In vivo mitochondrial matrix proteome profiling reveals RTN4IP1/OPA10 as an antioxidant NADPH oxidoreductase

Targeting proximity labeling enzymes to specific cellular locations is a viable strategy for profiling subcellular proteomes. Here, we generated transgenic mice expressing a mitochondrial matrix-targeted ascorbate peroxidase (MAX-Tg) to analyze tissue-specific matrix proteomes. Desthiobiotin-phenol labeling of muscle tissues from MAX-Tg mice allowed for efficient profiling of mitochondrial-localized proteins in these tissues. Comparative analysis of matrix proteomes from MAX-Tg muscle tissues revealed differential enrichment of mitochondrial proteins related to energy production in between different muscle groups. Reticulon 4 interacting protein 1 (RTN4IP1), also known as Optic Atrophy-10 (OPA10), was highly enriched in the cardiac and soleus muscles and was found to localize to the mitochondrial matrix via a strong mitochondrial targeting sequence at its N-terminus. Protein structure analysis revealed that RTN4IP1 is an NADPH oxidoreductase with structural homology to bacterial quinone oxidoreductase. Enzymatic activity assays, interactome analysis, and metabolite profiling confirmed a function for RTN4IP1 in coenzyme Q (CoQ) biosynthesis. Rtn4ip1-knockout C2C12 cells had reduced CoQ9 levels, were vulnerable to oxidative stress, and had decreased oxygen consumption rates and ATP production. Collectively, RTN4IP1 is a mitochondrial antioxidant NADPH oxidoreductase supporting oxidative phosphorylation activity in muscle tissue.

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Biocompatibility Evaluation of Electrospun Scaffolds of Poly (L-Lactide) with Pure and Grafted Hydroxyapatite

Journal of the Mexican Chemical Society ◽

10.29356/jmcs.v58i4.53 ◽

2017 ◽

Vol 58 (4) ◽

Author(s):

José Manuel Cornejo-Bravo ◽

Luis Jesús Villarreal-Gómez ◽

Ricardo Vera-Graziano ◽

María Raquel Vega-Ríos ◽

José Luis Pineda-Camacho ◽

...

Keyword(s):

Bacterial Adhesion ◽

Neutral Red ◽

C2c12 Cells ◽

Surrounding Tissue ◽

Neutral Red Assay ◽

Electrospun Scaffolds ◽

Muscle Tissues ◽

Hematoxylin Eosin

The objective of this work was to evaluate the biocompatibility of scaffolds of poly(L-lactide) with pure and grafted hydroxyapatite, at various concentrations of reinforcement. The biocompatibility tests were carried out in vivo in Wistar rats by implanting the material into the subcutaneous and muscle tissues from 1 to 14 weeks and evaluating the surrounding tissue stained with hematoxylin-eosin. For in vitro assays, MTT and neutral red assay were used to evaluate any cytotoxicity in Mioblast Muscle C2C12 Cells (ATCC® CRL-1772™) and Bovine Coronary Artery Endothelial Cells (BCAEC); Escherichia coli and Staphylococcus aureus were used to evaluate bacterial adhesion. All variants of scaffolds provoked a mild inflammatory response, without showing necrosis. No evidence of cytotoxicity was presented in cell viability tests and good bacterial cell adhesion was visualized for all of the materials studied.

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Bicalutamide Elicits Renal Damage by Causing Mitochondrial Dysfunction via ROS Damage and Upregulation of HIF-1

International Journal of Molecular Sciences ◽

10.3390/ijms21093400 ◽

2020 ◽

Vol 21 (9) ◽

pp. 3400

Author(s):

Kuan-Chou Chen ◽

Chang-Rong Chen ◽

Chang-Yu Chen ◽

Kai-Yi Tzou ◽

Chiung-Chi Peng ◽

...

