Protein Tom40 Research Articles

Abstract 1324: TRPM2 promotes leukemia proliferation and survival through modulation of ROS and autophagy

Abstract Acute myeloid leukemia (AML) is a heterogeneous group of leukemias that arise in hematopoietic precursors. Understanding the mechanisms involved in AML proliferation and survival is key to development of new therapies. Transient receptor potential melastatin 2 (TRPM2), a calcium permeable ion channel, modulates cell proliferation, survival and oxidative stress-induced cell death and is highly expressed in many cancer cells including AML. To investigate the role of TRPM2 in AML, we depleted the TRPM2 gene in the human AML cell line U937 using clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR) technology. RT-PCR, western blot, and patch clamp analyses confirmed that TRPM2 was depleted in U937 knockout (KO) cells. Cell proliferation and viability measured by XTT and trypan blue exclusion assays were significantly reduced in KO cells after doxorubicin treatment. Expression of wild type TRPM2 (TRPM2-L) restored cell growth and cell survival to doxorubicin-treated TRPM2 KO cells, whereas the TRPM2 channel pore mutant (E960D) or empty vector did not. In a xenograft mouse model, mice were injected with TRPM2 KO or control U937 cells which express luciferase. Mice injected with TRPM2 KO U937 cells showed significantly reduced leukemia growth and enhanced sensitivity to doxorubicin. To examine the mechanisms, mitochondrial ROS were measured with confocal microscopy and MitoSOX Red. ROS were significantly elevated in TRPM2 KO cells compared to scrambled control, and further increased when KO cells were treated with doxorubicin. ATP production, oxygen consumption rate (OCR), and mitochondrial Ca2+ uptake were significantly reduced and mitochondrial membrane potential (ΔΨ) significantly increased in TRPM2 KO cells compared to scrambled control cells treated with doxorubicin, demonstrating dysfunctional mitochondria. Western blot analysis showed that expressions of HIF1/2α, FOXO3a and Nrf2 were downregulated in TRPM2 KO cells, further contributing to the increase in ROS. Expression of autophagy proteins ULK1, Atg7 and Atg5 was significantly reduced and mitochondrial protein Tom20 significantly increased in TRPM2 KO cells, suggesting that depletion of TRPM2 significantly reduced autophagy in AML cells. Reconstitution of wild type TRPM2 but not E960D restored ULK1 expression. In conclusion, depletion of TRPM2 significantly inhibits AML proliferation and increases sensitivity to doxorubicin by reducing mitochondrial function, increasing ROS, and impairing autophagy. TRPM2 contributes significantly to AML survival and may be a novel therapeutic target in leukemia therapy. Citation Format: Shu-Jen Chen, Lei Bao, Kerry Keefer, Longgui Chen, JuFang Wang, Xue-Qian Zhang, Santhanam Shanmughapriya, Iwona Hirschler-Laszkiewicz, Muniswamy Madesh, Joseph Y. Cheung, Hong-Gang Wang, Barbara A. Miller. TRPM2 promotes leukemia proliferation and survival through modulation of ROS and autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1324.

Induction of mitochondrial biogenesis protects against acetaminophen hepatotoxicity

Mitochondrial biogenesis (MB) is an adaptive response to maintain metabolic homeostasis after mitochondrial dysfunction. Induction of MB during APAP hepatotoxicity has not been studied. To investigate this, mice were treated with toxic doses of APAP and euthanized between 0 and 96 h. At early time points, APAP caused both mitochondrial dysfunction and reduction of mitochondrial mass, indicated by reduced activity of electron transport chain (ETC) complexes I and IV and depletion of mitochondrial DNA (mtDNA), respectively. Both ETC activity and mtDNA gradually recovered after 12 h, suggesting that MB occurs at late time points after APAP overdose. Immunofluorescent staining of mitochondria with mitochondrial outer membrane protein Tom20 further demonstrated that MB occurs selectively in hepatocytes surrounding necrotic areas. MB signaling mediators including PPARγ co-activator 1-α (Pgc-1α), nuclear respiratory factor-1 (Nrf-1) and mitochondrial fission protein dynamin-related protein-1 (Drp-1) were induced. Pgc-1α was selectively increased in hepatocytes surrounding necrotic areas. In addition, the time course of MB induction coincides with increased liver regeneration. Post-treatment with the known MB inducer SRT1720 increased Pgc-1α expression and liver regeneration, resulting in protection against late liver injury after APAP overdose. Thus, induction of MB is an important feature during APAP hepatotoxicity and liver regeneration.

