Involvement of Caveolin-1 in Repair of DNA Damage through Both Homologous Recombination and Non-Homologous End Joining

Caveolin-1 (Cav-1), the major component of caveolae, is a 21-24 kDa integral membrane protein that interacts with a number of signaling molecules. By acting as a scaffolding protein, Cav-1 plays crucial roles in the regulation of various physiologic and patho-physiologic processes including oncogenic transformation and tumorigenesis, and tumor invasion and metastasis.In the present study we sought to explore the role of Cav-1 in response to DNA damage and the mechanism involved. We found that the level of Cav-1 was up-regulated rapidly in cells treated with ionizing radiation. The up-regulation of Cav-1 following DNA damage occurred only in cells expressing endogenous Cav-1, and was associated with the activation of DNA damage response pathways. Furthermore, we demonstrated that the expression of Cav-1 protected cells against DNA damage through modulating the activities of both the homologous recombination (HR) and non-homologous end joining (NHEJ) repair systems, as evidenced by the inhibitory effects of the Cav-1-targeted siRNA on cell survival, HR frequency, phosphorylation of DNA-dependent protein kinase (DNA-PK), and nuclear translocation of epidermal growth factor receptor (EGFR) following DNA damage, and by the stimulatory effect of the forced expression of Cav-1 on NHEJ frequency.Our results indicate that Cav-1 may play a critical role in sensing genotoxic stress and in orchestrating the response of cells to DNA damage through regulating the important molecules involved in maintaining genomic integrity

The role of SNARE proteins – syntaxin 4 and syntaxin 17 in the HCV life cycle

The Hepatitis C virus (HCV) infects more than 170 million individuals worldwide and causes challenging HCV-related diseases. Unfortunately, there is no vaccine available. Therefore, a better understanding of the HCV life cycle is urgently needed to develop more effective and better tolerated therapies. It has been reported that the secretory pathway plays an essential role for the release of HCV, and the SNARE complexes are a central factor controlling intracellular vesicular trafficking. Recently, our group observed that α-taxilin that binds to free syntaxin 4 prevents the SNARE complex formation and exerts an inhibitory effect on the release of HCV particles. Therefore, it was analyzed whether the t-SNARE protein syntaxin 4 is involved in the HCV life cycle. An increased intracellular amount of syntaxin 4 was found in HCV-positive cells, while the level of syntaxin 4-specific transcripts was decreased as observed in HCV-positive Huh7.5 cells and in HCV-infected primary human hepatocytes (PHH). Since in HCV-positive cells a significant longer half-life of syntaxin 4 was found, the decreased expression is overcompensated, leading to the elevated amount of syntaxin 4. Overexpression of syntaxin 4 increases the amount of secreted infectious viral particles, while silencing of syntaxin 4 expression decreases the number of released viral particles, which indicates that HCV could use the SNARE-dependent secretory pathway for viral release. Confocal immunofluorescence microscopy and co-immunoprecipitation experiments revealed that syntaxin 4 interacts with HCV core and NS5A. To identify the binding domain, various mutants of syntaxin 4 were generated. Based on these mutants, it was found that the H3 domain of syntaxin 4 interacts with core. These data show that the t-SNARE protein syntaxin 4 is an essential cellular factor for HCV morphogenesis and secretion. HCV induces autophagy, and in HCV-infected cells a major fraction of the de novo synthesized viral particles is not released but intracellularly degraded. Syntaxin 17 is an autophagosomal SNARE required for the fusion of autophagosomes with lysosomes to form autolysosomes and thereby to deliver the enclosed contents for degradation. Therefore, we aim to investigate whether syntaxin 17 is a relevant factor for the HCV life cycle by regulating the fusion between autophagosomes and lysosomes. It was found that HCV-positive cells possess a decreased amount of syntaxin 17, and HCV reduces the intracellular level of syntaxin 17 by NS5A-mediated interruption of c-Raf signaling, which triggers the syntaxin 17 transcription, and by HCV-dependently induced autophagy. Overexpression of syntaxin 17 decreases the intracellular amount of viral particles and reduces the number of released infectious viral particles by favoring the formation of autolysosomes, in which HCV particles can be degraded. Vice versa, inhibition of syntaxin 17 expression by specific siRNAs results in an elevated amount of intracellular viral particles and increases the number of released viral particles by impaired autophagosome-lysosome fusion. Confocal immunofluorescence microscopy analyses show a fraction of core protein in autophagosomes as stained by lysotracker and the autophagy maker p62. These data identify syntaxin 17 as a novel factor controlling the release of HCV and reveal the autophagosome-autolysosome fusion as an essential step affecting the equilibrium between the release of infectious viral particles and lysosomal degradation of intracellular viral particles. Taken together, these data identify the t-SNARE proteins syntaxin 4 and syntaxin 17 as essential cellular factors for HCV morphogenesis and secretion.Weltweit sind mehr als 170 Millionen Menschen chronisch mit dem Hepatitis-C-Virus (HCV) infiziert, das schwere Leberschäden hervorruft. Derzeit ist leider keine präventive Vakzinierung verfügbar. Deshalb ist es besonders wichtig, den HCV-Lebenszyklus besser zu verstehen, um effektivere und tolerierbarere Therapien entwickeln zu können..

