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The mitochondrial enzyme methylenetetrahydrofolate dehydrogenase (MTHFD2) is involved in purine and thymidine synthesis via 1C metabolism. MTHFD2 is exclusively overexpressed in cancer cells but absent in most healthy adult human tissues. However, the two close homologs of MTHFD2 known as MTHFD1 and MTHFD2L are expressed in healthy adult human tissues and share a great structural resemblance to MTHFD2 with 54% and 89% sequence similarity, respectively. It is therefore notably challenging to find selective inhibitors of MTHFD2 due to the structural similarity, in particular protein binding site similarity with MTHFD1 and MTHFD2L. Tricyclic coumarin-based compounds (substrate site binders) and xanthine derivatives (allosteric site binders) are the only selective inhibitors of MTHFD2 reported till date. Nanomolar potent diaminopyrimidine-based inhibitors of MTHFD2 have been reported recently, however, they also demonstrate significant inhibitory activities against MTHFD1 and MTHFD2L. In this study, we have employed extensive computational modeling involving molecular docking and molecular dynamics simulations in order to investigate the binding modes and key interactions of diaminopyrimidine-based inhibitors at the substrate binding sites of MTHFD1, MTHFD2 and MTHFD2L, and compare with the tricyclic coumarin-based selective MTHFD2 inhibitor. The outcomes of our study provide significant insights into desirable and undesirable structural elements for rational structure-based design of new and selective inhibitors of MTHFD2 against cancer., (© 2024. The Author(s).)

Myostatin is a widely recognized inhibitory factor of skeletal muscle growth and significantly influences muscle development and metabolism. In mice, myostatin inhibition improves insulin sensitivity, increases glucose uptake by skeletal muscle, and reduces body fat. Furthermore, Mss51 is downregulated in response to myostatin inhibition, and its deletion appears to improve the metabolic state of skeletal muscle and reduce adipose tissue, which makes Mss51 a potential target for the treatment of obesity and type 2 diabetes. Here, we report a computationally predicted and validated three-dimensional structure of Mss51. Computational screening was used to identify naturally occurring compounds from the Herbal and Specs chemical database that might inhibit Mss51, based on binding affinities and physiochemical and ADMET properties. ZINC00338371, ZINC95099599 and ZINC08214878 were found to bind to Mss51 with high binding affinity and specificity. In addition, 100 ns molecular dynamics simulations were conducted to assess the stabilities of the interactions between the three compounds and Mss51. MD simulation demonstrated that all three compounds bind to the active pocket site of Mss51 stably and cause conformation changes. ZINC00338371 was found to bind most stably with binding free energy -229.022 ± 13.776 kJ/mol to Mss51, suggesting that it has therapeutic potential as a treatment option for obesity and type 2 diabetes.Communicated by Ramaswamy H. Sarma.

Background: Uncontrolled cell growth and proliferation, which originate from lung tissue often lead to lung carcinoma and are more likely due to smoking as well as inhaled environmental toxins. It is widely recognized that tumour cells evade the ability of natural programmed death (apoptosis) and facilitates tumour progression and metastasis. Therefore investigating and targeting the apoptosis pathway is being utilized as one of the best approaches for decades., Objective: This review describes the emergence of SMAC mimetic drugs as a treatment approach, its possibilities to synergize the response along with current limitations as well as future perspective therapy for lung cancer., Method: Articles were analysed using search engines and databases namely Pubmed and Scopus., Result: Under cancerous circumstances, the level of Inhibitor of Apoptosis Proteins (IAPs) gets elevated, which suppresses the pathway of programmed cell death, plus supports the proliferation of lung cancer. As it is a major apoptosis regulator, natural drugs that imitate the IAP antagonistic response like SMAC mimetic agents/Diablo have been identified to trigger cell death. SMAC i.e. second mitochondria activators of caspases is a molecule produced by mitochondria, stimulates apoptosis by neutralizing/inhibiting IAP and prevents its potential responsible for the activation of caspases. Various preclinical data have proven that these agents elicit the death of lung tumour cells. Apart from inducing apoptosis, these also sensitize the cancer cells toward other effective anticancer approaches like chemo, radio, or immunotherapies. There are many SMAC mimetic agents such as birinapant, BV-6, LCL161, and JP 1201, which have been identified for diagnosis as well as treatment purposes in lung cancer and are also under clinical investigation., Conclusion: SMAC mimetics acts in a restorative way in the prevention of lung cancer., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Ferroptosis is evolving as a highly promising approach to combat difficult-to-treat tumour entities including therapy-refractory and dedifferentiating cancers 1-3 . Recently, ferroptosis suppressor protein-1 (FSP1), along with extramitochondrial ubiquinone or exogenous vitamin K and NAD(P)H/H + as an electron donor, has been identified as the second ferroptosis-suppressing system, which efficiently prevents lipid peroxidation independently of the cyst(e)ine-glutathione (GSH)-glutathione peroxidase 4 (GPX4) axis 4-6 . To develop FSP1 inhibitors as next-generation therapeutic ferroptosis inducers, here we performed a small molecule library screen and identified the compound class of 3-phenylquinazolinones (represented by icFSP1) as potent FSP1 inhibitors. We show that icFSP1, unlike iFSP1, the first described on-target FSP1 inhibitor 5 , does not competitively inhibit FSP1 enzyme activity, but instead triggers subcellular relocalization of FSP1 from the membrane and FSP1 condensation before ferroptosis induction, in synergism with GPX4 inhibition. icFSP1-induced FSP1 condensates show droplet-like properties consistent with phase separation, an emerging and widespread mechanism to modulate biological activity 7 . N-terminal myristoylation, distinct amino acid residues and intrinsically disordered, low-complexity regions in FSP1 were identified to be essential for FSP1-dependent phase separation in cells and in vitro. We further demonstrate that icFSP1 impairs tumour growth and induces FSP1 condensates in tumours in vivo. Hence, our results suggest that icFSP1 exhibits a unique mechanism of action and synergizes with ferroptosis-inducing agents to potentiate the ferroptotic cell death response, thus providing a rationale for targeting FSP1-dependent phase separation as an efficient anti-cancer therapy., (© 2023. The Author(s).)

Glutamate excitotoxicity may contribute to retinal ganglion cell (RGC) degeneration in glaucoma and other optic neuropathies, leading to irreversible blindness. Growing evidence has linked impaired mitochondrial quality control with RGCs degeneration, while parkin, an E3 ubiquitin ligase, has proved to be protective and promotes mitophagy in RGCs against excitotoxicity. The purpose of this study was to explore whether a small molecule S3 could modulate parkin-mediated mitophagy and has therapeutic potential for RGCs. The results showed that as an inhibitor of deubiquitinase USP30, S3 protected cultured RGCs and improved mitochondrial health against NMDA-induced excitotoxicity. Administration of S3 promoted the parkin expression and its downstream mitophagy-related proteins in RGCs. An upregulated ubiquitination level of Mfn2 and protein level of OPA1 were also observed in S3-treated RGCs, while parkin knockdown resulted in a major loss of the protective effect of S3 on RGCs under excitotoxicity. These findings demonstrated that S3 promoted RGC survival mainly through enhancing parkin-mediated mitophagy against excitotoxicity. The neuroprotective value of S3 in glaucoma and other optic neuropathies deserves further investigation.

Endoplasmic Reticulum (ER) stress is the response that occurs after the dysfunction of ER and its structure. Activated UPR triggers a stress response using ER membrane proteins such as PERK, IRE-1, GRP78, ATF5 ve ATF6. Sirtuins are enzymes that carry out post-translational modifications such as deacetylation and ADP-ribosylation. In our previous study, we identified Calreticulin as a SIRT4-interacting protein via mass spectrometry. Calreticulin binds to misfolded proteins, prevents them from leaving ER, which results in the reduction of ER stress. In this study, we aimed to investigate the interaction between SIRT4 and Calreticulin during ER stress in glia cells (IHA-immortalized human astrocytes). To trigger ER stress in glia cells, we first optimized the dose and the duration of the Tunicamycin which is 2.5 μg/ml concentration for 16 h. SIRT4 gene was silenced with lentiviral particles using 4 MOI (Multiplicity of Infection). In SIRT4-silenced cells, when treated with 2.5 μg/ml Tunicamycin for 16 h, the increase in the expressions of ATF6, GRP78 and the ratio of spliced/unspliced XBP1 mRNA were reduced. This shows that silencing SIRT4 may decrease ER stress. SIRT4-Calreticulin interaction was shown both in control and ER-stress induced glia cells. Additionally, this interaction did not change with the ER stress. SIRT4 only ADP-ribosylates Calreticulin during ER stress. Normally, SIRT4 ADP-ribosylates and deactivates Calreticulin during ER stress condition. When SIRT4 is silenced, the ADP-ribosylation level of Calreticulin decreases resulting in the activation of Calreticulin and the reduction of ER stress. In summary, SIRT4 inhibitors may be investigated as protective agents or drug candidates in neurodegenerative diseases where ER stress mostly underlies as one of the molecular mechanisms., (Copyright © 2021. Published by Elsevier B.V.)

