Phosphorylation of p62 activates the Keap1-Nrf2 pathway during selective autophagy.

The Keap1-Nrf2 system and autophagy are both involved in the oxidative-stress response, metabolic pathways, and innate immunity, and dysregulation of these processes is associated with pathogenic processes. However, the interplay between these two pathways remains largely unknown. Here, we show that phosphorylation of the autophagy-adaptor protein p62 markedly increases p62's binding affinity for Keap1, an adaptor of the Cul3-ubiquitin E3 ligase complex responsible for degrading Nrf2. Thus, p62 phosphorylation induces expression of cytoprotective Nrf2 targets. p62 is assembled on selective autophagic cargos such as ubiquitinated organelles and subsequently phosphorylated in an mTORC1-dependent manner, implying coupling of the Keap1-Nrf2 system to autophagy. Furthermore, persistent activation of Nrf2 through accumulation of phosphorylated p62 contributes to the growth of human hepatocellular carcinomas (HCCs). These results demonstrate that selective autophagy and the Keap1-Nrf2 pathway are interdependent, and that inhibitors of the interaction between phosphorylated p62 and Keap1 have potential as therapeutic agents against human HCC.

SNP Discrimination by Tolane-Modified Peptide Nucleic Acids: Application for the Detection of Drug Resistance in Pathogens.

During the treatment of viral or bacterial infections, it is important to evaluate any resistance to the therapeutic agents used. An amino acid substitution arising from a single base mutation in a particular gene often causes drug resistance in pathogens. Therefore, molecular tools that discriminate a single base mismatch in the target sequence are required for achieving therapeutic success. Here, we synthesized peptide nucleic acids (PNAs) derivatized with tolane via an amide linkage at the N-terminus and succeeded in improving the sequence specificity, even with a mismatched base pair located near the terminal region of the duplex. We assessed the sequence specificities of the tolane-PNAs for single-strand DNA and RNA by UV-melting temperature analysis, thermodynamic analysis, an in silico conformational search, and a gel mobility shift assay. As a result, all of the PNA-tolane derivatives stabilized duplex formation to the matched target sequence without inducing mismatch target binding. Among the different PNA-tolane derivatives, PNA that was modified with a naphthyl-type tolane could efficiently discriminate a mismatched base pair and be utilized for the detection of resistance to neuraminidase inhibitors of the influenza A/H1N1 virus. Therefore, our molecular tool can be used to discriminate single nucleotide polymorphisms that are related to drug resistance in pathogens.

FYCO1 Contains a C-terminally Extended, LC3A/B-preferring LC3-interacting Region (LIR) Motif Required for Efficient Maturation of Autophagosomes during Basal Autophagy.

FYCO1 (FYVE and coiled-coil protein 1) is a transport adaptor that binds to phosphatidylinositol 3-phosphate, to Rab7, and to LC3 (microtubule-associated protein 1 light chain 3) to mediate transport of late endosomes and autophagosomes along microtubules in the plus end direction. We have previously shown that FYCO1 binds to LC3B via a 19-amino acid sequence containing a putative core LC3-interacting region (LIR) motif. Here, we show that FYCO1 preferentially binds to LC3A and -B. By peptide array-based two-dimensional mutational scans of the binding to LC3B, we found FYCO1 to contain a C-terminally extended LIR domain. We determined the crystal structure of a complex between a 13-amino acid LIR peptide from FYCO1 and LC3B at 1.53 Å resolution. By combining the structural information with mutational analyses, both the basis for the C-terminally extended LIR and the specificity for LC3A/B binding were revealed. FYCO1 contains a 9-amino acid-long F-type LIR motif. In addition to the canonical aromatic residue at position 1 and the hydrophobic residue at position 3, an acidic residue and a hydrophobic residue at positions 8 and 9, respectively, are important for efficient binding to LC3B explaining the C-terminal extension. The specificity for binding to LC3A/B is due to the interaction between Asp(1285) in FYCO1 and His(57) in LC3B. To address the functional significance of the LIR motif of FYCO1, we generated FYCO1 knock-out cells that subsequently were reconstituted with GFP-FYCO1 WT and LIR mutant constructs. Our data show that FYCO1 requires a functional LIR motif to facilitate efficient maturation of autophagosomes under basal conditions, whereas starvation-induced autophagy was unaffected.

