Endoscopic mucosal resection and endoscopic submucosal dissection with an external additional working channel (EMR+ and ESD+) are equivalent to using a double-channel endoscope: a systematic evaluation in a porcine ex vivo model.

BACKGROUND AND AIMS: With an external additional working channel (AWC) endoscopic mucosal resection (EMR) as well as endoscopic submucosal dissection (ESD) can be extended to techniques termed "EMR+" and "ESD+." These novel techniques are systematically compared to EMR and ESD under the use of a double-channel endoscope (DC). METHODS: Our trial was conducted prospectively in a pre-clinical porcine animal model (EASIE-R simulator) with standardized gastric lesions measuring 3 or 4 cm. RESULTS: EMR+ and EMR DC showed both good results for 3 cm lesions with no adverse events and an en bloc resection rate of 73.33% (EMR+) and 60.00% (EMR DC, p = 0.70). They came to their limits in 4 cm lesions with muscularis damages of 20.00% (EMR+), 13.33% (EMR DC, p ≥ 0.99) and decreasing en bloc resection rates of 60.00% (EMR+) and 46.67% (EMR DC, p = 0.72). ESD+ and ESD DC were both reliable concerning en bloc resection rates (100% in all groups) and adverse events (0.00% in 3 cm lesions, 12.50% muscularis damages in both ESD+ and ESD DC in 4 cm lesions). Resection time was slightly shorter in all groups with the AWC compared to DC although only reaching significance in 3 cm ESD lesions (p < 0.05*). CONCLUSIONS: With the AWC, a standard endoscope can easily be transformed to double-channel functionality. We could show that EMR+ and ESD+ are non-inferior to EMR and ESD under the use of a double-channel endoscope. Consequently, the AWC presents an affordable alternative to a double-channel endoscope for both EMR and ESD.

Enhancer of Zeste Homolog 2 (EZH2) Is a Marker of High-Grade Neuroendocrine Neoplasia in Gastroenteropancreatic and Pulmonary Tract and Predicts Poor Prognosis.

Tumor grading is a robust prognostic predictor in patients with neuroendocrine neoplasms (NEN) and guides therapy, especially in tumors with high proliferation. NEN can be separated into well-differentiated and poorly differentiated types. The more aggressive NEN have been further separated into neuroendocrine tumors (NET G3) with a better prognosis and neuroendocrine carcinomas (NEC) with a worse prognosis. Despite this distinction's tremendous clinical and therapeutic relevance, optimal diagnostic biomarkers are still lacking. In this study, we analyzed the protein expression and prognostic impact of Enhancer of Zeste Homolog 2 (EZH2) by immunohistochemistry in 219 tissue samples of gastroenteropancreatic (GEP-NEN) and pulmonary NEN (P-NEN). EZH2 was almost exclusively expressed in NEN with a proliferation rate above 20% (G3), while all low-grade tumors were nearly negative. Among high-grade NEN, 65% showed high and 35% low expression of EZH2. In this group, the high expression of EZH2 was significantly associated with poor overall survival and NEC histology. Interestingly, EZH2 seems to act independently of Polycomb Repressive Complex 2 (PRC2) in NEN. In conclusion, we propose EZH2 as a robust biomarker for distinguishing between NET G3 and NEC among gastroenteropancreatic and pulmonary NEN.

NFATc1 signaling drives chronic ER stress responses to promote NAFLD progression.

OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) can persist in the stage of simple hepatic steatosis or progress to steatohepatitis (NASH) with an increased risk for cirrhosis and cancer. We examined the mechanisms controlling the progression to severe NASH in order to develop future treatment strategies for this disease. DESIGN: NFATc1 activation and regulation was examined in livers from patients with NAFLD, cultured and primary hepatocytes and in transgenic mice with differential hepatocyte-specific expression of the transcription factor (Alb-cre, NFATc1c.a . and NFATc1Δ/Δ ). Animals were fed with high-fat western diet (WD) alone or in combination with tauroursodeoxycholic acid (TUDCA), a candidate drug for NAFLD treatment. NFATc1-dependent ER stress-responses, NLRP3 inflammasome activation and disease progression were assessed both in vitro and in vivo. RESULTS: NFATc1 expression was weak in healthy livers but strongly induced in advanced NAFLD stages, where it correlates with liver enzyme values as well as hepatic inflammation and fibrosis. Moreover, high-fat WD increased NFATc1 expression, nuclear localisation and activation to promote NAFLD progression, whereas hepatocyte-specific depletion of the transcription factor can prevent mice from disease acceleration. Mechanistically, NFATc1 drives liver cell damage and inflammation through ER stress sensing and activation of the PERK-CHOP unfolded protein response (UPR). Finally, NFATc1-induced disease progression towards NASH can be blocked by TUDCA administration. CONCLUSION: NFATc1 stimulates NAFLD progression through chronic ER stress sensing and subsequent activation of terminal UPR signalling in hepatocytes. Interfering with ER stress-responses, for example, by TUDCA, protects fatty livers from progression towards manifest NASH.

Tunnelled Peritoneal Catheter for Malignant Ascites-An Open-Label, Prospective, Observational Trial.

Treatment of recurrent malignant ascites in cancer patients is a challenge. Evidence based guidelines regarding the best treatment strategy are lacking. The aim of this prospective study was to investigate the safety and efficacy of a tunnelled peritoneal catheter (PleurX) in cancer patients with symptomatic ascites. Patients with symptomatic, diuretics-refractory ascites and indication for the implantation of a tunnelled peritoneal PleurX catheter were prospectively enrolled between August 2018 and July 2020. The number of catheter days, complications, amount of drained ascites and ascites-associated symptoms and hospitalization rate pre- and post-PleurX insertion were analysed. 51 Patients (64.7% male) were prospectively enrolled. The mean age was 66.6 (±7.9) years. The most common cause of ascites was pancreatic adenocarcinoma (n = 10) followed by cholangiocellular carcinoma (n = 9) and hepatocellular carcinoma (n = 8). The technical success rate of PleurX implantation was 100%. The mean volume of weakly drained ascites was 5.44l (±4.08). Major complications included cellulitis (n = 2), peritonitis and drainage dislocation (each n = 1). The mean catheter days per patient was 59.8 (±107.4) (Min 4, Max 668). Abdominal discomfort, impaired mobility, dyspnoea, fatigue, nausea and vomiting were significantly reduced 30 days after PleurX insertion (p < 0.05). Moreover, hospitalization rate was significantly reduced (p < 0.001; 27.08% of days preimplantation vs. 11.27% postimplantation). We conclude that implantation of a tunnelled ascites catheter is a safe and effective method for the treatment of refractory ascites in cancer patients with advanced disease. Serious complications are rare. Burdensome ascites-associated symptoms and hospitalization rates can be significantly reduced over a longer period of time.

Enhancer of Zeste Homolog 2 in Colorectal Cancer Development and Progression.

BACKGROUND: Colorectal cancer (CRC) is the leading gastrointestinal malignancy. The development from premalignant intraepithelial lesions leading to invasive cancer is paradigmatic for the stepwise carcinogenesis of epithelial cancers, but the knowledge of the underlying mechanism of carcinogenesis and progression of CRC is still incomplete. The understanding of epigenetic mechanisms of carcinogenesis has led to new therapeutic approaches during the last years. Enhancer of zeste homolog 2 (EZH2) is one central epigenetic silencer of the polycomb repressor complex 2 (PRC2) that is already in clinical use as a novel drug target and is associated with poorer prognosis in several cancer entities. PATIENTS AND METHODS: The protein expression of EZH2 and other members of the PRC2 as well as resulting posttranslational modifications were investigated by immunohistochemistry in 187 patients with CRC and in 94 patients with premalignant colorectal lesions and correlated with their clinical outcome. Furthermore, the corresponding mRNA expression levels were analyzed in 217 patients with rectal cancer that were enrolled in a prospective clinical trial. RESULTS: We found a weak expression of EZH2 in normal colon mucosa that increased in low grade, peaked in high grade intraepithelial neoplasia, and decreased again in invasive CRC. The posttranslational modification caused by EZH2 as a measure of EZH2 activity showed the same behavior. Strong protein and mRNA expression of EZH2 were significantly correlated with favorable prognosis in both investigated cohorts. CONCLUSION: The expression and activity of EZH2 are associated with colorectal carcinogenesis and most expressed in intraepithelial high-grade lesions. Strong expression of EZH2 is associated with a significantly favorable prognosis in patients suffering from CRC.

