MUL1-RING recruits the substrate, p53-TAD as a complex with UBE2D2-UB conjugate.

The RING domain of MUL1 (RINGMUL1 ) alone mediates ubiquitylation of the p53-transactivation domain (TADp53 ). To elucidate the mechanism underlying the simultaneous recruitment of UBE2D2 and the substrate TADp53 by RINGMUL1 , we determined the complex structure of RINGMUL1 :UBE2D2 and studied the interaction between RINGMUL1 and TADp53 in the presence of UBE2D2-UB thioester (UBE2D2~UB) mimetics. The RINGMUL1 -binding induced the closed conformation of UBE2D2S22R/C85S -UBK48R oxyester (UBE2D2RS -UBR OE ), and strongly accelerated its hydrolysis, which was suppressed by the additional N77A-mutation of UBE2D2. Interestingly, UBE2D2S22R/N77A/C85S -UBK48R oxyester (UBE2D2RAS -UBR OE ) already formed a closed conformation in the absence of RINGMUL1 . Although TADp53 exhibited weak binding for RINGMUL1 or UBE2D2 alone, its binding affinity was enhanced and even further for RINGMUL1 :UBE2D2 and RINGMUL1 :UBE2D2RAS -UBR OE , respectively. The recognition of TADp53 by RINGMUL1 as a complex with UBE2D2~UB is related to the multivalency of the binding events and underlies the ability of RINGMUL1 to ubiquitylate the intrinsically disordered protein, TADp53 .

The RING domain of mitochondrial E3 ubiquitin ligase 1 and its complex with Ube2D2: crystallization and X-ray diffraction.

Mitochondrial E3 ubiquitin ligase 1 (MUL1) is located in the mitochondrial outer membrane and regulates various biological processes, including apoptosis, cell growth, mitophagy and mitochondrial dynamics. The C-terminal region of MUL1 faces the cytoplasm and contains the RING domain (MUL1-RING) where the Ub~E2 thioester binds. Unlike most RING-type E3 enzymes, MUL1-RING alone does not have an additional region that recruits a substrate protein, yet is still able to ubiquitylate the substrate, the p53 protein. Nevertheless, the exact mechanism of the ubiquitylation of p53 by MUL1-RING has not yet been elucidated. In order to understand this novel ubiquitylation mechanism, it is necessary to determine the three-dimensional structures of MUL1-RING and of its complex with the cognate E2 enzyme. Here, Ube2D2 was validated as a functional E2 enzyme for the ubiquitylation of the p53 transactivation domain (p53-TAD) by MUL1-RING, and purification and crystallization processes for MUL1-RING and the MUL1-RING-Ube2D2 complex are reported.

Electron Beam Irradiation Isolates Cellulose Nanofiber from Korea "Tall Goldenrod" Invasive Alien Plant Pulp.

This work investigates the possibility of isolating cellulose nanofibers from pulps of tall goldenrod plant, which are invasive plants in Korea, by a convenient method, without strong acids or high-pressure homogenization, using electron beam irradiation (EBI). The obtained cellulose nanofibers were characterized by scanning electron microscopy (SEM), ultraviolet-visible (UV-vis) spectroscopy, X-ray diffraction (XRD), thermogravimetric analysis (TGA), and in terms of their mechanical properties. SEM showed that the initially isolated 10-µm-diameter cellulose fibers became more finely separated with increasing EBI dose, and that cellulose fibers treated with 300 kGy of EBI were separated into long cellulose nanofibers of around 160 nm in diameter. In addition, the paper samples prepared from more finely separated fibers generated by using higher doses of EBI had enhanced UV-vis transmittance. Via the XRD analysis, we observed that cellulose I in the EBI-treated cellulose fibers were gradually converted into a different type of cellulose similar to cellulose type II, as the EBI dose increased. Meanwhile, the TGA demonstrated that the finely separated cellulose fibers observed after administering the high EBI dose had lowered thermal stability due to the reduction of cellulose I but higher char yield. In addition, tensile strengths of paper samples increased with decreasing the diameters of their constituent fibers that result from the different EBI doses used in the preparation of the paper pulp.

Solution structure of MUL1-RING domain and its interaction with p53 transactivation domain.

