respiratory chain complex
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Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3329-3329
Author(s):  
Yue Feng ◽  
Yulia Jitkova ◽  
Harjeet Soor ◽  
Suzanne Trudel ◽  
Kaitlin Lees ◽  
...  

Abstract The mitochondrial serine protease, ClpXP, regulates the integrity of the respiratory chain by degrading damaged and/or misfolded proteins. This protease is over-expressed in a subset of AML and inhibiting or hyperactivating it kills leukemic cells and stem cells in vitro and in vivo. Yet, it is unknown how the mitochondrial ClpXP recognizes proteins for degradation. In Bacillus subtilis, the bacterial ClpXP homologue recognizes proteins tagged with phospho-arginine for degradation. To determine if phosphorylated amino acids influence mitochondrial ClpXP-mediated protein degradation, we incubated recombinant ClpXP with its unnatural substrate FITC-casein and increasing concentrations of phospho-serine (pSer), phospho-threonine (pThr), phospho-arginine (pArg), or phospho-tyrosine (pTyr) in a cell-free assay and measured release of fluorogenic FITC. In a dose-dependent manner, pSer and pThr free amino acids inhibited casein cleavage by ClpXP while pTyr, pArg, and the dephosphorylated amino acids had no effect on ClpXP activity. Likewise, ApSA, RRApSVA, and ApTA peptides inhibited ClpXP enzyme activity, while the non-phosphorylated version (ASA, RRASVA, and ATA) had no effect. Next, we tested whether the phosphorylation state of full length proteins would influence their degradation by ClpXP. Using gel-based cell-free assays, the phosphorylation enriched α-casein and β-casein were degraded by recombinant ClpXP. In contrast, κ-casein with low levels of phosphorylation and dephosphorylated α-casein were not cleaved by ClpXP. As ClpX is an AAA ATPase, we asked if pSer and pThr acted on the ATPase of the enzyme. pSer and pThr did not inhibit the ATPase activity of ClpX. We also measured the effect of pSer and pThr on the peptidase activity of ClpP alone without its regulatory subunit ClpX. Neither pSer or pThr inhibited ClpP peptidase activity. We investigated if pSer and pThr could bind to ClpX. Using thermal shift binding assays, we demonstrated that pSer and pThr but not pTyr and pArg bound ClpX and none of the phosphorylated amino acids bound ClpP. Similarly, ApSA, RRApSVA, and ApTA peptides bound ClpX, while the non-phosphorylated ASA, RRASVA, and ATA did not bind ClpX. Previously we showed that ClpP interacted with respiratory chain complex II subunit SDHA, and ClpP knockdown in AML cells impaired respiratory chain complex II activity and reactive oxygen species increased. Therefore, we tested how ClpXP knockdown impacts levels of phospho-serine SDHA. Using shRNA, we knocked down ClpP and ClpX individually in OCI-AML2 cells. After target knockdown, we pulled down pSer proteins and then probed for SDHA. Knockdown of both ClpP and ClpX increased the abundance of serine phosphorylated SDHA comparing to control, suggesting that ClpXP degrades serine phosphorylated SDHA. Finally, as a chemical approach, we generated small molecules that mimic pSer and demonstrated that they inhibited ClpXP-mediated degradation of FITC-casein and bound ClpX with a potency similar to pSer. In summary, we discovered that ClpX binds pSer and pThr and phosphorylation of these amino acids mark proteins for degradation by the ClpXP mitochondrial protease. This work highlights a new strategy to develop inhibitors of ClpXP for the treatment of AML. Disclosures Schimmer: Takeda Pharmaceuticals: Consultancy, Research Funding; Medivir AB: Research Funding; Otsuka Pharmaceuticals: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; UHN: Patents & Royalties.


2021 ◽  
pp. 127546
Author(s):  
Libing Yu ◽  
Zhaozhu Lin ◽  
Xuedan Cheng ◽  
Jian Chu ◽  
Xijian Li ◽  
...  

Toxins ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 595
Author(s):  
Jiayu Liu ◽  
Qi Liu ◽  
Jiahui Han ◽  
Jiayu Feng ◽  
Tianmin Guo ◽  
...  