Endoscopic submucosal dissection with an additional working channel (ESD+): a novel technique to improve procedure time and safety of ESD.

BACKGROUND AND AIMS: A new external additional working channel (AWC) was recently introduced by which endoscopic submucosal dissection (ESD) can be converted to a technique termed "ESD+ ". We aim to systematically evaluate this novel technique in flat gastric lesions and compare it to classical ESD. METHODS: The study was prospectively conducted in a pre-clinical ex vivo animal model (EASIE-R simulator) with porcine stomachs. Prior to intervention, we set standardized lesions measuring 3 cm or 4 cm in antegrade as well as in retrograde positions. RESULTS: Overall, 64 procedures were performed by an experienced endoscopist. Both techniques were reliable and showed en bloc resection rates of 100%. Overall, ESD+ reduced time of procedure compared to ESD (24.5 vs. 32.5 min, p = 0.025*). Particularly, ESD+ was significantly faster in retrograde lesions with a median of 22.5 vs. 34.0 min in 3 cm retrograde lesions (p = 0.002*) and 34.5 vs. 41.0 min (p = 0.011*) in 4 cm retrograde lesions. There were 0 perforations with both techniques. In ESD+ , 1 muscularis damage occurred (3.13%) compared to 6 muscularis damages with ESD (18.75%, p = 0.045*). CONCLUSIONS: By its grasp-and-mobilize technique, ESD+ allows potentially faster and safer resections of flat gastric lesions compared to conventional ESD in an ex vivo porcine model. The potential advantages of ESD+ in terms of procedure time may be particularly relevant for difficult lesions in retrograde positions.

Diagnostic accuracy of B-Mode ultrasound and Hepatorenal Index for graduation of hepatic steatosis in patients with chronic liver disease.

BACKGROUND/AIMS: The aim of our study was to evaluate the diagnostic accuracy of B-Mode ultrasound and Hepatorenal Index (HRI) by high-end devices for the detection and classification of hepatic steatosis in patients with various causes of chronic liver disease (CLD). METHODS: We retrospectively enrolled patients with CLD who underwent liver biopsy and baseline ultrasound between March 2016 and May 2019. Sonographic graduation of steatosis (0°-III°) using B-Mode criteria and HRI were correlated with the histological graduation (S0 (<5% fat), S1 (≥5-33%), S2 (>33-66%) and S3 (>66%). Interobserver agreement was calculated. RESULTS: 157 patients were evaluated. B-Mode ultrasound had a sensitivity of 75.6% and a specificity of 76.0% to differentiate between steatosis and no steatosis (AUROC 0.758). Using B-Mode criteria for advanced steatosis (≥II°), specificity for presence of histological steatosis was ≥98.7%. For detection of advanced steatosis (≥S2), sensitivity of B-mode criteria was 90.9%. In a subgroup of patients with advanced liver fibrosis, sensitivity of B-mode criteria was 95.0% for detection of advanced steatosis (S≥2). A HRI cut-off-value of 1.46 differentiates between patients with steatosis and patients without steatosis with a sensitivity of 42.7% and a specificity of 90.7% (AUROC 0.680). Interobserver agreement of both B-Mode and HRI was good to excellent. CONCLUSION: B-Mode ultrasound using high-end devices is an excellent method to detect advanced steatosis in patients with various CLD. For diagnosis of mild steatosis, modern ultrasound devices may have higher sensitivity but at the expense of specificity. Stage of fibrosis and etiology of CLD seem not to impact on diagnostic accuracy. The additional calculation of HRI seems to have no additional benefit with regard to detect or grade hepatic steatosis in our study population.

