Green tea epigallocatechin gallate binds to and inhibits respiratory complexes in swelling but not normal rat hepatic mitochondria

Epigallocatechin gallate (EGCG), the major flavonoid in green tea, is consumed via tea products and dietary supplements, and has been tested in clinical trials. However, EGCG can cause hepatotoxicity in humans and animals by unknown mechanisms. Here EGCG effects on rat liver mitochondria were examined. EGCG showed negligible effects on oxidative phosphorylation at 7.5–100 μM in normal mitochondria. However, respiratory chain complexes (RCCs) were profoundly inhibited by EGCG in mitochondria undergoing Ca²⁺ overload-induced mitochondrial permeability transition (MPT). As RCCs are located in mitochondrial inner membranes (IM) and matrix, it was reasoned that EGCG could not readily pass through IM to affect RCCs in normal mitochondria but may do so when IM integrity is compromised. This speculation was substantiated in three ways. (1) Purified EGCG-bound proteins were barely detectable in normal mitochondria and contained no RCCs as determined by Western blotting, but swelling mitochondria contained about 1.5-fold more EGCG-bound proteins which included four RCC subunits together with cyclophilin D that locates in mitochondrial matrix. (2) Swelling mitochondria consumed more EGCG than normal ones. (3) The MPT blocker cyclosporine A diminished the above-mentioned difference. Among four subunits of RCC II, only SDHA and SDHB which locate in mitochondrial matrix, but not SDHC or SDHD which insert into the IM, were found to be EGCG targets. Interestingly, EGCG promoted Ca²⁺ overload-induced MPT only when moderate MPT already commenced. This study identified hepatic RCCs as targets for EGCG in swelling but not normal mitochondria, suggesting EGCG may trigger hepatotoxicity by worsening pre-existing mitochondria abnormalities.     

Characterization of the C584R variant in the mtDNA depletion syndrome gene FBXL4, reveals a novel role for FBXL4 as a regulator of mitochondrial fusion

Mutations in FBXL4 (F-Box and Leucine rich repeat protein 4), a nuclear-encoded mitochondrial protein with an unknown function, cause mitochondrial DNA depletion syndrome. We report two siblings, from consanguineous parents, harbouring a previously uncharacterized homozygous variant in FBXL4 (c.1750 T > C; p.Cys584Arg). Both patients presented with encephalomyopathy, lactic acidosis and cardiac hypertrophy, which are reported features of FBXL4 impairment. Remarkably, dichloroacetate (DCA) administration to the younger sibling improved metabolic acidosis and reversed cardiac hypertrophy. Characterization of FBXL4 patient fibroblasts revealed severe bioenergetic defects, mtDNA depletion, fragmentation of mitochondrial networks, and abnormalities in mtDNA nucleoids. These phenotypes, observed with other pathogenic FBXL4 variants, confirm the pathogenicity of the p.Cys584Arg variant. Although treating FBXL4 fibroblasts with DCA improved extracellular acidification, in line with reduced lactate levels in patients, DCA treatment did not improve any of the other mitochondrial functions. Nonetheless, we highlight DCA as a potentially effective drug for the management of elevated lactate and cardiomyopathy in patients with pathogenic FBXL4 variants. Finally, as the exact mechanism through which FBXL4 mutations lead to mtDNA depletion was unknown, we tested the hypothesis that FBXL4 promotes mitochondrial fusion. Using a photo-activatable GFP fusion assay, we found reduced mitochondrial fusion rates in cells harbouring a pathogenic FBXL4 variant. Meanwhile, overexpression of wildtype FBXL4, but not the p.Cys584Arg variant, promoted mitochondrial hyperfusion. Thus, we have uncovered a novel function for FBXL4 in promoting mitochondrial fusion, providing important mechanistic insights into the pathogenic mechanism underlying FBXL4 dysfunction.     

