ATG9A supports Chlamydia trachomatis infection via autophagy-independent mechanisms.

Chlamydia trachomatis infection can be regulated by autophagy-related (ATG) genes. Here, we found that the depletion of ATG9A, one of the core ATG genes, in HeLa cells suppressed C. trachomatis growth in the inclusion. The growth was restored by re-expressing ATG9A or an ATG9A mutant impairing lipid scramblase activity in ATG9A-knockout (KO) cells. Moreover, the depletion of lipid transfer proteins ATG2A/B, responsible for isolation membrane expansion together with ATG9A, did not significantly alter the growth, suggesting that the non-autophagic function of ATG9A supports C. trachomatis infection. ATG9A-KO cells showed no infection-induced redistribution of the Golgi from the perinuclear region to inclusion, which was restored by re-expressing the mutant but not the ATG9A mutant lacking an N-terminal adapter protein-binding domain. Re-expression of the N-terminal deletion mutant in ATG9A-KO cells did not rescue C. trachomatis growth, suggesting the importance of this domain for its growth. Although ATG9A-KO cells showed enhanced TBK1 activation, interferon (IFN)-ß was not significantly increased, excluding the possibility that upregulation of stimulator of IFN genes (STING) signaling suppressed bacterial growth. Taken together, these findings suggest that the proper trafficking, rather than the isolation membrane expansion function, of ATG9A assists C. trachomatis growth in the inclusion. IMPORTANCE ATG9A is an autophagy-related gene that functions during the isolation membrane expansion process to form autophagosomes, but it also has other functions independent of autophagy. In this study, we employed ATG9A-deficient HeLa cells and found that the absence of ATG9A negatively impacted proliferation of Chlamydia trachomatis in inclusions. Furthermore, rescue experiments using ATG9A mutants revealed that this action was mediated not by its autophagic function but by its binding ability to clathrin adapter proteins. These findings suggest that the proper trafficking of ATG9A assists C. trachomatis growth in the inclusion.

Chlamydia repurposes the actin-binding protein EPS8 to disassemble epithelial tight junctions and promote infection.

Invasive microbial pathogens often disrupt epithelial barriers, yet the mechanisms used to dismantle tight junctions are poorly understood. Here, we show that the obligate pathogen Chlamydia trachomatis uses the effector protein TepP to transiently disassemble tight junctions early during infection. TepP alters the tyrosine phosphorylation status of host proteins involved in cytoskeletal regulation, including the filamentous actin-binding protein EPS8. We determined that TepP and EPS8 are necessary and sufficient to remodel tight junctions and that the ensuing disruption of epithelial barrier function promotes secondary invasion events. The genetic deletion of EPS8 renders epithelial cells and endometrial organoids resistant to TepP-mediated tight junction remodeling. Finally, TepP and EPS8 promote infection in murine models of infections, with TepP mutants displaying defects in ascension to the upper genital tract. These findings reveal a non-canonical function of EPS8 in the disassembly of epithelial junctions and an important role for Chlamydia pathogenesis.

Hepatocyte-specific depletion of ubiquitin regulatory X domain containing protein 8 accelerates fibrosis in a mouse non-alcoholic steatohepatitis model.

Ubiquitin regulatory X domain-containing protein 8 (UBXD8) is engaged in the degradation of lipidated apolipoprotein B in hepatocytes. We previously showed that hepatocyte-specific UBXD8-deficient mice (U8-HKO) fed a moderately high-fat diet (31 kcal % fat) showed periportal macrovesicular steatosis along with a decrease in very low-density lipoprotein secretion, but did not develop fibrosis. In the present study, we examined whether U8-HKO mice show NASH-like phenotypes when fed a very high-fat diet (60 kcal % fat). U8-HKO mice and their age-matched littermates (control) were fed with two NASH model diets: choline-sufficient very high-fat diet and choline-deficient very high-fat diet. After being fed a very high-fat diet for 2 weeks, U8-HKO mice showed hepatic fibrosis in a significantly wider area than in the control. Fibrosis in U8-HKO mouse liver was further enhanced under a very high-fat diet depleted of choline (the liver surface was lumpy). Concomitant administration of an angiotensin 2 type 1 receptor blocker reduced the hepatic fibrosis caused by the very high-fat diet, suggesting the existence of inflammation. Carbon tetrachloride also induced hepatic fibrosis but the severity was comparable in the control and U8-HKO mice. In conjunction with our previous finding, the results indicate that although UBXD8 functionality can be largely compensated in the normal setting, it is crucial to sustain VLDL secretion when exposed to a dietary challenge of high fat. U8-HKO mice that develop fibrosis within 2 weeks of high-fat feeding can be used as a model to study NAFLD/NASH disease progression.