Keyword(s):

Messenger Rna ◽

Renal Damage ◽

Mesangial Cells ◽

Cell Model ◽

Hypoxia Inducible Factor ◽

Kidney Injury ◽

Mitochondrial Potential ◽

Interacting Protein ◽

Atp Production

Combined androgen blockade using bicalutamide (Bic) is a therapeutic choice for treating prostate cancer (PCa). However, even at regular clinical dosages, Bic frequently shows adverse effects associated with cardiovascular and renal damage. Previously, we found that Bic selectively damaged mesangial cells compared to tubular cells and in an in vivo rat model, we also found renal damage caused by Bic. In the present study, a rat mesangial cell model was used to further the investigation. Results indicated that Bic enhanced lactate dehydrogenase release, reactive oxygen species (ROS) production, lysosome population and kidney injury molecule-1 and decreased N-cadherin. Bic elicited mitochondrial swelling and reduced the mitochondrial potential, resulting in severe suppression of the oxygen consumption rate (OCR), maximum respiration and ATP production. The hypoxia-inducible factor (HIF)-1 transcriptional activity and messenger RNA were significantly upregulated in dose-dependent manners. The HIF-1 protein reached a peak value at 24 h then rapidly decayed. BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 and cleaved caspase-3 were dose-dependently upregulated by Bic (60 M) and that eventually led to cell apoptosis. It is suggested that Bic induces renal damage via ROS and modulates HIF-1 pathway and clinically, some protective agents like antioxidants are recommended for co-treatment.

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Tanshinone I exerts cardiovascular protective effects in vivo and in vitro through inhibiting necroptosis via Akt/Nrf2 signaling pathway

Chinese Medicine ◽

10.1186/s13020-021-00458-7 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Youqiong Zhuo ◽

Renyikun Yuan ◽

Xinxin Chen ◽

Jia He ◽

Yangling Chen ◽

...

Keyword(s):

Protein Kinase ◽

Protective Effect ◽

Heme Oxygenase 1 ◽

Related Factor ◽

H9c2 Cells ◽

Quinone Oxidoreductase ◽

Interacting Protein ◽

Tnf Α

Abstract Background Tanshinone I (TI) is a primary component of Salvia miltiorrhiza Bunge (Danshen), which confers a favorable role in a variety of pharmacological activities including cardiovascular protection. However, the exact mechanism of the cardiovascular protection activity of TI remains to be illustrated. In this study, the cardiovascular protective effect and its mechanism of TI were investigated. Methods In this study, tert-butyl hydroperoxide (t-BHP)-stimulated H9c2 cells model was employed to investigate the protective effect in vitro. The cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) kit. The reactive-oxygen-species (ROS) level and mitochondrial membrane potential (MMP) were investigated by the flow cytometry and JC-1 assay, respectively. While in vivo experiment, the cardiovascular protective effect of TI was determined by using myocardial ischemia–reperfusion (MI/R) model including hematoxylin–eosin (H&E) staining assay and determination of superoxide dismutase (SOD) and malondialdehyde (MDA). Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) release were detected by Enzyme-linked immunosorbent assay (ELISA). Receptor interacting protein kinase 1 (RIP1), receptor interacting protein kinase 3 (RIP3), receptor interacting protein kinase 3 (MLKL), protein kinase B (Akt), Nuclear factor erythroid 2 related factor 2 (Nrf2), Heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase-1 (NQO-1) were determined by western blotting. Results Our data demonstrated that TI pretreatment attenuated t-BHP and MI/R injury-induced necroptosis by inhibiting the expression of p-RIP1, p-RIP3, and p-MLKL. TI activated the Akt/Nrf2 pathway to promote the expression of antioxidant-related proteins such as phosphorylation of Akt, nuclear factor erythroid 2 related factor 2 (Nrf2), quinone oxidoreductase-1 (NQO-1) and heme oxygenase-1 (HO-1) expression in t-BHP-stimulated H9c2 cells. TI relieved oxidative stress by mitigating ROS generation and reversing MMP loss. In vivo experiment, TI made electrocardiograph (ECG) recovery better and lessened the degree of myocardial tissue damage. The counts of white blood cell (WBC), neutrophil (Neu), lymphocyte (Lym), and the release of TNF-α and IL-6 were reversed by TI treatment. SOD level was increased, while MDA level was decreased by TI treatment. Conclusion Collectively, our findings indicated that TI exerted cardiovascular protective activities in vitro and in vivo through suppressing RIP1/RIP3/MLKL and activating Akt/Nrf2 signaling pathways, which could be developed into a cardiovascular protective agent.