The accumulation of aggregated α-synuclein (α-syn) has been identified as the primary component of Lewy bodies that are the pathological hallmarks of Parkinson's disease (PD). Several preclinical studies have shown α-syn aggregation, and particularly the intermediates formed during the aggregation process to be toxic to cells. Current PD treatments only provide symptomatic relief, and α-syn serves as a promising target to develop a disease-modifying therapy for PD. Our previous studies have revealed that a small-molecular inhibitor for prolyl oligopeptidase (PREP), KYP-2047, increases α-syn degradation by accelerating macroautophagy (MA) leading to disease-modifying effects in preclinical PD models. However, α-syn is also degraded by chaperone-mediated autophagy (CMA). In the present study, we tested the effects of PREP inhibition or deletion on CMA activation and α-syn degradation. HEK-293 cells were transfected with α-syn and incubated with 1 & 10 µM KYP-2047 for 24 h. Both 1 & 10 µM KYP-2047 increased LAMP-2A levels, induced α-syn degradation and reduced the expression of Hsc70, suggesting that the PREP inhibitor prevented α-syn aggregation by activating the CMA pathway. Similarly, KYP-2047 increased the LAMP-2A immunoreactivity and reduced the Hsc70 levels in mouse primary cortical neurons. When LAMP-2A was silenced by a siRNA, KYP-2047 increased the LC3BII/LC3BI ratio and accelerated the clearance of α-syn. Additionally, KYP-2047 induced CMA effectively also when MA was blocked by bafilomycin A1. Based on our results, we suggest that PREP might function as a core network node in MA-CMA crosstalk, and PREP inhibition can reduce α-syn levels via both main autophagy systems., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The mitochondrial protein LonP1 is an ATP-dependent protease that mitigates cell stress and calibrates mitochondrial metabolism and energetics. Biallelic mutations in the LONP1 gene are known to cause a broad spectrum of diseases, and LonP1 dysregulation is also implicated in cancer and age-related disorders. Despite the importance of LonP1 in health and disease, specific inhibitors of this protease are unknown. Here, we demonstrate that 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and its -methyl and -imidazole derivatives reversibly inhibit LonP1 by a noncompetitive mechanism, blocking ATP-hydrolysis and thus proteolysis. By contrast, we found that CDDO-anhydride inhibits the LonP1 ATPase competitively. Docking of CDDO derivatives in the cryo-EM structure of LonP1 shows these compounds bind a hydrophobic pocket adjacent to the ATP-binding site. The binding site of CDDO derivatives was validated by amino acid substitutions that increased LonP1 inhibition and also by a pathogenic mutation that causes cerebral, ocular, dental, auricular and skeletal (CODAS) syndrome, which ablated inhibition. CDDO failed to inhibit the ATPase activity of the purified 26S proteasome, which like LonP1 belongs to the AAA + superfamily of ATPases Associated with diverse cellular Activities, suggesting that CDDO shows selectivity within this family of ATPases. Furthermore, we show that noncytotoxic concentrations of CDDO derivatives in cultured cells inhibited LonP1, but not the 26S proteasome. Taken together, these findings provide insights for future development of LonP1-specific inhibitors with chemotherapeutic potential., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Increasing evidence suggests that the pathogenesis of chronic obstructive pulmonary disease (COPD) is associated with FUN14 domain protein 1 (FUNDC1)-mediated mitophagy. Recently, studies have reported that puerarin has protective effects against excessive oxidative damage in cells. Therefore, we hypothesized that puerarin may be involved in COPD progression via regulating FUNDC1 mediated mitophagy. We found that the viability of cigarette smoke extract (CSE)-stimulated human bronchial epithelial cells (HBECs) was enhanced and apoptosis was reduced after treatment with different concentrations of puerarin. Puerarin reversed mitochondrial membrane potential (MMP) levels and ATP content, and decreased reactive oxygen species (ROS) content in CSE stimulated HBECs. Moreover, puerarin significantly inhibited apoptosis related proteins, as well as the expression of mitophagy related proteins. After inhibition of FUNDC1 phosphorylation by protein phosphatase inhibitor (PH0321), puerarin restored MMP level, decreased ROS content, promoted ATP synthesis, and downregulated autophagy related protein expression in HBECs. In addition, mitochondrial division inhibitor (Mdivi) inhibited the expression of autophagy related proteins and reduced apoptosis after blocking cell autophagy, which was the same as the inhibition of puerarin. Finally, puerarin activated the PI3K/Akt/mTOR signaling pathway to participate in COPD progression by up regulating the phosphorylation levels of PI3K, Akt and mTOR.

Diabetes is a prevalent disease worldwide that can result in several complications, including renal failure, blindness, and amputation. Diabetic foot ulcers, which have the characteristics of chronic wounds, are a devastating component of diabetes progression that can lead to lower extremity amputation. In this study, we set out to investigate the mechanisms involved in wound healing of diabetic foot ulcers. The expression of USP30 in skin tissues of patients with diabetic foot ulcers and HSF2 human skin fibroblasts treated with advanced glycation end (AGE) products was detected by qRT-PCR, and CCK-8, cell scratch and ELISA assay were used to detect cell viability, migration and levels of Col I, Col III, MMP-2, MMP-9, IL-1β and IL-18. The interaction between USP30 and NLRP3 was verified by co-immunoprecipitation and ubiquitination assays. The expression of USP30, NLRP3 and caspase-1 p20 was detected by Western blot. USP30 inhibitor MF-094 was used to treat diabetic rat model established by streptozotocin (STZ). We found that USP30, a deubiquitinase, was upregulated in skin tissues of patients with diabetic foot ulcers compared with normal skin tissues. In vitro, we found that treatment of HSF2 human skin fibroblasts with advanced glycation end (AGE) products, known to contribute to diabetic complications, resulted in suppressed viability and migration of HSF2 cells, as well as increased levels of USP30 mRNA and protein. Functionally, downregulation of USP30 via shRNA-mediated knockdown or treatment with the USP30 inhibitor MF-094, restored viability and migration of AGE-treated HSF2 cells. We identified the NLRP3 inflammasome as a critical target of USP30 in AGE-induced functions. Mechanistically, we demonstrate that USP30 activates the NLRP3 inflammasome by deubiquitinating NLRP3. Finally, we show that inhibition of USP30 via MF-094 treatment facilitated wound healing in diabetic rats and resulted in decreased protein levels of NLRP3 and its downstream target caspase-1 p20, thus establishing the physiological importance of the identified USP30-NLRP3 link. Together, our findings suggest a therapeutic potential for USP30 in diabetic foot ulcers., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In the liver, contact sites between the endoplasmic reticulum (ER) and mitochondria (named MAMs) may be crucial hubs for the regulation of lipid metabolism, thus contributing to the exacerbation or prevention of fatty liver. We hypothesized that tether proteins located at MAMs could play a key role in preventing triglyceride accumulation in hepatocytes and nonalcoholic fatty liver disease (NAFLD) occurrence. To test this, we explored the role of two key partners in building MAM integrity and functionality, the glucose-regulated protein 75 (Grp75) and mitofusin 2 (Mfn2), which liver contents are altered in obesity and NAFLD. Grp75 or Mfn2 expression was either silenced using siRNA or overexpressed with adenoviruses in Huh7 cells. Silencing of Grp75 and Mfn2 resulted in decreased ER-mitochondria interactions, mitochondrial network fusion state and mitochondrial oxidative capacity, while overexpression of the two proteins induced mirror impacts on these parameters. Furthermore, Grp75 or Mfn2 silencing decreased cellular cholesterol content and enhanced triglyceride secretion in ApoB100 lipoproteins, while their overexpression led to reverse effects. Cellular phosphatidylcholine/phosphatidylethanolamine ratio was decreased only upon overexpression of the proteins, potentially contributing to altered ApoB100 assembly and secretion. Despite the opposite differences, both silencing and overexpression of Grp75 or Mfn2 induced triglyceride storage, although a fatty acid challenge was required to express the alteration upon protein silencing. Among the mechanisms potentially involved in this phenotype, ER stress was closely associated with altered triglyceride metabolism after Grp75 or Mfn2 overexpression, while blunted mitochondrial FA oxidation capacity may be the main defect causing triglyceride accumulation upon Grp75 or Mfn2 silencing. Further studies are required to decipher the link between modulation of Grp75 or Mfn2 expression, change in MAM integrity and alteration of cholesterol content of the cell. In conclusion, Grp75 or Mfn2 silencing and overexpression in Huh7 cells contribute to altering MAM integrity and cholesterol storage in opposite directions, but all promote triglyceride accumulation through distinct cellular pathways. This study also highlights that besides Mfn2, Grp75 could play a central role in hepatic lipid and cholesterol metabolism in obesity and NAFLD., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background and Aims: The hepatic mitogen-activated protein kinase (MAPK) cascade leading to c-Jun N-terminal kinase (JNK) activation has been implicated in the pathogenesis of nonalcoholic fatty liver (NAFL)/NASH. In acute hepatotoxicity, we previously identified a pivotal role for mitochondrial SH3BP5 (SAB; SH3 homology associated BTK binding protein) as a target of JNK, which sustains its activation through promotion of reactive oxygen species production. Therefore, we assessed the role of hepatic SAB in experimental NASH and metabolic syndrome., Approach and Results: In mice fed high-fat, high-calorie, high-fructose (HFHC) diet, SAB expression progressively increased through a sustained JNK/activating transcription factor 2 (ATF2) activation loop. Inducible deletion of hepatic SAB markedly decreased sustained JNK activation and improved systemic energy expenditure at 8 weeks followed by decreased body fat at 16 weeks of HFHC diet. After 30 weeks, mice treated with control-antisense oligonucleotide (control-ASO) developed steatohepatitis and fibrosis, which was prevented by Sab-ASO treatment. Phosphorylated JNK (p-JNK) and phosphorylated ATF2 (p-ATF2) were markedly attenuated by Sab-ASO treatment. After 52 weeks of HFHC feeding, control N-acetylgalactosamine antisense oligonucleotide (GalNAc-Ctl-ASO) treated mice fed the HFHC diet exhibited progression of steatohepatitis and fibrosis, but GalNAc-Sab-ASO treatment from weeks 40 to 52 reversed these findings while decreasing hepatic SAB, p-ATF2, and p-JNK to chow-fed levels., Conclusions: Hepatic SAB expression increases in HFHC diet-fed mice. Deletion or knockdown of SAB inhibited sustained JNK activation and steatohepatitis, fibrosis, and systemic metabolic effects, suggesting that induction of hepatocyte Sab is an important driver of the interplay between the liver and the systemic metabolic consequences of overfeeding. In established NASH, hepatocyte-targeted GalNAc-Sab-ASO treatment reversed steatohepatitis and fibrosis., (© 2021 by the American Association for the Study of Liver Diseases.)