Regulation of lipid metabolism via a connection between the endoplasmic reticulum and lipid droplets.

Lipid droplets (LDs) are ubiquitous organelles that store and supply lipids to regulate cellular lipid homeostasis. Fatty acids are packaged as triglyceride and cholesterol ester into endoplasmic reticulum (ER) membranes to synthesize LDs. Cytosolic LDs move dynamically and interact with organelles, including other LDs. In this process, functional proteins for metabolism are also transferred to LDs. In this review, I focus on interactions between the ER and LDs related to lipid metabolism.

[A Case of HER2-Positive Esophagogastric Junction Cancer with Perforation Curatively Resected after Neoadjuvant Chemotherapy plus Trastuzumab].

A 60-year-old man was diagnosed with adenocarcinoma of the esophagogastric junction with lymph node metastasis along the left gastric artery. The clinical stage was determined to be T4b, N1, M0, Stage IIIB, and a neoadjuvant chemotherapy (NAC)regimen of capecitabine/CDDP plus trastuzumab was selected for treatment. Before 3 courses of chemotherapy, the patient developed perforated gastric cancer. With conservative therapy, we were able to obtain closure of the perforation without affecting the curability of the cancer. We changed the chemotherapy regimen to S-1/CDDP plus trastuzumab, and the patient underwent curative resection.

New endoscopic classification of the cardiac orifice in esophageal achalasia: Champagne glass sign.

BACKGROUND AND AIM: Endoscopy, barium esophagram and manometry are used in the diagnosis of achalasia. In the case of early achalasia, characteristic endoscopic findings are difficult to recognize. As a result, the diagnosis of achalasia is often made several years after symptom onset. Therefore, we examined the endoscopic findings of the cardiac orifice in achalasia and propose a new classification. METHODS: A total of 400 patients with spastic esophageal motility disorders who underwent peroral endoscopic myotomy (POEM) at our hospital between March 2014 and August 2015 were screened for this study. Champagne glass sign (CG) was defined as when the distal end of the lower esophageal sphincter relaxation failure (LESRF) was proximal to the squamocolumnar junction (SCJ) and the SCJ was dilated in the retroflex view. Specifically, CG-1 was defined as a distance from the SCJ to the lower end of LESRF of <1 cm, and CG-2 was defined as a distance ≥1 cm. RESULTS: CG-0 was seen in 73 patients (28.0%), whereas the CG sign was seen in 186 patients (71.3%), of whom 170 (65.1%) were CG-1 and 16 (6.1%) were CG-2. CONCLUSIONS: The CG sign is often observed in esophageal achalasia patients. CG-0 (equal to Maki-tsuki) was observed in 28.0% of achalasia patients only. Its absence with dilated SCJ cannot be used to rule out achalasia. Barium esophagram and manometry should be done if esophageal achalasia is strongly suspected.

Hepatocyte-Specific Depletion of UBXD8 Induces Periportal Steatosis in Mice Fed a High-Fat Diet.

We showed previously that UBXD8 plays a key role in proteasomal degradation of lipidated ApoB in hepatocarcinoma cell lines. In the present study, we aimed to investigate the functions of UBXD8 in liver in vivo. For this purpose, hepatocyte-specific UBXD8 knockout (UBXD8-LKO) mice were generated. They were fed with a normal or high-fat diet, and the phenotypes were compared with those of littermate control mice. Hepatocytes obtained from UBXD8-LKO and control mice were analyzed in culture. After 26 wk of a high-fat diet, UBXD8-LKO mice exhibited macrovesicular steatosis in the periportal area and microvesicular steatosis in the perivenular area, whereas control mice exhibited steatosis only in the perivenular area. Furthermore, UBXD8-LKO mice on a high-fat diet had significantly lower concentrations of serum triglyceride and VLDL than control mice. A Triton WR-1339 injection study revealed that VLDL secretion from hepatocytes was reduced in UBXD8-LKO mice. The decrease of ApoB secretion upon UBXD8 depletion was recapitulated in cultured primary hepatocytes. Accumulation of lipidated ApoB in lipid droplets was observed only in UBXD8-null hepatocytes. The results showed that depletion of UBXD8 in hepatocytes suppresses VLDL secretion, and could lead to periportal steatosis when mice are fed a high-fat diet. This is the first demonstration that an abnormality in the intracellular ApoB degradation mechanism can cause steatosis, and provides a useful model for periportal steatosis, which occurs in several human diseases.

