Leishmania and Trypanosoma, belonging to the same Trypanosomatidae family, are the causative agents of Leishmaniasis, Chagas disease, and human African trypanosomiasis. Overall, these infections affect millions of people worldwide, posing a serious health issue as well as socio-economical concern. Current treatments are inadequate, mainly due to poor efficacy, toxicity, and emerging resistance; therefore, there is an urgent need for new drugs. Among several molecular targets proposed, trypanothione reductase (TR) is of particular interest for its critical role in controlling the parasite’s redox homeostasis and several classes of active compounds that inhibit TR have been proposed so far. This review provides a comprehensive overview of TR’s structural characterization. In particular, we discuss all the structural features of TR relevant for drug discovery, with a focus on the recent advances made in the understanding of inhibitor binding. The reported cases show how, on the basis of the detailed structural information provided by the crystallographic analysis, it is possible to rationally modify molecular scaffolds to improve their properties.The protozoans  Leishmania (L.), Trypanosoma (T.) cruzi, and Trypanosoma brucei, respectively.Leishmaniasis, Chagas disease, and human African trypanosomiasis (HAT), also known as sleeping sickness, are vector borne zoonosis that affect millions of people worldwide and lead to the death of about 100,000 humans per year. These diseases are caused by infection with the trypanosomatids Leishmania parasites, transmitted by the bite of infected female phlebotomine sandflies, cause three main forms of leishmaniases: visceral (VL), cutaneous (CL), and mucocutaneous (MCL). There are an estimated 700,000 to 100,000,000 new cases of Leishmaniases annually in the world, widely distributed in tropical and subtropical climate zones, which lead to 26,000 to 65,000 deaths thiophen-2-yl)cyclohexyl)piperidine (BTCP), another class of compounds able to bind MBS, are probably, to date, the most explored compounds for structure-based development. Identified by HTS together with many other tricycles , the leating HAT . The firting HAT . Previouting HAT . Howeveryl rings . Two keyFurther efforts for improving properties and potency of this class have been recently undertaken by De Gasparo and colleagues ,46. TheyT. brucei TR with an inhibition constant Ki of 73 nM and is fully ineffective against human GR, even if its toxicity against mammalian cells is relatively high [2 cavity -2 (M9J in PDB), claimed to be the most effective non-covalent inhibitor of TR ever reported, inhibits ely high . Two maj2 cavity .T. brucei TR, was found to inhibit both the recombinant enzyme and the enzyme in cell lysate, as well as parasite proliferation in the low micromolar range (2–5 µM) while being inactive on human GR. Crystallographic studies confirmed the hot-spots for interaction already found for BTCP-derivatives  by competing with TS2, and to affect parasites in the micromolar range (IC50 29 μM). The crystal structure of RDS777 (RDS in PDB) in complex with L. infantum TR revealed the binding of 4 inhibitor molecules, one of which lays at the bottom of TS2 cavity, in direct contact with catalytic site by establishing hydrogen bonds with the residues involved in catalysis, namely Glu466’, Cys57, and Cys52. A second molecule is found in one out of two cavities, placed closer to the entrance, engaged in a stacking interaction with the first one. The other two molecules interact with the NADPH-binding site and are discussed later. Based on the structural information, a series of new derivatives have been synthesized, one of which has a higher activity on parasite cultures (IC50 11 μM) and is able to decrease the reduced-T(SH)2 concentration in cell [i 12 μM) and docking studies suggest that it prefers the second outermost binding site, indicating that it likely has other intracellular targets besides TR.Other compounds have shown the capability to bind the inner part of the active site. Recently, screening of an in-house collection detected a novel class of diarylsulfides active on e and TR . In partOther diaryl sulfides have been proposed previously and the binding site, predicted using docking, was different from RDS777, corresponding to the MBP and Z-site ,51. Howei 1.5 μM) by forming a stable complex with the residues involved in catalysis, namely the two cysteines (Cys52 and Cys57), His461 (the residue that together with Glu466′ activates the Cys52 similar to the cysteine proteases), and Thr335. Besides antimony, silver and gold were proven to bind TR in a similar way but even more efficiently [i down to 20 nM. Particularly interesting is the case of auranofin, a gold-containing drug used to treat rheumatoid arthritis [Leishmania TR and parasites [i 0.15 μM) and, most crucially, it acts via a double mode of action. In fact, besides expected gold complexation, the thiosugar moiety of auranofin contributes to inhibition by binding the inner part of TS2 site. This finding suggests the opportunity to combine scaffolds that are able to bind the outer TS2 cavity with auranofin or other metal-coordinating moieties to exploit double inhibition mechanism and to promote a selective targeting of otherwise poorly specific metal inhibitors . Crystallographic analysis of the complex revealed that the compound binds at the entrance of NADPH site, similar to RDS777, in a pocket not conserved in human GR  and activity in the low micromolar range as well as Leishmania viability in vitro [The disruption of the functional dimer of TR by targeting PPI has been recently proposed as an intriguing strategy alternative to competitive inhibition. Starting from the analysis of the dimerization interface of in vitro . Later, in vitro .Among the many pathways proposed as potential targets for antitrypanosomatid drug development, trypanothione metabolism is one of the most explored due to its critical role in redox homeostasis and its peculiarity. TR has been considered a promising target since its discovery because it satisfies most requirements for candidates, being essential, unique and druggable. Various inhibitor series have been identified and proposed as lead compounds but, to our knowledge, none of them has been advanced to clinical trials. Common reasons for that are sub-optimal potency, poor selectivity leading to toxicity, low bioavailability or biodistribution causing inactivity on animal models.In this context, structural characterization of inhibitor binding offers precious aid to improve inhibitor performances through structure-based design. In the last few years, several studies explored TR-inhibitor interaction by X-ray crystallography, revealing important information for binding rationalization and future development. Indeed, most inhibitors characterized so far locate to the wide trypanothione cavity, mainly at the entrance of the so-called mepacrine binding site (MBS). The MBS resulted to be quite promiscuous; in fact, besides the polyamine moiety of the substrate, it is able to bind different aromatic scaffolds. Binding site promiscuity can be an advantage in drug development because it favors polypharmacology approaches, known to decrease emergence of resistance. Moreover, it allows researchers to select molecules with convenient characteristics, as is the case for two identified scaffolds, BTCP and spiro-moiety, that are able to cross the BBB, a desirable feature for the treatment of HAT.Besides the MBS, other hot-spots have been identified in the trypanothione cavity. A hydrophobic subpocket, located deeper in the site, accommodates the hydrophobic arm of two different inhibitors, one of which displays nanomolar activity on TR. Finally, metal ions such as Sb(III), Ag(I), and Au(I) have proven to target redox-active cysteines in the catalytic site, confirming one of the proposed mechanisms of action for antileishmanial antimonial therapy ,14.The broadness of the trypanotione cavity is believed to be responsible for the relatively low potency of most inhibitors identified to date, that show inhibition constants in the low micromolar range, not enough for effective action. However, the identification of multiple hot-spots for interaction provides the chance to merge different scaffolds in one molecule, as in the serendipitous case of auranofin, in order to increase efficacy and selectivity, keeping in mind the size limitations for drug-like compounds.Despite of the most rational approach, recent results have shown that the search for lead compounds should not focus solely on the trypanothione cavity: the identification of an inhibitor addressing the NADPH cavity, selective against the main off-target GR, as well as peptides and peptidomimetics interfering with TR dimerization give a proof of concept for the idea that other sites can be exploited for TR inactivation.T. cruzi and T. brucei TR, a fragment-based screen campaign could reveal new small organic molecules suitable as lead compounds, targeting already known sites or unexplored hot-spots for new mechanisms of inhibition.It cannot be excluded that unexpected effective binding sites exist. Given the availability of well diffracting crystals for both