The deubiquitylase USP30 is an actionable target considered for treatment of conditions associated with defects in the PINK1-PRKN pathway leading to mitophagy. We provide a detailed cell biological characterization of a benzosulphonamide molecule, compound 39, that has previously been reported to inhibit USP30 in an in vitro enzymatic assay. The current compound offers increased selectivity over previously described inhibitors. It enhances mitophagy and generates a signature response for USP30 inhibition after mitochondrial depolarization. This includes enhancement of TOMM20 and SYNJ2BP ubiquitylation and phosphoubiquitin accumulation, alongside increased mitophagy. In dopaminergic neurons, generated from Parkinson disease patients carrying loss of function PRKN mutations, compound 39 could significantly restore mitophagy to a level approaching control values. USP30 is located on both mitochondria and peroxisomes and has also been linked to the PINK1-independent pexophagy pathway. Using a fluorescence reporter of pexophagy expressed in U2OS cells, we observe increased pexophagy upon application of compound 39 that recapitulates the previously described effect for USP30 depletion. This provides the first pharmacological intervention with a synthetic molecule to enhance peroxisome turnover., (© 2021 Rusilowicz-Jones et al.)

Mitochondrial, autophagic impairment, excitotoxicity, and also neuroinflammation are implicated in Alzheimer's disease (AD) pathophysiology. We postulated that inhibiting the mitochondrial pyruvate carrier-1 (MPC-1), which inhibits the activation of the mammalian target of rapamycin (mTOR), may ameliorate the neurodegeneration of hippocampal neurons in the rat AD model. To assess this, we used lapatinib ditosylate (LAP), an anti-cancer drug that inhibits MPC-1 through suppression of estrogen-related receptor-alpha (ERR-α), in D-galactose/ovariectomized rats. AD characteristics were developed in ovariectomized (OVX) rats following an 8-week injection of D-galactose (D-gal) (150 mg/kg, i.p.). The human epidermal growth factor receptor-2 (HER-2) inhibitor, LAP (100 mg/kg, p.o.) was daily administered for 3 weeks. LAP protected against D-gal/OVX-induced changes in cortical and hippocampal neurons along with improvement in learning and memory, as affirmed using Morris water maze (MWM) and novel object recognition (NOR) tests. Furthermore, LAP suppressed the hippocampal expression of Aβ1-42, p-tau, HER-2, p-mTOR, GluR-II, TNF-α, P38-MAPK, NOX-1, ERR-α, and MPC-1. Also, LAP treatment leads to activation of the pro-survival PI3K/Akt pathway. As an epilogue, targeting MPC-1 in the D-gal-induced AD in OVX rats resulted in the enhancement of autophagy, and suppression of neuroinflammation and excitotoxicity. Our work proves that alterations in metabolic signaling as a result of inhibiting MPC-1 were anti-inflammatory and neuroprotective in the AD model, revealing that HER-2, MPC-1, and ERR-α may be promising therapeutic targets for AD., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has become a global health emergency. Although new vaccines have been generated and being implicated, discovery and application of novel preventive and control measures are warranted. We aimed to identify compounds that may possess the potential to either block the entry of virus to host cells or attenuate its replication upon infection. Using host cell surface receptor expression (angiotensin-converting enzyme 2 (ACE2) and Transmembrane protease serine 2 (TMPRSS2)) analysis as an assay, we earlier screened several synthetic and natural compounds and identified candidates that showed ability to down-regulate their expression. Here, we report experimental and computational analyses of two small molecules, Mortaparib and MortaparibPlus that were initially identified as dual novel inhibitors of mortalin and PARP-1, for their activity against SARS-CoV-2. In silico analyses showed that MortaparibPlus, but not Mortaparib, stably binds into the catalytic pocket of TMPRSS2. In vitro analysis of control and treated cells revealed that MortaparibPlus caused down-regulation of ACE2 and TMPRSS2; Mortaparib did not show any effect. Furthermore, computational analysis on SARS-CoV-2 main protease (Mpro) that also predicted the inhibitory activity of MortaparibPlus. However, cell-based antiviral drug screening assay showed 30-60% viral inhibition in cells treated with non-toxic doses of either MortaparibPlus or Mortaparib. The data suggest that these two closely related compounds possess multimodal anti-COVID-19 activities. Whereas MortaparibPlus works through direct interactions/effects on the host cell surface receptors (ACE2 and TMPRSS2) and the virus protein (Mpro), Mortaparib involves independent mechanisms, elucidation of which warrants further studies., (© 2021 The Author(s).)

While the biochemistry of rhomboid proteases has been extensively studied since their discovery two decades ago, efforts to define the physiological roles of these enzymes are ongoing and would benefit from chemical probes that can be used to manipulate the functions of these proteins in their native settings. Here, we describe the use of activity-based protein profiling (ABPP) technology to conduct a targeted screen for small-molecule inhibitors of the mitochondrial rhomboid protease PARL, which plays a critical role in regulating mitophagy and cell death. We synthesized a series of succinimide-containing sulfonyl esters and sulfonamides and discovered that these compounds serve as inhibitors of PARL with the most potent sulfonamides having submicromolar affinity for the enzyme. A counterscreen against the bacterial rhomboid protease GlpG demonstrates that several of these compounds display selectivity for PARL over GlpG by as much as two orders of magnitude. Both the sulfonyl ester and sulfonamide scaffolds exhibit reversible binding and are able to engage PARL in mammalian cells. Collectively, our findings provide encouraging precedent for the development of PARL-selective inhibitors and establish N-[(arylsulfonyl)oxy]succinimides and N-arylsulfonylsuccinimides as new molecular scaffolds for inhibiting members of the rhomboid protease family., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