Endoscopic mucosal resection with an additional working channel (EMR+) in a porcine ex vivo model: a novel technique to improve en bloc resection rate of snare polypectomy.

Background and study aims Recently, a new external additional working channel (AWC) was introduced by which conventional endoscopic mucosal resection (EMR) can be improved to a technique termed "EMR+". We first evaluated this novel technique in comparison to classical EMR in flat lesions. Methods The trial was prospectively conducted in an ex vivo animal model with porcine stomachs placed into the EASIE-R simulator. Prior to intervention, standardized lesions were set by coagulation dots, measuring 1, 2, 3 or 4 cm. Results Overall, 152 procedures were performed. EMR and EMR+ were both very reliable in 1-cm lesions, each showing en bloc resection rates of 100 %. EMR+ en bloc resection rate was significantly higher in 2-cm lesions (95.44 % vs. 54.55 %, P  = 0.02), in 3-cm lesions (86.36 % vs. 18.18 %, P  < 0.01) and also in 4-cm lesions (60.00 % vs. 0 %, P  < 0.01). Perforations occurred only in EMR+ procedures in 4-cm lesions (3 of 20; 15 %). Conclusions With its grasp-and-snare technique, EMR+ facilitates en bloc resection of larger lesions compared to conventional EMR. In lesions 2 cm and larger, EMR+ has demonstrated advantages, especially concerning en bloc resection rate. At 3 cm, EMR+ reaches its best discriminatory power whereas EMR+ has inherent limits at 4 cm and in lesions of that size, other techniques such as ESD or surgery should be considered.

Noninvasive assessment of liver fibrosis in a real-world cohort of patients with known or suspected chronic liver disease using 2D-shear wave elastography.

OBJECTIVES: We aimed to investigate the diagnostic accuracy of liver stiffness measurement (LSM) by 2D-shear wave elastography (2D-SWE, GE, Logiq E9) in patients with known or suspected chronic liver disease and to define cutoff values for the different stages of fibrosis. METHODS: First, we retrospectively enrolled 21 patients in a pilot study and validated the results in a prospective cohort of 70 patients between May 2017 and February 2019. In all patients, LSM and liver biopsy were performed. We analyzed the diagnostic accuracy of LSM for the different fibrosis stages and examined the impact of additional clinical parameters on LSM. RESULTS: The success rate of LSM was 88.6%. In the prospective cohort, optimal cutoff values for F ≥ 1, F ≥ 2, F ≥ 3 and F = 4 were 6.24, 7.86, 8.05 and 10.74 kPa [area under the receiver operating characteristic curve (AUROC) 0.831, 0.913, 0.996 and 0.954]. In both cohorts and in the subgroup of patients with nonalcoholic fatty liver disease (NAFLD) (n = 35), a cutoff value of 8.05 kPa differentiates patients with advanced fibrosis (F ≥ 3) and patients with no or mild fibrosis (F0-F2) with high diagnostic accuracy (AUROC 0.995-1.000). Parameters such as age, sex, BMI, bilirubin- and alanine aminotransferase-level had no significant impact on LSM. CONCLUSION: LSM by 2D-SWE is an excellent method to differentiate between patients with advanced fibrosis (F ≥ 3) and patients with no or mild fibrosis (F ≤ 2). We were able to show this also in a subgroup of patients with NAFLD.

Testing of Helicobacter pylori by Endoscopic Biopsy: The Clinical Dilemma of Suppressive Conditions.