Case Report: Genetic Alterations Associated with the Progression of Carotid Paraganglioma

Paragangliomas (PGLs) are rare neuroendocrine tumors that can develop from any paraganglion across the body. The carotid body is the most often location of PGLs in the head and neck region. Carotid PGLs (CPGLs) are characterized by predominantly non-aggressive behavior; however, all tumors have the potential to metastasize. To date, molecular mechanisms of paraganglioma progression remain elusive. We report a case of a 38-year-old woman with metastatic CPGL manifesting as a recurrent tumor with lymph node metastasis. The tumor was fast-growing and had a high Ki-67 proliferation index. Immunohistochemical (IHC) examination and whole-exome sequencing were performed for both recurrent tumor and metastasis. A germline pathogenic splice acceptor variant in the SDHB gene was found in the patient. Immunoreactivity of the SDHB subunit was weak diffuse in both samples, indicating deficiency of the succinate dehydrogenase. Moreover, the recurrent tumor exhibited loss of heterozygosity (LOH) at the SDHB locus, that is according to Knudson’s "two-hit" hypothesis of cancer causation. We also identified a rare somatic promotor mutation in the TERT gene associated with the tumor progression. Obtained results confirmed the indicative role of the germline SDHB mutation for metastatic CPGLs, as well as the potential prognostic value of the TERT promoter mutation.     

抑制线粒体复合体II诱导线粒体自噬影响细胞增殖的研究

线粒体复合体II,也被称为琥珀酸脱氢酶,参与线粒体呼吸作用及代谢重编程的调控过程。复合体II由四个亚基构成,其突变与肿瘤的发生密切相关。本论文探讨了复合体II与线粒体自噬调控及细胞增殖之间的关系。本实验采用复合体II的特异性抑制剂TTFA或敲除复合体II的B亚基SDHB使其功能缺失。结果发现,复合体II功能的缺失显著引起线粒体形态的片段化进而发生线粒体自噬,导致线粒体蛋白水平减少,抑制ATP生成,由于线粒体功能受到抑制,细胞葡萄糖消耗及乳酸产生水平增加,并显著抑制细胞的细胞的增殖。综上所述,复合体II功能缺失可能通过调控线粒体自噬而影响细胞增殖,从而在肿瘤发生中起重要作用。     

Simvastatin induces autophagic flux to restore cerulein-impaired phagosome-lysosome fusion in acute pancreatitis

BackgroundDuring pancreatitis, autophagy is activated, but lysosomal degradation of dysfunctional organelles including mitochondria is impaired, resulting in acinar cell death. Retrospective cohort analyses demonstrated an association between simvastatin use and decreased acute pancreatitis incidence.MethodsWe examined whether simvastatin can protect cell death induced by cerulein and the mechanisms involved during acute pancreatitis. Mice were pretreated with DMSO or simvastatin (20 mg/kg) for 24 h followed by 7 hourly cerulein injections and sacrificed 1 h after last injection to harvest blood and tissue for analysis.ResultsPancreatic histopathology revealed that simvastatin reduced necrotic cell death, inflammatory cell infiltration and edema. We found that cerulein triggered mitophagy with autophagosome formation in acinar cells. However, autophagosome-lysosome fusion was impaired due to altered levels of LAMP-1, AMPK and ULK-1, resulting in autophagosome accumulation (incomplete autophagy). Simvastatin abrogated these effects by upregulating LAMP-1 and activating AMPK which phosphorylated ULK-1, resulting in increased formation of functional autolysosomes. In contrast, autophagosomes accumulated in control group during pancreatitis. The effects of simvastatin to promote autophagic flux were inhibited by chloroquine. Mitochondria from simvastatin-treated mice were resistant to calcium overload compared to control, suggesting that simvastatin induced mitochondrial quality control to eliminate susceptible mitochondria. Clinical specimens showed a significant increase in cell-free mtDNA in plasma during pancreatitis compared to normal controls. Furthermore, genetic deletion of parkin abrogated the benefits of simvastatin.ConclusionOur findings reveal the novel role of simvastatin in enhancing autophagic flux to prevent pancreatic cell injury and pancreatitis.     

