Cholesterol transport through lysosome-peroxisome membrane contacts.

Cholesterol is dynamically transported among organelles, which is essential for multiple cellular functions. However, the mechanism underlying intracellular cholesterol transport has remained largely unknown. We established an amphotericin B-based assay enabling a genome-wide shRNA screen for delayed LDL-cholesterol transport and identified 341 hits with particular enrichment of peroxisome genes, suggesting a previously unappreciated pathway for cholesterol transport. We show dynamic membrane contacts between peroxisome and lysosome, which are mediated by lysosomal Synaptotagmin VII binding to the lipid PI(4,5)P2 on peroxisomal membrane. LDL-cholesterol enhances such contacts, and cholesterol is transported from lysosome to peroxisome. Disruption of critical peroxisome genes leads to cholesterol accumulation in lysosome. Together, these findings reveal an unexpected role of peroxisome in intracellular cholesterol transport. We further demonstrate massive cholesterol accumulation in human patient cells and mouse model of peroxisomal disorders, suggesting a contribution of abnormal cholesterol accumulation to these diseases.

Cholesterol Modification of Smoothened Is Required for Hedgehog Signaling.

Hedgehog (Hh) has been known as the only cholesterol-modified morphogen playing pivotal roles in development and tumorigenesis. A major unsolved question is how Hh signaling regulates the activity of Smoothened (SMO). Here, we performed an unbiased biochemical screen and identified that SMO was covalently modified by cholesterol on the Asp95 (D95) residue through an ester bond. This modification was inhibited by Patched-1 (Ptch1) but enhanced by Hh. The SMO(D95N) mutation, which could not be cholesterol modified, was refractory to Hh-stimulated ciliary localization and failed to activate downstream signaling. Furthermore, homozygous SmoD99N/D99N (the equivalent residue in mouse) knockin mice were embryonic lethal with severe cardiac defects, phenocopying the Smo-/- mice. Together, the results of our study suggest that Hh signaling transduces to SMO through modulating its cholesterylation and provides a therapeutic opportunity to treat Hh-pathway-related cancers by targeting SMO cholesterylation.

Cardiomyocyte-specific overexpression of FPN1 diminishes cardiac hypertrophy induced by chronic intermittent hypoxia.

The significance of iron in myocardial mitochondria function cannot be underestimated, because deviations in iron levels within cardiomyocytes may have profound detrimental effects on cardiac function. In this study, we investigated the effects of ferroportin 1 (FPN1) on cardiac iron levels and pathological alterations in mice subjected to chronic intermittent hypoxia (CIH). The cTNT-FPN1 plasmid was administered via tail vein injection to induce the mouse with FPN1 overexpression in the cardiomyocytes. CIH was established by exposing the mice to cycles of 21%-5% FiO2 for 3 min, 8 h per day. Subsequently, the introduction of hepcidin resulted in a reduction in FPN1 expression, and H9C2 cells were used to establish an IH model to further elucidate the role of FPN1. First, FPN1 overexpression ameliorated CIH-induced cardiac dysfunction, myocardial hypertrophy, mitochondrial damage and apoptosis. Second, FPN1 overexpression attenuated ROS levels during CIH. In addition, FPN1 overexpression mitigated CIH-induced cardiac iron accumulation. Moreover, the administration of hepcidin resulted in a reduction in FPN1 levels, further accelerating the CIH-induced levels of ROS, LIP and apoptosis in H9C2 cells. These findings indicate that the overexpression of FPN1 in cardiomyocytes inhibits CIH-induced cardiac iron accumulation, subsequently reducing ROS levels and mitigating mitochondrial damage. Conversely, the administration of hepcidin suppressed FPN1 expression and worsened cardiomyocyte iron toxicity injury.

Circular RNA circPHF16 enhances IL-17A expression and secretion by sequestering miR-378a-3p to activate the IL6ST axis in Graves' disease.