In vitro, BJF significantly improved cigarette smoke extract-induced mitochondrial functional impairment in L6 skeletal muscle cells through effects on mitochondrial membrane potential, mitochondrial permeability transition pores, adenosine triphosphate production, and mitochondrial respiration. In addition, BJF led to upregulated expression of mitochondrial biogenesis markers, including AMPK-α, PGC-1α, and TFAM and downregulation of mitophagy markers, including LC3B, ULK1, PINK1, and Parkin, with increased expression of downstream markers of the AMPK pathway, including mTOR, PPARγ, and SIRT1. In conclusion, BJF significantly improved skeletal muscle and mitochondrial function in COPD rats and L6 cells by promoting mitochondrial biogenesis and suppressing mitophagy via the AMPK pathway. This study suggests that BJF may have therapeutic potential for prophylaxis and treatment of skeletal muscle dysfunction in patients with COPD.Skeletal muscle dysfunction, a striking systemic comorbidity of chronic obstructive pulmonary disease (COPD), is associated with declines in activities of daily living, reductions in health status and prognosis, and increases in mortality. Bufei Jianpi formula (BJF), a traditional Chinese herbal formulation, has been shown to improve skeletal muscle tension and tolerance via inhibition of cellular apoptosis in COPD rat models. This study aimed to investigate the mechanisms by which BJF regulates the adenosine monophosphate-activated protein kinase (AMPK) pathway to improve mitochondrial function and to suppress mitophagy in skeletal muscle cells. Our study showed that BJF repaired lung function and ameliorated pathological impairment in rat lung and skeletal muscle tissues. BJF also improved mitochondrial function and reduced mitophagy via the AMPK signaling pathway in rat skeletal muscle tissue.  Chronic obstructive pulmonary disease (COPD), a common and preventable disease, is characterized by persistent airflow limitations caused by chronic inflammatory responses to noxious particles and gases . In ChinDevelopment of SMD is likely multifactorial, with inflammation and mitochondrial dysfunction playing predominant roles. Other pathophysiological factors, including oxidative stress, imbalance of protein synthesis/degradation, hypoxia, and cellular apoptosis have also been postulated to contribute to the muscle wasting experienced by COPD patients . Recent Decreased mitochondrial biogenesis and enhanced mitophagy play particularly important roles in mitochondrial impairment. Peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α has been shown to be a master regulator of mitochondrial biogenesis . PGC-1α Mitophagy, on the other hand, is closely associated with the AMPK pathway. AMPK directly facilitates phosphorylation of UNC-51-like kinase (ULK1), which stimulates the mitophagic activity of ULK1 . The PTETraditional Chinese medicine (TCM) has been successfully used to treat COPD for thousands of years. Lung-spleen qi deficiency syndrome is believed to be the main underlying pathologic mechanism for the SMD observed in patients with COPD . Bufei Jin vivo COPD rat model and an in vitro cigarette smoke extract (CSE)-induced skeletal muscle cell line to identify potential underlying mechanisms and to provide a basis for further research.In this study, we aimed to explore the mechanism by which BJF affects mitochondrial regulation via the AMPK pathway in an Klebsiella pneumoniae (strain: 46114) was obtained from the National Center for Medical Culture Collections . HongqiQu® filter cigarettes  were purchased from Henan Tobacco Industry . Aminophylline (APL) tablets were obtained from Shandong Xinhua Pharmaceutical Co., LTD. (lot H37020630) . 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) was purchased from Selleck Chemicals (lot 2627-69-2) . Dorsomorphin dihydrochloride (BML-275 dihydrochloride) was purchased from MedChemExpress (lot 1219168-18-9) .Thirty-six male and 36 female Sprague-Dawley (SD) rats with an average body weight of 200 ± 20 g were purchased from the Experimental Animal Center of Henan Province . The rats were housed in specific pathogen-free cages under standard temperature (25 ± 2°C) and light conditions, with a 12-h light/dark cycle, and free access to food and water. All animal studies were conducted in accordance with the Institute’s Guide for the Care and Use of Laboratory Animals and were approved by the Experimental Animal Care and Ethics Committee of the First Affiliated Hospital of the Henan University of Chinese Medicine.BJF, consisting of 12 herbs , was preSixty Sprague-Dawley rats were randomized into control and Bufei Jianpi groups. Rats in the Bufei Jianpi group were administered BJF (4.5 1g/kg/d) intragastrically 10 times once per day for 7 days. Rats in control group were administrated distilled water . On day 7, all rats were narcotized with 4% chloral hydrate and blood samples were collected, centrifugated at 3,500 rpm for 15 min, inactivated at 56°C, and stored in a −80°C freezer for further study.Klebsiella pneumonia suspension  was slowly dropped into the nasal cavities of rats every 5 days for the first 8 weeks. The successful generation of a COPD rat model was evaluated based on symptoms, lung function, and pulmonary pathology , 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), Bufei Jianpi Formula + 5-Aminoimidazole-4-carboxamide ribonucleotide (BJF + AICAR), and Aminophylline (APL), with 12 animals in each group. The detailed procedures for creation of the COPD rat model have been described in a previous report ; howeverathology .D represents dose, I represents body shape index, and W represents body weight.Starting from week 9, rats in the control and model groups were intragastrically administered normal saline . BJF (4.51 g/kg/d) was administered to rats in the BJF and BJF + AICAR groups, while APL (2.7 mg/kg/d) was administered to rats in the APL group twice per day for 12 weeks. AICAR (25 mg/kg/d) was intraperitoneally administered to rats in the AICAR and BJF + AICAR groups once a day during week 20. The equivalent dosages of BJF and APL were calculated using the equation:All rats underwent necropsy after intraperitoneal injection of 10% chloral hydrate at 100 mg/kg of body weight at the end of week 20, and lung and quadriceps muscle tissues were collected for further analysis.Lung function was measured by a whole-body plethysmography system  at weeks 0, 4, 8, 12, 16, and 20. Tidal volume (TV), peak expiratory flow (PEF), and 50% tidal volume expiratory flow (EF50) were evaluated.After being fixed in 10% formalin for 72 h, left lung and quadriceps muscle tissues were embedded with paraffin, cut into 4-μm thick sections, and stained with hematoxylin and eosin (HE) for light microscopy.After being cut into sections with 1-mm thicknesses, fresh quadriceps muscle tissue was fixed in 2.5% glutaraldehyde for 2 h followed by 1% osmium