The biological foundation of the genetic association of TOMM40 with late-onset Alzheimer's disease

A variable-length poly-T variant in intron 6 of the TOMM40 gene, rs10524523, is associated with risk and age-of-onset of sporadic (late-onset) Alzheimer's disease. In Caucasians, the three predominant alleles at this locus are Short (S), Long (L) or Very long (VL). On an APOE ε3/3 background, the S/VL and VL/VL genotypes are more protective than S/S. The '523 poly-T has regulatory properties, in that the VL poly-T results in higher expression than the S poly-T in luciferase expression systems. The aim of the current work was to identify effects on cellular bioenergetics of increased TOM40 protein expression. MitoTracker Green fluorescence and autophagic vesicle staining was the same in control and over-expressing cells, but TOM40 over-expression was associated with increased expression of TOM20, a preprotein receptor of the TOM complex, the mitochondrial chaperone HSPA9, and PDHE1a, and increased activities of the oxidative phosphorylation complexes I and IV and of the TCA member α-ketoglutaric acid dehydrogenase. Consistent with the complex I findings, respiration was more sensitive to inhibition by rotenone in control cells than in the TOM40 over-expressing cells. In the absence of inhibitors, total cellular ATP, the mitochondrial membrane potential, and respiration were elevated in the over-expressing cells. Spare respiratory capacity was greater in the TOM40 over-expressing cells than in the controls. TOM40 over-expression blocked Ab-elicited decreases in the mitochondrial membrane potential, cellular ATP levels, and cellular viability in the control cells. These data suggest elevated expression of TOM40 may be protective of mitochondrial function.

Study on autophagy in nucleated red blood cells in patients with myelodysplastic syndromes

目的研究骨髓增生异常综合征（MDS）患者有核红细胞的自噬水平。方法54例MDS患者为实验组，对照组33例。采用透射电镜观察自噬过程，流式细胞术检测骨髓细胞中GlycoA+有核红细胞自噬相关蛋白微管相关蛋白轻链3B（LC3B）的表达水平，Real-time PCR法检测GlycoA+有核红细胞自噬相关基因AMPK、ULK1和mTOR mRNA的表达，Western blot法检测GlycoA+有核红细胞线粒体外膜蛋白TOM20的表达。结果MDS患者有核红细胞中不易观察到自噬体及自噬溶酶体。相对高危组MDS患者有核红细胞LC3B表达（0.22±0.12）显著低于对照组（0.43±0.22，P<0.001）及相对低危组（0.40±0.16，P=0.001）。相对高危组患者AMPK、ULK1 mRNA表达水平明显低于对照组[AMPK：0.26（0.60）对1.00（2.07），ULK1：0.27（3.31）对1.07（4.41），P值均<0.017]，mTOR mRNA表达水平明显高于对照组[1.82（3.74）对1.26（1.38），P<0.017]。相对高危组MDS患者有核红细胞LC3B水平与HGB呈正相关（r=0.529，P=0.009）。相对高危组MDS患者有核红细胞TOM20蛋白（9.42±4.42）表达升高。结论高危MDS患者有核红细胞自噬水平下降。

Mitochondrial protein import - Functional analysis of the highly diverged Tom22 orthologue of Trypanosoma brucei

The β-barrel protein Tom40 and the α-helically anchored membrane protein Tom22 are the only universally conserved subunits of the protein translocase of the mitochondrial outer membrane (TOM). Tom22 has an N-terminal cytosolic and a C-terminal intermembrane space domain. It occurs in two variants: one typified by the yeast protein which has a cytosolic domain containing a cluster of acidic residues, and a shorter variant typified by the plant protein that lacks this domain. Yeast-type Tom22 functions as a secondary protein import receptor and is also required for the stability of the TOM complex. Much less is known about the more widespread short variant of Tom22, which is also found in the parasitic protozoan Trypanosoma brucei. Here we show that the intermembrane space domain of trypanosomal Tom22 binds mitochondrial precursor proteins and that it is essential for normal growth and mitochondrial protein import. Moreover, complementation experiments indicate that the intermembrane space domain cannot be replaced by the corresponding regions of the yeast or plant Tom22 orthologues. Lack or replacement of the short cytosolic domain, however, does not interfere with protein function. Finally, we show that only the membrane-spanning domain of trypanosomal Tom22 is essential for assembly of the trypanosomal TOM complex analogue.

Treadmill exercise alleviates motor deficits and improves mitochondrial import machinery in an MPTP-induced mouse model of Parkinson's disease

Alpha-synuclein (α-Syn) accumulation is significantly correlated with motor deficits and mitochondrial dysfunction in Parkinson's disease (PD), but the molecular mechanism underlying its pathogenesis is unclear. In this study, we investigated the effects of treadmill exercise on motor deficits and mitochondrial dysfunction in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. Treadmill exercise inhibited dopaminergic neuron loss by promoting the expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT) and seemed to improve cell survival by reducing α-Syn expression. Most importantly, treadmill exercise increased expression of the mitochondrial import machinery proteins TOM-40, TOM-20, and TIM-23. This was associated with decreased α-Syn expression and subsequent upregulation of the mitochondrial proteins COX-I, COX-IV, and mtHSP70. Taken together, these results indicate that treadmill exercise may ameliorate motor deficits and improve mitochondrial dysfunction by reducing α-Syn expression in the MPTP-induced mouse model of PD.