Structural determinants in GABARAP required for the selective binding and recruitment of ALFY to LC3B-positive structures.

Several autophagy proteins contain an LC3-interacting region (LIR) responsible for their interaction with Atg8 homolog proteins. Here, we show that ALFY binds selectively to LC3C and the GABARAPs through a LIR in its WD40 domain. Binding of ALFY to GABARAP is indispensable for its recruitment to LC3B-positive structures and, thus, for the clearance of certain p62 structures by autophagy. In addition, the crystal structure of the GABARAP-ALFY-LIR peptide complex identifies three conserved residues in the GABARAPs that are responsible for binding to ALFY. Interestingly, introduction of these residues in LC3B is sufficient to enable its interaction with ALFY, indicating that residues outside the LIR-binding hydrophobic pockets confer specificity to the interactions with Atg8 homolog proteins.

Conserved Mode of Interaction between Yeast Bro1 Family V Domains and YP(X)nL Motif-Containing Target Proteins.

Yeast Bro1 and Rim20 belong to a family of proteins which possess a common architecture of Bro1 and V domains. Alix and His domain protein tyrosine phosphatase (HD-PTP), mammalian Bro1 family proteins, bind YP(X)nL (n = 1 to 3) motifs in their target proteins through their V domains. In Alix, the Phe residue, which is located in the hydrophobic groove of the V domain, is critical for binding to the YP(X)nL motif. Although the overall sequences are not highly conserved between mammalian and yeast V domains, we show that the conserved Phe residue in the yeast Bro1 V domain is important for binding to its YP(X)nL-containing target protein, Rfu1. Furthermore, we show that Rim20 binds to its target protein Rim101 through the interaction between the V domain of Rim20 and the YPIKL motif of Rim101. The mutation of either the critical Phe residue in the Rim20 V domain or the YPIKL motif of Rim101 affected the Rim20-mediated processing of Rim101. These results suggest that the interactions between V domains and YP(X)nL motif-containing proteins are conserved from yeast to mammalian cells. Moreover, the specificities of each V domain to their target protein suggest that unidentified elements determine the binding specificity.

Pba3-Pba4 heterodimer acts as a molecular matchmaker in proteasome α-ring formation.

Eukaryotic proteasome assembly is assisted by multiple dedicated chaperones. In yeast, formation of the heteroheptameric ring composed of α1-α7 subunits is promoted by the heterodimeric chaperone Pba3-Pba4. Here we reveal that in the absence of this dimeric chaperone, α2 replaces α4 during α-ring assembly, thereby giving rise to a non-productive complex that lacks α4, ß1, ß5, ß6, and ß7 subunits and aggregates of α4. Furthermore, our structure-guided mutational data demonstrate that the Pba3-Pba4 heterodimer acts as molecular matchmaker reinforcing the interaction between α4 and α5, which is the crucial step in the α-ring formation.

A case of robot-assisted resection for cecum cancer with anomalous venous confluence.

There are many reports on the positional relationship between the ileocolic artery and superior mesenteric vein (SMV). However, there have been no reports of anomalous venous confluence in the ileocecal vessel area. A 69-year-old man was diagnosed with cecal cancer on a preoperative examination of a lung tumor. We planned to perform surgery for the cecal cancer. Computed tomography angiography revealed an anomalous vein confluence in the ileocolic region. We performed robot-assisted ileocecal resection. Although the small intestinal vein was misidentified as the SMV at first, we confirmed the misidentification, identified the SMV on the dorsal side of the ileocolic artery, and ligated the ileocolic vessels with precise forceps manipulation during robotic surgery. Especially for cases with vascular anomalies revealed by preoperative computed tomography angiography, robotic surgery may be useful, as flexible forceps manipulation prevents vascular injury.