tetroxide. Sections were then stained with uranyl acetate, dehydrated in a methanol series and propylene oxide, and embedded with Epon-812. Ultrathin sections were obtained, and ultrastructural changes and mitophagy of skeletal muscle cells were examined using a JEM-1400 transmission electron microscope .Mitochondrial characteristics, including mitochondrial membrane potentials (MMP), ATP production, and opening of mitochondrial permeability transition pores (MPTPs) were measured with a JC-1 kit , an ATP assay kit , and an MPTP assay , respectively, according to the manufacturer’s instruction and as previously described .2-humidified atmosphere at 37°C. When cells had grown by 70–90%, they were plated on dishes at a density of 1 × 105 cells/mL and incubated in 5% CO2-95% air for 24 h. On the next day, cells were treated with 10% cigarette smoke extract (CSE), 10% CSE + AICAR (2.5 ng/mL), or 10% CSE +10% BJF-containing serum in DMEM and harvested 24 h later.Rat L6 myoblasts  were cultured in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% fetal bovine serum (FBS) in a 5% COCSE was prepared using a modified version of Sheridan’s method . BrieflyMitochondrial respiration was measured with a Seahorse XF Cell Mito Stress Test Kit  on a Seahorse XF24 Extracellular Flux Analyzer  according to the manufacturer’s instruction. L6 cells were seeded  onto Seahorse assay plates in DMEM media and treated with 10% CSE for 24 h. During the experiment, cells were switched to an XF assay medium (HCO3-free modified DMEM) supplemented with 10 mM of L-glutamine, 10 mM of glucose, and 10 mM of pyruvate, while a pH of 7.4 was maintained at 37 °C. After baseline measurement of oxygen combustion rate (OCR), a mitochondrial respiration test was performed by sequential additions of 1.0 μM oligomycin, 2.0 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone, and 0.5 μM rotenone/antimycin A. Several mitochondrial parameters were determined, including basal respiration, maximal respiratory capacity, proton leak, and non-mitochondrial respiration. The results were expressed as OCRs (pmol/min/lg protein).5 cells per well and treated with 10% CSE, 10% CSE + AICAR, or 10% CSE +10% BJF-containing serum for 24 h. Before the experiment, cells were treated with carbonyl cyanide m-chlorophenyl hydrazone (CCCP)  for 1 h as a positive control. Changes in mitochondrial membrane potentials of L6 cells in different groups were then assessed using the JC-1 mitochondrial membrane potential detection kit according to the manufacturer's instruction. Red fluorescence represented JC-1 aggregates, whereas green fluorescence represented the monomeric form of JC-1. Fluorescence intensity was analyzed by a Varioskan® Flash Microplate Reader . MMPs in each group were calculated as the ratio of red/green fluorescence. In addition, cells were visualized using confocal laser scanning microscopy at a 514-nm excitation and a 529-nm emission for green and a 585-nm excitation and a 590-nm emission for red.For measurement of mitochondrial membrane potentials, L6 cells were seeded in 6-well plates with a density of 1 ×105 cells per well for 24 h. After treatment with study drugs, cells were lyzed using a cell lysis reagent, and the ATP content in cell lysates was measured using an ATP assay kit  following the manufacturer’s protocol. The total and mitochondrial protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit .For measurement of ATP levels, L6 cells were seeded in 6-well plates with a density of 1 ×10GAPDH) was used as an internal standard.Expression levels of mRNA in quadriceps muscle tissuewere analyzed by semiquantitative polymerase chain reactions (qPCR). Total RNA was extracted from the quadriceps muscle using a total RNA isolation kit  according to the manufacturer's protocol. Real-time (RT)-qPCR was performed using a reverse transcriptase kit  and a SYBR Green Master Mix . To normalize the amount of total RNA in each reaction, a reference gene , anti-sirtuin 1 (SIRT1), anti-peroxisome proliferator-activated receptor (PPAR)γ , or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH)  at 4°C overnight. After incubation with the HRP-coupled secondary antibody  at room temperature for 2 h, signals were detected using a super-enhanced chemiluminescence plus reagent and scanned and quantified by a ChemiDoc MP imaging system .Total proteins from cells or tissue samples were lyzed in a lysis buffer and quantified using a BCA assay. Ten-cell lysates were loaded into each well in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked in 5% nonfat milk in tris-buffered saline and polysorbate 20 (TBST) for 2 h at room temperature and incubated with the indicated primary antibodies, including anti-AMPK-α, anti-P-values of < 0.05.Data are expressed as mean ± SD. Statistical differences between the groups were identified by one-way analysis of variance (ANOVA). Differences were considered to be significant at Epimedium A (0.6869 mg/g), Icariin (1.1195 mg/g), Nobiletin (0.0951 mg/g), BaohuosidⅠ (0.0806 mg/g) were identified and their structures had also been shown.Fingerprint of BJF The retention time values of the identified compounds were compared with that of the reference substances. A representative chromatogram of BJF was shown in To assess potential effects on skeletal and lung tissues, BJF was orally administered to COPD rats for 12 weeks using APL as a positive control. We found that BJF attenuated declines in body weight and respiratory function in COPD rats. As shown in As shown in We also found that BJF obviously reduced lung injury identified on histopathological examination. As shown in BJF also obviously improved morphological and ultrastructural changes in skeletal muscle of COPD rats. As shown in To confirm the effect of BJF on mitochondrial function, we measured the levels of mitochondrial functional indicators. As shown in BJF Promoted Expression of Mitochondrial Biogenesis Factors via Regulation of the AMPK Signaling Pathway in COPD RatsAMPK-α, PGC-α, and TFAM were markedly decreased in model rats. While treatment with BJF and APL significantly promoted expression of AMPK-α, PGC-α, and TFAM mRNA, AICAR upregulated levels of AMPK-α and PGC-α but not TFAM. As shown in p-AMPK-α, PGC-α, and TFAM were lowered in model rats compared with control rats. BJF, AICAR, and APL treatment resulted in dramatic increases in AMPK-α, p-AMPK-α, PGC-α, and TFAM protein expression.To explore the mitochondrial biogenesis-related mechanisms of BJF, we measured mRNA and protein levels of AMPK-α, PGC-α, and TFAM in skeletal muscle tissues of COPD rats. As shown in BJF Downregulated Expression of Mitophagic Factors via Regulation of the AMPK Signaling Pathway in COPD RatsLC3B, ULK1, PINK1, and Parkin mRNA levels were significantly upregulated in COPD rats in comparison to control rats. While treatment with BJF, AICAR, and APL downregulated expression of LC3B, ULK1, PINK1, and Parkin mRNA, AICAR upregulated levels of all the above-mentioned indicators. Additionally, protein expression of LC3B, ULK1, PINK1, and Parkin were markedly decreased in COPD rats compared with control rats. While BJF and APL treatment upregulated protein expression of LC3B, ULK1, PINK1, and Parkin, AICAR showed no effect on the level of LC3B . Our study measured the respiratory characteristics of mitochondria under cellular baseline and stress conditions. there is evidence indicated that mitochondrial aerobic respiration was inhibited in L6 skeletal muscle cells exposed to high-dose fructose, with significantly reduced oxygen consumption rate, basal and maximal respiration, and ATP synthesis . NeverthDifferent factors related to mitochondrial dysfunction, including abnormalities in mitochondrial biogenesis and mitophagy, may contribute to the loss of skeletal muscle strength that leads to functional declines in patients with COPD . AMPK, ap-ULK have also been observed in COPD patients  and the Scientific Research Fund for doctors of Henan University of Chinese Medicine (RSBSJJ2018-03).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.


