Endoplasmic reticulum-specific BH3-only protein BNIP1 induces mitochondrial fragmentation in a Bcl-2- and Drp1-dependent manner

Bcl-2/adenovirus E1B 19-kDa interacting protein 1 (BNIP1), which is predominantly localized to the endoplasmic reticulum (ER), is a pro-apoptotic Bcl-2 homology domain 3 (BH3)-only protein. Here, we show that the expression of BNIP1 induced not only a highly interconnected ER network but also mitochondrial fragmentation in a BH3 domain-dependent manner. Functional analysis demonstrated that BNIP1 expression increased dynamin-related protein 1 (Drp1) expression followed by the mitochondrial translocation of Drp1 and subsequent mitochondrial fission. Both BNIP1-induced mitochondrial fission and the translocation of Drp1 were abrogated by Bcl-2 overexpression. These results collectively indicate that ER-specific BNIP1 plays an important role in mitochondrial dynamics by modulating the mitochondrial fission protein Drp1 in a BH3 domain-dependent fashion.     

HIV-1 Tat suppresses gp120-specific T cell response in IL-10-dependent manner

A large number of multicomponent vaccine candidates are currently in clinical evaluation, many of which also include the HIV-1 Tat protein, an important regulatory protein of the virus. However, whether Tat, a known immune effector molecule with a well-conserved sequence among different HIV subtypes, affects the immune response to a coimmunogen is not well understood. In this study, using a bicistronic vector expressing both gp120 and Tat, we have analyzed the role of Tat in elicitation of the gp120-specific immune response. The T cell responses to gp120 were greatly diminished in mice coimmunized with Tat as compared with mice immunized with gp120 alone. This immunosuppressive activity of Tat was not confined to viral Ag only because it also suppressed the immune response of unrelated Ag. Analysis of the cytokine profile suggests that Tat induces IL-10 and since IL-10 has been demonstrated to have appreciable T cell inhibitory activity, it is plausible that IL-10 could be responsible for Tat-mediated immunosuppression. Finally, the immunosuppressive effect of Tat was not observed in IL-10-deficient mice, confirming the role of IL-10 in Tat-mediated immunosuppression. Thus, our results demonstrate for the first time that the immunosuppressive effect of Tat is mediated through IL-10 and suggests that Tat-induced IL-10-mediated immune suppression seems to cripple immune surveillance during HIV-1 infection.     

E2F1 inhibits MDM2 expression in a p53-dependent manner

MDM2 expression is down-regulated upon E2F1 over-expression, but the mechanism is not well defined. In the current study, we found that E2F1 inhibits MDM2 expression by suppressing its promoter activity. Although E2F1 binds to the MDM2 promoter, the inhibitory effect of E2F1 on the MDM2 promoter does not require the direct binding. We demonstrate that E2F1 inhibits MDM2 promoter activity in a p53-dependent manner. Knockdown of p53 in U2OS cells impairs the inhibitory effect of E2F1 on the MDM2 promoter. Consistent with this observation, E2F1 does not inhibit MDM2 promoter activity in p53-deficient H1299 cells, and the inhibition is restored when p53 is expressed exogenously. Both E2F1 and p53 are up-regulated after DNA damage stimulation. We show that such stimulation induces E2F1 to inhibit MDM2 promoter activity and promote p53 accumulation. Furthermore, inhibition of MDM2 by E2F1 promotes E2F1 induced apoptosis. These data suggest that E2F1 regulates the MDM2-p53 pathway by inhibiting p53 induced up-regulation of MDM2.     

Identification of a novel mitochondrial complex containing mitofusin 2 and stomatin-like protein 2

A reverse genetics approach was utilized to discover new proteins that interact with the mitochondrial fusion mediator mitofusin 2 (Mfn2) and that may participate in mitochondrial fusion. In particular, in vivo formaldehyde cross-linking of whole HeLa cells and immunoprecipitation with purified Mfn2 antibodies of SDS cell lysates were used to detect an approximately 42-kDa protein. This protein was identified by liquid chromatography and tandem mass spectrometry as stomatin-like protein 2 (Stoml2), previously described as a peripheral plasma membrane protein of unknown function associated with the cytoskeleton of erythrocytes (Wang, Y., and Morrow, J. S. (2000) J. Biol. Chem. 275, 8062-8071). Immunoblot analysis with anti-Stoml2 antibodies showed that Stoml2 could be immunoprecipitated specifically with Mfn2 antibody either from formaldehyde-cross-linked and SDS-lysed cells or from cells lysed with digitonin. Subsequent immunocytochemistry and cell fractionation experiments fully supported the conclusion that Stoml2 is indeed a mitochondrial protein. Furthermore, demonstration of mitochondrial membrane potential-dependent import of Stoml2 accompanied by proteolytic processing, together with the results of sublocalization experiments, suggested that Stoml2 is associated with the inner mitochondrial membrane and faces the intermembrane space. Notably, formaldehyde cross-linking revealed a "ladder" of high molecular weight protein species, indicating the presence of high molecular weight Stoml2-Mfn2 hetero-oligomers. Knockdown of Stoml2 by the short interfering RNA approach showed a reduction of the mitochondrial membrane potential, without, however, any obvious changes in mitochondrial morphology.     