A case of MCA arising from ICA: a case report.

BACKGROUND: Complete mesocolic excision (CME) and central vascular detachment are very important procedures in surgery for colorectal cancer. Preoperative and intraoperative assessments of the anatomy of major colorectal vessels are necessary to avoid massive bleeding, especially in endoscopic surgery. A case with a rare anomaly in which the middle colic artery (MCA) and ileocolic artery (ICA) had a common trunk is reported. CASE PRESENTATION: The patient was a 73-year-old woman diagnosed with ascending colon cancer on colonoscopy. Preoperative abdominal contrast-enhanced computed tomography confirmed that the MCA and ICA had a common trunk. She underwent laparoscopic ileocecal resection for the ascending colon cancer with D3 lymph node dissection. Intraoperative indocyanine green fluorescence imaging was conducted. After confirming vessel bifurcation, the ICA was dissected at the distal end of the MCA bifurcation. The patient has been followed as an outpatient, with no signs of recurrence as of 2 years postoperatively. CONCLUSION: A case of an ascending colon cancer with a unique vascular bifurcation pattern was presented. Preoperative and intraoperative evaluations of the major colorectal vessels are very important for preventing perioperative and postoperative complications.

Structural basis for specific recognition of Rpt1p, an ATPase subunit of 26 S proteasome, by proteasome-dedicated chaperone Hsm3p.

The 26 S proteasome is a 2.5-MDa molecular machine that degrades ubiquitinated proteins in eukaryotic cells. It consists of a proteolytic core particle and two 19 S regulatory particles (RPs) composed of 6 ATPase (Rpt) and 13 non-ATPase (Rpn) subunits. Multiple proteasome-dedicated chaperones facilitate the assembly of the proteasome, but little is known about the detailed mechanisms. Hsm3, a 19 S RP dedicated chaperone, transiently binds to the C-terminal domain of the Rpt1 subunit and forms a tetrameric complex, Hsm3-Rpt1-Rpt2-Rpn1, during maturation of the ATPase ring of 19 S RP. To elucidate the structural basis of Hsm3 function, we determined the crystal structures of Hsm3 and its complex with the C-terminal domain of the Rpt1 subunit (Rpt1C). Hsm3 has a C-shaped structure that consists of 11 HEAT repeats. The structure of the Hsm3-Rpt1C complex revealed that the interacting surface between Hsm3 and Rpt1 is a hydrophobic core and a complementary charged surface. Mutations in the Hsm3-Rpt1 surface resulted in the assembly defect of the 26 S proteasome. Furthermore, a structural model of the Hsm3-Rpt ring complex and an in vitro binding assay suggest that Hsm3 can bind Rpt2 in addition to Rpt1. Collectively, our results provide the structural basis of the molecular functions of Hsm3 for the RP assembly.

Structural insight into the recognition of the linear ubiquitin assembly complex by Shigella E3 ligase IpaH1.4/2.5.

Pathogenic bacteria deliver virulence factors called effectors into host cells in order to facilitate infection. The Shigella effector proteins IpaH1.4 and IpaH2.5 are members of the 'novel E3 ligase' (NEL)-type bacterial E3 ligase family. These proteins ubiquitinate the linear ubiquitin assembly complex (LUBAC) to inhibit nuclear factor (NF)-κB activation and, concomitantly, the inflammatory response. However, the molecular mechanisms underlying the interaction and recognition between IpaH1.4 and IpaH2.5 and LUBAC are unclear. Here we present the crystal structures of the substrate-recognition domains of IpaH1.4 and IpaH2.5 at resolutions of 1.4 and 3.4 Å, respectively. The LUBAC-binding site on IpaH1.4 was predicted based on structural comparisons with the structures of other NEL-type E3s. Structural and biochemical data were collected and analysed to determine the specific residues of IpaH1.4 that are involved in interactions with LUBAC and influence NF-κB signaling. The new structural insight presented here demonstrates how bacterial pathogens target innate immune signaling pathways.