However, little is known about regulation of the membrane-bound protein import machinery. Here, we report that Pex14, a central component of the protein translocation complex in peroxisomal membrane, is phosphorylated in response to oxidative stresses such as H2O2 in mammalian cells. The H2O2-induced phosphorylation of Pex14 at Ser232 suppresses peroxisomal import of catalase in vivo and selectively impairs in vitro the interaction of catalase with the Pex14-Pex5 complex. A phosphomimetic mutant Pex14-S232D elevates the level of cytosolic catalase, but not canonical PTS1-proteins, conferring higher cell resistance to H2O2. We thus suggest that the H2O2-induced phosphorylation of Pex14 spatiotemporally regulates peroxisomal import of catalase, functioning in counteracting action against oxidative stress by the increase of cytosolic catalase.Most of peroxisomal matrix proteins including a hydrogen peroxide (H In Phos-tag PAGE, phosphorylated proteins can be distinguished as slower-migrating bands from the corresponding non-phosphorylated form . We founsphatase . Furthersphatase , where tt, ZP161 .2O2, peaked at 30 min to 1 hr, and gradually decreased to a nearly basal level within 5 hr  analysis. Four phosphorylated peptides corresponding to the amino acids at alignment positions 228–237 (QFPPpSPSAPK)    , 2–25 [ApSPSAPK)  were ideectively , suggests target . Ser232 s target . In contncrease) . H2O2-ints (MEF) , suggest2O2-treatment. His-Pex14-WT showed two phosphorylated bands in Phos-tag PAGE of H2O2-treated Fao cells and S232A mutation eliminated both bands  and its mutants, phosphorylation-defective Pex14-S232A (SA-13) and phosphomimetic Pex14-S232D (SD-30)  . In thes (SD-30) . AOx B-c14 ZP161 , hence s14 ZP161 . As in C14 ZP161 . However14 ZP161 , where P14 ZP161 , which w14 ZP161 . We furt14 ZP161 . Consist14 ZP161 , a part 14 ZP161 . Catalas14 ZP161 , lane 7.14 ZP161 , suggest14 ZP161 . Precurs14 ZP161 . Only ma14 ZP161 , demonst14 ZP161  as in th14 ZP161 , showing2O2 treatment in vivo, Fao cells were pulse-labeled with 35S-labeled methionine and cysteine for 1 hr and fractionated into the cytosolic and organelle fractions. Immunoprecipitated 35S-catalase was more detected in the cytosolic fraction than that in the organelle fraction at the start of chase , an enzyme with a typical PTS1, was increased in the organelle fractions during 1 hr chase in the absence or presence of H2O2  and 0.5 mM, where both concentrations of H2O2 gave rise to phosphorylation of Pex14 at Ser232 to a similar extent  reported here. Although the study with kinase inhibitors suggests the ERK-mediated phosphorylation of Pex14 at Ser247 and Ser252 .ROS activates various cellular signaling pathways. ATM is reported as a peroxisome-localized kinase activated by ROS, mediating Pex5 phosphorylation and induction of pexophagy . An ATM 93 cells  showed nao cells . Therefod Ser252 , furtheried form , where tied form . This is2O2-induced phosphorylation of Pex14 predominately suppresses peroxisomal import of catalase more efficiently than typical PTS1 proteins. Together with the findings that H2O2 treatment transiently induces Pex14 phosphorylation  followed by selection with Zeocin (Invitrogen), as described Pex5S and ClPex5L , β-actin (MBL), and Tom20 , and goat anti-lactate dehydrogenase antibody (Rockland). Kinase inhibitors, U0126 and SB203580, were purchased from Cell Signaling. KU5933 and Compound C were from Abcam and Merck, respectively.Antibodies used were rabbit polyclonal antibodies each to C-terminal 19-amino acid residues of Pex14 , Pex5 O, Pex13 , Pex3 G, acyl-Co2 for the lysates of mouse tissues and 25 µM Phos-tag and 50 µM MnCl2 for those of cultured cells , and 1 mM dithiothreitol) containing 0.5% Nonidet P-40% and 0.1% SDS by ten strokes of homogenization with an Elvehjem-Potter homogenizer . After ced cells .6 cells) were lysed in RIPA buffer  supplemented with a complete protease inhibitor cocktail (Roche) and Phos-stop phosphatase inhibitor cocktail (Sigma-Aldrich) and were flash-frozen in liquid nitrogen. After thawing on ice and centrifugation at 20,000 g for 15 min at 4 °C, supernatant fractions were subjected to immunoprecipitation with anti-Pex14 antibody immobilized on SureBeads Protein G magnetic beads (Bio-Rad) for 3 hr at 4 °C with rotation. After washing with RIPA buffer four times and then with 50 mM ammonium bicarbonate twice, proteins on the beads were digested by adding 400 ng trypsin/Lys-C mix (Promega) for 16 hr at 37 °C. The digests were acidified and desalted using GL-Tip SDB (GL Sciences). The eluates were evaporated in a SpeedVac concentrator and dissolved in 3% acetonitrile (ACN) and 0.1% trifluoroacetic acid.Fao cells  target of 1 × 106 and a mass range from 350 to 1,500 m/z. HCD MS/MS spectra were acquired at a resolution of 17,500, an AGC target of 5 × 104, an isolation window of 2.0 m/z, a maximum injection time of 60 ms and a normalized collision energy of 27. Dynamic exclusion was set to 10 s. Raw data were directly analyzed against the SwissProt database restricted to H. sapiens using Proteome Discoverer version 2.3 (Thermo Fisher Scientific) with Mascot search engine version 2.5 (Matrix Science) for identification and label-free precursor ion quantification. The search parameters were as follows: (i) trypsin as an enzyme with up to two missed cleavages; (ii) precursor mass tolerance of 10 ppm; (iii) fragment mass tolerance of 0.02 Da; (iv) carbamidomethylation of cysteine as a fixed modification; and (v) acetylation of the protein N-terminus, oxidation of methionine and phosphorylation of serine, threonine, and tyrosine as variable modifications. Peptides were filtered at a false-discovery rate of 1% using the percolator node. Normalization was performed such that the total sum of abundance values for each sample over all peptides was the same.LC-MS/MS analysis of the resultant peptides was performed on an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer equipped with a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a 75 µm inner diameter x 150 mm C18 reversed-phase column (Nikkyo Technos) with a linear 4Immunostaining of cells was performed as described  with 4% 3VO4, and 6 mM β-glycerophosphate) for 5 min at room temperature as described  in buffer H and centrifuged at 800 g for 10 min at 4°C to yield post nuclear supernatant (PNS) fraction. Organelle fraction was separated by ultracentrifugation of the PNS fraction at 100,000 g for 30 min at 4°C. The organelle pellet was lysed in buffer L containing 0.5% CHAPS, 1 mM NaF, 1 mM Na3VO4, and 6 mM β-glycerophosphate for 30 min at 4°C. After centrifugation at 20,000 g for 10 min at 4°C, resulting supernatants were incubated with antibodies to Pex14, Pex14-pS232, and Pex13 in buffer L containing 0.5% CHAPS for 2 hr at 4°C. Antibody-antigen complexes were recovered by incubating for 1 hr at 4°C with Protein A-Sepharose CL-4B  and eluted with Laemmli sample buffer. For detection of mono-ubiquitinated Pex5 in organelle fraction of Fao cells, subcellular fractionation was performed with H buffer containing 5 mM N-ethylmaleimide and no DTT as described , as described  were seeded in a 96-well plate and grown for 24 hr and then treated for 14 hr with vehicle alone, 0.8 mM H2O2 alone, or each together with 20 mM 3-aminotriazole (3-AT). After treatment, cells were incubated for additional 2 hr with Celltiter 96 aqueous one solution reagent (Promega). Cell viability was determined by the absorbance of media at 490 nm as described in the manufacturer’s protocol and represented as percentages relative to that of each mock-treated and H2O2-untreated cells.Cell viability was measured with a tetrazolium-based toxicology assay kit (Promega). CHO cells . To chase the 35S-labeled proteins, cells were washed twice and further incubated for 1 hr with DMEM supplemented with 10% FBS and 10 mM methionine. 