Search

Objective To investigate the effect of mTORC1/2 inhibitor AZD2014 induced autophagy on cell proliferation in HCCLM3 cell lines in vitro. Methods HCCLM3 cells were cultured in the presence or absence of AZD2014 (100 nmol/L) or rapamycin (100 nmol/L) or 3-methyladenine (10 nmol/L). To detect autophagy vacuoles, HCCLM3 cells were divided into the control group, rapamycin group, AZD2014 group, control+3-MA group, rapamycin+3-MA group, and AZD2014+3-MA group, and auto-fluorescent dye monodansylcadaverine (MDC) was used to monitor autophagy vacuoles. To semi-quantify the expression of autophagy-related proteins, HCCLM3 cells were divided into the control group, rapamycin group, and AZD2014 group, and Western blotting analysis was carried out to detect the expression of autophagy-related proteins, LC3B-II and Beclin-1. To evaluate the effect of autophagy induced by AZD2014 on cell proliferation, HCCLM3 cells were divided into the control group, 3-MA group, AZD2014 group, and AZD2014+3-MA group, Cell Counting Kit-8 was used. Results AZD2014-treated HCC cells showed an increase in the number of MDC-labeled vacuoles as well as in their size. AZD2014 treatment increased the levels of LC3B-Ⅱ and Beclin-1 (P

The correct and quick operation of backup power automatic switching（BPAS）devices is an effective guarantee of reliable power supply to the system. However, when transformer substations are connected to small-capacity distributed power like the local small hydropower stations, the conventional BPAS devices usually fail to get into operation, which leads to the loss of power in some parts of the system. To solve this shortage，this article comes up with an improved scheme of BPAS, which has been proved through PSCAD simulation to be effective in enhancing the stability and reliability of the power grid

This study aims to assess the treatment effects of the molecular adsorbent recirculating system (MARS) in patients with acute and acute-on-chronic liver failure. We searched MEDLINE, EMBASE, and the Cochrane Controlled Trials Registry database between January 1966 and January 2014. We included randomized controlled trials, which compared the treatment effects of MARS with standard medical treatment. Study quality assessed according to Consolidated Standards of Reporting Trials (CONSORT) criteria. The risk ratio was used as the effect-size measure according to a fixed-effects model. The search strategy revealed 72 clinical studies, 10 of which were randomized controlled trials that met the criteria and were included. Four addressed ALF (93 patients) and six addressed AOCLF (453 patients). The mean CONSORT score was 15 (range 10-20). By meta-analysis, MARS significantly improved survival in ALF (risk ratio 0.61; 95% CI 0.38, 0.97; P = 0.04). There was no significant survival benefit in AOCLF (risk ratio 0.88; 95% CI 0.74, 1.06; P = 0.16). MARS significantly improved survival in patients with acute liver failure, however, there is no evidence that it improved survival in patients with acute-on-chronic liver failure. In conclusion, the present meta-analysis indicates that MARS therapy can improve survival in patients with ALF. It is necessary to develop MARS treatment because of the increasing demand for liver transplantation and the risk of liver failure.

TDP-43 (TAR DNA-binding protein of 43 kDa) is a major deposited protein in amyotrophic lateral sclerosis and frontotemporal dementia with ubiquitin. A great number of genetic mutations identified in the flexible C-terminal region are associated with disease pathologies. We investigated the molecular determinants of TDP-43 aggregation and its underlying mechanisms. We identified a hydrophobic patch (residues 318–343) as the amyloidogenic core essential for TDP-43 aggregation. Biophysical studies demonstrated that the homologous peptide formed a helix-turn-helix structure in solution, whereas it underwent structural transformation from an α-helix to a β-sheet during aggregation. Mutation or deletion of this core region significantly reduced the aggregation and cytoplasmic inclusions of full-length TDP-43 (or TDP-35 fragment) in cells. Thus, structural transformation of the amyloidogenic core initiates the aggregation and cytoplasmic inclusion formation of TDP-43. This particular core region provides a potential therapeutic target to design small-molecule compounds for mitigating TDP-43 proteinopathies.

To compare the operative techniques of single-incision laparoscopic cholecystectomy (SILC) via suture-suspension versus three-device method.Retrospective analysis was performed for a total of 300 patients undergoing umbilical single-incision laparoscopic cholecystectomy from June 2008 to November 2011 at our hospital. The procedures were of suture-suspension (n = 200) and three-device (n = 100). Operative duration, estimated intra-operative blood loss, exposure extent of Calot's triangle, postoperative pain score, hospital stay and complications were compared respectively between two groups. Both groups were matched for age, gender, body mass index (BMI), diagnoses and American Society of Anesthesiology (ASA) class.All procedures were completed by the same surgeon. Comparison between two groups showed insignificant differences in blood loss (mean: (15.6 ± 9.5) vs (16.8 ± 7.4) ml; t = 1.266, P = 0.207), postoperative complications (number of case, incision contusion:4 vs 2, P = 1.000;incision hemorrhage:2 vs 2, P = 0.603) and hospitalization duration (mean: (1.6 ± 0.5) vs (1.6 ± 0.5) d; t = 0.653, P = 0.514), but significant differences in operative duration (mean:(40.5 ± 16.0) vs (51.5 ± 18.0) min; t = 5.381, P = 0.000), postoperative pain (mean: 2.0 ± 1.7 vs 3.7 ± 1.6; t = 8.324, P = 0.000) and exposure of Calot's triangle (number of case, 197 vs 68; χ(2) = 60.178, P = 0.000). Thus the suture-suspension method was superior to the three-device counterpart.The suture-suspension method of SILC is safe, economic and easy-to-handle in clinical practice.