Patulin (PAT) belongs to the family of food-borne mycotoxins. Our previous studies revealed that PAT caused cytotoxicity in human embryonic kidney cells (HEK293). In the present research, we systematically explored the detailed mechanism of ROS production and ROS clearance in PAT-induced HEK293 cell apoptosis. Results showed that PAT treatment (2.5, 5, 7.5, 10 μM) for 10 h could regulate the expression of genes and proteins involved in the mitochondrial respiratory chain complex, resulting in dysfunction of mitochondrial oxidative phosphorylation and induction of ROS overproduction. We further investigated the role of N-acetylcysteine (NAC), an ROS scavenger, in promoting the survival of PAT-treated HEK293 cells. NAC improves PAT-induced apoptosis of HEK293 cells by clearing excess ROS, modulating the expression of mitochondrial respiratory chain complex genes and proteins, and maintaining normal mitochondrial function. In addition, NAC protects the activity of antioxidant enzymes, maintains normal GSH content, and relieves oxidative damage. Additionally, 4 mM NAC alleviated 7.5 μM PAT-mediated apoptosis through the caspase pathway in HEK293 cells. In summary, our study demonstrated that ROS is significant in PAT-mediated cytotoxicity, which provides valuable insight into the management of PAT-associated health issues.


Author(s):  
Eleanor Hay ◽  
Louise C. Wilson ◽  
Bethan Hoskins ◽  
Martin Samuels ◽  
Pinki Munot ◽  
...  

AbstractWe report a patient with profound congenital hypotonia, central hypoventilation, poor visual behaviour with retinal hypopigmentation, and significantly decreased mitochondrial respiratory chain complex I activity in muscle, who died at 7 months of age having made minimal developmental progress. Biallelic predicted truncating P4HTM variants were identified following trio whole-genome sequencing, consistent with a diagnosis of hypotonia, hypoventilation, intellectual disability, dysautonomia, epilepsy and eye abnormalities (HIDEA) syndrome. Very few patients with HIDEA syndrome have been reported previously and mitochondrial abnormalities were observed in three of four previous cases who had a muscle biopsy, suggesting the possibility that HIDEA syndrome represents a primary mitochondrial disorder. P4HTM encodes a transmembrane prolyl 4-hydroxylase with putative targets including hypoxia inducible factors, RNA polymerase II and activating transcription factor 4, which has been implicated in the integrated stress response observed in cell and animal models of mitochondrial disease, and may explain the mitochondrial dysfunction observed in HIDEA syndrome.


2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Simon X. M. Dong ◽  
Frederick S. Vizeacoumar ◽  
Kalpana K. Bhanumathy ◽  
Nezeka Alli ◽  
Cristina Gonzalez-Lopez ◽  
...  

Abstract Background Macrophages, besides resting latently infected CD4+ T cells, constitute the predominant stable, major non-T cell HIV reservoirs. Therefore, it is essential to eliminate both latently infected CD4+ T cells and tissue macrophages to completely eradicate HIV in patients. Until now, most of the research focus is directed towards eliminating latently infected CD4+ T cells. However, few approaches have been directed at killing of HIV-infected macrophages either in vitro or in vivo. HIV infection dysregulates the expression of many host genes essential for the survival of infected cells. We postulated that exploiting this alteration may yield novel targets for the selective killing of infected macrophages. Methods We applied a pooled shRNA-based genome-wide approach by employing a lentivirus-based library of shRNAs to screen novel gene targets whose inhibition should selectively induce apoptosis in HIV-infected macrophages. Primary human MDMs were infected with HIV-eGFP and HIV-HSA viruses. Infected MDMs were transfected with siRNAs specific for the promising genes followed by analysis of apoptosis by flow cytometry using labelled Annexin-V in HIV-infected, HIV-exposed but uninfected bystander MDMs and uninfected MDMs. The results were analyzed using student’s t-test from at least four independent experiments. Results We validated 28 top hits in two independent HIV infection models. This culminated in the identification of four target genes, Cox7a2, Znf484, Cstf2t, and Cdk2, whose loss-of-function induced apoptosis preferentially in HIV-infected macrophages. Silencing these single genes killed significantly higher number of HIV-HSA-infected MDMs compared to the HIV-HSA-exposed, uninfected bystander macrophages, indicating the specificity in the killing of HIV-infected macrophages. The mechanism governing Cox7a2-mediated apoptosis of HIV-infected macrophages revealed that targeting respiratory chain complex II and IV genes also selectively induced apoptosis of HIV-infected macrophages possibly through enhanced ROS production. Conclusions We have identified above-mentioned novel genes and specifically the respiratory chain complex II and IV genes whose silencing may cause selective elimination of HIV-infected macrophages and eventually the HIV-macrophage reservoirs. The results highlight the potential of the identified genes as targets for eliminating HIV-infected macrophages in physiological environment as part of an HIV cure strategy.