35S-labeled cells were harvested and incubated for 5 min in buffer H containing 50 μg/ml digitonin at room temperature as described .Fao cells growing in DMEM supplemented with 10% FBS in 6-well plate were washed twice with PBS, incubated in cysteine- and methionine-free DMEM (Gibco) supplemented with 10% FBS that had been dialyzed for 1 hr in excess PBS with a Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific). Cells were then pulse-labeled for 1 hr by adding 100 μCi/ml escribed . After cescribed . 35S-labhttp://www.r-project.org). Quantitative data were represented as means ± SEM from at least three independent experiments. Statistical significance was determined using a two-tailed unpaired Student’s t test for comparisons between two groups or one-way ANOVA with Dunnett’s post hoc test for more than two groups. P-values of <0.05 were considered statistically significant.Statistical analysis was performed using R software As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is 'in revision at Summary:2O2, that a phosphomimetic mutation of this site impairs catalase import, and that the reduced import of catalase during H2O2 treatment is important to maintaining viability during the stress. in vitro interaction studies suggest that the phosphomimetic Pex14 mutant can bind Pex5L but does not form a stable ternary complex with Pex5L and catalase. The other phosphorylation sites seem to have less of an effect but may affect other PTS1 import substrates. The primary conceptual advance here is that peroxisome import machinery can be regulated by phosphorylation to affect import of some, but not other substrates. The referees agreed that the conceptual advance of identifying a new regulatory aspect of peroxisomal import is appropriate for publication in eLife, but that the data are currently insufficiently complete to fully support the manuscript's claims.This study investigates whether peroxisomal import is regulated by phosphorylation. The authors initially identify peroxide-triggered phosphorylation on Pex14 using Phos-tag gels, then identify the sites, and use mutations to probe their importance. The key discovery is that S232 on Pex14 is phosphorylated (among other sites) in response to HEssential revisions:1) The mechanism proposed by the authors for regulation of catalase import involves Pex14 phosphorylation. Yet it is Pex5 that recognizes catalase in the cytosol and is required for chaperoning catalase across the peroxisome membrane. Thus, to understand the mechanism of regulation, their crucial in vitro experiments examining substrate-Pex5-Pex14 interactions need to use the appropriate substrate-Pex5 complexes. Mammals have two Pex5's, a short Pex5 responsible for PTS1 import, and Pex5L, which binds to Pex7 and helps guide PTS2-containing proteins into the peroxisome. The authors do not provide any justification in the manuscript for why Pex5L was used in the in vitro binding experiments, and they do not provide any comparative experiments using the short Pex5. The authors must address this concern in order to justify the extrapolation of the in vitro experiments to the situation in cells.2O2 treatment. The dynamics of Pex14 turnover and its contribution to peroxisome import dynamics is not explored by the authors but has important implications for their hypothesis. The authors should carefully consider the possibility that phosphorylation regulates Pex14 turnover, which impacts import dynamics. If the authors have data on the turnover of Pex14 and its mutants under different conditions, this would be important to include. At the very least, this alternative explanation for regulation should be discussed in a revised manuscript.2) The phospho-serine rich site identified by the authors is predicted to be a PEST sequence by bioinformatic searches using the sequence for rat Pex14. PEST sequences are typically found on short-lived proteins and act as a signal for turnover by the proteasome or calcium-dependent calpain proteases. In several instances, Figure 1B, Figure 2A, Figure 2D, etc. it appears that oxidative stress results in a reduction of Pex14, consistent with a hypothesis that this proline and serine rich site is functioning like a PEST sequence. In Figure 4F, phosphorylated Pex14 is detected in the cytosolic fraction, which the authors claim is non-specific. An alternative explanation is that Pex14 is being extracted from the peroxisome and turned over upon H3) The microscopy experiments present in Figure 3 and Figure 4 are not very convincing and are incomplete. It is difficult to see catalase in the cytosol in the S-to-D mutants. The control images stained for SKL are not shown, confounding the analysis. Further, the localization of the Pex14 mutants, while appearing punctate in the images, was not confirmed by colocalization with another PMP. Finally, equal expression of the different mutants relative to wild type was not verified . To make the experiment more complete, control SKL images need to be presented, the subcellular localization of the Pex14 mutants verified by colocalization with another PMP, and equal expression of the mutants verified by either quantification of the microscopy or SDS-PAGE.4) Loading controls for the experiment in Figure 4D are needed to make this fully interpretable. Quantification of EGFP-PTS1 and HA-catalase in Figure 5C would be helpful to a reader.5) Figure 4F is not convincing because the differences claimed are not very easy to appreciate and the degree of reproducibility of the small effects is not clear. To be convincing, this experiment needs to be quantified from multiple replicates and should be accompanied by total samples to show the levels of the proteins in each sample before fractionation.6) The anti-His blot in Figure 2D is of poor quality and cannot be interpreted with confidence. The Pex14 blot is clear but is complicated by co-expression and partial co-migration of endogenous and exogenous Pex14 species. This experiment would be improved by either improving the quality of the anti-His blot, or perhaps if the authors preformed