Ataxin-7 (Atx7) is a component of the nuclear transcription co-activator complex; its polyglutamine (polyQ) expansion may cause nuclear accumulation and recruit numerous proteins to the intranuclear inclusion bodies. Full-length R85 (R85FL) is such a protein sequestered by polyQ-expanded Atx7. Here, we report that Atx7 specifically interacts with the third SH3 domain (SH3C) of R85FL through its second portion of proline-rich region (PRR). NMR structural analysis of the SH3C domain and its complex with PRR revealed that SH3C contains a large negatively charged surface for binding with the RRTR motif of Atx7. Microscopy imaging demonstrated that sequestration of R85FL by the polyQ-expanded Atx7 in cell is mediated by this specific SH3C–PRR interaction, which is implicated in the pathogenesis of spinocerebellar ataxia 7.

The J-domain co-chaperones work together with the heat shock protein 70 (HSP70) chaperone to regulate many cellular events, but the mechanism underlying the J-domain-mediated HSP70 function remains elusive. We studied the interaction between human-inducible HSP70 and Homo sapiens J-domain protein (HSJ1a), a J domain and UIM motif-containing co-chaperone. The J domain of HSJ1a shares a conserved structure with other J domains from both eukaryotic and prokaryotic species, and it mediates the interaction with and the ATPase cycle of HSP70. Our in vitro study corroborates that the N terminus of HSP70 including the ATPase domain and the substrate-binding β-subdomain is not sufficient to bind with the J domain of HSJ1a. The C-terminal helical α-subdomain of HSP70, which was considered to function as a lid of the substrate-binding domain, is crucial for binding with the J domain of HSJ1a and stimulating the ATPase activity of HSP70. These fluctuating helices are likely to contribute to a proper conformation of HSP70 for J-domain binding other than directly bind with the J domain. Our findings provide an alternative mechanism of allosteric activation for functional regulation of HSP70 by its J-domain co-chaperones.

Deubiquitination is a reverse process of cellular ubiquitination important for many biological events. Ubiquitin (Ub)-specific protease 13 (USP13) is an ortholog of USP5 implicated in catalyzing hydrolysis of various Ub chains, but its enzymatic properties and catalytic regulation remain to be explored. Here we report studies of the roles of the Ub-binding domains of USP13 in regulatory catalysis by biochemical and NMR structural approaches. Our data demonstrate that USP13, distinct from USP5, exhibits a weak deubiquitinating activity preferring to Lys63-linked polyubiquitin (K63-polyUb) in a non-activation manner. The zinc finger (ZnF) domain of USP13 shares a similar fold with that of USP5, but it cannot bind with Ub, so that USP13 has lost its ability to be activated by free Ub. Substitution of the ZnF domain with that of USP5 confers USP13 the property of catalytic activation. The tandem Ub-associated (UBA) domains of USP13 can bind with different types of diUb but preferentially with K63-linked, providing a possible explanation for the weak activity preferring to K63-polyUb. USP13 can also regulate the protein level of CD3δ in cells, probably depending on its weak deubiquitinating activity and the Ub-binding properties of the UBA domains. Thus, the non-activating catalysis of USP13 for K63-polyUb chains implies that it may function differently from USP5 in cellular deubiquitination processes.

To explore a new approach to the management of malignant biliary obstruction using percutaneous transhepatic biliary radiofrequency and endoprothesis.Percutaneous transhepatic biliary radiofrequency and endoprothesis were performed in 2 cases of malignant biliary obstruction, including 1 of hilar cholangiocarcinoma and 1 of pancreatic head carcinoma. The tumor was ablated with radiofrequency followed by placement of matched metal stents into the biliary duct.The surgical procedures were carried out smoothly in the 2 cases. The symptoms of the patients were obviously improved after the operation with a significant decrease in the serum levels of total bilirubin, and CA-199 level decreased to the normal level in 1 case.This new approach is safe for management of malignant biliary obstruction. Compared with the more conventional interventional therapy, radiofrequency can reduce the intraoperative bleeding and arrest the local tumor growth to promote the patency of the stent as well as the postoperative survival of the patients.