Impaired Mitophagy of Erythrocytes Leads to Anemia in Patients with Myelodysplastic Syndromes

Objective: To investigate the change of mitophagy level of bone marrow nucleated red blood cell in anemia patients with myelodysplastic syndromes (MDS), to explore its significance in the anemic pathogenesis of MDS.Methods: Fifty anemia patients with MDS and twenty-six normal controls were enrolled in this study. The mitophagy were observed by transmission electron microscopy (TEM). The level of autophagy-associated protein LC3B, the number of mitochondria, mitochondrial membrane potential, the level of ROS in GlycoA + nucleated red blood cell were measured by Flow cytometry .The expression of AMPK, ULK1, mTOR mRNA of GlycoA + nucleated red blood cell were measured by real time PCR. The expression of the mitochondrial outer membrane protein TOM20 in GlycoA + nucleated red blood cell were detected by Western blotting.Results: (1) Autophagosome or autolysosome including mitochondria were scarcely observed by TEM. (2) The expression of LC3B in GlycoA + nucleated red blood cell in high-risk MDS patients(0.22±0.12) was significantly lower than that in normal controls (0.43±0.22, P<0.01), and lower than that in low-risk MDS patients (0.40±0.16, P<0.01), there were no significant difference in expression of LC3B between normal controls and low-risk MDS patients (P>0.05); (3) The number of mitochondria in GlycoA + nucleated red blood cell in high-risk MDS patients (937.17±707.85) was significantly higher than that in normal controls (513.49±372.33, P<0.05), and higher than that in low-risk MDS patients (461.74±438.02, P<0.01), there were no significant difference in number of mitochondria between normal controls and low-risk MDS patients (P>0.05); (4) The level of mitochondrial membrane potential in GlycoA + nucleated red blood cell in high-risk MDS patients(4.24±3.00)was significantly higher than that in normal controls (2.04±1.11, P<0.01), and higher than that in low-risk MDS patients (2.11±1.10, P<0.01), there were no significant difference in the level of mitochondrial membrane potential between normal controls and low-risk MDS patients(P>0.05); (5) The level of ROS in GlycoA + nucleated red blood cell in high-risk MDS patients(438.65±322.83)was significantly higher than that in normal controls (242.77±136.87, P<0.01), and higher than that in low-risk MDS patients(197.40+95.07, P<0.01), there were no significant difference in the level of ROS between normal controls and low-risk MDS patients(P>0.05); (6) The expression of AMPK mRNA in GlycoA + nucleated red blood cell in high-risk MDS patients(1.06±1.51) was significantly lower than that in low-risk MDS patients (4.35±4.79, P>0.05), there were no significant difference with normal controls(P>0.05). (7) The exception of ULK1 mRNA in GlycoA + nucleated red blood cell in high-risk MDS patients (0.64±0.91) was significantly lower than that in normal controls (2.70±3.27, P<0.01), and lower than that in low-risk MDS patients(4.82±4.81, P<0.01);(8)The level of mTOR mRNA in GlycoA + nucleated red blood cell in high-risk MDS patients (2.81±2.80) was significantly higher than that in normal controls (1.28±0.81, P<0.05),and higher than that in low-risk MDS patients (0.85±0.74, P<0.05), there were no significant difference in expression of AMPK,ULK1and mTOR mRNA between low-risk MDS patients and normal controls (P>0.05). (9) The expression of mitochondrial outer membrane protein TOM20 in high-risk MDS patients was higher than that in normal controls.Conclusion: The pathogenesis of anemia in MDS may be correlated with declining mitophagy level, increasing damaged mitochondria, increasing ROS, DNA damage, and shortening the life of nucleated red blood cells, it may be an effective method to induce mitophagy to correct anemia in MDS. DisclosuresNo relevant conflicts of interest to declare.

Counteracting PINK/Parkin Deficiency in the Activation of Mitophagy: A Potential Therapeutic Intervention for Parkinson's Disease.