Antioxidants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1003
Author(s):  
Lukas Babylon ◽  
Rekha Grewal ◽  
Pascal-L. Stahr ◽  
Ralph W. Eckert ◽  
Cornelia M. Keck ◽  
...  

Mitochondrial dysfunction represents a hallmark of both brain aging and age-related neurodegenerative disorders including Alzheimer disease (AD). AD-related mitochondrial dysfunction is characterized by an impaired electron transport chain (ETC), subsequent decreased adenosine triphoshpate (ATP) levels, and elevated generation of reactive oxygen species (ROS). The bioactive citrus flavanone hesperetin (Hst) is known to modulate inflammatory response, to function as an antioxidant, and to provide neuroprotective properties. The efficacy in improving mitochondrial dysfunction of Hst nanocrystals (HstN) with increased bioavailability has not yet been investigated. Human SH-SY5Y cells harboring neuronal amyloid precursor protein (APP695) acted as a model for the initial phase of AD. MOCK-transfected cells served as controls. The energetic metabolite ATP was determined using a luciferase-catalyzed bioluminescence assay. The activity of mitochondrial respiration chain complexes was assessed by high-resolution respirometry using a Clarke electrode. Expression levels of mitochondrial respiratory chain complex genes were determined using quantitative real-time polymerase chain reaction (qRT-PCR). The levels of amyloid β-protein (Aβ1-40) were measured using homogeneous time-resolved fluorescence (HTRF). ROS levels, peroxidase activity, and cytochrome c activity were determined using a fluorescence assay. Compared to pure Hst dissolved in ethanol (HstP), SH-SY5Y-APP695 cells incubated with HstN resulted in significantly reduced mitochondrial dysfunction: ATP levels and respiratory chain complex activity significantly increased. Gene expression levels of RCC I, IV, and V were significantly upregulated. In comparison, the effects of HstN on SY5Y-MOCK control cells were relatively small. Pure Hst dissolved in ethanol (HstP) had almost no effect on both cell lines. Neither HstN nor HstP led to significant changes in Aβ1-40 levels. HstN and HstP were both shown to lower peroxidase activity significantly. Furthermore, HstN significantly reduced cytochrome c activity, whereas HstP had a significant effect on reducing ROS in SH-SY5Y-APP695 cells. Thus, it seems that the mechanisms involved may not be linked to altered Aβ production. Nanoflavonoids such as HstN have the potential to prevent mitochondria against dysfunction. Compared to its pure form, HstN showed a greater effect in combatting mitochondrial dysfunction. Further studies should evaluate whether HstN protects against age-related mitochondrial dysfunction and thus may contribute to late-onset AD.


2021 ◽  
Author(s):  
Ionica Masgras ◽  
Giuseppe Cannino ◽  
Francesco Ciscato ◽  
Carlos Sanchez-Martin ◽  
Marco Pizzi ◽  
...  

Neurofibromin loss drives neoplastic growth and a rewiring of mitochondrial metabolism. Here, we report that neurofibromin ablation dampens expression and activity of NADH dehydrogenase, the respiratory chain complex I, in an ERK-dependent fashion. This provides cells with resistance to pro-oxidants targeting complex I and decreases both respiration and intracellular NAD+. Expression of the alternative NADH dehydrogenase NDI1 raises NAD+/NADH ratio, enhances the activity of the mitochondrial NAD+-dependent deacetylase SIRT3 and interferes with tumorigenicity in neurofibromin-deficient cells. This anti-neoplastic effect is mimicked both in vitro and in vivo by administration of NAD+ precursors or by rising expression of the NAD+ deacetylase SIRT3, and is synergistic with ablation of the mitochondrial chaperone TRAP1, which augments succinate dehydrogenase activity further contributing to block pro-neoplastic metabolic changes of these cells. These findings shed light on chemotherapeutic resistance and on bioenergetic adaptations of tumors lacking neurofibromin, linking complex I inhibition to mitochondrial NAD+/NADH unbalance and SIRT3 inhibition, as well as to down-regulation of succinate dehydrogenase. This metabolic rewiring could unveil attractive therapeutic targets for neoplasms related to neurofibromin loss.