Interleukin-17A (IL-17A) plays a pivotal role in the pathogenesis of Graves' disease (GD), an autoimmune disorder affecting thyroid function, but the detailed regulatory mechanisms remain elusive. Circular RNAs (circRNAs) have emerged as key regulators of IL-17A expression and secretion in autoimmune diseases, yet their specific role in GD, especially within CD4 + T lymphocytes, are not well understood. In this study, a circRNA, circPHF16 (hsa_circ_0090364) was found to be highly expressed in the peripheral blood mononuclear cells and serum of GD patients. In vitro experiments in Jurkat T cells revealed that silencing of circPHF16 suppressed IL-17A expression and secretion, while overexpression of circPHF16 had the opposite effect. Furthermore, bioinformatics analysis demonstrated a circPHF16/miR-378a-3p/IL6ST pathway, in which circPHF16 regulates IL6ST expression, which, in turn, influences IL-17A expression and secretion by interacting with miR-378a-3p. In vivo studies in a mouse model of GD showed similar trends in molecular expression levels, consistent with competitive endogenous RNA interactions. Together the results of the study identify circPHF16 as a potential target in the development of new strategies for GD diagnosis and treatment, and thus, offer a theoretical foundation for clinical therapeutic approaches in GD.

Potentiating the antitumour response of CD8(+) T cells by modulating cholesterol metabolism.

CD8(+) T cells have a central role in antitumour immunity, but their activity is suppressed in the tumour microenvironment. Reactivating the cytotoxicity of CD8(+) T cells is of great clinical interest in cancer immunotherapy. Here we report a new mechanism by which the antitumour response of mouse CD8(+) T cells can be potentiated by modulating cholesterol metabolism. Inhibiting cholesterol esterification in T cells by genetic ablation or pharmacological inhibition of ACAT1, a key cholesterol esterification enzyme, led to potentiated effector function and enhanced proliferation of CD8(+) but not CD4(+) T cells. This is due to the increase in the plasma membrane cholesterol level of CD8(+) T cells, which causes enhanced T-cell receptor clustering and signalling as well as more efficient formation of the immunological synapse. ACAT1-deficient CD8(+) T cells were better than wild-type CD8(+) T cells at controlling melanoma growth and metastasis in mice. We used the ACAT inhibitor avasimibe, which was previously tested in clinical trials for treating atherosclerosis and showed a good human safety profile, to treat melanoma in mice and observed a good antitumour effect. A combined therapy of avasimibe plus an anti-PD-1 antibody showed better efficacy than monotherapies in controlling tumour progression. ACAT1, an established target for atherosclerosis, is therefore also a potential target for cancer immunotherapy.

Magnetocaloric Effect and Thermal Conductivity of a 3D Coordination Polymer of [Gd(HCOO)(C2O4)]n.

A 3D coordination polymer, [Gd(HCOO)(C2O4)]n was prepared. Its magnetocaloric effect (MCE) (32.7 J K-1 kg-1 at 2 K and 2 T) is significantly larger than that of commercial Gd3Ga5O12 (GGG) (14.6 J kg-1 K-1 at 2 K and 2 T), while its thermal conductivity (9.9 W m-1 K-1 at 3 K) is comparable to that of the commercial GGG (about 10 W m-1 K-1 at 3 K).

Myeloid Acat1/Soat1 KO attenuates pro-inflammatory responses in macrophages and protects against atherosclerosis in a model of advanced lesions.