Parkinson’s Disease (PD) related genes PINK1, a protein kinase [1], and Parkin, an E3 ubiquitin ligase [2], operate within the same pathway [3-5], which controls, via specific elimination of dysfunctional mitochondria, the quality of the organelle network [6]. Parkin translocates to impaired mitochondria and drives their elimination via autophagy, a process known as mitophagy [6]. PINK1 regulates Parkin translocation through a not yet completely understood mechanism [7, 8]. Mitochondrial outer membrane proteins Mitofusin (MFN), VDAC, Fis1 and TOM20 were found to be targets for Parkin mediated ubiquitination [9-11]. By adding ubiquitin molecules to its targets expressed on mitochondria, Parkin tags and selects dysfunctional mitochondria for clearance, contributing to maintain a functional and healthy mitochondrial network. Abnormal accumulation of misfolded proteins and unfunctional mitochondria is a characteristic hallmark of PD pathology. Therefore a therapeutic approach to enhance clearance of misfolded proteins and potentiate the ubiquitin-proteosome system (UPS) could be instrumental to ameliorate the progression of the disease. Recently, much effort has been put to identify specific de-ubiquitinating enzymes (DUBs) that oppose Parkin in the ubiquitination of its targets. Similar to other post-translational modifications, such as phosphorylation and acetylation, ubiquitination is also a reversible modification, mediated by a large family of DUBs [12]. DUBs inhibitors or activators can affect cellular response to stimuli that induce mitophagy via ubiquitination of mitochondrial outer membrane proteins MFN, VDAC, Fis1 and TOM20. In this respect, the identification of a Parkin-opposing DUB in the regulation of mitophagy, might be instrumental to develop specific isopeptidase inhibitors or activators that can modulate the fundamental biological process of mitochondria clearance and impact on cell survival.

Exposure to low-dose rotenone precipitates synaptic plasticity alterations in PINK1 heterozygous knockout mice

Heterozygous mutations in the PINK1 gene are considered a susceptibility factor to develop early-onset Parkinson's disease (PD), as supported by dopamine hypometabolism in asymptomatic mutation carriers and subtle alterations of dopamine-dependent striatal synaptic plasticity in heterozygous PINK1 knockout (PINK1+/−) mice. The aim of the present study was to investigate whether exposure to low-dose rotenone of heterozygous PINK1+/− mice, compared to their wild-type PINK1+/+ littermates, could impact on dopamine-dependent striatal synaptic plasticity, in the absence of apparent structural alterations.Mice were exposed to a range of concentrations of rotenone (0.01–1mg/kg). Chronic treatment with concentrations of rotenone up to 0.8mg/kg did not cause manifest neuronal loss or changes in ATP levels both in the striatum or substantia nigra of PINK1+/− and PINK1+/+ mice. Moreover, rotenone (up to 0.8mg/kg) treatment did not induce mislocalization of the mitochondrial membrane protein Tom20 and release of cytochrome c in PINK1+/− striata. Accordingly, basic electrophysiological properties of nigral dopaminergic and striatal medium spiny neurons (MSNs) were normal. Despite the lack of gross alterations in neuronal viability in chronically-treated PINK1+/−, a complete loss of both long-term depression (LTD) and long-term potentiation (LTP) was recorded in MSNs from PINK1+/− mice treated with a low rotenone (0.1mg/kg) concentration. Even lower concentrations (0.01mg/kg) blocked LTP induction in heterozygous PINK1+/− MSNs compared to PINK1+/+ mice. Of interest, chronic pretreatment with the antioxidants alpha-tocopherol and Trolox, a water-soluble analog of vitamin E and powerful antioxidant, rescued synaptic plasticity impairment, confirming that, at the doses we utilized, rotenone did not induce irreversible alterations.In this model, chronic exposure to low-doses of rotenone was not sufficient to alter mitochondrial integrity and ATP production, but profoundly impaired the expression of long-term plasticity at corticostriatal synapses in PINK1 heterozygous knockout mice, suggesting that disruption of synaptic plasticity may represent an early feature of a pre-manifesting state of the disease, and a potential tool to test novel neuroprotective agents.

Cryo-slicing Blue Native-Mass Spectrometry (csBN-MS), a Novel Technology for High Resolution Complexome Profiling

Blue native (BN) gel electrophoresis is a powerful method for protein separation. Combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), it enables large scale identification of protein complexes and their subunits. Current BN-MS approaches, however, are limited in size resolution, comprehensiveness, and quantification. Here, we present a new methodology combining defined sub-millimeter slicing of BN gels by a cryo-microtome with high performance LC-MS/MS and label-free quantification of protein amounts. Application of this cryo-slicing BN-MS approach to mitochondria from rat brain demonstrated a high degree of comprehensiveness, accuracy, and size resolution. The technique provided abundance-mass profiles for 774 mitochondrial proteins, including all canonical subunits of the oxidative respiratory chain assembled into 13 distinct (super-)complexes. Moreover, the data revealed COX7R as a constitutive subunit of distinct super-complexes and identified novel assemblies of voltage-dependent anion channels/porins and TOM proteins. Together, cryo-slicing BN-MS enables quantitative profiling of complexomes with resolution close to the limits of native gel electrophoresis.