2021 ◽  
Vol 22 (9) ◽  
pp. 4684
Author(s):  
Min Li ◽  
Li Chen ◽  
Aziz Khan ◽  
Xiangjun Kong ◽  
Muhammad Rabnawaz Khan ◽  
...  

Cytoplasmic male sterility (CMS) is important for large-scale hybrid seed production. Rearrangements in the mitochondrial DNA (mtDNA) for the cotton (Gossypium hirsutum L.) CMS line J4A were responsible for pollen abortion. However, the expression patterns of nuclear genes associated with pollen abortion and the molecular basis of CMS for J4A are unknown, and were the objectives of this study by comparing J4A with the J4B maintainer line. Cytological evaluation of J4A anthers showed that microspore abortion occurs during meiosis preventing pollen development. Changes in enzyme activity of mitochondrial respiratory chain complex IV and mitochondrial respiratory chain complex V and the content of ribosomal protein and ATP during anther abortion were observed for J4A suggesting insufficient synthesis of ATP hindered pollen production. Additionally, levels of sucrose, starch, soluble sugar, and fructose were significantly altered in J4A during the meiosis stage, suggesting reduced sugar metabolism contributed to sterility. Transcriptome and miRNAomics analyses identified 4461 differentially expressed mRNAs (DEGs) and 26 differentially expressed microRNAs (DEMIs). Pathway enrichment analysis indicated that the DEMIs were associated with starch and sugar metabolism. Six deduced target gene regulatory pairs that may participate in CMS were identified, ghi-MIR7484-10/mitogen-activated protein kinase kinase 6 (MAPKK6), ghi-undef-156/agamous-like MADS-box protein AGL19 (AGL19), ghi-MIR171-1-22/SNF1-related protein kinase regulatory subunit gamma-1 and protein trichome birefringence-like 38, and ghi-MIR156-(8/36)/WRKY transcription factor 28 (WRKY28). Overall, a putative CMS mechanism involving mitochondrial dysfunction, the ghi-MIR7484-10/MAPKK6 network, and reduced glucose metabolism was suggested, and ghi-MIR7484-10/MAPKK6 may be related to abnormal microspore meiosis and induction of excessive sucrose accumulation in anthers.


2021 ◽  
Author(s):  
Arianna Franca Anzmann ◽  
Olivia Sniezek ◽  
Alexandra Pado ◽  
Veronica F. Busa ◽  
Frederic M Vaz ◽  
...  

Barth syndrome (BTHS) is an X-linked genetic condition caused by defects in TAZ, which encodes a transacylase involved in the remodeling of the inner mitochondrial membrane phospholipid, cardiolipin (CL). As such, CL has been implicated in numerous mitochondrial functions, and the role of defective CL in the clinical pathology of BTHS is under intense investigation. We used untargeted proteomics, shotgun lipidomics, gene expression analysis, and targeted metabolomics to identify novel areas of mitochondrial dysfunction in a new model of TAZ deficiency in HEK293 cells. Functional annotation analysis of proteomics data revealed abnormal regulation of mitochondrial respiratory chain complex I (CI), driven by the reduced abundance of 6 CI associated proteins in TAZ-deficient HEK293 cells: MT-ND3, NDUFA5, NDUFAB1, NDUFB2, NDUFB4, and NDUFAF1. This resulted in reduced assembly and function of CI in TAZ-deficient HEK293 cells as well as BTHS patient derived lymphoblast cells. We also identified increased abundance of PARL, a rhomboid protein involved in the regulation of mitophagy and apoptosis, and abnormal downstream processing of PGAM5, another mediator of mitochondrial quality control, in TAZ-deficient cells. Lastly, we modulated CL via the phospholipase inhibitor bromoenol lactone and the CL targeted SS-peptide, SS-31, and showed that each is able to remediate abnormalities in CI abundance as well as PGAM5 processing. Thus, mitochondrial respiratory chain CI and PARL/PGAM5 regulated mitochondrial quality control, both of whose functions localize to the inner mitochondrial membrane, are dysregulated due to TAZ deficiency and are partially remediated via modulation of CL.


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