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Multi-omic mitoprotease profiling defines a role for Oct1p in coenzyme Q production

10.1101/155044 ◽

2017 ◽

Author(s):

Mike T. Veling ◽

Andrew G. Reidenbach ◽

Elyse C. Freiberger ◽

Nicholas W. Kwiecien ◽

Paul D. Hutchins ◽

...

Keyword(s):

Coenzyme Q ◽

Strong Association ◽

Single Mutation ◽

Mitochondrial Matrix ◽

Recognition Motif ◽

Mitochondrial Functions ◽

Efficient Exploration ◽

Edman Sequencing

SUMMARYMitoproteases are becoming recognized as key regulators of diverse mitochondrial functions, although their direct substrates are often difficult to discern. Through multi-omic profiling of diverse Saccharomyces cerevisiae mitoprotease deletion strains, we predicted numerous associations between mitoproteases and distinct mitochondrial processes. These include a strong association between the mitochondrial matrix octapeptidase Oct1p and coenzyme Q (CoQ) biosynthesis—a pathway essential for mitochondrial respiration. Through Edman sequencing, and in vitro and in vivo biochemistry, we demonstrated that Oct1p directly processes the N-terminus of the CoQ-related methyltransferase, Coq5p, which markedly improves its stability. A single mutation to the Oct1p recognition motif in Coq5p disrupted its processing in vivo, leading to CoQ deficiency and respiratory incompetence. This work defines the Oct1p processing of Coq5p as an essential post-translational event for proper CoQ production. Our custom data visualization tool enables efficient exploration of mitoprotease profiles that can serve as the basis for future mechanistic investigations.

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Mitochondrial matrix calcium ions regulate energy production in myocardium: A cytochemical study

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100134429 ◽

1990 ◽

Vol 48 (2) ◽

pp. 166-167

Author(s):

W.A. Jacob ◽

R. Hertsens ◽

A. Van Bogaert ◽

M. De Smet

Keyword(s):

Energy Metabolism ◽

Calcium Ions ◽

Energy Production ◽

Regulation Of Respiration ◽

Free Calcium ◽

Mitochondrial Matrix ◽

Cytochemical Study ◽

Nmr Techniques

In the past most studies of the control of energy metabolism focus on the role of the phosphorylation potential ATP/ADP.Pi on the regulation of respiration. Studies using NMR techniques have demonstrated that the concentrations of these compounds for oxidation phosphorylation do not change appreciably throughout the cardiac cycle and during increases in cardiac work. Hence regulation of energy production by calcium ions, present in the mitochondrial matrix, has been the object of a number of recent studies.Three exclusively intramitochondnal dehydrogenases are key enzymes for the regulation of oxidative metabolism. They are activated by calcium ions in the low micromolar range. Since, however, earlier estimates of the intramitochondnal calcium, based on equilibrium thermodynamic considerations, were in the millimolar range, a physiological correlation was not evident. The introduction of calcium-sensitive probes fura-2 and indo-1 made monitoring of free calcium during changing energy metabolism possible. These studies were performed on isolated mitochondria and extrapolation to the in vivo situation is more or less speculative.

