SMAD4 endows TGF-ß1-induced highly invasive tumor cells with ferroptosis vulnerability in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive malignancy prone to recurrence and metastasis. Studies show that tumor cells with increased invasive and metastatic potential are more likely to undergo ferroptosis. SMAD4 is a critical molecule in the transforming growth factor ß (TGF-ß) pathway, which affects the TGF-ß-induced epithelial-mesenchymal transition (EMT) status. SMAD4 loss is observed in more than half of patients with PDAC. In this study, we investigated whether SMAD4-positive PDAC cells were prone to ferroptosis because of their high invasiveness. We showed that SMAD4 status almost determined the orientation of transforming growth factor ß1 (TGF-ß1)-induced EMT via the SMAD4-dependent canonical pathway in PDAC, which altered ferroptosis vulnerability. We identified glutathione peroxidase 4 (GPX4), which inhibited ferroptosis, as a SMAD4 down-regulated gene by RNA sequencing. We found that SMAD4 bound to the promoter of GPX4 and decreased GPX4 transcription in PDAC. Furthermore, TGF-ß1-induced high invasiveness enhanced sensitivity of SMAD4-positive organoids and pancreas xenograft models to the ferroptosis inducer RAS-selective lethal 3 (RSL3). Moreover, SMAD4 enhanced the cytotoxic effect of gemcitabine combined with RSL3 in highly invasive PDAC cells. This study provides new ideas for the treatment of PDAC, especially SMAD4-positive PDAC.

Structural transformation of the amyloidogenic core region of TDP-43 protein initiates its aggregation and cytoplasmic inclusion.

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 ß-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.

The C-terminal helices of heat shock protein 70 are essential for J-domain binding and ATPase activation.

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 ß-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.

[Comparison of operative techniques in single-incision laparoscopic cholecystectomy: suture-suspension versus three-device method].

OBJECTIVE: To compare the operative techniques of single-incision laparoscopic cholecystectomy (SILC) via suture-suspension versus three-device method. METHODS: 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. RESULTS: 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. CONCLUSION: The suture-suspension method of SILC is safe, economic and easy-to-handle in clinical practice.

Occult colon cancer with sepsis as the primary manifestation identified by bone marrow puncture: A case report.

BACKGROUND: Bone marrow metastasis is common in liver and lung cancer, but there are few reports on bone marrow metastasis in colon cancer. To date, there are no such reports from mainland China, and reports of bone marrow metastasis with septic shock as the main manifestation are even rarer. CASE SUMMARY: A 71-year-old woman with sepsis as the first symptom presented with high fever, low blood pressure and high inflammation indicators. Computed tomography (CT) examination revealed mild inflammation of the lungs and no obvious abnormalities in the abdomen. Blood culture suggested Escherichia coli, Aeromonas hydrophila and Aeromonas caviae infection. Antibiotic treatment significantly improved the patient's sepsis symptoms; however, her thrombocytopenia (TCP) could not be corrected despite repeated platelet transfusions. Many malignant cells were ultimately found following a bone marrow puncture smear, and further positron emission tomography/CT (PET/CT) examination confirmed that the malignant tumor in the ascending colon was accompanied by multiple metastases, including the liver and bones. Colon adenocarcinoma was confirmed by autopsy. CONCLUSION: Patients with advanced colon cancer may not have typical clinical symptoms, and sepsis may be the first symptom. When patients have severe TCP that cannot be explained by sepsis of intestinal origin, it is necessary to be aware of the possibility of bone marrow metastasis of intestinal tumors. As such patients often cannot tolerate endoscopy, bone marrow biopsy smears or biopsy tests for specialized cells can help obtain a diagnosis, especially in less developed countries where PET/CT is scarce.

Interaction with synphilin-1 promotes inclusion formation of alpha-synuclein: mechanistic insights and pathological implication.

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.

Single-Port Laparoscopic Surgery Is Feasible and Safe for Hepatic Left Lateral Sectionectomy for Benign Liver Lesions.