Mitochondrial E3 ubiquitin ligase 1 (MUL1) is a multifunctional mitochondrial protein involved in various biological processes such as mitochondrial dynamics, cell growth, apoptosis, and mitophagy. MUL1 mediates the ubiquitylation of mitochondrial p53 for proteasomal degradation. Although the interaction of MUL1-RING domain with its substrate, p53, is a unique mechanism in RING-mediated ubiquitylation, the molecular basis of this process remains unknown. In this study, we determined the solution structure of the MUL1-RING domain and characterized its interaction with the p53 transactivation domain (p53-TAD) by nuclear magnetic resonance (NMR) spectroscopy. The overall structure of the MUL1-RING domain is similar to those of RING domains of other E3 ubiquitinases. The MUL1-RING domain adopts a ßßαß fold with three anti-parallel ß-strands and one α-helix, containing a canonical cross-brace motif for the ligation of two zinc ions. Through NMR chemical shift perturbation experiments, we determined the p53-TAD-binding site in the MUL1-RING domain and showed that the MUL1-RING domain interacts mainly with the p53-TAD2 subdomain composed of residues 39-57. Taken together, our results provide a molecular basis for the novel recognition mechanism of the p53-TAD substrate by the MUL1-RING domain.

A novel mechanism of irinotecan targeting MDM2 and Bcl-xL.

Irinotecan is a strong anticancer drug whose mechanism of action has been reported only for the inhibition of DNA topoisomerase I (Topo I) through its active metabolite SN-38. In this study, we present a new mechanism of Irinotecan which inhibits the activities of MDM2, an E3 ligase of tumour suppressor p53, and Bcl-xL, an anti-apoptotic protein, through direct binding. In our structure modelling study, Irinotecan could fit to the binding sites of MDM2 and Bcl-xL for their known drugs, Nutlin-3 and ABT-737, with a better binding affinity than to Topo I. The direct binding of Irinotecan to both proteins was confirmed through a NMR study. We further showed that Irinotecan increased the amount of p53 only in the presence of MDM2 and inhibited the physical interaction of Bcl-xL with Bim, a core pro-apoptotic protein. In addition, we demonstrated that Irinotecan induced the down regulation of proliferation and strong G2/M arrest in HCT116 colon cancer cells shortly after treatment. Collectively, we suggest a new mechanism of action for Irinotecan as a dual target inhibitor of MDM2 and Bcl-xL facilitating the anticancer activities mediated by p53 and Bcl-xL interaction partners.

Latent Heat Storage and Thermal Efficacy of Carboxymethyl Cellulose Carbon Foams Containing Ag, Al, Carbon Nanotubes, and Graphene in a Phase Change Material.

Carbon foam was prepared from carboxymethyl cellulose (CMC) and Ag, Al and carbon nanotubes (CNTs), and graphene was added to the foam individually, to investigate the enhancement effects on the thermal conductivity. In addition, we used the vacuum method to impregnate erythritol of the phase change material (PCM) into the carbon foam samples to maximize the latent heat and minimize the latent heat loss during thermal cycling. Carbon foams containing Ag (CF-Ag), Al (CF-Al), CNT (CF-CNT) and graphene (CF-G) showed higher thermal conductivity than the carbon foam without any nano thermal conducting materials (CF). From the variations in temperature with time, erythritol added to CF, CF-Ag, CF-Al, CF-CNT, and CF-G was observed to decrease the time required to reach the phase change temperature when compared with pure erythritol. Among them, erythritol added to CF-G had the fastest phase change temperature, and this was related to the fact that this material had the highest thermal conductivity of the carbon foams used in this study. According to differential scanning calorimetry (DSC) analyses, the materials in which erythritol was added (CF, CF-Ag, CF-Al, CF-CNT, and CF-G) showed lower latent heat values than pure erythritol, as a result of their supplementation with carbon foam. However, the latent heat loss of these supplemented materials was less than that of pure erythritol during thermal cycling tests because of capillary and surface tension forces.

Influence of Cooking, Storage Period, and Re-heating on Production of Cholesterol Oxides in Chicken Meat.

The objective of present study was to investigate the effect of cooking and their combinations with re-heating methods on the formation of cholesterol oxidation products (COPs) in stored chicken thigh meat. Pan roasting, steaming, oven grilling, charcoal grilling, and microwaving were used for cooking. Re-heating of samples was done using the same cooking methods or microwaving after 3 and 6 d of refrigerated storage. Cooking and re-heating resulted in reduction of crude fat and cholesterol contents of chicken thigh meat depending on storage period before re-heating. Cooking and storage period had no influence on the total amount of COPs. The highest total amount of COPs was observed in meat samples cooked by steaming and reheated by microwaving after 6 d of storage, which showed similar value to raw chicken meat stored for 6 days. However, different re-heating methods formed different types of COPs depending on storage period before re-heating. The high amount (p<0.05) of 25-hydroxycholesterol or α-epoxide was detected in meat samples reheated by steaming or microwaving at 3 or 6 d of storage after steamed cooking, respectively. As a result, the combination of steaming and re-heating with microwaving could increase the total amount of COPs in chicken thigh meat and different cooking/re-heating methods could form different types of COPs, even though no significant difference in the total amount of COPs depending on storage period.