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Mechanism of CK2.3, a Novel Mimetic Peptide of Bone Morphogenetic Protein Receptor Type IA, Mediated Osteogenesis

International Journal of Molecular Sciences ◽

10.3390/ijms20102500 ◽

2019 ◽

Vol 20 (10) ◽

pp. 2500 ◽

Cited By ~ 3

Author(s):

Vrathasha Vrathasha ◽

Hilary Weidner ◽

Anja Nohe

Keyword(s):

Signaling Pathways ◽

Treatment Options ◽

Time Frame ◽

Bmp Signaling ◽

C2c12 Cells ◽

Molecular Sequence ◽

Sequence Of Events ◽

Protein Receptor

Background: Osteoporosis is a degenerative skeletal disease with a limited number of treatment options. CK2.3, a novel peptide, may be a potential therapeutic. It induces osteogenesis and bone formation in vitro and in vivo by acting downstream of BMPRIA through releasing CK2 from the receptor. However, the detailed signaling pathways, the time frame of signaling, and genes activated remain largely unknown. Methods: Using a newly developed fluorescent CK2.3 analog, specific inhibitors for the BMP signaling pathways, Western blot, and RT-qPCR, we determined the mechanism of CK2.3 in C2C12 cells. We then confirmed the results in primary BMSCs. Results: Using these methods, we showed that CK2.3 stimulation activated OSX, ALP, and OCN. CK2.3 stimulation induced time dependent release of CK2β from BMPRIA and concurrently CK2.3 colocalized with CK2α. Furthermore, CK2.3 induced BMP signaling depends on ERK1/2 and Smad1/5/8 signaling pathways. Conclusion: CK2.3 is a novel peptide that drives osteogenesis, and we detailed the molecular sequence of events that are triggered from the stimulation of CK2.3 until the induction of mineralization. This knowledge can be applied in the development of future therapeutics for osteoporosis.

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miR-125b enhances metastasis and progression of cancer via the TXNIP and HIF1α pathway in pancreatic cancer

Cancer Biomarkers ◽

10.3233/cbm-203112 ◽

2021 ◽

pp. 1-12

Author(s):

Pengli Wang ◽

Dan Zheng ◽

Hongyang Qi ◽

Qi Gao

Keyword(s):

Pancreatic Cancer ◽

Hypoxia Inducible Factor ◽

Immunofluorescence Assay ◽

Luciferase Assay ◽

Interacting Protein ◽

Over Expression ◽

Thioredoxin Interacting Protein ◽

Cellular Processes ◽

And Migration

BACKGROUND: MicroRNAs (miRNAs) play potential role in the development of various types of cancer conditions including pancreatic cancer (PC) targeting several cellular processes. Present study was aimed to evaluate function of miR-125b and the mechanism involved in PC. METHODS: Cell migration, MTT and BrdU study was done to establish the migration capability, cell viability and cell proliferation respectively. Binding sites for miR-125b were recognized by luciferase assay, expression of protein by western blot and immunofluorescence assay. In vivo study was done by BALB/c nude xenograft mice for evaluating the function of miR-125b. RESULTS: The study showed that expression of miR-125b was elevated in PC cells and tissues, and was correlated to proliferation and migration of cells. Also, over-expression of miR-125b encouraged migration, metastasis and proliferation of BxPC-3 cells, the suppression reversed it. We also noticed that thioredoxin-interacting protein (TXNIP) was the potential target of miR-125b. The outcomes also suggested that miR-125b governed the expression of TXNIP inversely via directly attaching to the 3′-UTR activating hypoxia-inducible factor 1α (HIF1α). Looking into the relation between HIF1α and TXNIP, we discovered that TXNIP caused the degradation and export of HIF1α by making a complex with it. CONCLUSION: The miR-125b-TXNIP-HIF1α pathway may serve useful strategy for diagnosing and treating PC.

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EBV-LMP1 promotes radioresistance by inducing protective autophagy through BNIP3 in nasopharyngeal carcinoma

Cell Death and Disease ◽

10.1038/s41419-021-03639-2 ◽

2021 ◽

Vol 12 (4) ◽

Author(s):

San Xu ◽

Zhuan Zhou ◽

Xingzhi Peng ◽

Xuxiu Tao ◽

Peijun Zhou ◽

...