We have recently reported on the development and trypanocidal activity of a class of inhibitors of Trypanosome Alternative Oxidase (TAO) that are targeted to the mitochondrial matrix by coupling to lipophilic cations via C14 linkers to enable optimal interaction with the enzyme's active site. This strategy resulted in a much-enhanced anti-parasite effect, which we ascribed to the greater accumulation of the compound at the location of the target protein, i.e. the mitochondrion, but to date this localization has not been formally established. We therefore synthesized a series of fluorescent analogues to visualize accumulation and distribution within the cell. The fluorophore chosen, julolidine, has the remarkable extra feature of being able to function as a viscosity sensor and might thus additionally act as a probe of the cellular glycerol that is expected to be produced when TAO is inhibited. Two series of fluorescent inhibitor conjugates incorporating a cationic julolidine-based viscosity sensor were synthesized and their photophysical and biological properties were studied. These probes display a red emission, with a high signal-to-noise ratio (SNR), using both single- and two-photon excitation. Upon incubation with T. brucei and mammalian cells, the fluorescent inhibitors 1a and 2a were taken up selectively in the mitochondria as shown by live-cell imaging. Efficient partition of 1a in functional isolated (rat liver) mitochondria was estimated to 66 ± 20% of the total. The compounds inhibited recombinant TAO enzyme in the submicromolar (1a, 2c, 2d) to low nanomolar range (2a) and were effective against WT and multidrug-resistant trypanosome strains (B48, AQP1-3 KO) in the submicromolar range. Good selectivity (SI > 29) over mammalian HEK cells was observed. However, no viscosity-related shift could be detected, presumably because the glycerol was produced cytosolically, and released through aquaglyceroporins, whereas the probe was located, virtually exclusively, in the trypanosome's mitochondrion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Propofol (PRO) protects against hepatic ischemia/reperfusion (I/R) injury. Bnip3 is involved in the I/R-induced injury. This study investigated whether the effect of PRO on hepatic hypoxia/reoxygenation (H/R) injury was realized through regulating Bnip3. After establishing a hepatic ischemia reperfusion (I/R ) injury model in mice, the serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined by an automatic biochemical analyzer. The histopathology and apoptosis of liver tissues were detected by hematoxylin-eosin and TUNEL staining. After the H/R liver cells were cultured and treated with PRO, the viability, apoptosis, reactive oxygen species (ROS) production, and the levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), TNF-α, and IL-6 were detected by MTT, flow cytometry, colorimetry, and ELISA. The expressions of Bnip3 and apoptosis-related factors in I/R mouse liver tissues and H/R cells were determined by immunohistochemical assay, immunofluorescence, Western blot, or RT-qPCR. PRO ameliorated the abnormal histopathology, reduced cell apoptosis and the levels of AST, ALT, Bnip3, Cleaved Caspase-3, and Bax, but upregulated the Bcl-2 level in the liver tissues of I/R mice. In H/R liver cells, PRO promoted the cell viability, downregulated the levels of LDH, MDA, TNF-α, IL-6, and reduced ROS production. Moreover, PRO promoted the downregulated expressions of cytosolic Bnip3, total Bni3p, Cleaved Caspase-3, and Bax and upregulated the Bcl-2 level. siBnip3 reversed the effect of H/R on the liver cells, and its overexpression also reversed the effect of PRO on H/R-induced liver cells. PRO protects against hepatic I/R injury via inhibiting Bnip3., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.)

Candidemia caused by Candida spp. is a serious threat in hospital settings being a major cause of acquired infection and death and a possible contributor to Covid-19 mortality. Candidemia incidence has been rising worldwide following increases in fungicide-resistant pathogens highlighting the need for more effective antifungal agents with novel modes of action. The membrane-bound enzyme alternative oxidase (AOX) promotes fungicide resistance and is absent in humans making it a desirable therapeutic target. However, the lipophilic nature of the AOX substrate (ubiquinol-10) has hindered its kinetic characterisation in physiologically-relevant conditions. Here, we present the purification and expression of recombinant AOXs from C. albicans and C. auris in a self-assembled proteoliposome (PL) system. Kinetic parameters (K m and V max ) with respect to ubiquinol-10 have been determined. The PL system has also been employed in dose-response assays with novel AOX inhibitors. Such information is critical for the future development of novel treatments for Candidemia., (© 2021. The Author(s).)

Mitochondrial biogenesis relies on different protein import machinery, as mitochondrial proteins are imported from the cytosol. The mitochondrial intermembrane space assembly (MIA) pathway consists of GFER/ALR and CHCHD4/Mia40, responsible for importing proteins and their oxidative folding inside the mitochondria. The MIA pathway plays an essential role in complex IV (COX IV) biogenesis via importing copper chaperone COX17, associated with the respiratory chain. BPA, an environmental toxicant, found in consumable plastics, causes neurotoxicity via impairment in mitochondrial dynamics, neurogenesis, and cognitive functions. We studied the levels of key regulatory proteins of mitochondrial import pathways and mitochondrial biogenesis after BPA exposure in the rat hippocampus. BPA caused a significant reduction in the levels of mitochondrial biogenesis proteins (PGC1α, and TFAM) and mitochondrial import protein (GFER). Immunohistochemical analysis showed reduced co-localization of NeuN with GFER, PGC-1α, and TFAM suggesting impaired mitochondrial biogenesis and protein import. BPA exposure resulted in damaged mitochondria with distorted cristae in neurons and caused a significant reduction in GFER localization inside IMS as depicted by immunogold electron microscopy. The reduced levels of GFER resulted in defective COX17 import. The translocation of cytochrome c into the cytosol and increased cleaved caspase-3 levels triggered apoptosis due to BPA toxicity. Overall, our study implicates GFER as a potential target for impaired mitochondrial protein machinery, biogenesis, and apoptosis against BPA neurotoxicity in the rat hippocampus., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Adiponectin is an adipocytokine that plays a key regulatory role in glucose and lipid metabolism in obesity. The prevalence of obesity has led to an increase in the incidence of obesity-related glomerulopathy (ORG). This study aimed to identify the protective role of adiponectin in ORG., Methods: Small-interfering RNA (siRNA) against the gene encoding adiponectin was transfected into podocytes. The oxidative stress level was determined using a fluorometric assay. Apoptosis was analyzed by flow cytometry. The expressions of podocyte markers and pyrin domain containing protein 3 (NLRP3) inflammasome-related proteins were measured by qRT-PCR, immunohistochemistry, and Western blot., Results: Podocytes treated with palmitic acid (PA) showed downregulated expressions of podocyte markers, increased apoptosis, upregulated levels of NLRP3 inflammasome-related proteins, increased production of inflammatory cytokines (IL-18 and IL-1β), and induced activation of NF-κB as compared to the vehicle-treated controls. Decreased adiponectin expression was observed in the serum samples from high fat diet (HFD)-fed mice. Decreased podocin expression and upregulated NLRP3 expression were observed in the kidney samples from high fat diet (HFD)-fed mice. Treatment with adiponectin or the NLRP3 inflammasome inhibitor, MCC950, protected cultured podocytes against podocyte apoptosis and inflammation. Treatment with adiponectin protected mouse kidney tissues against decreased podocin expression and upregulated NLRP3 expression. The knockout of adiponectin gene by siRNA increased ROS production, resulting in the activation of NLRP3 inflammasome and the phosphorylation of NF-κB in podocytes. Pyrrolidine dithiocarbamate, an NF-κB inhibitor, prevented adiponectin from ameliorating FFA-induced podocyte injury and NLRP3 activation., Conclusions: Our study showed that adiponectin ameliorated PA-induced podocyte injury in vitro and HFD-induced injury in vivo via inhibiting the ROS/NF-κB/NLRP3 pathway. These data suggest the potential use of adiponectin for the prevention and treatment of ORG.

The human mitochondrial AAA+ protein LONP1 is a critical quality control protease involved in regulating diverse aspects of mitochondrial biology including proteostasis, electron transport chain activity, and mitochondrial transcription. As such, genetic or aging-associated imbalances in LONP1 activity are implicated in pathologic mitochondrial dysfunction associated with numerous human diseases. Despite this importance, the molecular basis for LONP1-dependent proteolytic activity remains poorly defined. Here, we solved cryo-electron microscopy structures of human LONP1 to reveal the underlying molecular mechanisms governing substrate proteolysis. We show that, like bacterial Lon, human LONP1 adopts both an open and closed spiral staircase orientation dictated by the presence of substrate and nucleotide. Unlike bacterial Lon, human LONP1 contains a second spiral staircase within its ATPase domain that engages substrate as it is translocated toward the proteolytic chamber. Intriguingly, and in contrast to its bacterial ortholog, substrate binding within the central ATPase channel of LONP1 alone is insufficient to induce the activated conformation of the protease domains. To successfully induce the active protease conformation in substrate-bound LONP1, substrate binding within the protease active site is necessary, which we demonstrate by adding bortezomib, a peptidomimetic active site inhibitor of LONP1. These results suggest LONP1 can decouple ATPase and protease activities depending on whether AAA+ or both AAA+ and protease domains bind substrate. Importantly, our structures provide a molecular framework to define the critical importance of LONP1 in regulating mitochondrial proteostasis in health and disease.

The human mitochondrial ClpXP protease complex (HsClpXP) has recently attracted major attention as a target for novel anti-cancer therapies. Despite its important role in disease progression, the cellular role of HsClpXP is poorly characterized and only few small molecule inhibitors have been reported. Herein, we screened previously established S. aureus ClpXP inhibitors against the related human protease complex and identified potent small molecules against human ClpXP. The hit compounds showed anti-cancer activity in a panoply of leukemia, liver and breast cancer cell lines. We found that the bacterial ClpXP inhibitor 334 impairs the electron transport chain (ETC), enhances the production of mitochondrial reactive oxygen species (mtROS) and thereby promotes protein carbonylation, aberrant proteostasis and apoptosis. In addition, 334 induces cell death in re-isolated patient-derived xenograft (PDX) leukemia cells, potentiates the effect of DNA-damaging cytostatics and re-sensitizes resistant cancers to chemotherapy in non-apoptotic doses.