ELMOD2 is anchored to lipid droplets by palmitoylation and regulates adipocyte triglyceride lipase recruitment.

Adipocyte triglyceride lipase (ATGL) is the major enzyme involved in the hydrolysis of triglycerides. The Arf1-coat protein complex I (COPI) machinery is known to be engaged in the recruitment of ATGL to lipid droplets (LDs), but the regulatory mechanism has not been clarified. In the present study, we found that ELMOD2, a putative noncanonical Arf-GTPase activating protein (GAP) localizing in LDs, plays an important role in controlling ATGL transport to LDs. We showed that knockdown of ELMOD2 by RNA interference induced an increase in the amount of ATGL existing in LDs and decreased the total cellular triglycerides. These effects of ELMOD2 knockdown were canceled by transfection of small interfering RNA-resistant cDNA of wild-type ELMOD2 but not by that of mutated ELMOD2 lacking the Arf-GAP activity. ELMOD2 was distributed in the endoplasmic reticulum and mitochondria as well as in LDs, but palmitoylation was required only for distribution to LDs. An ELMOD2 mutant deficient in palmitoylation failed to reconstitute the ATGL transport after the ELMOD2 knockdown, indicating that distribution in LDs is indispensable to the functionality of ELMOD2. These results indicate that ELMOD2 regulates ATGL transport and cellular lipid metabolism by modulating the Arf1-COPI activity in LDs.

Inhibition of Niemann-Pick-type C1-like1 by ezetimibe activates autophagy in human hepatocytes and reduces mutant α1-antitrypsin Z deposition.

UNLABELLED: Autophagy can degrade aggregate-prone proteins, but excessive autophagy can have adverse effects. It would be beneficial if autophagy could be enhanced in a cell type-specific manner, but this has been difficult because the basic mechanism of autophagy is common. In the present study we found that inhibition of Niemann-Pick-type C1-like 1 (NPC1L1) by ezetimibe activates autophagy only in hepatocytes and small intestinal epithelia, but not in other cells. Ezetimibe induced accumulation of free cholesterol in the late endosome/lysosome and increased partitioning of a Ragulator component, LAMTOR1, in rafts. The latter change led to down-regulation of mammalian target of rapamycin (mTOR)C1 activity by decreasing mTOR recruitment to the late endosome/lysosome and activated autophagy. A primary effect of ezetimibe was found to be a decrease of free cholesterol in the plasma membrane, because all the results caused by ezetimibe were suppressed by supplementation of cholesterol as a methyl-ß-cyclodextrin complex. By enhancing autophagy in human primary hepatocytes with ezetimibe, insoluble mutant α1-antitrypsin Z was reduced significantly. CONCLUSION: Inhibition of NPC1L1 by ezetimibe activates autophagy in human hepatocytes by modulating cholesterol homeostasis. Ezetimibe may be used to ameliorate liver degeneration in α1-antitrypsin deficiency.

Open questions in lipid droplet biology.

Lipid droplets (LDs) have been the focus of intense research for the past decade because of their active engagement in lipid metabolism and relationship with diseases. In contrast to other intracellular organelles, LDs are composed of a mass of hydrophobic lipid esters that is covered with a phospholipid monolayer. The unique architecture makes the LD a formidable object to study by the methods available today, and many fundamental questions remain unanswered. This review focuses on some of those questions, such as how LDs form and grow, how proteins move to and from LDs, and how LDs are related to protein degradation; we will also discuss what is not known about LDs. We think that small LDs that have thus far eluded analysis are the key to resolving many of the above-mentioned questions.

Imaging lipid droplets by electron microscopy.