BACKGROUND AND OBJECTIVES: The feasibility and safety of single-port laparoscopic surgery for left lateral liver lobectomy are largely unknown. This study is aimed at comparing the effectiveness and safety between single-port laparoscopic (SPL) and conventional multiport laparoscopic (CL) surgeries for hepatic left lateral sectionectomy. METHODS: A total of 65 patients receiving laparoscopic hepatic left lateral sectionectomy between January 2008 and July 2015 were included and divided into the SPL group (n = 40) and the CL group (n = 25). RESULTS: There was no significant difference in the operative time, estimated intraoperative blood loss, length of hospital stay, and incidences of postoperative complications (biliary leakage, hemorrhage, and contusion at incision) between groups (all P > 0.05). However, the SPL group had a significantly lower VAS pain score (at 24 h but not 7 days postoperation) and higher cosmetic satisfaction scores (at both 2 months and 6 months postoperation) than the CL group (all P < 0.01). Moreover, multivariate linear regression analysis further confirmed the superior pain score and cosmetic outcome in the SPL group. CONCLUSIONS: Single-port laparoscopic hepatic left lateral sectionectomy is a safe and feasible treatment for patients with lesions in the left hepatic lobe. Patients with benign lesions in the left hepatic lobe are more suitable to receive single-port laparoscopic hepatic left lateral sectionectomy than those with malignancies.

[Value of detection of serum glypican-3 level in diagnosis and therapeutic effect evaluation of primary hepatocellular carcinoma].

OBJECTIVE: To explore the clinical value of detecting serum glypican-3 in the diagnosis and therapeutic effect evaluation of primary hepatocellular carcinoma (PHC). METHODS: Using sandwich ELISA, we detected serum glypican-3 levels in 60 patients with PHC, 60 with metastatic liver cancer, 50 with liver cirrhosis, 50 with chronic viral hepatitis, 20 with hepatic cyst, 20 with fatty liver, 20 with hepatic hemangioma and 20 with drug-induced hepatitis as well as in 40 healthy subjects (control). We also analyzed the changes in serum levels of glypican-3 and alpha fetoprotein (AFP) in PHC patients after treatment. RESULTS: PHC patients had significantly higher serum levels of glypican-3 than patients with other liver diseases and the control subjects (P<0.05). The levels of serum glypican-3 were significantly higher in patients with metastatic liver cancer, liver cirrhosis and viral hepatitis than in those with other benign liver diseases and the control subjects (P<0.05). Glypican-3 level was not associated with AFP level or liver function in PHC patients, in whom the positivity rates for glypican-3 and AFP were 65% and 56.7%, respectively. The detection rate of PHC increased to 85% by a combined detection of AFP and glypican-3. In the 23 PHC patients who responded positively to treatments, serum glypican-3 level showed a steady decline compared with that in 15 patients before treatment, while serum AFP level showed a similar decrease only in 10 patients. CONCLUSION: Combined detection of glypican-3 and AFP is expected to improve the early diagnosis rate of PHC. The different thresholds of serum glypican-3 may play a role in the differential diagnosis of PHC and other various liver diseases. Glypican-3 may serve as a better marker than AFP with a high specificity and sensitivity for evaluating the therapeutic effect in PHC patients.

Artificial liver support in pigs with acetaminophen-induced acute liver failure.

AIM: To establish a reversible porcine model of acute liver failure (ALF) and treat it with an artificial liver system. METHODS: Sixteen pigs weighing 30-35 kg were chosen and administered with acetaminophen (APAP) to induce ALF. ALF pigs were then randomly assigned to either an experimental group (n = 11), in which a treatment procedure was performed, or a control group (n = 5). Treatment was started 20 h after APAP administration and continued for 8 h. Clinical manifestations of all animals, including liver and kidney functions, serum biochemical parameters and survival times were analyzed. RESULTS: Twenty hours after APAP administration, the levels of serum aspartate aminotransferase, total bilirubin, creatinine and ammonia were significantly increased, while albumin levels were decreased (P < 0.05). Prothrombin time was found to be extended with progression of ALF. After continuous treatment for 8 h (at 28 h), aspartate aminotransferase, total bilirubin, creatinine, and ammonia showed a decrease in comparison with the control group (P < 0.05). A cross-section of livers revealed signs of vacuolar degeneration, nuclear fragmentation and dissolution. Concerning survival, porcine models in the treatment group survived for longer times with artificial liver system treatment (P < 0.05). CONCLUSION: This model is reproducible and allows for quantitative evaluation of new liver systems, such as a bioartificial liver. The artificial liver system (ZHJ-3) is safe and effective for the APAP-induced porcine ALF model.

