, we demonstrate the unique ability of a new class of molecules, containing the caged Garcinia xanthone (CGX) motif, to induce spheroidal dissolution and apoptosis at IC50 values of 0.42 +/-0.02 μM for gambogic acid and 0.66 +/-0.02 μM for MAD28. On the other hand, treatment of spheroids(MARY-X) with various currently approved chemotherapeutics of solid and blood-borne cancer types failed to induce any response as indicated by high dissolution indices and subsequent poor IC50 values, such as 7.8 +/-3.1 μM for paclitaxel. Our studies highlight the significance of the spheroids(MARY-X) model in drug screening and underscore the potential of the CGX motif as a promising anticancer pharmacophore

Should Diaphragmatic Involvement Preclude Resection of Large Hepatic Tumors?

Treatment of peripherally located liver tumors with diaphragmatic invasion is technically demanding but does not preclude resection for cure. The aim of the present study was to compare patients undergoing combined liver and diaphragmatic resection with those submitted to hepatectomy alone so as to evaluate the safety, effectiveness, and value of this complex surgical procedure. From January 2000 to September 2011, 36 consecutive patients underwent en bloc liver-diaphragm resection (group A). These were individually matched for age, gender, tumor size, pathology, and co-morbitidies with 36 patients who underwent hepatectomy alone during the same time (group B). Operative time, warm ischemia time, blood loss, required transfusions, postoperative complications, and long-term survival were evaluated. Mean operative time was significantly longer in group A than in group B (165 vs 142 min; P = 0.004). The two groups were comparable regarding warm ischemia time, intraoperative blood loss, required transfusions, and postoperative laboratory value fluctuations. Some 33 % of group A patients developed complications postoperatively as opposed to 23 % of group B patients (P = 0.03). The mortality rate was 2.8 % in group A compared to 0 % in group B. Postoperative follow-up demonstrated 60 % 1-year survival for group A patients as opposed to 80 % 1-year survival for group B patients, a difference that is practically eliminated the longer the follow-up period is extended (35 vs 40 % 3-year survival and 33 vs 37 % 5-year survival for group A and group B patients, respectively). En bloc diaphragmatic and liver resection is a challenging but safe surgical procedure that is fully justified when diaphragmatic infiltration cannot be ruled out and the patient is considered fit enough to undergo surgery

Evaluation of Ischemia-Reperfusion Liver Injury by Near-Infrared Spectroscopy in an Experimental Swine Model: The Effect of Desferoxamine

Introduction: Ischemia-reperfusion (I-R) injury has long been regarded a primary factor for the physiological dysfunction that can occur following major liver resection performed under vascular control. The aim of our study was to assess the effect of treatment with desferoxamine (DFO), a potent antioxidative agent, monitoring the I-R injury on a porcine model of major hepatectomy. Materials and Methods: Twelve female pigs were allocated to control (n = 6) and DFO groups (n = 6) and underwent 30 min of liver ischemia, during which a &gt;= 30% hepatectomy was performed, followed by six hours of postoperative monitoring. The DFO group animals were preconditioned with a continuous iv solution of DFO to a total dose of 100 mg/kg during their postoperative period. Liver remnants (approximate to 70% of initial liver volume) were evaluated by means of infrared spectroscopy, serum lactate measurement of the systemic, portal and hepatic vein blood, and by immunohistochemical assessment of apoptosis in consecutive liver biopsies. Results: DFO group demonstrated considerably faster restoration of tissue oxygenation (92.33% vs. 80%, p &lt; .05) and serum lactate values (1.23 mmol/l vs. 2.27 mmol/l, p &lt; .05). Moreover, apoptosis as estimated by TUNEL and caspase-3 staining was significantly lower in the DFO group (0.06% vs. 1.17% and 1.17% vs. 2%, respectively, p &lt; .05). The severity of the I-R injury showed a linear correlation to the restoration of tissue oxygenation, as estimated by infrared-spectroscopy (r(2) = 0.81, p &lt; .01). Conclusion: Iron chelation with DFO appears to attenuate I-R injury of the liver remnant following hepatectomy, as reflected by faster restoration of tissue oxygenation and lower apoptotic activity