Evidence of Distinct Channel Conformations and Substrate Binding Affinities for the Mitochondrial Outer Membrane Protein Translocase Pore Tom40

Nearly all mitochondrial proteins are coded by the nuclear genome and must be transported into mitochondria by the translocase of the outer membrane complex. Tom40 is the central subunit of the translocase complex and forms a pore in the mitochondrial outer membrane. To date, the mechanism it utilizes for protein transport remains unclear. Tom40 is predicted to comprise a membrane-spanning β-barrel domain with conserved α-helical domains at both the N and C termini. To investigate Tom40 function, including the role of the N- and C-terminal domains, recombinant forms of the Tom40 protein from the yeast Candida glabrata, and truncated constructs lacking the N- and/or C-terminal domains, were functionally characterized in planar lipid membranes. Our results demonstrate that each of these Tom40 constructs exhibits at least four distinct conductive levels and that full-length and truncated Tom40 constructs specifically interact with a presequence peptide in a concentration- and voltage-dependent manner. Therefore, neither the first 51 amino acids of the N terminus nor the last 13 amino acids of the C terminus are required for Tom40 channel formation or for the interaction with a presequence peptide. Unexpectedly, substrate binding affinity was dependent upon the Tom40 state corresponding to a particular conductive level. A model where two Tom40 pores act in concert as a dimeric protein complex best accounts for the observed biochemical and electrophysiological data. These results provide the first evidence for structurally distinct Tom40 conformations playing a role in substrate recognition and therefore in transport function.

Abstract 3340: Intraoral administration of mitochondrial targeted GS-nitroxide (JP4-039) radioprotects the oral mucosa but not orthotopic tumors in Fancd2-/- mice

Abstract Fanconi Anemia (FA) patients are at risk for head and neck squamous cell carcinomas. Their normal tissue radiosensitivity often presents a challenge to deliver radiotherapy. Locally applied GS-nitroxide JP4-039 radioprotects the oral mucosa of Fancd2-/-(129/Sv) mice from single fraction or fractionated irradiation (Berhane et al, Radiation Research 182:35-49, 2014). We sought to prove that JP4-039 targets the mitochondria of the oral mucosal cells, and protects normal tissue in vivo, but does not prevent radiocontrolability of tumors. Fancd2+/+, Fancd2+/- and Fancd2-/- mice of a different background strain (C57BL/6), received intraoral flurochrome labelled bodipy-JP4-039 in F15 liposomes, were sacrificed 2 hr later and the oral mucosa removed sectioned, and stained with an antibody to mitochondrial protein TOM-20 and examined microscopically for co-localization of bodipy-JP4-039. Mitochondria were isolated from explanted oral mucosal cells and uptake of JP4-039 compared to non-mitochondrial targeted Tempo, measured using EPR. Fancd2+/+, Fancd2+/- and Fancd2-/- mice with orthotopic TC-1 murine squamous cell tumors were treated with JP-4-039/F15, then irradiated. Mitochondrial uptake in normal tissue but not tumors was demonstrated by co-localization of Bodipy-JP4-039 and mitochondrial protein TOM-20. EPR analysis of purified mitochondria from explanted oral cavity cells of Fancd2+/+ and Fancd2-/- mice treated with F15-JP4-039 showed a 15.6 and 19.1 fold increased uptake of nitroxide signal in JP4-039 treated mice, respectively, compared to Tempo uptake in mitochondria . There was no significant difference in tumor control between the JP4-039/F15 treated then irradiated compared to control irradiated mice, in any of the 3 genotypes: Fancd2+/+ group, p = 0.6851; Fancd2+/− group p = 0.7174 and p = 0.7559 in the Fancd2−/− group. JP4-039/F15 administration prior to either single fraction 28 Gy or prior to each fraction of fractionated head and neck irradiation (8 Gy x 4) showed significant normal tissue protection (decreased ulceration) but not tumor radioprotection. Irradiation reduced tumor volume in mice of all genotypes with no detectable effect of JP4-039/F15. Specifically, with wild type Fancd2+/+ mice after 28 Gy, there was no radioprotective effect of JP4-039/F15 on tumor regrowth (p = 0.7520). Tumor bearing Fancd2−/− (p = 0.1843) and Fancd2+/− mice also showed normal tissue but not tumor protection (p = 0.4106 and p = 0.1843, respectively). The data support use of normal mucosal radioprotective JP4-039/F15 during radiotherapy of FA patients with head and neck cancer. Citation Format: Michael W. Epperly, Ashwin Shinde, Hebist Berhane, Byung Han Rhieu, Ronny Kalash, Karen Xu, Darcy Franicola, Xichen Zhang, Tracy Dixon, Donna Shields, Hong Wang, Peter Wipf, Kalindi Parmar, Eva Guinan, Valerian Kagan, Yulia Tyurina, Robert L. Ferris, Song Li, Joel S. Greenberger. Intraoral administration of mitochondrial targeted GS-nitroxide (JP4-039) radioprotects the oral mucosa but not orthotopic tumors in Fancd2-/- mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3340. doi:10.1158/1538-7445.AM2015-3340