Tripartite comparison of single-incision and conventional laparoscopy in cholecystectomy: A multicenter trial.

AIM: To compare the characteristics of two single-incision methods, and conventional laparoscopy in cholecystectomy, and demonstrate the safety and feasibility. METHODS: Three hundred patients with gallstones or gallbladder polyps were admitted to two clinical centers from January 2013 to January 2014 and were randomized into three groups of 100: single-incision three-device group, X-Cone group, and conventional group. The operative time, intraoperative blood loss, complications, postoperative pain, cosmetic score, length of hospitalization, and hospital costs were compared, with a follow-up duration of 1 mo. RESULTS: A total of 142 males (47%) and 158 females (53%) were enrolled in this study. The population characteristics of these three groups is no significant differences exist in terms of age, sex, body mass index and American Society of Anesthesiology (P > 0.05). In results, there were no significant differences in blood loss, length of hospitalization, postoperative complications.The operative time in X-Cone group was significantly longer than other groups.There were significant differences in postoperative pain scores and cosmetic scores at diffent times after surgery (P < 0.05). CONCLUSION: This study shows that this two single-incision methods are safe and feasible. Both methods are superior to the conventional procedure in cosmetic and pain scores.

Meta-analysis of survival with the molecular adsorbent recirculating system for liver failure.

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.

A ubiquitin shuttle DC-UbP/UBTD2 reconciles protein ubiquitination and deubiquitination via linking UbE1 and USP5 enzymes.

The ubiquitination levels of protein substrates in eukaryotic cells are delicately orchestrated by various protein cofactors and enzymes. Dendritic cell-derived ubiquitin (Ub)-like protein (DC-UbP), also named as Ub domain-containing protein 2 (UBTD2), is a potential Ub shuttle protein comprised of a Ub-like (UbL) domain and a Ub-binding domain (UBD), but its biological function remains largely unknown. We identified two Ub-related enzymes, the deubiquitinating enzyme USP5 and the Ub-activating enzyme UbE1, as interacting partners of DC-UbP from HEK 293T cells. Biochemical studies revealed that the tandem UBA domains of USP5 and the C-terminal Ub-fold domain (UFD) of UbE1 directly interacted with the C-terminal UbL domain of DC-UbP but on the distinct surfaces. Overexpression of DC-UbP in HEK 293T cells enhanced the association of these two enzymes and thus prompted cellular ubiquitination, whereas knockdown of the protein reduced the cellular ubiquitination level. Together, DC-UbP may integrate the functions of USP5 and UbE1 through interacting with them, and thus reconcile the cellular ubiquitination and deubiquitination processes.

Structural basis for recognition of the third SH3 domain of full-length R85 (R85FL)/ponsin by ataxin-7.

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.

Laparoendoscopic single-site cholecystectomy vs three-port laparoscopic cholecystectomy: a large-scale retrospective study.