Cholesterol esters are a key ingredient of foamy cells in atherosclerotic lesions; their formation is catalyzed by two enzymes: acyl-CoA:cholesterol acyltransferases (ACATs; also called sterol O-acyltransferases, or SOATs) ACAT1 and ACAT2. ACAT1 is present in all body cells and is the major isoenzyme in macrophages. Whether blocking ACAT1 benefits atherosclerosis has been under debate for more than a decade. Previously, our laboratory developed a myeloid-specific Acat1 knockout (KO) mouse (Acat1-M/-M), devoid of ACAT1 only in macrophages, microglia, and neutrophils. In previous work using the ApoE KO (ApoE-/-) mouse model for early lesions, Acat1-M/-M significantly reduced lesion macrophage content and suppressed atherosclerosis progression. In advanced lesions, cholesterol crystals become a prominent feature. Here we evaluated the effects of Acat1-M/-M in the ApoE KO mouse model for more advanced lesions and found that mice lacking myeloid Acat1 had significantly reduced lesion cholesterol crystal contents. Acat1-M/-M also significantly reduced lesion size and macrophage content without increasing apoptotic cell death. Cell culture studies showed that inhibiting ACAT1 in macrophages caused cells to produce less proinflammatory responses upon cholesterol loading by acetyl low-density lipoprotein. In advanced lesions, Acat1-M/-M reduced but did not eliminate foamy cells. In advanced plaques isolated from ApoE-/- mice, immunostainings showed that both ACAT1 and ACAT2 are present. In cell culture, both enzymes are present in macrophages and smooth muscle cells and contribute to cholesterol ester biosynthesis. Overall, our results support the notion that targeting ACAT1 or targeting both ACAT1 and ACAT2 in macrophages is a novel strategy to treat advanced lesions.

Bifunctional Squaramide-Catalyzed Asymmetric [3 + 2] Cyclization of 2-(1-Methyl-2-oxoindolin-3-yl)malononitriles with Unsaturated Pyrazolones To Construct Spirooxindole-Fused Spiropyrazolones.

The present paper reports a highly stereoselective synthesis of spirooxindole-fused spiropyrazolones through the asymmetric [3 + 2] cyclization reaction of 2-(1-methyl-2-oxoindolin-3-yl)malononitriles with unsaturated pyrazolones under mild conditions. With only a 1 mol % bifunctional squaramide catalyst, a series of chiral dispirocyclic pyrazolone derivatives were attained in high yields (85-97%) with excellent stereoselectivities (up to >99% ee and in all cases >20:1 dr). Moreover, gram-scale synthesis and further transformation of the products were also demonstrated.

Squaramide-catalyzed asymmetric Mannich reactions between 3-fluorooxindoles and pyrazolinone ketimines.

An enantioselective Mannich reaction between 3-fluorooxindoles and pyrazolinone ketimines has been developed for the construction of amino-pyrazolone-oxindoles containing stereogenic C-F units. Based on this new protocol that allows for the generation of two adjacent tetrasubstituted stereocenters, a variety of structurally diverse fluorinated amino-pyrazolone-oxindoles were obtained in good to excellent yields with excellent diastereoselectivities and enantioselectivities (up to 98% yield, >20 : 1 dr and >99% ee). What's more, good yield and high stereoselectivities were obtained in the gram-scale reaction.

Numb directs the subcellular localization of EAAT3 through binding the YxNxxF motif.

Excitatory amino acid transporter type 3 (EAAT3, also known as SLC1A1) is a high-affinity, Na(+)-dependent glutamate carrier that localizes primarily within the cell and at the apical plasma membrane. Although previous studies have reported proteins and sequence regions involved in EAAT3 trafficking, the detailed molecular mechanism by which EAAT3 is distributed to the correct location still remains elusive. Here, we identify that the YVNGGF sequence in the C-terminus of EAAT3 is responsible for its intracellular localization and apical sorting in rat hepatoma cells CRL1601 and Madin-Darby canine kidney (MDCK) cells, respectively. We further demonstrate that Numb, a clathrin adaptor protein, directly binds the YVNGGF motif and regulates the localization of EAAT3. Mutation of Y503, N505 and F508 within the YVNGGF motif to alanine residues or silencing Numb by use of small interfering RNA (siRNA) results in the aberrant localization of EAAT3. Moreover, both Numb and the YVNGGF motif mediate EAAT3 endocytosis in CRL1601 cells. In summary, our study suggests that Numb is a pivotal adaptor protein that mediates the subcellular localization of EAAT3 through binding the YxNxxF (where x stands for any amino acid) motif.

Inhibition of the sterol regulatory element-binding protein pathway suppresses hepatocellular carcinoma by repressing inflammation in mice.