BACKGROUND AND AIMS: Testing for Helicobacter pylori is frequently conducted during esophagogastroduodenoscopy (EGD). Suppressive conditions such as the intake of proton-pump inhibitors (PPIs), preceded antibiotic treatment or recent upper gastrointestinal bleeding impair H. pylori test quality. The aim of our study was to evaluate the frequency and pattern of H. pylori suppressive conditions in a large patient collective undergoing elective EGD in a German university hospital. METHODS: The trial was performed as a single-center study. Only elective EGD from inpatients and outpatients were included. Prior to endoscopy, H. pylori suppressive conditions were collected using a standardized questionnaire. If H. pylori testing was indicated according to the guidelines, always both histology and helicobacter urease test were performed in analogy to the Sydney classification. RESULTS: One thousand six hundred and thirty-one patients were included (median 61 years, 36.0% outpatients, 64.0% inpatients). Overall, 76.5% of patients were under H. pylori suppressive conditions. The main suppressive condition was the intake of PPIs (70.7%). In 819 (50.2%) of all included cases, H. pylori testing was performed. The following were the results: 17.3% (142) had a positive H. pylori testing and 82.7% (677) were negative. Of those with negative result, 70.0% were tested under suppressive conditions. CONCLUSION: Guidelines recommend H. pylori testing under non-suppressive conditions. However, this does not always meet the clinical practice. Our data show that de facto, many patients undergoing elective EGD are tested for H. pylori under suppressive conditions coming along with a higher risk of potentially false negative results. Particularly, concerning this issue, further research is needed to improve and clarify everyday clinical practice.

Cytomegalovirus colitis in inflammatory bowel disease and after haematopoietic stem cell transplantation: diagnostic accuracy, predictors, risk factors and disease outcome.

BACKGROUND: Concurrent cytomegalovirus (CMV) colitis in inflammatory bowel disease (IBD) and after haematopoietic stem cell transplantation (HSCT) is an important clinical entity associated with high rates of morbidity and mortality. METHODS: A retrospective study of 47 patients with IBD and 61 HSCT patients was performed regarding the evaluation of diagnostic accuracy of applied methods, predictors, risk factors for CMV disease manifestation, the proportion of patients with antiviral treatment and disease outcome. RESULTS: The sensitivity of quantitative PCR (qPCR) with a cut-off value of >250 copies/mg for CMV colitis in patients with IBD and HSCT patients was 79% and 92%, respectively. Predictors for CMV colitis in the IBD cohort were anaemia and the presence of endoscopic ulcers. Glucocorticoids, calcineurin inhibitors and >2 concurrent lines of treatment with immunosuppressive drugs could be identified as risk factors for CMV colitis in the IBD cohort with an OR of 7.1 (95% CI 1.7 to 29.9), 21.3 (95% CI 2.4 to 188.7) and 13.4 (95% CI 3.2 to 56.1), respectively. Predictors and risk factors for CMV gastroenteritis in the HSCT cohort was the presence of endoscopic ulcers (OR 18.6, 95% CI 3.3 to 103.7) and >2 concurrent lines of treatment with immunosuppressive drugs. Antiviral therapy was administered in 70% of patients with IBD and 77% of HSCT patients with CMV disease. 71% of antiviral-treated patients with IBD showed an improvement of their disease activity and 14% underwent colectomy. The mortality rate of HSCT patients was 21% irrespective of their CMV status. CONCLUSIONS: In addition to the implementation of histological methods, qPCR may be performed in patients with suspected high-risk IBD and HSCT patients for CMV colitis. Independent validations of these results in further prospective studies are needed.

Evaluation of liver stiffness by 2D-SWE in combination with non-invasive parameters as predictors for esophageal varices in patients with advanced chronic liver disease.