New crystal structure of the proteasome-dedicated chaperone Rpn14 at 1.6†Šresolution.

The 26S proteasome is an ATP-dependent protease responsible for selective degradation of polyubiquitylated proteins. Recent studies have suggested that proteasome assembly is a highly ordered multi-step process assisted by specific chaperones. Rpn14, an assembly chaperone for ATPase-ring formation, specifically recognizes the ATPase subunit Rpt6. The structure of Rpn14 at 2.0 Šresolution in space group P6(4) has previously been reported, but the detailed mechanism of Rpn14 function remains unclear. Here, a new crystal structure of Rpn14 with an E384A mutation is presented in space group P2(1) at 1.6 Šresolution. This high-resolution structure provides a framework for understanding proteasome assembly.

Jejunogastric intussusception after pancreaticoduodenectomy: a case report.

BACKGROUND: Jejunogastric intussusception (JGI) is a rare, but potentially fatal complication that can occur following gastric surgery, and the reported incidence of JGI is as low as 0.1%. Early diagnosis and treatment are critical for JGI to prevent major complications such as bowel necrosis and death. Although emergency surgery is the standard treatment, endoscopic reduction has also been reported to be effective in JGI patients without bowel necrosis. Several early recurrent cases treated with surgical or endoscopic reduction have been reported. We report an extremely rare case of JGI after pancreaticoduodenectomy (PD) using Child's procedure that was successfully treated with surgical reduction and fixation. CASE PRESENTATION: An 81-year-old man who had undergone PD using Child's procedure 3 years ago presented to our hospital with epigastric pain and nausea. His vital signs were stable, and abdominal examination revealed mild tenderness with a palpable mass in the mid-epigastrium. Abdominal computed tomography (CT) and gastroscopy revealed a JGI of the efferent loop, and exploratory laparotomy was immediately performed. During the operation, the efferent loop showed no adhesions and was intussuscepted through the gastrojejunostomy into the gastric lumen. An incision in the anterior wall of the stomach revealed no evidence of ischemia of the intussusceptum. The efferent loop was reduced using Hutchinson's maneuver and fixed to the afferent loop to prevent a recurrence. The postoperative course was uneventful, and there was no sign of recurrence 12 months postoperatively. CONCLUSIONS: JGI after PD is an extremely rare, but has severe complications. Surgery might be the optimal treatment for JGI in terms of preventing recurrence, even in cases without bowel necrosis.

Crystal structure of yeast rpn14, a chaperone of the 19 S regulatory particle of the proteasome.

The ubiquitin-proteasome pathway is a major proteolytic system in eukaryotic cells and regulates various cellular processes. The 26 S proteasome, the central enzyme of this pathway, consists of a proteolytic core particle and two 19 S regulatory particles (RPs) composed of ATPase (Rpt) and non-ATPase (Rpn) subunits. Growing evidence indicates that proteasome assembly is assisted by a variety of chaperones. In particular, it has been reported recently that Nas2, Nas6, Rpn14, and Hsm3 bind specific Rpt subunits, thereby contributing to the formation of 19 S RP. Rpn14 transiently binds to the C-terminal domain of the Rpt6 subunit (Rpt6-C) during maturation of the ATPase ring of 19 S RP. In this study, we determined the crystal structure of yeast Rpn14 at 2.0 A resolution, which revealed that this chaperone consists of a unique N-terminal domain with unknown function and a C-terminal domain assuming a canonical seven-bladed beta-propeller fold. The Rpt6-binding site on Rpn14 was predicted based on structural comparison with the complex formed between Nas6 and Rpt3-C. The top face of Rpn14 exhibits a highly acidic surface area, whereas the putative interacting surface of Rpt6-C is basic. By inspection of structural data along with genetic and biochemical data, we determined the specific residues of Rpn14 and Rpt6 for complementary charge interactions that are required for 19 S RP assembly.

[Prediction of serum albumin levels by non-invasive factors among elderly female patients].