Structural Insight into the Critical Role of the N-Terminal Region in the Catalytic Activity of Dual-Specificity Phosphatase 26.

Human dual-specificity phosphatase 26 (DUSP26) is a novel target for anticancer therapy because its dephosphorylation of the p53 tumor suppressor regulates the apoptosis of cancer cells. DUSP26 inhibition results in neuroblastoma cell cytotoxicity through p53-mediated apoptosis. Despite the previous structural studies of DUSP26 catalytic domain (residues 61-211, DUSP26-C), the high-resolution structure of its catalytically active form has not been resolved. In this study, we determined the crystal structure of a catalytically active form of DUSP26 (residues 39-211, DUSP26-N) with an additional N-terminal region at 2.0 Å resolution. Unlike the C-terminal domain-swapped dimeric structure of DUSP26-C, the DUSP26-N (C152S) monomer adopts a fold-back conformation of the C-terminal α8-helix and has an additional α1-helix in the N-terminal region. Consistent with the canonically active conformation of its protein tyrosine phosphate-binding loop (PTP loop) observed in the structure, the phosphatase assay results demonstrated that DUSP26-N has significantly higher catalytic activity than DUSP26-C. Furthermore, size exclusion chromatography-multiangle laser scattering (SEC-MALS) measurements showed that DUSP26-N (C152S) exists as a monomer in solution. Notably, the crystal structure of DUSP26-N (C152S) revealed that the N-terminal region of DUSP26-N (C152S) serves a scaffolding role by positioning the surrounding α7-α8 loop for interaction with the PTP-loop through formation of an extensive hydrogen bond network, which seems to be critical in making the PTP-loop conformation competent for phosphatase activity. Our study provides the first high-resolution structure of a catalytically active form of DUSP26, which will contribute to the structure-based rational design of novel DUSP26-targeting anticancer therapeutics.

Impact of Cooking, Storage, and Reheating Conditions on the Formation of Cholesterol Oxidation Products in Pork Loin.

This study investigates the effect of cooking, storage, and reheating conditions on the formation of cholesterol oxidation products (COPs) in pork loin. Samples of pork loin procured 24 h postmortem were initially processed and assessed for total fat and cholesterol content. The cooking methods evaluated were pan roasting, steaming, oven grilling, and microwaving. Cooked pork loin samples were stored at 4℃ and reheated after 3 and 6 d of storage using the original method of preparation or alternately, microwaving. Fat content increased significantly with cooking as a result of the loss in moisture but cholesterol content remained unchanged. Pan roasting and microwave cooking caused a significantly higher production of COPs, as with the process of reheating using microwave, pan roasting, and oven grilling methods. The major COPs found in pork loin were cholestanetriol, 20-hydroxycholesterol, and 25-hydroxycholesterol, whose concentrations varied according to the different cooking and reheating methods used. Moreover, the aerobic storage of cooked pork loin under a refrigerated condition also increased the formation of cholesterol oxides on reheating.

High-resolution crystal structure of the PDZ1 domain of human protein tyrosine phosphatase PTP-Bas.

Protein tyrosine phosphatase-Basophil (PTP-Bas) is a membrane-associated protein tyrosine phosphatase with five PDZ domains and is involved in apoptosis, tumorigenesis, and insulin signaling. The interaction between PTP-Bas and tandem-PH-domain-containing protein 1/2 (TAPP1/2) plays an essential role in the regulation of insulin signaling. Despite its high sequence homology with the other PDZ domains, only the PDZ1 domain of PTP-Bas showed distinct binding specificity for TAPP1/2. Although the interaction between PTP-Bas PDZ1 and TAPP1/2 is a therapeutic target for diabetes, the structural basis for the interaction has not been elucidated. In the present study, we determined the crystal structure of the PTP-Bas PDZ1 domain at 1.6 Å resolution. In addition, we calculated the structural models of complexes of PTP-Bas PDZ1 and the C-terminal peptides of TAPP1/2 (referred to as TAPP1p/2p). Structural comparison with the PTP-Bas PDZ2/RA-GEF2 peptide complex revealed a structural basis for distinct binding specificity of PTP-Bas PDZ1 for TAPP1p/2p peptides. Our high-resolution crystal structure of PTP-Bas PDZ1 will serve as a useful template for rational structure-based design of novel anti-diabetes therapeutics.