Background/aims: Esophageal varices (EV) are common complications in patients with advanced chronic liver disease (ACLD). Non-invasive parameters to exclude EV in patients with ACLD would be desirable. The aim of this study was the evaluation of liver stiffness measurement (LSM) using 2D-shear wave elastography (GE Logiq E9) and other non-invasive parameters as predictors for EV. Methods: Hundred patients with ACLD were enrolled. Abdominal sonography, including measurement of gall bladder wall thickness (GBWT), spleen diameter and LSM, gastroscopy and blood test results were evaluated. Statistical analyses were performed for the association between EV and non-invasive parameters. Results: Fifty-one per cent of the patients had EV. The mean LSM (14.6 kPa) and GBWT (3.88 mm) in the group with EV were significantly higher than in the group without EV (10.6 kPa; 2.94 mm; p < .01). Performing area under the receiver operating characteristic curve, LSM has a better diagnostic performance (0.781) than GBWT (0.707), spleen diameter (0.672) and platelet count (0.635). Combining LSM (cut-off 13.58 kPa) and GBWT (cut-off 3.07 mm) resulted in a sensitivity of 86.3% and a specificity of 71.4% for the presence of EV. A sensitivity of 100% (negative predictive value 1.0) was achieved at LSM >9 kPa or GBWT >4 mm. Following these criteria in our current study population, 18% of the gastroscopies could have been avoided. Conclusions: Combining LSM with non-invasive parameters, especially GBWT, improves the diagnostic accuracy for predicting EV. We suggest reconsidering screening gastroscopy in patients with ACLD who show LSM <9 kPa and GBWT <4 mm due to the very low risk of having varices.

The p97-Ufd1-Npl4 ATPase complex ensures robustness of the G2/M checkpoint by facilitating CDC25A degradation.

The p97-Ufd1-Npl4 ATPase complex is associated with the response to DNA damage and replication stress, but how its inactivation leads to manifestation of chromosome instability is unclear. Here, we show that p97-Ufd1-Npl4 has an additional direct role in the G2/M checkpoint. Upon DNA damage, p97-Ufd1-Npl4 binds CDC25A downstream of ubiquitination by the SCF-ßTrCP ligase and facilitates its proteasomal degradation. Depletion of Ufd1-Npl4 leads to G2/M checkpoint failure due to persistent CDC25 activity and propagation of DNA damage into mitosis with deleterious effects on chromosome segregation. Thus, p97-Ufd1-Npl4 is an integral part of G2/M checkpoint signaling and thereby suppresses chromosome instability.

Emerging functions of the VCP/p97 AAA-ATPase in the ubiquitin system.

The ATP-driven chaperone valosin-containing protein (VCP)/p97 governs critical steps in ubiquitin-dependent protein quality control and intracellular signalling pathways. It cooperates with diverse partner proteins to help process ubiquitin-labelled proteins for recycling or degradation by the proteasome in many cellular contexts. Recent studies have uncovered unexpected cellular functions for p97 in autophagy, endosomal sorting and regulating protein degradation at the outer mitochondrial membrane, and elucidated a role for p97 in key chromatin-associated processes. These findings extend the functional relevance of p97 to lysosomal degradation and reveal a surprising dual role in protecting cells from protein stress and ensuring genome stability during proliferation.

Endolysosomal sorting of ubiquitylated caveolin-1 is regulated by VCP and UBXD1 and impaired by VCP disease mutations.

The AAA-ATPase VCP (also known as p97) cooperates with distinct cofactors to process ubiquitylated proteins in different cellular pathways. VCP missense mutations cause a systemic degenerative disease in humans, but the molecular pathogenesis is unclear. We used an unbiased mass spectrometry approach and identified a VCP complex with the UBXD1 cofactor, which binds to the plasma membrane protein caveolin-1 (CAV1) and whose formation is specifically disrupted by disease-associated mutations. We show that VCP-UBXD1 targets mono-ubiquitylated CAV1 in SDS-resistant high-molecular-weight complexes on endosomes, which are en route to degradation in endolysosomes. Expression of VCP mutant proteins, chemical inhibition of VCP, or siRNA-mediated depletion of UBXD1 leads to a block of CAV1 transport at the limiting membrane of enlarged endosomes in cultured cells. In patient muscle, muscle-specific caveolin-3 accumulates in sarcoplasmic pools and specifically delocalizes from the sarcolemma. These results extend the cellular functions of VCP to mediating sorting of ubiquitylated cargo in the endocytic pathway and indicate that impaired trafficking of caveolin may contribute to pathogenesis in individuals with VCP mutations.