Anti- EFG1 2'- O MethylRNA is an antisense oligonucleotide (ASO) that has the ability to recognize and block the EFG1 gene and to control Candida albicans filamentation. However, it is important to protect the anti- EFG1 2'- O MethylRNA ASO from the environmental human body conditions and to ensure that they will be delivered to their site of action, and polyplex microparticles (MPs) represent a class of vehicles to ASO cargo with these functionalities. Thus, the goal of this work was to develop polyplexes based on porous poly(γ-butyrolactam) (PA4) or poly(ε-caprolactam) (PA6) MPs for the anti- EFG1 2'- O MethylRNA ASO cargo and delivery. Two types of polyplexes were prepared with payloads of anti- EFG1 2'- O MethylRNA molecules, either entrapped or immobilized on prefabricated polyamide MPs. Our data confirm that PA4 and PA6 polyplex MPs can be feasible carriers for anti- EFG1 2'- O MethylRNA ASO molecules, using either the entrapment or immobilization strategies, whereby the released ASO maintains its activity against C. albicans cells.

LONP1 is an AAA+ protease that maintains mitochondrial homeostasis by removing damaged or misfolded proteins. Elevated activity and expression of LONP1 promotes cancer cell proliferation and resistance to apoptosis-inducing reagents. Despite the importance of LONP1 in human biology and disease, very few LONP1 inhibitors have been described in the literature. Herein, we report the development of selective boronic acid-based LONP1 inhibitors using structure-based drug design as well as the first structures of human LONP1 bound to various inhibitors. Our efforts led to several nanomolar LONP1 inhibitors with little to no activity against the 20S proteasome that serve as tool compounds to investigate LONP1 biology.

Lithium hexafluorophosphate (LiPF 6 ) is one of the leading electrolytes in lithium-ion batteries, and its usage has increased tremendously in the past few years. Little is known, however, about its potential environmental and biological impacts. In order to improve our understanding of the cytotoxicity of LiPF 6 and the specific cellular response mechanisms to it, we performed a genome-wide screen using a yeast ( Saccharomyces cerevisiae ) deletion mutant collection and identified 75 gene deletion mutants that showed LiPF 6 sensitivity. Among these, genes associated with mitochondria showed the most enrichment. We also found that LiPF 6 is more toxic to yeast than lithium chloride (LiCl) or sodium hexafluorophosphate (NaPF 6 ). Physiological analysis showed that a high concentration of LiPF 6 caused mitochondrial damage, reactive oxygen species (ROS) accumulation, and ATP content changes. Compared with the results of previous genome-wide screening for LiCl-sensitive mutants, we found that oxidative phosphorylation-related mutants were specifically hypersensitive to LiPF 6 . In these deletion mutants, LiPF 6 treatment resulted in higher ROS production and reduced ATP levels, suggesting that oxidative phosphorylation-related genes were important for counteracting LiPF 6 -induced toxicity. Taken together, our results identified genes specifically involved in LiPF 6 -modulated toxicity, and demonstrated that oxidative stress and ATP imbalance maybe the driving factors in governing LiPF 6 -induced toxicity.

Calcium-dependent proteolytic calpains are implicated in a variety of physiological processes, as well as pathologies associated with calcium overload. However, the mechanism by which calpain is activated remains elusive since intracellular calcium levels under physiological conditions do not reach the high concentration range required to trigger calpain activation. From a candidate screening using the abundance of the calpain target glutamate receptor GluRIIA at the Drosophila neuromuscular junction as a readout, we uncovered that calpain activity was inhibited upon knockdown of Ttm50, a subunit of the Tim23 complex known to be involved in the import of proteins across the mitochondrial inner membrane. Unexpectedly, Ttm50 and calpain are co-localized at calcium stores Golgi and endoplasmic reticulum (ER), and Ttm50 interacts with calpain via its C-terminal domain. This interaction is required for calpain localization at Golgi/ER, and increases calcium sensitivity of calpain by roughly an order of magnitude. Our findings reveal the regulation of calpain activation by Ttm50, and shed new light on calpain-associated pathologies.

Lipids play essential roles in numerous cellular processes, including membrane remodeling, signal transduction, the modulation of hormone activity, and steroidogenesis. We chose steroidogenic MA-10 mouse tumor Leydig cells to investigate subcellular lipid localization during steroidogenesis. Electron microscopy showed that cAMP stimulation increased associations between the plasma membrane (PM) and the endoplasmic reticulum (ER) and between the ER and mitochondria. cAMP stimulation also increased the movement of cholesterol from the PM compared to untreated cells, which was partially inhibited when ATPase family AAA-domain containing protein 3 A (ATAD3A), which functions in ER and mitochondria interactions, was knocked down. Mitochondria, ER, cytoplasm, PM, PM-associated membranes (PAMs), and mitochondria-associated membranes (MAMs) were isolated from control and hormone-stimulated cells. Lipidomic analyses revealed that each isolated compartment had a unique lipid composition, and the induction of steroidogenesis caused the significant remodeling of its lipidome. cAMP-induced changes in lipid composition included an increase in phosphatidylserine and cardiolipin levels in PAM and PM compartments, respectively; an increase in phosphatidylinositol in the ER, mitochondria, and MAMs; and a reorganization of phosphatidic acid, cholesterol ester, ceramide, and phosphatidylethanolamine. Abundant lipids, such as phosphatidylcholine, were not affected by hormone treatment. Our data suggested that PM-ER-mitochondria tethering may be involved in lipid trafficking between organelles and indicated that hormone-induced acute steroid production involves extensive organelle remodeling.

Necrostatin-1 (Nec-1) has previously been shown to protect neurons from death in traumatic and ischemic brain injuries. This study tests the hypothesis that Nec-1 protects neural cells against traumatic and ischemic brain injuries through inhibition of the Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3). We have used biochemical and morphological techniques to determine the inhibition of Nec-1 on BNIP3-induced cell death and to identify its mechanism of action in in vivo and in vitro models of neurodegeneration. Here we show that Nec-1 significantly increased neuronal viability following prolonged exposure to hypoxia in vitro, and attenuated myelin damage and neuronal death in traumatic brain injury and cerebral ischemia in Sprague-Dawley rats. Nec-1 alleviated traumatic brain injury-induced up-regulation of BNIP3 in mature oligodendrocytes. In isolated mitochondria, Nec-1 prevented BNIP3 from integrating into mitochondria by modifying its binding sites on the mitochondria. Consequently, Nec-1 robustly inhibited BNIP3-induced collapse of mitochondrial membrane potential and reduced the opening probability of mitochondrial permeability transition pores. Nec-1 also preserved mitochondrial ultrastructure and suppressed BNIP3-induced nuclear translocation of apoptosis-inducing factor. In conclusion, Nec-1 protects neurons and oligodendrocytes against traumatic and ischemic brain injuries by targeting the BNIP3-induced cell death pathway, and is a novel inhibitor for BNIP3. Cover Image for this issue: https://doi.org/10.1111/jnc.15056., (© 2020 International Society for Neurochemistry.)

The alternative oxidase (AOX) is a monotopic diiron carboxylate protein that catalyses the oxidation of ubiquinol and the reduction of oxygen to water. Although a number of AOX inhibitors have been discovered, little is still known about the ligand-protein interaction and essential chemical characteristics of compounds required for a potent inhibition. Furthermore, owing to the rapidly growing resistance to existing inhibitors, new compounds with improved potency and pharmacokinetic properties are urgently required. In this study we used two computational approaches, ligand-protein docking and Quantitative Structure-Activity Relationships (QSAR) to investigate binding of AOX inhibitors to the enzyme and the molecular characteristics required for inhibition. Docking studies followed by protein-ligand interaction fingerprint (PLIF) analysis using the AOX enzyme and the mutated analogues revealed the importance of the residues Leu 122, Arg 118 and Thr 219 within the hydrophobic cavity. QSAR analysis, using stepwise regression analysis with experimentally obtained IC 50 values as the response variable, resulted in a multiple regression model with a good prediction accuracy. The model highlighted the importance of the presence of hydrogen bonding acceptor groups on specific positions of the aromatic ring of ascofuranone derivatives, acidity of the compounds, and a large linker group on the compounds on the inhibitory effect of AOX.

Tuberculosis (TB), caused by the infection of Mycobacterium tuberculosis (MTB), is one of the leading causes of death worldwide, especially in children. However, the mechanisms by which MTB infects its cellular host, activates an immune response, and triggers inflammation remain unknown. Mitochondria play important roles in the initiation and activation of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome, where mitochondria-associated endoplasmic reticulum membranes (MAMs) may serve as the platform for inflammasome assembly and activation. Additionally, mitofusin 2 (MFN2) is implicated in the formation of MAMs, but, the roles of mitochondria and MFN2 in MTB infection have not been elucidated. Using mircroarry profiling of TB patients and in vitro MTB stimulation of macrophages, we observed an up-regulation of MFN2 in the peripheral blood mononuclear cells of active TB patients. Furthermore, we found that MTB stimulation by MTB-specific antigen ESAT-6 or lysate of MTB promoted MFN2 interaction with NLRP3 inflammasomes, resulting in the assembly and activation of the inflammasome and, subsequently, IL-1β secretion. These findings suggest that MFN2 and mitochondria play important role in the pathogen-host interaction during MTB infection., (Copyright © 2020 © 2020 Xu et al. Published by Elsevier Inc. All rights reserved.)