AIM: To apply nutrition care management to elderly female patients, we predicted serum albumin (s-Alb) levels by non-invasive factors. METHODS: After excluding patients with lesions/diseases which were directly related to s-Alb levels, we investigated 147 elderly women aged 75-years or over who were taking meals orally and were hospitalized from April 2008 to April 2009 at a hospital in Toyota. The patients were classified into 2 groups, one of patients with s-Alb levels of 3.5 g/dl or below (n=80), and the other of those with s-Alb levels of over 3.5 g/dl (n=67). Between the 2 groups, we examined differences in age, body mass index (BMI), living arrangements, necessary nursing care level (NNCL), bed confinement level (BCL), OH scale level (OHSL), and dietary intake either by the Student t-test, Mann-Whitney U test or chi-square test. Pearson correlation coefficients were calculated among s-Alb levels and selected variables. Taking into account the correlation coefficients, we conducted multiple regression analysis adopting the s-Alb level as a dependent variable and non-invasive factors as independent variables. For all the performed tests and analyses, a p value of less than 0.05 (on two-tailed analysis) was assumed to represent a statistically significant difference. RESULTS: S-Alb level was significantly associated with variables, including age, BMI, NNCL, BCL, OHSL, and percentage of protein intake (PPI). Multiple regression analysis revealed 4 significant variables: age, BCL, OHSL, and PPI. The multiple regression equation was y=4.977-(0.098×OHSL)-(0.080×BCL)-(0.016×age)+(0.003×PPI), and the multiple correlation coefficient R(2) was 0.398 (p <0.001). CONCLUSIONS: S-Alb levels among elderly female patients may be predicted by 4 non-invasive variables: age, BCL, OHSL, and PPI.

Laparoscopic surgery for sigmoid colon cancer with complicated communication between the superior and inferior mesenteric arteries.

To perform complete mesocolic excision with central vessel ligation, it is important to recognize the vessel anomaly and the location of the tumor. For left-sided colon cancer, the variations in the course of the left colic artery and accessary middle colic artery must be recognized preoperatively. Here, we describe our experience with a 57-year-old man who was diagnosed with sigmoid colon cancer with complicated inter-mesenteric connections between the inferior mesenteric artery (IMA) and superior mesenteric artery (SMA), possibly due to median arcuate ligament syndrome. We performed laparoscopic sigmoidectomy with low ligation of the IMA to preserve the extremely enlarged left colic artery. The total operative time was 155 minutes, and the estimated total blood loss was 10 mL. The patient was discharged on postoperative day 9 without any postoperative complications. For patients with vascular anomalies in the left-sided mesocolon, preoperatively ruling out SMA stenosis by using angiography and 3-D CT might be important.

Involvement of nitric oxide in a rat model of carrageenin-induced pleurisy.

Some evidence indicates that nitric oxide (NO) contributes to inflammation, while other evidence supports the opposite conclusion. To clarify the role of NO in inflammation, we studied carrageenin-induced pleurisy in rats treated with an NO donor (NOC-18), a substrate for NO formation (L-arginine), and/or an NO synthase inhibitor (S-(2-aminoethyl) isothiourea or N(G)-nitro-L-arginine). We assessed inflammatory cell migration, nitrite/nitrate values, lipid peroxidation and pro-inflammatory mediators. NOC-18 and L-arginine reduced the migration of inflammatory cells and edema, lowered oxidative stress, and normalized antioxidant enzyme activities. NO synthase inhibitors increased the exudate formation and inflammatory cell number, contributed to oxidative stress, induced an oxidant/antioxidant imbalance by maintaining high O(2) (-), and enhanced the production of pro-inflammatory mediators. L-arginine and NOC-18 reversed the proinflammatory effects of NO synthase inhibitors, perhaps by reducing the expression of adhesion molecules on endothelial cells. Thus, our results indicate that NO is involved in blunting-not enhancing-the inflammatory response.

Argon plasma coagulation for successful treatment of bile leakage after subtotal cholecystectomy.