Abstract 1769: Rottlerin induces ER stress-mediated cell death in pancreatic stellate cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with a 5-year survival rate under 5%. Whereas activated pancreatic stellate cells (PaSC) are important players in PDAC stroma, simultaneously targeting both cancer and stromal cells can be beneficial for cancer therapy. We previously reported that the phytochemical rottlerin dose-dependently reduces PaSC viability through dysregulation of autophagy and induction of apoptosis, and attenuates tumor progression in PDAC experimental models. Interestingly, both rottlerin and metformin have been shown to affect mitochondrial function, and metformin has been demonstrated to induce AMPK-dependent, CHOP-mediated cell death. Here we further studied the mechanisms underlying cell death and autophagy in rottlerin-treated mouse PaSC. Rottlerin rapidly depolarized mitochondrial membrane, increased oxygen consumption and decreased cellular ATP levels and the expression of the mitochondrial transmembrane protein TOM20, suggesting mitochondrial damage. Rottlerin dysregulated autophagy as indicated by rapid and prolonged increasing in LC3I-LC3II conversion and p62/SQSTM1 accumulation. However, rottlerin-induced LC3II did not further change in the presence of inhibitors of autophagic flux, suggesting that rottlerin blocks autophagy progression in PaSC. In addition, rottlerin induced marked AMPK activation and inhibited mTOR signaling. In contrast to metformin, AMPKα1/2 knockdown had no effect on rottlerin-induced inhibition of mTOR signaling and cell viability. Furthermore, rottlerin induced ER stress as manifested by persistent activation of the PERK pathway and upregulation of proapoptotic transcription factor CHOP. Importantly, rottlerin-induced apoptosis was greatly reduced in PaSC isolated from CHOP null mice. In summary, these data indicate that rottlerin reduces PaSC viability through impairing mitochondrial function, inhibiting mTOR signaling and inducing prolonged ER stress. Specifically, CHOP induction plays an important role in determining cell-fate in mitochondria-targeting agents, such as rottlerin and metformin. Citation Format: Hsin-Yuan Su, Richard Waldron, Raymond Gong, Stephen Pandol, Aurelia Lugea. Rottlerin induces ER stress-mediated cell death in pancreatic stellate cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1769. doi:10.1158/1538-7445.AM2015-1769

Retro-translocation of mitochondrial intermembrane space proteins

The content of mitochondrial proteome is maintained through two highly dynamic processes, the influx of newly synthesized proteins from the cytosol and the protein degradation. Mitochondrial proteins are targeted to the intermembrane space by the mitochondrial intermembrane space assembly pathway that couples their import and oxidative folding. The folding trap was proposed to be a driving mechanism for the mitochondrial accumulation of these proteins. Whether the reverse movement of unfolded proteins to the cytosol occurs across the intact outer membrane is unknown. We found that reduced, conformationally destabilized proteins are released from mitochondria in a size-limited manner. We identified the general import pore protein Tom40 as an escape gate. We propose that the mitochondrial proteome is not only regulated by the import and degradation of proteins but also by their retro-translocation to the external cytosolic location. Thus, protein release is a mechanism that contributes to the mitochondrial proteome surveillance.

Subcellular localization of ataxin-3 and its effect on the morphology of cytoplasmic organoids

To explore the subcellular localization of ataxin-3 and the effect of polyglutamine (polyQ) expansion mutation on the morphology of mitochondrion, golgi apparatus and endoplasmic reticulum. Transient transfection was employed to build cell models expressing wild-type or mutant ataxin-3 proteins. Indirect immunofluorescence was applied to identify markers of organelle membrane. The results were observed under a laser scanning confocal microscope. No co-localization was observed for ataxin-3 protein and mitochondrial marker TOM20, but the percentage of cells with mitochondrial fragmentation has increased in cells expressing mutant ataxin-3 (P<0.05). No co-localization was observed for ataxin-3 protein and golgi marker GM130, and mutant ataxin-3 did not cause golgi fragmentation. Wide type and polyQ-expanded ataxin-3 both showed partial co-localization with ER marker calnexin. The latter showed more overlap with calnexin, and the overlapping signals were mostly located in the places where aggregates were situated. PolyQ-expanded ataxin-3 protein may indirectly affect the integrity of mitochondria, but may cause no effect on the structure and functions of golgi apparatus. Endoplasmic reticulum may be another place where extended ataxin-3 protein can induce cytotoxicity in addition to the nucleus.