Objective: To assess the effects of 17β-estradiol (E2) on proliferation, apoptosis, and protein expressions of fibroblasts at different concentrations and time intervals to reveal the mechanism of E2 in the treatment of pelvic organ prolapse (POP)., Study Design: The uterosacral ligament fibroblasts were collected from seven POP patients for primary culture of fibroblasts. The culture media containing 0, 10 -6 , 10 -7 , 10 -8 , and 10 -9 mol/L E2 were used for 24, 48, 72, and 96 h., Main Outcome Measures: The cells were collected for cell counting kit-8 (CCK-8), apoptosis, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and Western blotting assays., Results: Compared with the control group, in the values of fibroblasts cultured in 10 -8 mol/L E2 for 72 h, the proliferation, mRNA and protein expression of Mitofusin-2 (Mfn2) separately increased (P < 0.05), decreased (P<0.001) and decreased (P<0.001). However, the expression level of procollagen 1A1/1A2/3A1 and cyclinD1 markedly increased (P<0.001, all), which was consistent with the results of protein level. What's more, the expression of estrogen receptor α(ERα), estrogen receptor β(ERβ) and G protein-coupled receptor 30(GPR30) were significantly increased in 10 -8 mol/L E2 group., Conclusions: E2 can inhibit the progress of POP by inhibiting the expression level of Mfn2, as well as promoting expression of procollagens and proliferation of fibroblasts. This effect is time- and concentration-dependent. Only when the estrogen concentration reaches 10 -8 mol/L, the therapeutic effect is the greatest after 72 h., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Wang, He, Xiao and Lu.)

The alternative oxidase (AOX) catalyzes the transfer of electrons from ubiquinol to oxygen without the translocation of protons across the inner mitochondrial membrane. This enzyme has been proposed to participate in the regulation of cell growth, sporulation, yeast-mycelium transition, resistance to reactive oxygen species, infection, and production of secondary metabolites. Two approaches have been used to evaluate AOX function: incubation of cells for long periods of time with AOX inhibitors or deletion of AOX gene. However, AOX inhibitors might have different targets. To test non-specific effects of n-octyl gallate (nOg) and salicylhydroxamic acid (SHAM) on fungal physiology we measured the growth and respiratory capacity of two fungal strains lacking (Ustilago maydis-Δaox and Saccharomyces cerevisiae) and three species containing the AOX gene (U. maydis WT, Debaryomyces hansenii, and Aspergillus nidulans). For U. maydis, a strong inhibition of growth and respiratory capacity by SHAM was observed, regardless of the presence of AOX. Similarly, A. nidulans mycelial growth was inhibited by low concentrations of nOg independently of AOX expression. In contrast, these inhibitors had no effect or had a minor effect on S. cerevisiae and D. hansenii growth. These results show that nOg and SHAM have AOX independent effects which vary in different microorganisms, indicating that studies based on long-term incubation of cells with these inhibitors should be considered as inconclusive., (Copyright © 2020 Elsevier Inc. All rights reserved.)

NAD(P) + transhydrogenase (NNT) is located in the inner mitochondrial membrane and catalyzes a reversible hydride transfer between NAD(H) and NADP(H) that is coupled to proton translocation between the intermembrane space and mitochondrial matrix. NNT activity has an essential role in maintaining the NADPH supply for antioxidant defense and biosynthetic pathways. In the present report, we evaluated the effects of chemical compounds used as inhibitors of NNT over the last five decades, namely, 4-chloro-7-nitrobenzofurazan (NBD-Cl), N,N'-dicyclohexylcarbodiimide (DCC), palmitoyl-CoA, palmitoyl-l-carnitine, and rhein, on NNT activity and mitochondrial respiratory function. Concentrations of these compounds that partially inhibited the forward and reverse NNT reactions in detergent-solubilized mouse liver mitochondria significantly impaired mitochondrial respiratory function, as estimated by ADP-stimulated and nonphosphorylating respiration. Among the tested compounds, NBD-Cl showed the best relationship between NNT inhibition and low impact on respiratory function. Despite this, NBD-Cl concentrations that partially inhibited NNT activity impaired mitochondrial respiratory function and significantly decreased the viability of cultured Nnt -/- mouse astrocytes. We conclude that even though the tested compounds indeed presented inhibitory effects on NNT activity, at effective concentrations, they cause important undesirable effects on mitochondrial respiratory function and cell viability., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The alternative oxidase (AOX) is a monotopic di‑iron carboxylate protein which acts as a terminal respiratory chain oxidase in a variety of plants, fungi and protists. Of particular importance is the finding that both emerging infectious diseases caused by human and plant fungal pathogens, the majority of which are multi-drug resistant, appear to be dependent upon AOX activity for survival. Since AOX is absent in mammalian cells, AOX is considered a viable therapeutic target for the design of specific fungicidal and anti-parasitic drugs. In this work, we have mutated conserved residues within the hydrophobic channel (R96, D100, R118, L122, L212, E215 and T219), which crystallography has indicated leads to the active site. Our data shows that all mutations result in a drastic reduction in V max and catalytic efficiency whilst some also affected the K m for quinol and oxygen. The extent to which mutation effects inhibitor sensitivity was also investigated, with mutation of R118 and T219 leading to a complete loss of inhibitor potency. However, only a slight reduction in IC 50 values was observed when R96 was mutated, implying that this residue is less important in inhibitor binding. In silico modelling has been used to provide insight into the reason for such changes, which we suggest is due to disruptions in the proton transfer network, resulting in a reduction in overall reaction kinetics. We discuss our results in terms of the structural features of the ubiquinol binding site and consider the implications of such findings on the nature of the catalytic cycle. SIGNIFICANCE: The alternative oxidase is a ubiquinol oxidoreductase enzyme that catalyses the oxidation of ubiquinol and the reduction of oxygen to water. It is widely distributed amongst the plant, fungal and parasitic kingdoms and plays a central role in metabolism through facilitating the turnover of the TCA cycle whilst reducing ROS production., Competing Interests: Declaration of competing interest L.Y., B.M. and A.L.M. hold patents and financial interests in the development of phytopathogenic fungicides., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mitochondrial dysfunction has a recognised role in the progression of Alzheimer's disease (AD) pathophysiology. Cerebral perfusion becomes increasingly inefficient throughout ageing, leading to unbalanced mitochondrial dynamics. This effect is exaggerated by amyloid β (Aβ) and phosphorylated tau, two hallmark proteins of AD pathology. A neuroprotective role for the adipose-derived hormone, leptin, has been demonstrated in neuronal cells. However, its effects with relation to mitochondrial function in AD remain largely unknown. To address this question, we have used both a glucose-serum-deprived (CGSD) model of ischaemic stroke in SH-SY5Y cells and a Aβ 1-42 -treatment model of AD in differentiated hippocampal cells. Using a combination of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and MitoRed staining techniques, we show that leptin prevents depolarisation of the mitochondrial membrane and excessive mitochondrial fragmentation induced by both CGSD and Aβ 1-42 . Thereafter, we used ELISAs and a number of activity assays to reveal the biochemical underpinnings of these processes. Specifically, leptin was seen to inhibit up-regulation of the mitochondrial fission protein Fis1 and down-regulation of the mitochondrial fusion protein, Mfn2. Furthermore, leptin was seen to up-regulate the expression and activity of the antioxidant enzyme, monoamine oxidase B. Herein we provide the first demonstration that leptin is sufficient to protect against aberrant mitochondrial dynamics and resulting loss of function induced by both CGSD and Aβ 1-42 . We conclude that the established neuroprotective actions of leptin may be facilitated through regulation of mitochondrial dynamics., (© 2020 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Mitochondrial transcription factor A (TFAM), which is required for mitochondrial DNA (mtDNA) transcription, has been linked to metabolic changes that contribute to tumorigenesis and chemoresistance. In this work, we investigated the expression pattern and role of TFAM in hepatocellular carcinoma (HCC). TFAM expression level is similar in 18 out of 20 paired normal liver and HCC tissues with only 2 HCC tissues showing 1.8-fold increase in TFAM. Similar phenomenon was observed in HCC cell lines compared to normal liver lines. Interestingly, TFAM expression is upregulated in resistant HCC cells regardless of the differential TFAM expression level in their parental lines and mechanism of resistance. TFAM depletion led to inhibition of growth and survival but not migration, and sensitization to doxorubicin and sorafenib treatment, through AMPK activation, reduction of nucleoside triphosphates and mitochondrial respiration in HCC cells. In addition, we demonstrated that resistant HCC cell lines were more sensitive to TFAM inhibition than parental lines, and this might be due to the increased mitochondrial biogenesis in resistant HCC cell lines. Our work reveals the preferential role of TFAM in HCC cell response to standard of care drugs, which suggests a potential sensitizing therapeutic target for HCC treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Recanalization therapy by intravenous thrombolysis or endovascular therapy is critical for the treatment of cerebral infarction. However, the recanalization treatment will also exacerbate acute brain injury and even severely threatens human life due to the reperfusion injury. So far, the underlying mechanisms for cerebral ischaemia-reperfusion injury are poorly understood and effective therapeutic interventions are yet to be discovered. Therefore, in the research, we subjected SK-N-BE(2) cells to oxygen-glucose deprivation/reperfusion (OGDR) insult and performed a pooled genome-wide CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) knockout screen to discover new potential therapeutic targets for cerebral ischaemia-reperfusion injury. We used Metascape to identify candidate genes which might involve in OGDR resistance. We found that the genes contributed to OGDR resistance were primarily involved in neutrophil degranulation, mitochondrial translation, and regulation of cysteine-type endopeptidase activity involved in apoptotic process and response to oxidative stress. We then knocked down some of the identified candidate genes individually. We demonstrated that MRPL19, MRPL32, MRPL52 and MRPL51 inhibition increased cell viability and attenuated OGDR-induced apoptosis. We also demonstrated that OGDR down-regulated the expression of MRPL19 and MRPL51 protein. Taken together, our data suggest that genome-scale screening with Cas9 is a reliable tool to analyse the cellular systems that respond to OGDR injury. MRPL19 and MRPL51 contribute to OGDR resistance and are supposed to be promising targets for the treatment of cerebral ischaemia-reperfusion damage., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: Esophageal squamous cell carcinoma (ESCC) is one of the most lethal forms of adult cancer with poor prognosis. Substantial evidence indicates that reactive oxygen species (ROS) are important modulators of aggressive cancer behavior. However, the mechanism by which ESCC cells integrate redox signals to modulate carcinoma progression remains elusive., Methods: The expression of interferon alpha inducible protein 6 (IFI6) in clinical ESCC tissues and cell lines was detected by RT-PCR and Western blotting. The correlation between IFI6 expression levels and aggressive ESCC disease stage was examined by immunohistochemistry. Bioinformatic analysis was conducted to explore the potential function of IFI6 in ESCC. ESCC cell lines stably depleted of IFI6 and ectopically expressing IFI6 were established using lentiviruses expressing shRNAs and an IFI6 expression plasmid, respectively. The effects of IFI6 on ESCC cells were determined by cell-based analyses, including EdU assay, apoptotic assay, cellular and mitochondria-specific ROS detection, seahorse extracellular flux, and mitochondrial calcium flux assays. Blue native-polyacrylamide gel electrophoresis was used to determine mitochondrial supercomplex assembly. Transcriptional activation of NADPH oxidase 4 (NOX4) via ATF3 was confirmed by dual luciferase assay. In vivo tumor growth was determined in mouse xenograft models., Results: We find that the expression of IFI6, an IFN-stimulated gene localized in the inner mitochondrial membrane, is markedly elevated in ESCC patients and a panel of ESCC cell lines. High IFI6 expression correlates with aggressive disease phenotype and poor prognosis in ESCC patients. IFI6 depletion suppresses proliferation and induces apoptosis by increasing ROS accumulation. Mechanistically, IFI6 ablation induces mitochondrial calcium overload by activating mitochondrial Ca 2+ uniporter and subsequently ROS production. Following IFI6 ablation, mitochondrial ROS accumulation is also induced by mitochondrial supercomplex assembly suppression and oxidative phosphorylation dysfunction, while IFI6 overexpression produces the opposite effects. Furthermore, energy starvation induced by IFI6 inhibition drives endoplasmic reticulum stress through disrupting endoplasmic reticulum calcium uptake, which upregulates NOX4-derived ROS production in an ATF3-dependent manner. Finally, the results in xenograft models of ESCC further corroborate the in vitro findings., Conclusion: Our study unveils a novel redox homeostasis signaling pathway that regulates ESCC pathobiology and identifies IFI6 as a potential druggable target in ESCC.