BACKGROUND: Subtotal cholecystectomy is an effective surgical method to decrease the risk of complications for gallbladders that are difficult to remove. However, there is a risk for postoperative refractory bile leakage through the gallbladder stump. Here, we report a new management technique involving the use of argon plasma coagulation (APC) to stop bile leakage after a subtotal cholecystectomy. CASE PRESENTATION: A 74-year-old man was referred to our hospital for abdominal pain and fever. Contrast-enhanced computed tomography of the abdomen showed fluid collection, such as an abscess, surrounding the gallbladder and hepatic flexure colon. The patient was diagnosed with colonic perforative peritonitis, and he underwent emergency surgery. On laparotomy, the abscess was observed outside of the hepatic flexure colon and gallbladder necrosis was detected. The neck of the gallbladder and the area close to the hepatoduodenal ligament was severely inflamed prohibiting dissection. The hepatic flexure colon was part of the abscess wall, and resection was needed. A subtotal cholecystectomy and right hemicolectomy confirmed peritonitis caused by cholecystic perforation. The mucous membrane of the gallbladder neck that remained was necrotic or detached. Therefore, the stump of the gallbladder was closed by primary sutures without cauterization of the mucosa. On postoperative day 6, bile leakage from the gallbladder stump was revealed. Percutaneous and endoscopic retrograde cholangiography drainage were performed. However, the liquid, which seemed to be secreted from the mucosa of the remnant gallbladder, was continuously obtained. We used APC to cauterize the gallbladder mucosa through the fistula of the abdominal drainage tube. Bile leakage and mucus discharge were improved after three rounds of APC cauterization. CONCLUSIONS: APC effectively treated refractory bile leakage from a gallbladder stump after subtotal cholecystectomy for severe cholecystitis.

Increase in P-glycoprotein accompanied by activation of protein kinase Calpha and NF-kappaB p65 in the livers of rats with streptozotocin-induced diabetes.

It is known that protein kinase C (PKC) signal transduction is enhanced in a diabetic state, and that PKC activator phorbol esters increase the gene expression of MDR1 in human tumor cells. To clarify the expression of the liver transporters under diabetic conditions and the roles of PKCalpha and the transcription factor NF-kappaB, we investigated the expression levels of Mdr1a, Mdr1b, Mdr2, Mrp2, Bcrp, Bsep, Oct1, Oat2, and Oat3 transporters, PKCalpha, IkappaB, and NF-kappaB in the liver of rats with STZ-induced hyperglycemia. A selective increase in the gene expression of Mdr1b was detected by RT-PCR. Western blotting with C219 antibody revealed an increase in P-glycoprotein. Although the mRNA level of PKCalpha was not affected, translocation of PKCalpha to the microsomal fraction was detected. NF-kappaB p65, IkappaBalpha and IkappaBbeta mRNA levels were increased as was the level of nuclear NF-kappaB p65. From these findings, it was suggested that STZ-induced hyperglycemia caused the upregulation of Mdr1b P-gp expression through the activation of PKCalpha and NF-kappaB.

Urinary excretion of 3-phenoxybenzoic acid in middle-aged and elderly general population of Japan.

Limited data are available on the background levels of exposure to synthetic pyrethroid (PYR) in Japan, despite their frequent application for agriculture and indoor extermination and possible effects of chronic and/or low-dose PYR exposure on human health. This study was conducted to describe the level and distribution of one of the major PYR metabolites, 3-phenoxybenzoic acid (3-PBA), in urine samples collected from a general population in Japan. The subjects were 535 individuals (184 men and 351 women; 61.5+/-9.8 years of age, mean+/-S.D.) residing in a town in Hokkaido, a dairy and agricultural area. Urinary 3-PBA was found detectable in 98% of samples above the limit of detection of 0.02 microg/l. The geometric mean values of urinary 3-PBA in occupationally exposed farmers (n=87) and the remaining general group without occupational exposure (n=448) were 0.38 and 0.29 microg/l, respectively, ranging from