Cooking, storage, and reheating effect on the formation of cholesterol oxidation products in processed meat products.

BACKGROUND: Cholesterol is an important biological compound; however, its oxidation products have been proven to be harmful to human health. Cooking, storage, and reheating methods significantly affect the safety of meat products, as they contribute to the production of cholesterol oxidation products (COPs). METHODS: Three cooking methods were used to cook sausages, loin ham, bacon, luncheon meat, and pressed ham, in order to investigate the effect of cooking, storage, and reheating on total cholesterol and on the formation of COPs. Cooked samples were stored at 4 °C and reheated after 3 and 6 storage days by the same cooking method or by microwaving. The samples were assessed for total lipids, cholesterol, and cholesterol oxides. RESULTS: The average cholesterol content in the processed meat varied from 76.0 mg/100 g to 201.70 mg/100 g. Microwaved ham showed the lowest cholesterol content compared to that of other processed meat products. Significant differences were found in cholesterol content and cholesterol oxidation products depending on cooking, storage, and reheating methods. Six cholesterol oxides were found in processed meat, of which 7ß-hydroxycholesterol and α-epoxides were detected as the major oxidation products. CONCLUSIONS: Microwaving and oven grilling resulted in higher production of COPs in processed meat as compared with other cooking methods. Refrigerated storage tended to significantly increase the COPs content.

Monitoring the formation of cholesterol oxidation products in model systems using response surface methodology.

BACKGROUND: Cholesterol oxidation products (COPs) are produced during thermal processing of animal origin foods and are considered to have negative health impacts. The model systems are helpful to understand the impact of various factors on oxidation changes in foods during cooking process. METHODS: The study presented herein investigates the effects of pH, presence of unsaturated fatty acids, and heat on the formation of cholesterol oxidation products (COPs). Two model systems were designed to investigate the formation of cholesterol oxidation products in different lipid environments. The cholesterol oxides produced were quantified using gas chromatography. RESULTS: The level of cholesterol oxidation products decreased significantly at higher pH (above 5.8) and shorter heating time (3 h). The presence of unsaturated fatty acids (linoleic and oleic acids) significantly increased the amount of COPs under low-temperature heating conditions (100 °C and 1 h) but did not affect the production of COPs at higher temperature (150 °C). Increasing the temperature to 200 °C significantly increased the amount of COPs during the first hour of heating and this amount decreased upon further heating. The most frequently observed COPs in samples were α-epoxide, 20α-hydroxycholesterol, and 25-hydroxycholesterol. CONCLUSIONS: In conclusion, pH below 5.8, presence of unsaturated fatty acid, and high cooking temperature (>150 °C) leads to increased production of cholesterol oxidation products.

Colonoscopy-induced ischemic colitis in patients without risk factors.

Ischemic colitis is the most common form of intestinal ischemia. It is a condition that is commonly seen in the elderly and among individuals with risk factors for ischemia. Common predisposing conditions for ischemic colitis are major vascular occlusion, small vessel disorder, shock, some medications, colonic obstructions and hematologic disorders. Ischemic colitis following colonoscopy is rare. Here, we report two cases of ischemic colitis after a routine screening colonoscopy in patients without risk factors for ischemia.

A case of variceal bleeding from the jejunum in liver cirrhosis.

While esophagogastric varices are common manifestations of portal hypertension, variceal bleeding from the jejunum is a rare complication of liver cirrhosis. In addition, ectopic variceal bleeding occurs in the duodenum and at sites of previous bowel surgery in most cases, including of stomas. We report a case of obscure overt gastrointestinal bleeding from jejunal varices in a 55-year-old woman who had not previously undergone abdominal surgery, who had liver cirrhosis induced by the hepatitis C virus. Emergency endoscopy revealed the presence of esophageal varices without stigmata of recent bleeding, and no bleeding focus was found at colonoscopy. She continued to produce recurrent melena with hematochezia and received up to 21 units of packed red blood cells. CT angiography revealed the presence of jejunal varices, but no active bleeding was found. Capsule endoscopy revealed fresh blood in the jejunum. The patient submitted to embolization of the jejunal varices via the portal vein, after which she had a stable hemoglobin level and no recurrence of the melena. This is a case of variceal bleeding from the jejunum in a liver cirrhosis patient without a prior history of abdominal surgery.