The lipid droplet (LD) is different from other cellular organelles in that most of its volume is made of lipid esters and its surface is lined by a phospholipid monolayer. This uniquely lipid-dominant structure poses a problem for electron microscopy (EM) because the aldehydes commonly used as a fixative do not react with most lipids. To circumvent this difficulty and utilize the high resolving power of EM, many methods have been developed. In this chapter, we discuss methods that have been used and/or are potentially useful to study LDs. The methods include conventional EM to observe the LD core, cryoelectron microscopy to observe the LD surface, freeze-substitution, immunoelectron microscopy (pre-embedding, post-embedding, and cryosectioning methods), and freeze-fracture. Each method has strong and weak points and therefore some caution is necessary in interpreting the obtained results. In combination with methods of other disciplines, the electron microscopic techniques should contribute significantly to solving the remaining questions on LDs.

Organocatalyzed formal [2 + 2] cycloaddition of ketimines with allenoates: facile access to azetidines with a chiral tetrasubstituted carbon stereogenic center.

An enantioselective organocatalyzed aza-MBH-type reaction of ketimines and allenoates has been developed. The present formal [2 + 2] cycloaddition produces highly functionalized azetidines with a chiral tetrasubstituted carbon stereogenic center in good to excellent yields and high enantioselectivities.

Niobium(V) chloride catalyzed oxidation of dithioacetals with 30% hydrogen peroxide: a concise preparation of bissulfonylmethylene compounds.

The oxidation of dithioacetals with 16 eq of 30% hydrogen peroxide in the presence of 10 mol% niobium(V) chloride at room temperature provides bissulfonylmethylenes in high yields.

Protein kinase Cη is targeted to lipid droplets.

Protein kinase C (PKC) is a family of kinases that regulate numerous cellular functions. They are classified into three subfamilies, i.e., conventional PKCs, novel PKCs, and atypical PKCs, that have different domain structures. Generally, PKCs exist as a soluble protein in the cytosol in resting cells and they are recruited to target membranes upon stimulation. In the present study, we found that PKCη tagged with EGFP distributed in lipid droplets (LD) and induced a significant reduction in LD size. Two other novel PKCs, PKCδ and PKCε, also showed some concentration around LDs, but it was less distinct and less frequent than that of PKCη. Conventional and atypical PKCs (α, ßII, γ, and ζ) did not show any preferential distribution around LDs. 1,2-Diacylglycerol, which can activate novel PKCs without an increase of Ca(2+) concentration, is the immediate precursor of triacylglycerol and exists in LDs. The present results suggest that PKCη modifies lipid metabolism by phosphorylating unidentified targets in LDs.

Comparison of Clinicopathological Characteristics in the Patients with Cardiac Cancer with or without Esophagogastric Junctional Invasion: A Single-Center Retrospective Cohort Study.

Background. This study addresses clinicopathological differences between patients with gastric cardia and subcardial cancer with and without esophagogastric junctional invasion. Methods. We performed a single-center, retrospective cohort study. We studied patients who underwent curative surgery for gastric cardia and subcardial cancers. Tumors centered in the proximal 5 cm of the stomach were classed into two types, according to whether they did (Ge) or did not (G) invade the esophagogastric junction. Results. A total of 80 patients were studied; 19 (73.1%) of 26 Ge tumors and 16 (29.6%) of 54 G tumors had lymph nodes metastases. Incidence of nodal metastasis in pT1 tumors was significantly higher in the Ge tumor group. No nodal metastasis in cervical lymph nodes was recognized. Only two patients with Ge tumors had mediastinal lymph node metastases. Incidence of perigastric lymph node metastasis was significantly higher in those with Ge tumors. Ge tumors tended to be staged as progressive disease using the esophageal cancer staging manual rather than the gastric cancer staging manual. Conclusion. Because there are some differences in clinicopathological characteristics, it is thought to be adequate to distinguish type Ge from type G tumor.

Clinicopathological characteristics and optimal management for esophagogastric junctional cancer; a single center retrospective cohort study.