Obesity is a critical risk factor for hepatocellular carcinoma (HCC). However, it remains unknown whether inhibition of de novo lipid biosynthesis can suppress HCC. In this study, we blocked the sterol regulatory element-binding protein (SREBP) pathway, one of the key determinants of lipid homeostasis, by ablating 78-kDa cell-surface glycoprotein or SREBP cleavage-activating protein in hepatocytes, as well as by administering a chemical compound called betulin. We found that either genetically or pharmacologically inhibiting the SREBP pathway dramatically reduced diethylnitrosamine-induced HCC progression by down-regulating tumor-promoting cytokines, including interleukin (IL)-6, tumor necrosis factor alpha, and IL-1ß. CONCLUSION: Inhibition of de novo lipid biosynthesis by suppressing the SREBP pathway prevents HCC. This study identifies a previously underappreciated role of the SREBP pathway in HCC and suggests a novel metabolic strategy to control liver cancer. (Hepatology 2017;65:1936-1947).

Organocatalytic Asymmetric Synthesis of 3,3'-Pyrrolidinyl-bispirooxindoles via Michael/ N-Hemiketalization Cascade Reaction between 3-Aminooxindoles and Isatin-Derived ß,γ-Unsaturated α-Keto Esters.

A novel strategy for the construction of 3,3'-pyrrolidinyl-bispirooxindoles through a Michael/ N-hemiketalization cascade reaction of 3-aminooxindoles and isatin-derived ß,γ-unsaturated α-keto esters was developed. Under mild conditions, a series of 3,3'-pyrrolidinyl-bispirooxindoles were obtained in moderate to good yields with high diastereo- and enantioselectivities by using a cinchona-derived bifunctional squaramide organocatalyst. This work represents the first example using the 3-aminooxindoles for the construction of 3,3'-pyrrolidinyl-bispirooxindoles.

AAV9-NPC1 significantly ameliorates Purkinje cell death and behavioral abnormalities in mouse NPC disease.

Niemann-Pick type C (NPC) disease is a fatal inherited neurodegenerative disorder caused by loss-of-function mutations in the NPC1 or NPC2 gene. There is no effective way to treat NPC disease. In this study, we used adeno-associated virus (AAV) serotype 9 (AAV9) to deliver a functional NPC1 gene systemically into NPC1-/- mice at postnatal day 4. One single AAV9-NPC1 injection resulted in robust NPC1 expression in various tissues, including brain, heart, and lung. Strikingly, AAV9-mediated NPC1 delivery significantly promoted Purkinje cell survival, restored locomotor activity and coordination, and increased the lifespan of NPC1-/- mice. Our work suggests that AAV-based gene therapy is a promising means to treat NPC disease.

The role of metastasis-associated in colon cancer 1 (MACC1) in endometrial carcinoma tumorigenesis and progression.

Metastasis-associated in colon cancer-1 (MACC1), has recently been identified as a key regulator in the progression of many cancers. However, its role in endometrial carcinoma (EC) remains unknown. MACC1 expression was determined in EC and normal endometrial tissues by immunohistochemistry. EC cell phenotypes and related molecules were examined after MACC1 downregulation by Small interfering RNA (siRNA) or microRNA (miRNA) transfection. We found that MACC1 was highly expressed in EC tissues than normal samples, and was significantly different in FIGO staging (I and II vs. III and IV), the depth of myometrial infiltration (<1/2 vs. ≥1/2), lymph nodes metastasis (negative vs. positive), besides, MACC1 overexpression was correlated with lower cumulative and relapse-free survival rate. MACC1 downregulation by siRNA transfection significantly induced G1 phrase arrest, suppressed EC cell proliferation, migration, and invasion. In addition, MACC1 downregulation also reduced expression of Cyclin D1 and Cyclin-dependent Kinase 2 (CDK2), N-cadherin (N-Ca), α-SMA, matrix metalloproteinase 2 (MMP2), and MMP9, but increased expression of E-cadherin (E-Ca). Bioinformatic predictions and dual-luciferase reporter assays indicate that MACC1 is a possible target of miR-23b. MiR-23b overexpression reduced MACC1 expression in vitro and induced G1 phrase arrest, suppressed cell proliferation, migration, and invasion. MiR-23b transfection also reduced Cyclin D1 and CDK2, N-Ca, α-SMA, MMP2, MMP9 expression, but increased E-Ca expression. Furthermore, the nude mouse xenograft assay showed that miR-23b overexpression suppressed tumour growth through downregulating MACC1 expression. Taken together, our results demonstrate for the first time that MACC1 may be a new and important diagnosis and therapeutic target of endometrial carcinoma.