Nonalcoholic fatty liver disease (NAFLD) is considered the next major health epidemic with an estimated 25% worldwide prevalence. No drugs have yet been approved and NAFLD remains a major unmet need. Here, we identify MCJ (Methylation-Controlled J protein) as a target for non-alcoholic steatohepatitis (NASH), an advanced phase of NAFLD. MCJ is an endogenous negative regulator of the respiratory chain Complex I that acts to restrain mitochondrial respiration. We show that therapeutic targeting of MCJ in the liver with nanoparticle- and GalNAc-formulated siRNA efficiently reduces liver lipid accumulation and fibrosis in multiple NASH mouse models. Decreasing MCJ expression enhances the capacity of hepatocytes to mediate β-oxidation of fatty acids and minimizes lipid accumulation, which results in reduced hepatocyte damage and fibrosis. Moreover, MCJ levels in the liver of NAFLD patients are elevated relative to healthy subjects. Thus, inhibition of MCJ emerges as an alternative approach to treat NAFLD.

N-aryl-N'-ureido-O-sulfamates (AUSs) were recently reported as new class of Carbonic Anhydrase Inhibitors (CAIs), endowed of high potency and selectivity against hCA VII and XII. In this work, we extended the investigational study on this new class of CAIs profiling them against the mitochondrial CA isoforms hCA VA and VB. The results revealed a very interesting selectivity profile, with dramatic selectivity against hCA VB over the VA isoform observed for all the analyzed compounds 2-22. On derivative 15, selected as one of the most promising among the series, molecular modeling studies were conducted, highlighting the importance of small residue substitution between the two isoforms in substantially changing the tail orientation and interaction with the enzymes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Inflammation is critical in the progression from benign hepatic lipidosis to pathological hepatic steatosis. The objective of this study was to examine the potential role of the outer mitochondrial membrane protein mitofusin 2 (MFN2) in the etiology of hepatic steatosis in dairy cows during early lactation. Using a nested case-control design, we compared blood and liver samples from 10 healthy cows and 10 age-matched cows with moderate fatty liver. Cows with moderate fatty liver had high liver triacylglycerols, elevated plasma concentrations of free fatty acids (FFA) and β-hydroxybutyrate, and low concentrations of glucose. Cows with moderate fatty liver had overactivated inflammatory pathways in the liver, as indicated by increased abundance of phosphorylated nuclear factor κB (NF-κB) p65, NLR family pyrin domain containing 3 (NLRP3) and caspase-1 inflammasome protein, and elevated plasma concentrations and hepatic mRNA abundance of their molecular targets IL-1β, IL-6, and tumor necrosis factor α (TNF-α). In the cell culture model, we were able to replicate our findings in cows with moderate fatty liver: 1.2 mM exogenous FFA decreased the abundance of MFN2 and upregulated phosphorylation levels of the inhibitor of NF-κB (IκB) α and NF-κB p65, the IκB kinase β activity, and the abundance of NLRP3, caspase-1, IL-1β, IL-6, and TNF-α. Whereas MFN2 knockdown potentiated the FFA-induced activation of these inflammatory pathways, overexpression of MFN2 attenuated the detrimental effect of excess exogenous FFA by improving mitochondrial function and decreasing the release of reactive oxygen species, suggesting that MFN2 may be a potential therapeutic target for FFA-induced hepatic inflammation in dairy cows during early lactation., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Second mitochondria-derived activator of caspase (SMAC) mimetics (SMs) targeting inhibitor of apoptosis proteins (IAPs) activate cell death pathways, and are currently being evaluated in clinical trials. Their successful therapeutic implementation requires upfront identification of patients who could benefit from a SM-based treatment but biomarkers for SM sensitivity have not yet been described. Here, we analyzed the intrinsic activity of two monovalent (AT406 and LCL161) and two bivalent (Birinapant and BV6) SMs on unselected patient-derived pediatric precursor B-cell acute lymphoblastic leukemia (BCP-ALL) identifying a subset of patient samples to be particularly sensitive to SM-induced cell death. This subset was defined by a characteristic gene expression signature with 127 differentially regulated genes, amongst them TNFRSF1A encoding TNFR1, and a critical role of TNFR1 in SM-induced cell death in sensitive BCP-ALL was confirmed on the functional level. Interestingly, samples with intermediate or low sensitivity to SMs were sensitized to SM-induced cell death by inhibition of caspases using zVAD.fmk or Emricasan, a pan-caspase inhibitor in clinical trials. When we compared our expression data to published data sets, we identified an overlap of four genes to be commonly differentially regulated in SM-sensitive BCP-ALL, that is, TSPAN7, DIPK1C, MTX2 and, again, TNFRSF1A. Functional testing revealed that this set of genes identified samples with high sensitivity to SM treatment. In summary, our data suggest using this gene signature as biomarker predicting response to SM treatment and point to the development of new combinatorial treatments consisting of SMs and pan-caspase inhibitors for a successful clinical implementation of SMs in treatment of BCP-ALL., (© 2019 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Moniliophthora perniciosa is a fungal pathogen and causal agent of the witches' broom disease of cocoa, a threat to the chocolate industry and to the economic and social security in cocoa-planting countries. The membrane-bound enzyme alternative oxidase (MpAOX) is crucial for pathogen survival; however a lack of information on the biochemical properties of MpAOX hinders the development of novel fungicides. In this study, we purified and characterised recombinant MpAOX in dose-response assays with activators and inhibitors, followed by a kinetic characterization both in an aqueous environment and in physiologically-relevant proteoliposomes. We present structure-activity relationships of AOX inhibitors such as colletochlorin B and analogues which, aided by an MpAOX structural model, indicates key residues for protein-inhibitor interaction. We also discuss the importance of the correct hydrophobic environment for MpAOX enzymatic activity. We envisage that such results will guide the future development of AOX-targeting antifungal agents against M. perniciosa, an important outcome for the chocolate industry.