a His-pulldown followed by blotting to selectively visualize the exogenous proteins. The other option is to perform the experiment in cells lacking endogenous Pex14. Regardless of the approach taken, the authors should improve the quality of this important figure.7) The claimed role of Pex13 is not clear from the results in Figure 5A. This experiment can be improved if the authors perform IP with phospho-specific antibody to substantiate the claim that phosphorylation of Pex14 alters it complex formation with Pex13. Alternatively, an IP via Pex13 could also be performed and show that the pS232 is coming down with Pex13.8) The conclusion that phosphorylation is ERK mediated has been shown with a single inhibitor and should be extended to show this more directly by checking Pex14 in ERK knockout or siRNA cells. Summary:2O2, that a phosphomimetic mutation of this site impairs catalase import, and that the reduced import of catalase during H2O2 treatment is important to maintaining viability during the stress. in vitro interaction studies suggest that the phosphomimetic Pex14 mutant can bind Pex5L but does not form a stable ternary complex with Pex5L and catalase. The other phosphorylation sites seem to have less of an effect but may affect other PTS1 import substrates. The primary conceptual advance here is that peroxisome import machinery can be regulated by phosphorylation to affect import of some, but not other substrates. The referees agreed that the conceptual advance of identifying a new regulatory aspect of peroxisomal import is appropriate for publication in eLife, but that the data are currently insufficiently complete to fully support the manuscript's claims.This study investigates whether peroxisomal import is regulated by phosphorylation. The authors initially identify peroxide-triggered phosphorylation on Pex14 using Phos-tag gels, then identify the sites, and use mutations to probe their importance. The key discovery is that S232 on Pex14 is phosphorylated (among other sites) in response to HTo address the issues raised by the editors and reviewers, we performed additional experiments and provided the main revised points as follows:a) In vitro binding assays using Pex5S to show the similar property with Pex5L in the formation of Pex14-Pex5 complex with catalase and PTS1 protein .2O2-treatment, showing little effect of Pex14 phosphorylation in its turnover .b) Assessing the protein level of Pex14 upon Hc) Immunostaining images of catalase to clearly show the cytosolic catalase  and those of PTS1 and PMP70 to verify the peroxisomal import of PTS1 proteins and the co-localization of Pex14 variants in PMP70-positive peroxisomes .d) Several quantitative analyses to verify the peroxisomal import of catalase in the pulse-chase experiment  and the in vitro interaction of Pex14-Pex5 complex with catalase or PTS1 protein .e) A clear immunoblot image with anti-His antibody to assess the site-specific phosphorylation of Pex14 .2O2-treatment .f) Immunoprecipitation with antibodies to Pex13 and Pex14-p232 to verify the relationship between Pex14 and Pex13 upon H2O2-induced phosphorylation of Pex14 .g) ERK2 knockdown in Fao cells to address the involvement of ERK2 in the HEssential revisions:1) The mechanism proposed by the authors for regulation of catalase import involves Pex14 phosphorylation. Yet it is Pex5 that recognizes catalase in the cytosol and is required for chaperoning catalase across the peroxisome membrane. Thus, to understand the mechanism of regulation, their crucial in vitro experiments examining substrate-Pex5-Pex14 interactions need to use the appropriate substrate-Pex5 complexes. Mammals have two Pex5's, a short Pex5 responsible for PTS1 import, and Pex5L, which binds to Pex7 and helps guide PTS2-containing proteins into the peroxisome. The authors do not provide any justification in the manuscript for why Pex5L was used in the in vitro binding experiments, and they do not provide any comparative experiments using the short Pex5. The authors must address this concern in order to justify the extrapolation of the in vitro experiments to the situation in cells.While Pex5L is essential for PTS2-protein import as pointed out, both Pex5S and Pex5L are equally functional in PTS1-protein import and share the comparable interactions with the cargo PTS1-proteins and Pex14 . In the original manuscript, we mainly used Pex5L as a PTS1 receptor in in vitro binding assays  and also showed that Pex5S less efficiently forms a ternary complex with Pex14-S232D and catalase  as in the case of Pex5L . To further address this issue, we performed in vitro binding assays for interactions between a full series of Pex14 variants, Pex5S, and PTS1-proteins . Pex5S represented an interaction profile similar to Pex5L, demonstrating that Pex5S and Pex5L similarly mediate the complex formation of Pex14 with catalase and PTS1 protein accompanied by the phosphorylation-dependent regulation. These results further supported the situation in cells upon an oxidative stress.We added Figure 5—figure supplement 1B and revised the text involving this issue with additional introduction of two isoforms of Pex5 in the Results section and figure legends.2O2 treatment. The dynamics of Pex14 turnover and its contribution to peroxisome import dynamics is not explored by the authors but has important implications for their hypothesis. The authors should carefully consider the possibility that phosphorylation regulates Pex14 turnover, which impacts import dynamics. If the authors have data on the turnover of Pex14 and its mutants under different conditions, this would be important to include. At the very least, this alternative explanation for regulation should be discussed in a revised manuscript.2) The phospho-serine rich site identified by the authors is predicted to be a PEST sequence by bioinformatic searches using the sequence for rat Pex14. PEST sequences are typically found on short-lived proteins and act as a signal for turnover by the proteasome or calcium-dependent calpain proteases. In several instances, Figure 1B, Figure 2A, Figure 2D, etc. it appears that oxidative stress results in a reduction of Pex14, consistent with a hypothesis that this proline and serine rich site is functioning like a PEST sequence. In Figure 4F, phosphorylated Pex14 is detected in the cytosolic fraction, which the authors claim is non-specific. An alternative explanation is that Pex14 is being extracted from the peroxisome and turned over upon H2O2 treatment by re-analysis of the original Figure 1C using exactly the same samples as those used in the original figure. In contrast to ~70% of total Pex14 was phosphorylated with a peak at 1 hour post-H2O2 challenge, total Pex14 level remained nearly constant in SDS-PAGE for 2 hours and reduced by approximately 30% at 5 hours after the H2O2 treatment, where a cytosolic protein LDH indicated a similar pattern . We further verified any effect of H2O2 treatment on Pex14 stability and