The TOM Complex of Amoebozoans: the Cases of the Amoeba Acanthamoeba castellanii and the Slime Mold Dictyostelium discoideum

Protein import into mitochondria requires a wide variety of proteins, forming complexes in both mitochondrial membranes. The TOM complex (translocase of the outer membrane) is responsible for decoding of targeting signals, translocation of imported proteins across or into the outer membrane, and their subsequent sorting. Thus the TOM complex is regarded as the main gate into mitochondria for imported proteins. Available data indicate that mitochondria of representative organisms from across the major phylogenetic lineages of eukaryotes differ in subunit organization of the TOM complex. The subunit organization of the TOM complex in the Amoebozoa is still elusive, so we decided to investigate its organization in the soil amoeba Acanthamoeba castellanii and the slime mold Dictyostelium discoideum. They represent two major subclades of the Amoebozoa: the Lobosa and Conosa, respectively. Our results confirm the presence of Tom70, Tom40 and Tom7 in the A. castellanii and D. discoideum TOM complex, while the presence of Tom22 and Tom20 is less supported. Interestingly, the Tom proteins display the highest similarity to Opisthokonta cognate proteins, with the exception of Tom40. Thus representatives of two major subclades of the Amoebozoa appear to be similar in organization of the TOM complex, despite differences in their lifestyle.

Tom70 mediates Sendai virus-induced apoptosis on mitochondria.

Virus infection triggers immediate innate immune responses. Apoptosis represents another effective means to restrict virus invasion, besides robust expression of host cytokines and chemokines. IRF3 was recently demonstrated to be indispensable for Sendai virus (SeV)-induced apoptosis, but the underlying mechanism is not fully understood. Here we report that a dynamic protein complex, Tom70/Hsp90/IRF3/Bax, mediates SeV-induced apoptosis. The cytosolic proapoptotic protein Bax interacts specifically with IRF3 upon virus infection. The mitochondrial outer membrane protein Tom70 recruits IRF3 to mitochondria via Hsp90. Consequently, the relocation of Bax onto mitochondria induces the leakage of cytochrome c into the cytosol and initiates the corresponding apoptosis. Interestingly, IKK-i is essential for this apoptosis, whereas TBK1 is dispensable. Collectively, our study characterizes a novel protein complex that is important for SeV-induced apoptosis. Apoptosis is an effective means of sacrificing virus-infected cells and restraining the spread of virus. In this study, we demonstrate that IRF3 associates with Bax upon virus infection. Tom70 recruits this protein complex to the mitochondrial outer membrane through Hsp90, which thus induces the release of cytochrome c into the cytosol, initiating virus-induced apoptosis. Interestingly, IKK-i plays an essential role in this activation. This study uncovers a novel mechanism of SeV-induced apoptosis.

Proteomic analysis of mitochondria in respiratory epithelial cells infected with human respiratory syncytial virus and functional implications for virus and cell biology.

The aim of this study was to quantitatively characterise the mitochondrial proteome of airway epithelial cells infected with human respiratory syncytial virus (HRSV), a major cause of paediatric illness. Quantitative proteomics, underpinned by stable isotope labelling with amino acids in cell culture, coupled to LC-MS/MS, was applied to mitochondrial fractions prepared from HRSV-infected and mock-infected cells 12 and 24 h post-infection. Datasets were analysed using ingenuity pathway analysis, and the results were validated and characterised using bioimaging, targeted inhibition and gene depletion. The data quantitatively indicated that antiviral signalling proteins converged on mitochondria during HRSV infection. The mitochondrial receptor protein Tom70 was found to act in an antiviral manner, while its chaperone, Hsp90, was confirmed to be a positive viral factor. Proteins associated with different organelles were also co-enriched in the mitochondrial fractions from HRSV-infected cells, suggesting that alterations in organelle dynamics and membrane associations occur during virus infection. Protein and pathway-specific alterations occur to the mitochondrial proteome in a spatial and temporal manner during HRSV infection, suggesting that this organelle may have altered functions. These could be targeted as part of potential therapeutic strategies to disrupt virus biology.

RIP1-dependent Bid cleavage mediates TNFα-induced but Caspase-3-independent cell death in L929 fibroblastoma cells.