Cdc48/p97-Ufd1-Npl4 antagonizes Aurora B during chromosome segregation in HeLa cells.

During exit from mitosis in Xenopus laevis egg extracts, the AAA+ ATPase Cdc48/p97 (also known as VCP in vertebrates) and its adapter Ufd1-Npl4 remove the kinase Aurora B from chromatin to allow nucleus formation. Here, we show that in HeLa cells Ufd1-Npl4 already antagonizes Aurora B on chromosomes during earlier mitotic stages and that this is crucial for proper chromosome segregation. Depletion of Ufd1-Npl4 by small interfering RNA (siRNA) caused chromosome alignment and anaphase defects resulting in missegregated chromosomes and multi-lobed nuclei. Ufd1-Npl4 depletion also led to increased levels of Aurora B on prometaphase and metaphase chromosomes. This increase was associated with higher Aurora B activity, as evidenced by the partial resistance of CENP-A phosphorylation to the Aurora B inhibitor hesperadin. Furthermore, low concentrations of hesperadin partially rescued chromosome alignment in Ufd1-depleted cells, whereas, conversely, Ufd1-depletion partially restored congression in the presence of hesperadin. These data establish Cdc48/p97-Ufd1-Npl4 as a crucial negative regulator of Aurora B early in mitosis of human somatic cells and suggest that the activity of Aurora B on chromosomes needs to be restrained to ensure faithful chromosome segregation.

Cheating on ubiquitin with Atg8.

Macroautophagy sequesters superflous cytosol and organelles into double-membraned autophagosomes. Over 30 autophagy-related (ATG) genes have been identified without elucidating the molecular details of autophagosome biogenesis. All proposed models for autophagosome formation require membrane fusion events (Fig. 1). Previous studies assumed that the autophagic machinery mediates these membrane fusions in a SNARE-independent manner and identified the ubiquitin-like protein Atg8 as a key component especially for elongation of the forming autophagosome. However, if and how Atg8 mediates membrane fusion and why a ubiquitin-like protein is needed for autophagosome biogenesis remained open questions. Since nuclear envelope growth and fusion of Golgi fragments are topologically similar to autophagosome formation and depend on the AAA (+) ATPase p97/VCP and p47 we analyzed the involvement of their yeast homologues Cdc48 and Shp1 in macroautophagy.

Cdc48/p97 and Shp1/p47 regulate autophagosome biogenesis in concert with ubiquitin-like Atg8.

The molecular details of the biogenesis of double-membraned autophagosomes are poorly understood. We identify the Saccharomyces cerevisiae AAA-adenosine triphosphatase Cdc48 and its substrate-recruiting cofactor Shp1/Ubx1 as novel components needed for autophagosome biogenesis. In mammals, the Cdc48 homologue p97/VCP and the Shp1 homologue p47 mediate Golgi reassembly by extracting an unknown monoubiquitinated fusion regulator from a complex. We find no requirement of ubiquitination or the proteasome system for autophagosome biogenesis but detect interaction of Shp1 with the ubiquitin-fold autophagy protein Atg8. Atg8 coupled to phosphatidylethanolamine (PE) is crucial for autophagosome elongation and, in vitro, mediates tethering and hemifusion. Interaction with Shp1 requires an FK motif within the N-terminal non-ubiquitin-like Atg8 domain. Based on our data, we speculate that autophagosome formation, in contrast to Golgi reassembly, requires a complex in which Atg8 functionally substitutes ubiquitin. This, for the first time, would give a rationale for use of the ubiquitin-like Atg8 during macroautophagy and would explain why Atg8-PE delipidation is necessary for efficient macroautophagy.