Nanoreactor Design Based on Self-Assembling Protein Nanocages

Self-assembling proteins that form diverse architectures are widely used in material science and nanobiotechnology. One class belongs to protein nanocages, which are compartments with nanosized internal spaces. Because of the precise nanoscale structures, proteinaceous compartments are ideal materials for use as general platforms to create distinct microenvironments within confined cellular environments. This spatial organization strategy brings several advantages including the protection of catalyst cargo, faster turnover rates, and avoiding side reactions. Inspired by diverse molecular machines in nature, bioengineers have developed a variety of self-assembling supramolecular protein cages for use as biosynthetic nanoreactors that mimic natural systems. In this mini-review, we summarize current progress and ongoing efforts creating self-assembling protein based nanoreactors and their use in biocatalysis and synthetic biology. We also highlight the prospects for future research on these versatile nanomaterials

Cancer-Related Anemia Is a Risk Factor for Medium-Term Postoperative Cognitive Dysfunction in Laparoscopic Surgery Patients: An Observational Prospective Study

Anemia in the elderly may impair cognitive function. Our primary objective was to determine whether cancer-related anemia was associated with postoperative cognitive dysfunction (POCD) in nonelderly patients. We conducted an observational prospective study of 177 patients scheduled for laparoscopic surgery. Patients aged 18-64 were divided into two groups according to whether they were anemic due to cancer or not. The cognitive function was assessed by the Mini-Mental State Examination (MMSE) 1 day before and 1 week after operation. The cognitive function of the patients was evaluated by using the Telephone Interview for Cognitive Status-Modified (TICS-M) 3 months after operation. The quality of life of patients was evaluated after operation. The hemoglobin level and other clinical data were recorded before operation. Of the 170 patients, 100 without anemia and 70 anemia patients had been evaluated 1 week after operation. POCD was detected in 43 cases (25.3% of 170 cases) at 1 week and 30 cases (19% of 158 cases) at 3 months postoperatively. Anemia was an independent risk factor for 3-month POCD occurrence (P=0.034). The education level of the patients who had POCD at 1 week and 3 months after operation was lower (P<0.001, P=0.011, respectively). Age was independently associated with the incidence of POCD at 3 months (P=0.011). In general, these findings suggested that anemia may increase the incidence of medium-term POCD in cancer patients undergoing laparoscopic surgery

Carbon Nanotube Memory by the Self-Assembly of Silicon Nanocrystals as Charge Storage Nodes

A memory structure based on self-aligned silicon nanocrystals (Si NCs) grown over Al₂O₃-covered parallel-aligned carbon nanotubes (CNTs) by gas source molecular beam epitaxy is reported. Electrostatic force microscopy characterizations directly prove the charging and discharging of discrete NCs through the Al₂O₃ layer covering the CNTs. A CNT field effect transistor based on the NC/CNT structure is fabricated and characterized, demonstrating evident memory characteristics. Direct tunneling and Fowler–Nordheim tunneling phenomena are observed at different programming/erasing voltages. Retention is demonstrated to be on the order of 10⁴ s. Although there is still plenty of room to enhance the performance, the results suggest that CNT-based NC memory with diminutive CNTs and NCs could be an alternative structure to replace traditional floating gate memory

Silencing of Cav-1 expression decreases the IR-induced formation of BRCA1 foci.

<p>MDA-MB-468 cells were transfected with a Cav-1 siRNA or a non-targeting RNA. Forty-eight hours later, the cells were irradiated (5 Gy), and fixed for immunostaining with a BRCA1 antibody. BRCA1 foci were shown in green. DAPI was used for nucleus staining.</p

Treatment with IR stimulates the expression of Cav-1 protein.

<p>(<b>A</b>) MDA-MB-468 cells were irradiated (5 Gy) for the indicated period of time, and then the treated cells were collected for Western blot analysis of Cav-1. β-actin was used as a loading control. Expression of Cav-1 and β-actin were quantified using imageJ software, and Cav-1 level was normalized to that of β-actin. The normalized Cav-1 at the zero time point was arbitrarily set as 1. Bar represent mean ± S.D. of three separate experiments. (<b>B</b>) MCF-7, NCI/ADR-RES, PC-3, T98G and MCF-10A cells were treated or untreated with 5 Gy ionizing radiation, and Cav-1 expression was analyzed by Western blot. Results shown are the representative of three identical experiments.</p

Silencing of Cav-1 expression reduces the DSB repair by HR.

<p>(<b>A</b>) HT-1080 cells were irradiated (5 Gy) for the indicated period of time, and then cell lysates were prepared for Western blot analysis of Cav-1 and γ-H₂AX. β-actin was used as a loading control. (<b>B</b>) To determine the turnover of the silencing effect of Cav-1 siRNA in HT-1080 cells, we performed Western blot of analysis of Cav-1 at the indicated time after siRNA transfection. (<b>C</b>) HT-1080 cells were transfected with a non-targeting RNA or Cav-1 siRNA. Twenty-four hours after transfection, the cells were transfected with an HA tagged I-SceI endonuclease expressing vector (HA-I-SceI) or empty vector (HA) by electroporation, followed by Western blot analysis of Cav-1 and HA-I-SceI (upper panels), and by puromycin screening for HR frequency (lower panels). HR frequency was calculated as follows: the average numbers of colonies/dish were divided by the plating efficiency of transfection and divided by 85,000 (the total number of cells plated). The results shown are the representative of three similar experiments; each bar represents the mean±S.D. of triplicate determinations.</p