Keyword(s):

Nasopharyngeal Carcinoma ◽

Crucial Role ◽

Epstein Barr Virus ◽

Interacting Protein ◽

Multiple Tumors ◽

Barr Virus ◽

Signaling Axis ◽

Epstein Barr ◽

Adenovirus E1b

AbstractStudies have indicated that dysfunction of autophagy is involved in the initiation and progression of multiple tumors and their chemoradiotherapy. Epstein–Barr virus (EBV) is a lymphotropic human gamma herpes virus that has been implicated in the pathogenesis of nasopharyngeal carcinoma (NPC). EBV encoded latent membrane protein1 (LMP1) exhibits the properties of a classical oncoprotein. In previous studies, we experimentally demonstrated that LMP1 could increase the radioresistance of NPC. However, how LMP1 contributes to the radioresistance in NPC is still not clear. In the present study, we found that LMP1 could enhance autophagy by upregulating the expression of BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3). Knockdown of BNIP3 could increase the apoptosis and decrease the radioresistance mediated by protective autophagy in LMP1-positive NPC cells. The data showed that increased BNIP3 expression is mediated by LMP1 through the ERK/HIF1α signaling axis, and LMP1 promotes the binding of BNIP3 to Beclin1 and competitively reduces the binding of Bcl-2 to Beclin1, thus upregulating autophagy. Furthermore, knockdown of BNIP3 can reduce the radioresistance promoted by protective autophagy in vivo. These data clearly indicated that, through BNIP3, LMP1 induced autophagy, which has a crucial role in the protection of LMP1-positive NPC cells against irradiation. It provides a new basis and potential target for elucidating LMP1-mediated radioresistance.

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734 A novel NQO1 specific anti-tumor agent, SBSC-S3001, selectively regresses the growth of tumors with high NQO1 expression

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0734 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A778-A778

Author(s):

Minhyuk Yun ◽

Goo-Young Kim ◽

Sang Woo Jo ◽

Changhoon In ◽

Gyu-Young Moon ◽

...

Keyword(s):

Energy Metabolism ◽

Cancer Cells ◽

Poor Prognosis ◽

High Expression ◽

List Type ◽

Dependent Manner ◽

Breast Cancers ◽

Quinone Oxidoreductase ◽

Key Enzymes

BackgroundNAD(P)H-quinone oxidoreductase 1 (NQO1) is a cytosolic two-electron oxidoreductase overexpressed in many types of cancers, including breast cancer, pancreatic cancer, colorectal cancer, cholangiocarcinoma, uterine cervical cancer, melanoma, and lung cancer.1Up-regulation of NQO1 protects cells from oxidative stress and various cytotoxic quinones and is associated with late clinical stage, poor prognosis and lymph node metastasis.2 3 NQO1 increases stability of HIF-1α protein, which has been implicated in survival, proliferation, and malignance of cancer.1 Therefore, accumulating evidences suggest NQO1 as a promising therapeutic target for cancer. Accordingly, we have characterized the effect of a novel synthetic NQO1 substrate SBSC-S3001, and demonstrated its selective cytotoxic effects in cancer cells with high expression of NQO1.MethodsIn vitro cytotoxicity was determined by sulforhodamine B (SRB) assay in cancer cells with high NQO1 expression and CRISPR-mediated NQO1 knockout cells. The effect of SBSC-S3001 on the energy metabolism pathway was evaluated by western blot analysis of metabolism associated proteins from NQO1-overexpressed cancer cells treated with the compound for 24 hours. In vivo anti-tumor activity was evaluated in MC38 syngeneic and DLD-1 orthotopic mice models.ResultsSBSC-S3001 exhibited selective cytotoxicity in cancer cells with high expression of NQO1 in a dose-dependent manner. The cytotoxicity was observed in both normoxia and hypoxia conditions, correlating with the energy metabolism, mitochondrial biogenesis, and cancer proliferative pathways. Also, stronger cytotoxicity was observed in NQO1-overexpressed cancer cells treated with SBSC-S3001 compared to beta-lapachone and analogue treatment.4 When evaluated in vivo, SBSC-S3001 effectively inhibited the growth of syngeneic and orthotopic tumors when administered as a monotherapy. SBSC-S3001 treatment associated with reduction in key enzymes of the glycolytic pathway (LDHa and GAPDH) and HIF-1α and increase in levels of mitochondrial oxidative phosphorylation (OXPHOS) complex.ConclusionsTreatment of SBSC-S3001, a novel, NQO1-specific substrate reduces HIF-1α and key enzymes associated with glycolysis and suppresses the growth of tumors overexpressing NQO1. Further characterization of SBSC-S3001 as a novel metabolic anti-cancer agent for cancers with NQO1 overexpression is warranted.Ethics ApprovalThe study was approved by Samyang Biopharmaceuticals Institution’s Ethics Board, approval number SYAU2031.ReferencesOh ET, Kim JW, Kim JMet. al., NQO1 inhibits proteasome-mediated degradation of HIF-1α. Nat Commun 2016; 14:13593.Ma, Y. et al. NQO1 overexpression is associated with poor prognosis in squamous cell carcinoma of the uterine cervix. BMC Cancer 2014;14: 414Yang, Y. et al. Clinical implications of high NQO1 expression in breast cancers. J. Exp. Clin. Cancer Res 2014;33:144.Yang Y, Zhou X, Xu M, et al., β-lapachone suppresses tumour progression by inhibiting epithelial-to-mesenchymal transition in NQO1-positive breast cancers. Sci Rep 2017;7:2681.