Cancer stem cells (CSCs) have been proposed to be responsible for tumor recurrence, distant metastasis and drug-resistance, in the vast majority of cancer patients. Therefore, there is an urgent need to identify new drugs that can target and eradicate CSCs. To identify new molecular targets that are unique to CSCs, we previously compared MCF7 2D-monolayers with 3D-mammospheres, which are enriched in CSCs. We observed that 25 mitochondrial-related proteins were >100-fold over-expressed in 3D-mammospheres. Here, we used these 25 proteins to derive short gene signatures to predict distant metastasis (in N=1,395 patients) and tumor recurrence (in N=3,082 patients), by employing a large collection of transcriptional profiling data from ER(+) breast cancer patients. This analysis resulted in a 4-gene signature for predicting distant metastasis, with a hazard ratio of 1.91-fold (P=2.2e-08). This provides clinical evidence to support a role for CSC mitochondria in metastatic dissemination. Next, we employed a panel of mitochondrial inhibitors, previously shown to target mitochondria and selectively inhibit 3D-mammosphere formation in MCF7 cells and cell migration in MDA-MB-231 cells. Remarkably, these five mitochondrial inhibitors had only minor effects or no effect on MDA-MB-231 tumor formation, but preferentially and selectively inhibited tumor cell metastasis, without causing significant toxicity. Mechanistically, all five mitochondrial inhibitors have been previously shown to induce ATP-depletion in cancer cells. Since 3 of these 5 inhibitors were designed to target the large mitochondrial ribosome, we next interrogated whether genes encoding the large mitochondrial ribosomal proteins (MRPL) also show prognostic value in the prediction of distant metastasis in both ER(+) and ER(-) breast cancer patients. Interestingly, gene signatures composed of 6 to 9 MRPL mRNA-transcripts were indeed sufficient to predict distant metastasis, tumor recurrence and Tamoxifen resistance. These gene signatures could be useful as companion diagnostics to assess which patients may benefit most from anti-mito-ribosome therapy. Overall, our studies provide the necessary proof-of-concept, and in vivo functional evidence, that mitochondrial inhibitors can successfully and selectively target the biological process of cancer cell metastasis. Ultimately, we envision that mitochondrial inhibitors could be employed to develop new treatment protocols, for clinically providing metastasis prophylaxis, to help prevent poor clinical outcomes in cancer patients.

The transfer of metabolites through the mitochondrial membranes is a vital process that is highly controlled and regulated by the inner membrane. A variety of metabolites, nucleotides, and cofactors are transported across the inner mitochondrial membrane (IMM) by a superfamily of membrane transporters which are known as the mitochondrial carrier family (MCF) or the solute carrier family 25 (SLC25 protein family). In humans, the MCF has 53 members encoded by nuclear genes. Members of the SLC25 family of transporters, which is the largest group of solute carriers, are also known as mitochondrial carriers (MCs). Because MCs are nuclear-coded proteins, they must be imported into the IMM. When compared with normal cells, the mitochondria of cancer cells exhibit significantly increased transmembrane potentials and a number of their transporters are altered. SLC25 members were identified as potential biomarkers for various cancers. The objective of this review is to summarize what is currently known about the involvement of mitochondrial SLC25 carriers in associated diseases. This review suggests that the SLC25 family could be used for the development of novel points of attack for targeted cancer therapy.

Central post-stroke pain (CPSP) is a type of neuropathic pain for which the mechanism and relevant drug pathways remain unknown. Recently, it was reported that intracerebroventricular (ICV) administration of orexin-A suppresses pain and ischemia. In this study, we tested the role of orexin-A in CPSP induction in mice. Male ddY mice were subjected to 30 min of bilateral carotid artery occlusion (BCAO). CPSP was assessed by von Frey test. Colocalization of orexin 1 receptor (OX1R) with various neuron markers were determined by double-immunofluorescence. The hindpaw withdrawal responses to mechanical stimuli were significantly increased 3 days post-BCAO compared with those of sham groups. ICV injection of orexin-A dose-dependently suppressed BCAO-induced mechanical allodynia. These effects were inhibited by pre-treatment with SB334867 (an OX1R antagonist; ICV injection), yohimbine (a noradrenaline α 2 receptor antagonist; intrathecal (IT) injection), and WAY100635 (a serotonin 5-HT 1A receptor antagonist; IT injection), but not TCS OX2 29 (an OX2R antagonist; ICV injection). OX1R colocalized with TH (a noradrenergic neuron marker) and TPH (a serotonergic neuron marker) in the locus ceruleus (LC) and the rostral ventromedial medulla (RVM), respectively. The number of c-Fos positive cells in the LC and the RVM of BCAO mice was increased at 90 min after ICV injection of orexin-A compared to saline group. These results indicate that orexin-A/OX1R signaling plays an important role through activation of the descending pain control system in the induction of CPSP in mice., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest, financials or otherwise., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Conserved amongst all eukaryotes is a family of mitochondrial carrier proteins (SLC25A) responsible for the import of various solutes across the inner mitochondrial membrane. We previously reported that the human parasite Trypanosoma brucei possesses 26 SLC25A proteins (TbMCPs) amongst which two, TbMCP11 and TbMCP8, were predicted to function as phosphate importers. The transport of inorganic phosphate into the mitochondrion is a prerequisite to drive ATP synthesis by substrate level and oxidative phosphorylation and thus crucial for cell viability. In this paper we describe the functional characterization of TbMCP11. In procyclic form T. brucei, the RNAi of TbMCP11 blocked ATP synthesis on mitochondrial substrates, caused a drop of the mitochondrial oxygen consumption and drastically reduced cell viability. The functional complementation in yeast and mitochondrial swelling experiments suggested a role for TbMCP11 as inorganic phosphate carrier. Interestingly, procyclic form T. brucei cells in which TbMCP11 was depleted displayed an inability to either replicate or divide the kinetoplast DNA, which resulted in a severe cytokinesis defect., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Orexin neuropeptides are involved in opiate-induced physical dependence and expression of withdrawal signs following drug abstinence. Orexin type-1 receptors (OXR1) are expressed throughout the rostroventrolateral medulla (RVLM), particularly in the lateral paragigantocellularis (LPGi) nucleus. The present study examined whether blocking OXR1 in LPGi region could affect the development of morphine dependence and so behavioral manifestations induced by morphine withdrawal in rats. Male Wistar rats weighing 250-300 g were used. In order to induce drug dependence, morphine was injected subcutaneously (s.c.) (6, 16, 26, 36, 46, 56, and 66 mg/kg, 2 ml/kg) at an interval of 24 h for 7 days. Animals were divided into experimental groups in which the orexin type-1 receptor antagonist, SB-334867 (0.2 μl, 3 mM), or its vehicle were injected into the LPGi nucleus for 7 days before each morphine injection. On day 8, naloxone (2.5 mg/kg, i.p.) was administered and morphine withdrawal behaviors were monitored for 25 min. Our results indicated that the inhibition of OXR1 in LPGi nucleus significantly reduces the development of morphine dependence and behavioral signs elicited by the administration of naloxone in morphine-dependent rats., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Excessive proliferation and apoptosis-resistance are hallmarks of cancer. Increased dynamin-related protein 1 (Drp1)-mediated mitochondrial fission is one of the mediators of this phenotype. Mitochondrial fission that accompanies the nuclear division is called mitotic fission and occurs when activated Drp1 binds partner proteins on the outer mitochondrial membrane. We examine the role of Drp1-binding partners, mitochondrial dynamics protein of 49 and 51 kDa (MiD49 and MiD51), as drivers of cell proliferation and apoptosis-resistance in non-small cell lung cancer (NSCLC) and invasive breast carcinoma (IBC). We also evaluate whether inhibiting MiDs can be therapeutically exploited to regress cancer. We show that MiD levels are pathologically elevated in NSCLC and IBC by an epigenetic mechanism (decreased microRNA-34a-3p expression). MiDs silencing causes cell cycle arrest through (a) increased expression of cell cycle inhibitors, p27 Kip1 and p21 Waf1 , (b) inhibition of Drp1, and (c) inhibition of the Akt-mTOR-p70S6K pathway. Silencing MiDs leads to mitochondrial fusion, cell cycle arrest, increased apoptosis, and tumor regression in a xenotransplant NSCLC model. There are positive correlations between MiD expression and tumor size and grade in breast cancer patients and inverse correlations with survival in NSCLC patients. The microRNA-34a-3p-MiDs axis is important to cancer pathogenesis and constitutes a new therapeutic target., (© 2020 Federation of American Societies for Experimental Biology.)