found that the exposure to H2O2 for 5 hours significantly lowered the protein level of Pex14 and concomitantly decreased LDH to a similar extent, thereby showing the relatively unaltered protein level of Pex14 . As the cells in these experiments were lysed in a constant volume of the buffer, we interpreted these results to mean that significant decrease of Pex14 observed at 5 hours after the H2O2 treatment is most likely due to less number of the cells by H2O2 toxicity, not by a preferential degradation of phosphorylated Pex14. This is further supported by the findings that both phosphorylation-deficient and phosphomimetic mutants of Pex14 were similarly expressed as wild-type Pex14 even in H2O2-treated ZP161 cells for 2 hours . Taken together, we think that phosphorylation-dependent regulation of Pex14 turnover has little effect on peroxisomal protein import and that phosphorylated Pex14 upon H2O2 treatment is most likely de-phosphorylated by currently yet identified phosphatase(s). Thus, the putative PEST sequence does not appear to function in the Pex14 degradation.As the reviewers pointed out, a putative PEST sequence was identified in the C-terminal region of rat Pex14 (amino-acid residues at 259-278) by an algorithm, PESTfind. We addressed the protein level of Pex14 upon H2O2 treatment. These issues would be clarified in the future.Regarding the band detected in the cytosolic fraction in anti-phospho-Pex14 blot , it is most likely a non-specific band; 1) no cytosolic band was discernible with anti-Pex14 antibody at the position corresponding to that observed in anti-phospho-Pex14 blot and 2) the anti-phospho-Pex14-positive band in the cytosolic fraction seemed to migrate with slightly lower mobility than those in the organelle fractions in the respective conditions. We do not exclude the possibility that phosphorylation is involved in Pex14 turnover and a part of the phosphorylated Pex14 becomes extractable from the organelle membrane upon HWe added Figure 1—figure supplement 1A and revised Figure 1D  and the text addressing this issue in the Results section, Discussion section and figure legends.3) The microscopy experiments present in Figure 3 and Figure 4 are not very convincing and are incomplete. It is difficult to see catalase in the cytosol in the S-to-D mutants. The control images stained for SKL are not shown, confounding the analysis. Further, the localization of the Pex14 mutants, while appearing punctate in the images, was not confirmed by colocalization with another PMP. Finally, equal expression of the different mutants relative to wild type was not verified . To make the experiment more complete, control SKL images need to be presented, the subcellular localization of the Pex14 mutants verified by colocalization with another PMP, and equal expression of the mutants verified by either quantification of the microscopy or SDS-PAGE.We revised immunostaining images of catalase in ZP161 cells transiently or stably expressing Pex14 variants . These improved the images clearly showing cytosolic catalase in ZP161 cells expressing phosphomimetic S-to-D mutants of Pex14, in which Pex14 variants were equally expressed as assessed by immunoblot analysis . We provided representative images of PTS1 proteins  used for quantifying the activity of PTS1 import in Pex14 variants . Furthermore, we performed co-immunostaining of Pex14 variants with a peroxisomal membrane protein PMP70 in ZP161 cells and found that all Pex14 variants tested were co-localized with PMP70 in punctate structures, peroxisomes, as the wild-type Pex14 . In ZP161-stable cell lines, WT-6, SA-13, and SD-30, exclusive peroxisomal localization of PTS1  and co-localization of Pex14 variants with PMP70  were likewise observed as in CHO-K1 cells. Collectively, these results showed that both S-to-A and S-to-D mutations did not affect peroxisomal localization and a protein level of Pex14 variants, confirming the specific suppression of catalase import by the phosphomimetic Pex14 variant with S232D.We revised the text regarding New Figure 3A, New Figure 3—figure supplement 1BC, and Figure 4—figure supplement 1AB in the Results section and figure legends.4) Loading controls for the experiment in Figure 4D are needed to make this fully interpretable. Quantification of EGFP-PTS1 and HA-catalase in Figure 5C would be helpful to a reader.In Figure 4D, LDH was used as a loading control as well as a cytosolic protein for adequate subcellular fractionation. As the reviewers suggested, the recoveries of EGFP-PTS1 and HA-catalase in Figure 5C were quantified and represented at the bottom of these blots. We revised the text in the figure legends.5) Figure 4F is not convincing because the differences claimed are not very easy to appreciate and the degree of reproducibility of the small effects is not clear. To be convincing, this experiment needs to be quantified from multiple replicates and should be accompanied by total samples to show the levels of the proteins in each sample before fractionation.2O2 in catalase import. We think several reasons for this situation.We agree with the reviewers’ concern in regard to an apparently low effect of H2O2.1) Catalase was imported into peroxisomes with substantially less efficiency than typical PTS1 proteins in in vitro import system  and in cells , which is owing to the atypical PTS1 (KANL) of catalase. Therefore, the increase of catalase in organelle fraction was lower during 1 hour-chase even in the absence of H2O2 treatment. This would lead to a lower suppression effect of H2O2 on catalase import in pulse-chase experiment, where the experimental condition was best optimized including the essential processes for pulse-labeling. In the revised manuscript, we quantified 35S-catalase and 35S-DHAPAT and showed that the increase of peroxisomal import of catalase in normal condition was repressed by the H2O2-treatment . Unfortunately, we have been unable to perform further experiments by the restriction of our radioisotope-research facility under the epidemic of COVID-19. We hope that you would understand our situation. We think that in addition to the pulse-chase experiments, other several results including the H2O2-induced phosphorylation of endogenous Pex14 and suppressive phenotypes of phosphomimetic Pex14 variants in catalase import convincingly support the regulated import of catalase in cells in a Pex14 phosphorylation-dependent manner.2) We found in other experiments that medium changes with methionine- and cysteine-free medium and following washing steps attenuated the phosphorylation level of Pex14 upon HWe added Figure 4G and revised the text regarding this issue in the Results section and figure legends.6) The anti-His blot in Figure 2D is of poor quality and cannot be interpreted with confidence. The Pex14 blot is clear but is complicated by co-expression and partial co-migration of endogenous and exogenous Pex14 species. This experiment would be improved by either improving