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Bedaquiline, an FDA-approved drug, inhibits mitochondrial ATP production and metastasis in vivo, by targeting the gamma subunit (ATP5F1C) of the ATP synthase

Cell Death and Differentiation ◽

10.1038/s41418-021-00788-x ◽

2021 ◽

Author(s):

Marco Fiorillo ◽

Cristian Scatena ◽

Antonio Giuseppe Naccarato ◽

Federica Sotgia ◽

Michael P. Lisanti

Keyword(s):

Cell Migration ◽

Treatment Failure ◽

Atp Synthase ◽

Atp Depletion ◽

Multi Drug Resistance ◽

Gamma Subunit ◽

Primary Tumors ◽

Atp Production ◽

Mitochondrial Atp Synthase

AbstractHere, we provide evidence that high ATP production by the mitochondrial ATP-synthase is a new therapeutic target for anticancer therapy, especially for preventing tumor progression. More specifically, we isolated a subpopulation of ATP-high cancer cells which are phenotypically aggressive and demonstrate increases in proliferation, stemness, anchorage-independence, cell migration, invasion and multi-drug resistance, as well as high antioxidant capacity. Clinically, these findings have important implications for understanding treatment failure and cancer cell dormancy. Using bioinformatic analysis of patient samples, we defined a mitochondrial-related gene signature for metastasis, which features the gamma-subunit of the mitochondrial ATP-synthase (ATP5F1C). The relationship between ATP5F1C protein expression and metastasis was indeed confirmed by immunohistochemistry. Next, we used MDA-MB-231 cells as a model system to functionally validate these findings. Importantly, ATP-high MDA-MB-231 cells showed a nearly fivefold increase in metastatic capacity in vivo. Consistent with these observations, ATP-high cells overexpressed (i) components of mitochondrial complexes I–V, including ATP5F1C, and (ii) markers associated with circulating tumor cells (CTCs) and metastasis, such as EpCAM and VCAM1. Knockdown of ATP5F1C expression significantly reduced ATP-production, anchorage-independent growth, and cell migration, as predicted. Similarly, therapeutic administration of the FDA-approved drug, Bedaquiline, downregulated ATP5F1C expression in vitro and prevented spontaneous metastasis in vivo. In contrast, Bedaquiline had no effect on the growth of non-tumorigenic mammary epithelial cells (MCF10A) or primary tumors in vivo. Taken together, our results suggest that mitochondrial ATP depletion is a new therapeutic strategy for metastasis prophylaxis, to avoid treatment failure. In summary, we conclude that mitochondrial ATP5F1C is a promising new biomarker and molecular target for future drug development, for the prevention of metastatic disease progression.

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