Homo sapiens J domain protein (HSJ1) is a J-domain containing co-chaperone that is known to stimulate ATPase activity of HSP70 chaperone, while it also harbors two ubiquitin (Ub)-interacting motifs (UIMs) that may bind with ubiquitinated substrates and potentially function in protein degradation. We studied the effects of HSJ1a on the protein levels of both normal and the disease--related polyQ-expanded forms of ataxin-3 (Atx3) in cells. The results demonstrate that the N-terminal J-domain and the C-terminal UIM domain of HSJ1a exert opposite functions in regulating the protein level of cellular overexpressed Atx3. This dual regulation is dependent on the binding of the J-domain with HSP70, and the UIM domain with polyUb chains. The J-domain down-regulates the protein level of Atx3 through HSP70 mediated proteasomal degradation, while the UIM domain may alleviate this process via maintaining the ubiquitinated Atx3. We propose that co-chaperone HSJ1a orchestrates the balance of substrates in stressed cells in a Yin-Yang manner.

DC-UbP/UBTD2 is a ubiquitin (Ub) domain-containing protein first identified from dendritic cells, and is implicated in ubiquitination pathway. The solution structure and backbone dynamics of the C-terminal Ub-like (UbL) domain were elucidated in our previous work. To further understand the biological function of DC-UbP, we then solved the solution structure of the N-terminal domain of DC-UbP (DC-UbP_N) and studied its Ub binding properties by NMR techniques. The results show that DC-UbP_N holds a novel structural fold and acts as a Ub-binding domain (UBD) but with low affinity. This implies that the DC-UbP protein, composing of a combination of both UbL and UBD domains, might play an important role in regulating protein ubiquitination and delivery of ubiquitinated substrates in eukaryotic cells., link_to_OA_fulltext

alpha-Synuclein (alpha-Syn) is the major component of Lewy bodies (LBs) deposited in the brains of patients with Parkinson's disease. Synphilin-1 (Sph1) is a novel alpha-Syn-interacting protein also present in the LBs. However, the roles of alpha-Syn-Sph1 interaction in LB formation and in the related pathogenesis are still unclear. We have studied the interaction between alpha-Syn and Sph1 by biochemical and structural approaches and found that the central coiled-coil domain of Sph1 specifically interacts with the N-terminal stretch of alpha-Syn. When overexpressed in HEK 293T cells, Sph1 forms inclusions together with alpha-Syn, but the Sph1-positive inclusions cannot recruit the N-terminally truncated alpha-Syn. The central portion of Sph1 can also recruit alpha-Syn and induce inclusion formation through its coiled-coil domain. These observations demonstrate that the alpha-Syn-Sph1 interaction significantly promotes the formation of cytoplasmic alpha-Syn inclusions, which may have implications for LB formation in neural cells. We have also elucidated solution structure of the coiled-coil domain of Sph1 and its interaction with the N-terminal peptide of alpha-Syn. The specific interaction between alpha-Syn and Sph1 provides mechanistic insights into the inclusion-body formation in cells and pathological implication in Parkinson's disease.

Ubiquitin (Ub) is an essential modifier conserved in all eukaryotes from yeast to human. Phospholipase A(2)-activating protein (PLAA), a mammalian homolog of yeast DOA1/UFD3, has been proposed to be able to bind with Ub, which plays important roles in endoplasmic reticulum-associated degradation, vesicle formation, and DNA damage response. We have identified a core domain from the PLAA family ubiquitin-binding region of human PLAA (residues 386-465, namely PFUC) that can bind Ub and elucidated its solution structure and Ub-binding mode by NMR approaches. The PFUC domain possesses equal population of two conformers in solution by cis/trans-isomerization, whereas the two isomers exhibit almost equivalent Ub binding abilities. This domain structure takes a novel fold consisting of four beta-strands and two alpha-helices, and the Ub-binding site on PFUC locates in the surface of alpha2-helix, which is to some extent analogous to those of UBA, CUE, and UIM domains. This study provides structural basis and biochemical information for Ub recognition of the novel PFU domain from a PLAA family protein that may connect ubiquitination and degradation in endoplasmic reticulum-associated degradation.

The ubiquitin-interacting motif (UIM) is a short peptide with dual function of binding ubiquitin (Ub) and promoting ubiquitination. We elucidated the structures and dynamics of the tandem UIMs of ataxin-3 (AT3-UIM12) both in free and Ub-bound forms. The solution structure of free AT3-UIM12 consists of two α-helices and a flexible linker, whereas that of the Ub-bound form is much more compact with hydrophobic contacts between the two helices. NMR dynamics indicates that the flexible linker becomes rigid when AT3-UIM12 binds with Ub. Isothermal titration calorimetry and NMR titration demonstrate that AT3-UIM12 binds diUb with two distinct affinities, and the linker plays a critical role in association of the two helices in diUb binding. These results provide an implication that the tandem UIM12 interacts with Ub or diUb in a cooperative manner through an allosteric effect and dynamics change of the linker region, which might be related to its recognitions with various Ub chains and ubiquitinated substrates.