SDHB蛋白的表达、纯化及多克隆抗体制备

SDHB(succinate dehydrogenage complex,subunit B)基因可能介导呼吸链生物功能和调控细胞生长.采用PCR技术扩增出SDHB基因,并将其连接到pGEX-4T-1原核表达载体中,经酶切及测序鉴定后,转化BL21细菌,并用IPTG诱导表达融合蛋白,谷胱甘肽琼脂糖珠亲和纯化,将纯化的融合蛋白免疫新西兰大白兔制备多克隆抗体,并用Western blot检测抗体.获得了SDHB原核表达重组融合蛋白及高效价的特异性兔抗SDHB多克隆抗体,为SDHB进一步的功能研究奠定了基础.     

Trimethylamine N-oxide promotes apoE−/− mice atherosclerosis by inducing vascular endothelial cell pyroptosis via the SDHB/ROS pathway

Trimethylamine N-oxide (TMAO) is produced from the phosphatidylcholine metabolism of gut flora and acts as a risk factor of cardiovascular disease. However, the underlying mechanisms for its proatherogenic action remain unclear. This study aimed to observe the effect of TMAO on endothelial cell pyroptosis and explore the underlying mechanisms. Our results showed that TMAO promoted the progression of atherosclerotic lesions in apolipoprotein E-deficient (apoE^−/−) mice fed a high-fat diet. Pyroptosis and succinate dehydrogenase complex subunit B (SDHB) upregulation were detected in the vascular endothelial cells of apoE^−/− mice and in cultured human umbilical vein endothelial cells (HUVECs) treated with TMAO. Overexpression of SDHB in HUVECs enhanced pyroptosis and impaired mitochondria and high reactive oxygen species (ROS) level. Pyroptosis in the SDHB overexpression of endothelial cells was inhibited by the ROS scavenger NAC. In summary, TMAO promotes vascular endothelial cell pyroptosis via ROS induced through SDHB upregulation, thereby contributing to the progression of atherosclerotic lesions.     

Differences in Clinical Manifestations and Tumor Features Between Metastatic Pheochromocytoma/Paraganglioma Patients With and Without Germline SDHB Mutation

ObjectiveTo compare metastatic pheochromocytoma/paraganglioma (MPP) patients with germline SDHB mutations (SDHB MPP) and without SDHB mutations (non-SDHB MPP) in terms of baseline clinical manifestations, tumor characteristics, and outcomes.MethodsClinical data were retrospectively reviewed in 101 MPP patients, including 34 SDHB MPP patients and 61 non-SDHB MPP patients.ResultsSDHB MPP patients presented at a younger age at onset, diagnosis, or metastasis (25 ± 16 vs 36 ± 14, 28 ± 17 vs 38 ± 15, and 31 ± 17 vs 44 ± 14 years old, respectively, P < .01 for all) than non-SDHB patients. Compared with their non-SDHB counterparts, SDHB patients were more likely to have paragangliomas (83% vs 47%, P < .05), synchronous metastases (44% vs 23%, P < .05), bone metastases (80% vs 48%, P < .01), and a shorter progression-free survival (3 years vs 5 years, P < .01). The Ki-67 index was higher in SDHB tumors (P < .05). The 5- and 10-year survival rates were 79% and 74%, respectively, in all patients. Seventeen patients died from MPP, and the time from metastasis to death in patients who had received systemic therapy was significantly longer than in those who had not (3.1 ± 1.5 vs 1.4 ± 0.7 years, P < .01).ConclusionCompared with MPP patients without SDHB mutations, MPP patients with SDHB mutations were younger at onset, diagnosis, or metastasis; had a higher incidence of synchronous metastases, higher ratio of paraganglioma, and higher Ki-67 index; had a shorter postoperative progression-free survival; and were more likely to develop bone metastasis or sole liver metastasis. Our results suggest that patients with SDHB mutations should be identified early and monitored regularly to achieve optimal clinical outcomes.