L929 fibroblastoma cells (L929-A) and L929 fibrosarcoma cells (L929-N) are different cell lines that are commonly used to study the cytotoxicity of tumor necrosis factor alpha (TNFα). TNFα has been reported to induce necrosis in both of these cell lines. However, comparing the TNFα-induced cell death in these two cell lines, we found that, unlike the L929-N cells that show typical RIP3-dependent necrosis, TNFα-induced cell death in L929-A cells is pan-caspase inhibitor Z-VAD-FMK (Z-VAD)-sensitive, which does not depend on RIP3. We also confirmed that the cell death signal in the L929-A cells was initiated through cytosol-preassembled ripoptosome and that the knockdown of either Caspase-8 or RIP1 protein blocked cell death. Compared with the L929-N cells, the L929-A cell line had lower levels of constitutive and inducible TNFα autocrine production, and the pan-caspase inhibitors Z-VAD or Q-VD did not kill the L929-A cells as they affect the L929-N cells. Moreover, the L929-A cells expressed less RIP3 protein than the L929-N cells; therefore, TNFα failed to induce RIP3-dependent necroptosis. In addition, the ripoptosome-mediated cell death signal was transduced to the mitochondria through Caspase-8-mediated and RIP1 kinase activity-dependent Bid cleavage. The RIP1 kinase inhibitor Necrostatin-1 (Nec-1) or Caspase-8 knockdown completely blocked Bid cleavage, and the knockdown of Bid or Bax/Bak prevented TNFα-induced cell death in the L929-A cells. Although the activation of Bax/Bak decreased the mitochondrial membrane potential, the levels of mitochondrial intermembrane space proteins, including cytochrome-c (cyt-C) and Smac, declined, and western blotting and immunofluorescence staining analysis did not determine whether these proteins were redistributed to the cytosol. In addition, the mitochondrial outer membrane protein Tom20 was also reduced, indicating that the reduced mitochondria proteins may be induced by the reduced mitochondria numbers. No efficient cyt-C release was observed; therefore, the limited activation and cleavage of downstream caspases, including Caspase-9, Caspase-7, Caspase-6 and Caspase-3, was insufficient to kill the cells. The Caspase-9, Caspase-6 and Caspase-3/7 inhibitors or Caspase-9 and -3 knockdown also failed to block cell death, and the overexpression of Bcl-2 also did not abrogate cell death. Moreover, the dead cells showed necrotic-like but not apoptotic characteristics under transmission electronmicroscopy, and these features were significantly different from mitochondrial apoptosis, indicating that the effector caspases were not the executioners of cell death. These new discoveries show that TNFα-induced cell death in L929-A cells is different than typical RIP3-dependent necrosis and Caspase-8/Caspase-3-mediated apoptosis. These results highlight that caution is necessary when using different L929 cells as a model to investigate TNFα-induced cell death.

Abstract 2456: Overexpression of TOM40 (translocase in the outer mitochondrial membrane 40) inhibits the cell proliferation, invasion and migration abilities in ovarian cancer cell lines

Abstract Background: Mitochondria are essential for energy conversation and cellular metabolism in eukaryotic cell. Mitochondria contain mitochondrial proteins that are encoded in the nucleus and synthesized in cytosol. Synthesized mitochondrial proteins are specially sorted by sub-mitochondrial compartment and then transported to their functional location. If mitochondrial proteins are not transported to their specific regions, it is reported that Mitochondria has a tendency to be implicated in various diseases including cancer. Mitochondria consist of mitochondrial outer membrane and inner membrane, especially, TOM complex are integrated in mitochondrial outer membrane. TOM40 is a central component of TOM complex has General import pore (GIP) to pass mitochondrial pre-protein. The goal of this study is to investigate the function of TOM40 in ovarian cancer. Methods: At first, we performed microarray to identify new target gene in ovarian cancer for nine human ovarian cancer cell line and seven human ovarian surface epithelial cell lines (HOSEs). We investigated target gene expression level in ovarian cancer cell line by RT-PCR and Western blot. To evaluate the association of target gene expression level and clinicopatholigical parameter in ovarian cancer, we carried out immunohistochemistry by using normal and ovarian cancer tissue. Second, we constructed stable DOV-13 and RMUG-S cell lines for overexpression and knock down of TOM40 protein using lentiviral system. We carried out proliferation, invasion and migration assay of each individual stable cell line. Results: We identified TOM40 as a new target gene for ovarian cancer by microarray data that was overexpressed in all nine human ovarian cancer cell lines than HOSEs. TOM40 protein level in borderline and cancer tissue was higher than normal tissue and benign tissue. TOM40 immuno-staining score of normal ovarian tissues is 1.23 (95% CI, 0.44-2.01) that compare with TOM40 immuno-staining score of benign, borderline, and epithelial ovarian tumors is 1.23 (95% CI, 0.44-2.01), 1.41 (95% CI, 0.45-2.37), 3.42 (95% CI, 2.65-4.20), and 5.04 (95% CI, 4.82-5.27), respectively. We understood TOM40 expression level of DOV-13 cells and RMUG-S cells is lower and higher than other ovarian cancer cell line. TOM40 overexpression in DOV-13 cell line inhibited the cell proliferation, invasion and migration than that of control cell line. TOM40 knock down in RMUG-S cell line induced cell proliferation, invasion and migration abilities than in control RMUG-S cell line. Conclusion: It is important to transport pathways of mitochondrial proteins, actually, the paper discussing directly association of TOM complex involving mitochondrial proteins transport and various diseases including cancer is not found. Our results suggest that TOM40 can be a role as putative tumor suppressor for ovarian cancer. Citation Format: Sol Kim, Hanbyoul Cho, Wookyeom Yang, Hyunja Kwon, Ha yeon Shin, Eunju Lee, Eun-Suk Kang, Jae-hoon Kim. Overexpression of TOM40 (translocase in the outer mitochondrial membrane 40) inhibits the cell proliferation, invasion and migration abilities in ovarian cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2456. doi:10.1158/1538-7445.AM2014-2456