Loss of sirtuin 1 and mitofusin 2 contributes to enhanced ischemia/reperfusion injury in aged livers

Ischemia/reperfusion (I/R) injury is a causative factor contributing to morbidity and mortality during liver resection and transplantation. Livers from elderly patients have a poorer recovery from these surgeries, indicating reduced reparative capacity with aging. Mechanisms underlying this age‐mediated hypersensitivity to I/R injury remain poorly understood. Here, we investigated how sirtuin 1 (SIRT1) and mitofusin 2 (MFN2) are affected by I/R in aged livers. Young (3 months) and old (23–26 months) male C57/BL6 mice were subjected to hepatic I/R in vivo. Primary hepatocytes isolated from each age group were also exposed to simulated in vitro I/R. Biochemical, genetic, and imaging analyses were performed to assess cell death, autophagy flux, mitophagy, and mitochondrial function. Compared to young mice, old livers showed accelerated liver injury following mild I/R. Reperfusion of old hepatocytes also showed necrosis, accompanied with defective autophagy, onset of the mitochondrial permeability transition, and mitochondrial dysfunction. Biochemical analysis indicated a near‐complete loss of both SIRT1 and MFN2 after I/R in old hepatocytes, which did not occur in young cells. Overexpression of either SIRT1 or MFN2 alone in old hepatocytes failed to mitigate I/R injury, while co‐overexpression of both proteins promoted autophagy and prevented mitochondrial dysfunction and cell death after reperfusion. Genetic approaches with deletion and point mutants revealed that SIRT1 deacetylated K655 and K662 residues in the C‐terminus of MFN2, leading to autophagy activation. The SIRT1‐MFN2 axis is pivotal during I/R recovery and may be a novel therapeutic target to reduce I/R injury in aged livers.     

LncRNA H19 suppresses pyroptosis of cardiomyocytes to attenuate myocardial infarction in a PBX3/CYP1B1-dependent manner

Molecular and Cellular Biochemistry - Myocardial infarction (MI) is a major cause of cardiovascular disease which poses great healthy and financial burden for individuals. MI can be mainly induced...     

MARCH-V is a novel mitofusin 2- and Drp1-binding protein able to change mitochondrial morphology

Mitofusins and Drp1 are key components in mitochondrial membrane fusion and division, but the molecular mechanism underlying the regulation of their activities remains to be clarified. Here, we identified human membrane-associated RING-CH (MARCH)-V as a novel transmembrane protein of the mitochondrial outer membrane. Immunoprecipitation studies demonstrated that MARCH-V interacts with mitofusin 2 (MFN2) and ubiquitinated forms of Drp1. Overexpression of MARCH-V promoted the formation of long tubular mitochondria in a manner that depends on MFN2 activity. By contrast, mutations in the RING finger caused fragmentation of mitochondria. We also show that MARCH-V promotes ubiquitination of Drp1. These results indicate that MARCH-V has a crucial role in the control of mitochondrial morphology by regulating MFN2 and Drp1 activities.     

Mst1 contributes to nasal epithelium inflammation via augmenting oxidative stress and mitochondrial dysfunction in a manner dependent on Nrf2 inhibition

Nasal epithelium inflammation plays an important role in transmitting and amplifying damage signals for the lower airway. However, the molecular basis of nasal epithelium inflammation damage has not been fully addressed. Mst1 is reported to modulate inflammation via multiple effects. Thus, the aim of our study is to understand the pathological mechanism underlying Mst1-related nasal epithelium inflammation in vitro. Our result indicated that Mst1 expression was rapidly increased in response to tumor necrosis factor-α (TNF-α) treatment in vitro and this effect was a dose-dependent manner. Interestingly, knockdown of Mst1 via transfecting small interfering RNA markedly reversed cell viability in the presence of TNF-α. Further, we found that Mst1 deficiency reduced cellular oxidative stress and attenuated mitochondrial dysfunction, as evidenced by reversed mitochondrial complex-I activity, decreased mitochondrial permeability transition pore opening rate, and stabilized mitochondrial membrane potential. Besides, we found that Nrf2 expression was increased after deletion of Mst1 whereas silencing of Nrf2 abolished the protective effects of Mst1 deletion on nasal epithelium survival and mitochondrial homeostasis. Moreover, Nrf2 overexpression also protected nasal epithelium against TNF-α-induced inflammation damage. Altogether, our data confirm that the Mst1 activation and Nrf2 downregulation seem to be the potential mechanisms responsible for the inflammation-mediated injury in nasal epithelium via mediating mitochondrial damage and cell oxidative stress.     