The transcription factor FOXA1 induces epithelial ovarian cancer tumorigenesis and progression.

FOXA1 (forkhead box A1), a member of the FOXA transcription factor superfamily, plays an important role in tumor occurrence and development. However, the relationship between FOXA1 and ovarian cancer has not been reported. We examined normal ovarian tissue and ovarian cancer tissue and found increased FOXA1 expression in the cancer tissue. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry assays demonstrated that transfection with small interfering RNA to silence FOXA1 (si-FOXA1) in ovarian cancer cell lines decreased cell proliferation and induced apoptosis and S-phase arrest. In addition, si-FOXA1 transfection inhibited cell migration and invasion. Western blotting showed that si-FOXA1 transfection decreased the levels of YY1-associated protein 1, cyclin-dependent kinase 1, cyclin D1, phosphatidylinositol-3 kinase, E2F transcription factor 1, B-cell lymphoma 2, and vascular endothelial growth factor A protein. Based on these results, we suggest that FOXA1 plays a catalytic role in ovarian cancer pathogenesis and development by affecting the expression of the above-mentioned proteins.

Squaramide-Catalyzed Asymmetric Reactions.

Bifunctional squaramides have emerged as powerful hydrogen-bonding catalysts for promoting a wide array of useful asymmetric reactions, which provides convenient methods for the construction of complex molecular structures and chiral biologically active compounds. This review highlights the recent advances of our research group in the chiral squaramide-catalyzed asymmetric reactions, including Michael addition, Mannich reaction, aza-Henry reaction, Strecker reaction as well as cascade or sequential reactions.

Chiral squaramide-catalysed enantioselective Michael/cyclization cascade reaction of 3-hydroxyoxindoles with α,ß-unsaturated N-acylated succinimides.

A bifunctional squaramide-catalysed asymmetric Michael/cyclization cascade reaction of 3-hydroxyoxindoles with α,ß-unsaturated N-acylated succinimides is disclosed. With quinine-derived squaramide as the catalyst, a broad range of the desired spirooxindole lactone derivatives bearing two contiguous stereocenters were obtained in good yields (up to 89%) with high diastereoselectivities (up to >95 : 5 dr) and excellent enantioselectivities (up to 99% ee).

[Effects of vitamin E and ω-3 fatty acids on protecting ambient PM_(2. 5)-induced cardiovascular injury].