AIM: To perform a large-scale retrospective comparison of laparoendoscopic single-site cholecystectomy (LESSC) and three-port laparoscopic cholecystectomy (TPLC) in a single institution. METHODS: Data were collected from 366 patients undergoing LESSC between January 2005 and July 2008 and were compared with the data from 355 patients undergoing TPLC between August 2008 and November 2011 in our department. Patients with body mass index greater than 35 kg/m(2), a history of major upper abdominal surgery, signs of acute cholecystitis, such as fever, right upper quadrant tenderness with or without Murphy's sign, elevated white blood cell count, imaging findings suggestive of pericholecystic fluid, gallbladder wall thickening > 4 mm, and gallstones > 3 cm, were excluded to avoid bias. RESULTS: Altogether, 298 LESSC and 315 TPLC patients met the inclusion criteria. The groups were well matched with regard to demographic data. There were no significant differences in terms of postoperative complications (contusion: 19 vs 25 and hematoma at incision: 11 vs 19), hospital stay (mean ± SD, 1.4 ± 0.2 d vs 1.4 ± 0.7 d) and visual analogue pain score (mean ± SD, 8 h after surgery: 2.3 ± 1.4 vs 2.3 ± 1.3 and at day 1: 1.2 ± 0.4 vs 1.3 ± 1.2) between the LESSC and TPLC patients. Four patients required the addition of extra ports and 2 patients were converted to open surgery in the LESSC group, which was not significantly different when compared with TPLC patients converted to laparotomy (2 vs 2). LESSC resulted in a longer operating time (mean ± SD, 54.8 ± 11.0 min vs 33.5 ± 9.0 min), a higher incidence of intraoperative gallbladder perforation (56 vs 6) and higher operating cost (mean ± SD, 1933.7 ± 64.4 USD vs 1874.7 ± 46.2 USD) than TPLC. No significant differences in operating time (mean ± SD, 34.3 ± 6.0 min vs 32.7 ± 8.7 min) and total cost (mean ± SD, 1881.3 ± 32.8 USD vs 1876.2 ± 33.4 USD) were found when the last 100 cases in the two groups were compared. A correlation was observed between reduced operating time of LESSC and increased experience (Spearman rank correlation coefficient, -0.28). More patients in the LESSC group expressed satisfaction with the cosmetic result (98% vs 85%). CONCLUSION: LESSC is a safe and feasible procedure in selected patients with benign gallbladder diseases, with the significant advantage of cosmesis.

Single-incision vs three-port laparoscopic cholecystectomy: prospective randomized study.

AIM: To compare the clinical outcome of single-incision laparoscopic cholecystectomy (SILC) with three-port laparoscopic cholecystectomy (TPLC). METHODS: Between 2009 and 2011, one hundred and two patients with symptomatic benign gallbladder diseases were randomized to SILC (n = 49) or TPLC (n = 53). The primary end point was post operative pain score (at 6 h and 7 d). Secondary end points were blood loss, operation duration, overall complications, postoperative analgesic requirements, length of hospital stay, cosmetic result and total cost. Surgical techniques were standardized and all operations were performed by one experienced surgeon, who had performed more than 500 laparoscopic cholecystectomies. RESULTS: One patient in the SILC group required conversion to two-port LC. There were no open conversions or major complications in either treatment groups. There were no differences in terms of estimated blood loss (mean ± SD, 14 ± 6.0 mL vs 15 ± 4.0 mL), operation duration (mean ± SD, 41.8 ± 17.0 min vs 38.5 ± 22.0 min), port-site complications (contusion at incision: 5 cases vs 4 cases and hematoma at incision: 2 cases vs 1 case), total cost (mean ± SD, 12 075 ± 1047 RMB vs 11 982 ± 1153 RMB) and hospital stay (mean ± SD, 1.0 ± 0.5 d vs 1.0 ± 0.2 d) , respectively. TPLC had a significantly worse visual analogue pain score at 8 h after surgery (mean ± SD, 3.5 ± 1.6 vs 2.0 ± 1.5), however, the scores were similar on day 7 (mean ± SD, 2.5 ± 1.4 vs 2.0 ± 1.3). Cosmetic satisfaction, as determined by a survey at 2 mo follow-up favored SILC (mean ± SD, 8 ± 0.4 vs 6 ± 0.2). CONCLUSION: SILC is a safe and feasible approach in selected patients. The main advantages are a better cosmetic result and less pain.

[Percutaneous transhepatic biliary radiofrequency and endoprothesis: a new therapy for malignant biliary obstruction].

OBJECTIVE: To explore a new approach to the management of malignant biliary obstruction using percutaneous transhepatic biliary radiofrequency and endoprothesis. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Co-chaperone HSJ1a dually regulates the proteasomal degradation of ataxin-3.

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.

Domain analysis reveals that a deubiquitinating enzyme USP13 performs non-activating catalysis for Lys63-linked polyubiquitin.

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.

Solution structure of the N-terminal domain of DC-UbP/UBTD2 and its interaction with ubiquitin.

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.

Structural transformation of the tandem ubiquitin-interacting motifs in ataxin-3 and their cooperative interactions with ubiquitin chains.

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.