Chronically ischemic mouse skeletal muscle exhibits myopathy in association with mitochondrial dysfunction and oxidative damage

A myopathy characterized by mitochondrial pathology and oxidative stress is present in patients with peripheral arterial disease (PAD). Patients with PAD differ in disease severity, mode of presentation, and presence of comorbid conditions. In this study, we used a mouse model of hindlimb ischemia to isolate and directly investigate the effects of chronic inflow arterial occlusion on skeletal muscle microanatomy, mitochondrial function and expression, and oxidative stress. Hindlimb ischemia was induced by staged ligation/division of the common femoral and iliac arteries in C57BL/6 mice, and muscles were harvested 12 wk later. Muscle microanatomy was examined by bright-field microscopy, and mitochondrial content was determined as citrate synthase activity in muscle homogenates and ATP synthase expression by fluorescence microscopy. Electron transport chain (ETC) complexes I through IV were analyzed individually by respirometry. Oxidative stress was assessed as total protein carbonyls and 4-hydroxy-2-nonenal (HNE) adducts and altered expression and activity of manganese superoxide dismutase (MnSOD). Ischemic muscle exhibited histological features of myopathy and increased mitochondrial content compared with control muscle. Complex-dependent respiration was significantly reduced for ETC complexes I, III, and IV in ischemic muscle. Protein carbonyls, HNE adducts, and MnSOD expression were significantly increased in ischemic muscle. MnSOD activity was not significantly changed, suggesting MnSOD inactivation. Using a mouse model, we have demonstrated for the first time that inflow arterial occlusion alone, i.e., in the absence of other comorbid conditions, causes myopathy with mitochondrial dysfunction and increased oxidative stress, recapitulating the muscle pathology of PAD patients.     

Decreased cell adhesion promotes angiogenesis in a Pyk2-dependent manner

Angiogenesis is regulated by both soluble growth factors and cellular interactions with the extracellular matrix (ECM). While cell adhesion via integrins has been shown to be required for angiogenesis, the effects of quantitative changes in cell adhesion and spreading against the ECM remain less clear. Here, we show that angiogenic sprouting in natural and engineered three-dimensional matrices exhibited a biphasic response, with peak sprouting when adhesion to the matrix was limited to intermediate levels. Examining changes in global gene expression to determine a genetic basis for this response, we demonstrate a vascular endothelial growth factor (VEGF)-induced upregulation of genes associated with vascular invasion and remodeling when cell adhesion was limited, whereas cells on highly adhesive surfaces upregulated genes associated with proliferation. To explore a mechanistic basis for this effect, we turned to focal adhesion kinase (FAK), a central player in adhesion signaling previously implicated in angiogenesis, and its homologue, proline-rich tyrosine kinase 2 (Pyk2). While FAK signaling had some impact, our results suggested that Pyk2 can regulate both gene expression and endothelial sprouting through its enhanced activation by VEGF in limited adhesion contexts. We also demonstrate decreased sprouting of tissue explants from Pyk2-null mice as compared to wild type mice as further confirmation of the role of Pyk2 in angiogenic sprouting. These results suggest a surprising finding that limited cell adhesion can enhance endothelial responsiveness to VEGF and demonstrate a novel role for Pyk2 in the adhesive regulation of angiogenesis.     

Inhibition of c-Jun N-terminal kinase 2 expression suppresses growth and induces apoptosis of human tumor cells in a p53-dependent manner

c-Jun N-terminal kinase (JNK) plays a critical role in coordinating the cellular response to stress and has been implicated in regulating cell growth and transformation. To investigate the growth-regulatory functions of JNK1 and JNK2, we used specific antisense oligonucleotides (AS) to inhibit their expression. A survey of several human tumor cell lines revealed that JNKAS treatment markedly inhibited the growth of cells with mutant p53 status but not that of cells with normal p53 function. To further examine the influence of p53 on cell sensitivity to JNKAS treatment, we compared the responsiveness of RKO, MCF-7, and HCT116 cells with normal p53 function to that of RKO E6, MCF-7 E6, and HCT116 p53(-/-), which were rendered p53 deficient by different methods. Inhibition of JNK2 (and to a lesser extent JNK1) expression dramatically reduced the growth of p53-deficient cells but not that of their normal counterparts. JNK2AS-induced growth inhibition was correlated with significant apoptosis. JNK2AS treatment induced the expression of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1) in parental MCF-7, RKO, and HCT116 cells but not in the p53-deficient derivatives. That p21(Cip1/Waf1) expression contributes to the survival of JNK2AS-treated cells was supported by additional experiments demonstrating that p21(Cip1/Waf1) deficiency in HCT116 cells also results in heightened sensitivity to JNKAS treatment. Our results indicate that perturbation of JNK2 expression adversely affects the growth of otherwise nonstressed cells. p53 and its downstream effector p21(Cip1/Waf1) are important in counteracting these detrimental effects and promoting cell survival.