OBJECTIVE: To observe whether vitamin E( Ve) and ω-3 polyunsaturated fatty acids( ω-3 FA) could prevent the fine particulate matter( PM_(2. 5))-induced cardiovascular injury and explore the potential mechanism. METHODS: The SD rats were assigned randomly to 8 groups, those were control group, PM_(2. 5)group, Ve treatment groups( 3, 10, 30 mg/( kg·d)) and ω-3 FA treatment groups( 10, 30 and 90 mg/( kg·d)). The rats were pretreated with different concentration of Ve and ω-3 FA separately for 14 days, then were exposed to ambient PM_(2. 5) by intratracheal instillation( 10 mg/kg BW). All the rats were sacrificed after the last PM_(2. 5) exposure, then the arterial blood, lungs and cardiac tissues were collected. The expressions of tumor necrosis factor-α( TNF-α), interleukin-1ß( IL-1ß), interleukin-6( IL-6) in serum, bronchoalveolar lavage fluid and supernatant of cardiac tissue were detected by ELISA kits. The levels of malondialdehyde( MDA), superoxide dismutase( SOD) and glutathione-peroxidase( GSH-Px) in serum and myocardium were also measured. RESULTS: Compared with the severe injury of rats in PM_(2. 5) exposure group, the rats in Ve or ω-3 FA groups had a slighter injury in lung and cardiac tissue with the increase of Ve and ω-3 FA. Similarly, the levels of IL-1ß, IL-6 in bronchoalveolar lavage fluid had a decreasing trend with the increase of Ve and ω-3 FA compared with the PM_(2. 5) exposure groups. Meanwhile, the expressions of TNF-α in Ve and ω-3 FA high dose groups were significantly reduced when compared with the PM_(2. 5) exposure group( P <0. 05). In addition, the MDA levels in serum were markedly decreased and the activities of SOD were significantly increased compared with the PM_(2. 5)exposure group( P < 0. 05 or P < 0. 01) whereas the SOD activities were elevated only in the ω-3 FA high dose groups( P < 0. 05). Meanwhile, the levels of IL-6 and TNF-α in serum had an obvious decrease compared with the PM_(2. 5) exposure group( P < 0. 01). Similarly, compared with the PM_(2. 5)exposure group, the expressions of MDA were markedly decreased and the activities of SOD and GSH-Px in myocardium were significantly increased( P < 0. 05 or P < 0. 01) in the Ve treatment group. In addition, the activities of GSH-Px was found higher only in the ω-3 FA high treatment group compared with the PM_(2. 5)exposure group( P < 0. 05). Meanwhile, the levels of IL-1ß and TNF-α in cardiac tissue had an obvious decrease trend with the increase of Ve and ω-3 FA. CONCLUSION: PM_(2. 5) exposure may increase inflammatory response and oxidative stress, supplementation with Ve and ω-3 FA could prevent the PM_(2. 5)-induced inflammatory reaction and oxidative stress damage by increasing the activities of SOD and GSH-Px.

MicroRNA-505 functions as a tumor suppressor in endometrial cancer by targeting TGF-α.

BACKGROUND: Endometrial carcinoma (EC) is one of the most lethal gynecologic cancers. Patients frequently have regional or distant metastasis at diagnosis. MicroRNAs are small non-coding RNAs that participate in numerous biological processes. Recent studies have demonstrated that miR-505 is associated with several types of cancer; however, the expression and function of miR-505 have not been investigated in EC. METHODS: miR-505 expression in normal endometrial tissue, endometrial carcinomas were quantified by Quantitative reverse transcription PCR. The endometrial carcinoma cell lines HEC-1B and Ishikawa were each transfected with miR-505 or scrambled mimics, after which cell phenotype and expression of relevant molecules were assayed. Dual-luciferase reporter assay and a xenograft mouse model were used to examine miR-505 and its target gene TGF-α. RESULTS: RT-PCR results demonstrated that miR-505 was significantly downregulated in human EC tissues compared to normal endometrial tissues. Besides, miR-505 expression was negatively associated with FIGO stage (stage I-II vs. III-IV), and lymph node metastasis (negative vs. positive). In vitro, overexpression of miR-505 significantly suppressed EC cell proliferation, increased apoptosis and reduced migratory and invasive activity. A miR-505 binding site was identified in the 3' untranslated region of TGF-α mRNA (TGFA) using miRNA target-detecting software; a dual luciferase reporter assay confirmed that miR-505 directly targets and regulates TGFA. RT-PCR and Western-blotting results indicated that overexpressing miR-505 reduced the expression of TGF-α and the TGF-α-regulated proteins MMP2, MMP9, CDK2, while induced Bax and cleaved-PARP expression in EC cells. In vivo, overexpression of miR-505 reduced the tumorigenicity and inhibited the growth of xenograft tumors in a mouse model of EC. CONCLUSIONS: Taken together, this study demonstrates that miR-505 acts as tumor suppressor in EC by regulating TGF-α.