BACKGROUND: Esophagogastric junctional (EGJ) cancer occurs in the mucosa near the esophagogastric junction, and has characteristics of both esophageal and gastric malignancies; its optimal treatment strategy is controversial. METHODS: We conducted a single-center retrospective cohort study of the patients who underwent curative surgery with lymphadenectomy for EGJ cancer. Tumor specimens were categorized by histology and location into four types-centered in the esophagus < 5 cm from EGJ (type E), which were subtyped as (i) squamous-cell carcinoma (SQ) or (ii) adenocarcinoma (AD); (iii) any histological tumor centered in the stomach < 5 cm from EGJ, with EGJ invasion (type Ge); (iv) any histological tumor centered in the stomach < 5 cm from EGJ, without EGJ invasion (type G)-and classified by TNM system; these were compared to patients' clinicopathological characteristics and survival outcomes. RESULTS: A total of 92 EGJ cancer patients were studied. Median follow-up of surviving patients was 35.5 months. Tumors were categorized as 12 type E (SQ), 6 type E (AD), 27 type Ge and 47 type G; of these 7 (58.3%), 3 (50%), 19 (70.4%) and 14 (29.8%) and 23 patients, respectively, had lymph node metastases. No patients with type E (AD) and Ge tumors had cervical lymph node metastasis; those with type G tumors had no nodal metastasis at cervical and mediastinal lymph nodes. Multivariate analysis showed that type E (AD) tumor was an independent prognostic factor. CONCLUSIONS: We should distinguish type Ge tumor from type E (AD) tumor because of the clinicopathological and prognostic differentiation. Extended gastrectomy with or without lower esophagectomy according to tumor location and lower mediastinal and abdominal lymphadenectomy are recommended for EGJ cancer. TRIAL REGISTRATION: University Hospital Medical Information Network in Japan, UMIN000008596.

Histochemical detection of lipid droplets in cultured cells.

Cells store excess lipid as esters in the form of triglycerides and cholesterol esters. Most lipid esters are compartmentalized in globular structures called lipid droplets. Here we describe several methods of detecting lipid droplets by fluorescence microscopy. Lipid droplets can be visualized either by staining the lipid ester core using fluorescent dyes or by labeling lipid droplet-specific proteins using antibodies. The intracellular distribution of lipid droplets can be analyzed without much difficulty by these methods, but care must be taken to avoid certain pitfalls.

Translation inhibitors induce formation of cholesterol ester-rich lipid droplets.

Lipid droplets (LDs) in non-adipocytes contain triglycerides (TG) and cholesterol esters (CE) in variable ratios. TG-rich LDs are generated when unsaturated fatty acids are administered, but the conditions that induce CE-rich LD formation are less well characterized. In the present study, we found that protein translation inhibitors such as cycloheximide (CHX) induced generation of CE-rich LDs and that TIP47 (perilipin 3) was recruited to the LDs, although the expression of this protein was reduced drastically. Electron microscopy revealed that LDs formed in CHX-treated cells possess a distinct electron-dense rim that is not found in TG-rich LDs, whose formation is induced by oleic acid. CHX treatment caused upregulation of mTORC1, but the CHX-induced increase in CE-rich LDs occurred even when rapamycin or Torin1 was given along with CHX. Moreover, the increase in CE was seen in both wild-type and autophagy-deficient Atg5-null mouse embryonic fibroblasts, indicating that mTORC1 activation and suppression of autophagy are not necessary to induce the observed phenomenon. The results showed that translation inhibitors cause a significant change in the lipid ester composition of LDs by a mechanism independent of mTORC1 signaling and autophagy.

Derlin-1 and UBXD8 are engaged in dislocation and degradation of lipidated ApoB-100 at lipid droplets.

Apolipoprotein B-100 (ApoB) is the principal component of very low density lipoprotein. Poorly lipidated nascent ApoB is extracted from the Sec61 translocon and degraded by proteasomes. ApoB lipidated in the endoplasmic reticulum (ER) lumen is also subjected to proteasomal degradation, but where and how it dislocates to the cytoplasm remain unknown. In the present study, we demonstrate that ApoB after lipidation is dislocated to the cytoplasmic surface of lipid droplets (LDs) and accumulates as ubiquitinated ApoB in Huh7 cells. Depletion of UBXD8, which is almost confined to LDs in this cell type, decreases recruitment of p97 to LDs and causes an increase of both ubiquitinated ApoB on the LD surface and lipidated ApoB in the ER lumen. In contrast, abrogation of Derlin-1 function induces an accumulation of lipidated ApoB in the ER lumen but does not increase ubiquitinated ApoB on the LD surface. UBXD8 and Derlin-1 bind with each other and with lipidated ApoB and show colocalization around LDs. These results indicate that ApoB after lipidation is dislocated from the ER lumen to the LD surface for proteasomal degradation and that Derlin-1 and UBXD8 are engaged in the predislocation and postdislocation steps, respectively.