the quality of the anti-His blot, or perhaps if the authors preformed a His-pulldown followed by blotting to selectively visualize the exogenous proteins. The other option is to perform the experiment in cells lacking endogenous Pex14. Regardless of the approach taken, the authors should improve the quality of this important figure.We had a clear immunoblot image with anti-His antibody by re-analysis of exactly the same samples as those used in original manuscript. We revised the Figure 2D  by replacing the older anti-His blot with new, clear one that shows site-specific phosphorylation of Pex14.7) The claimed role of Pex13 is not clear from the results in Figure 5A. This experiment can be improved if the authors perform IP with phospho-specific antibody to substantiate the claim that phosphorylation of Pex14 alters it complex formation with Pex13. Alternatively, an IP via Pex13 could also be performed and show that the pS232 is coming down with Pex13.2O2-treated cells, while unmodified Pex14 was discernible at a similar level regardless of H2O2 treatment  to the unmodified Pex14 (open arrowhead) in Phos-tag PAGE (top panel). In immunoprecipitation with anti-Pex14-pS232 antibody, Pex13 was co-immunoprecipitated with phosphorylated Pex14, where a substantial amount of unmodified Pex14 was also associated with phosphorylated Pex14 . Together with the data of immunoprecipitation with anti-Pex13 antibody in the original manuscript , we concluded that H2O2-dependent phosphorylation of Pex14 increases its complex formation with Pex13 in peroxisomal membrane.As the reviewers suggested, we performed immunoprecipitation analyses with antibodies to Pex13 and Pex14-p232 . In immunoprecipitation of Pex13, phosphorylated Pex14 was more efficiently recovered from HWe revised Figure 5A by adding the immunoprecipitation data with antibodies to Pex13 and Pex14-p232 and the text regarding this issue in the Results section, Discussion section, Materials and methods section and figure legends.8) The conclusion that phosphorylation is ERK mediated has been shown with a single inhibitor and should be extended to show this more directly by checking Pex14 in ERK knockout or siRNA cells.ERK2 siRNA reduced the protein level of ERK2 by ~80%, but showed no apparent effect on phosphorylation level of Pex14 induced by H2O2 treatment . Upon H2O2 treatment, phosphorylation of ERK1 was instead induced in ERK2-depleted cells at the comparable level to those of ERK2 in control siRNA-transfected cells, suggesting the complementation of ERK2 depletion by ERK1. Collectively, ERK2-knockdown by siRNA transfection did not further support the possible role of ERK in H2O2-induced phosphorylation of Pex14 observed in the experiment using kinase inhibitors .As the reviewers suggested, we carried out knockdown of ERK by siRNA transfection. As simultaneous knockdown of ERK1 and ERK2 severely affected cell growth, ERK2, a mainly expressed ERK in Fao cells  was selected as a target. Transfection of 2O2-induced Pex14 phosphorylation. We revised the text regarding this issue in the Results section, Materials and methods section and figure legends.We added the data of ERK2-knockdown experiment as Figure 2—figure supplement 2. As described in the Discussion section in the initially submitted manuscript, addressing the kinase that directly phosphorylates Pex14 and the upstream kinase cascade is the most important task in the future works. At present, we think that more studies are required to show the involvement of ERK kinases in the H


The mitochondrial permeability transition pore (mPTP) plays a critical role in the pathogenesis of cardiovascular diseases, including ischemia/reperfusion injury. Although the pore structure is still unresolved, the mechanism through which cyclophilin D (CypD) regulates mPTP opening is the subject of intensive studies. While post-translational modifications of CypD have been shown to modulate pore opening, specific phosphorylation sites of CypD have not yet been identified. We hypothesized here that phosphorylation of CypD on a serine residue controls mPTP opening and subsequent cell death at reperfusion. We combined in silico analysis with in vitro and genetic manipulations to determine potential CypD phosphorylation sites and their effect on mitochondrial function and cell death. Importantly, we developed an in vivo intramyocardial adenoviral strategy to assess the effect of the CypD phosphorylation event on infarct size. Our results show that although CypD can potentially be phosphorylated at multiple serine residues, only the phosphorylation status at S191 directly impacts the ability of CypD to regulate the mPTP. Protein-protein interaction strategies showed that the interaction between CypD and oligomycin sensitivity-conferring protein (OSCP) was reduced by 45% in the phosphoresistant S191A mutant, whereas it was increased by 48% in the phosphomimetic S191E mutant cells. As a result, the phosphoresistant CypD S191A mutant was protected against 18 h starvation whereas cell death was significantly increased in phosphomimetic S191E group, associated with mitochondrial respiration alteration and ROS production. As in vivo proof of concept, in S191A phosphoresistant rescued CypD-KO mice developed significantly smaller infarct as compared to WT whereas infarct size was drastically increased in S191E phosphomimetic rescued mice. We conclude that CypD phosphorylation at S191 residue leads to its binding to OSCP and thus sensitizes mPTP opening for the subsequent cell death. Mitochondrial permeability transition pore (mPTP) opening is recognized to replay a crucial role in this phenomenon5. The identity of the current pore-forming protein(s) of mPTP is still debated6. Although recent publications show that mPTP formation does not require the c, b, or oligomycin sensitivity-conferring protein (OSCP) subunits of the ATP synthase7, other studies suggest that subunit c is the conducting core9, or the interface between two ATP synthases forms the conductive mPTP10. It cannot be excluded that in addition to a core‐conducting structure, several alternative structures exist as regulatory components.During myocardial infarction, reperfusion injury represents a significant amount of the resultant irreversible damage11. CypD was identified as a target of the cyclosporine A (CsA) drug, which inhibits mPTP opening12. In vitro, the addition of CsA inhibits mitochondrial swelling and increases mitochondrial calcium retention capacity (CRC)13, conferring protection against lethal stress. In vivo, the inhibition of CypD has been reported to reduce infarct size in preclinical studies17, but failed to be translated in human clinics19, highlighting the need for new CypD inhibitors. Additionally, the molecular mechanism of how CypD binds to the mPTP still remains unknown.The prolyl isomerase cyclophilin D (CypD) in the matrix is an important regulator of mPTP opening24. It was reported that Glycogen Synthase Kinase-3β (GSK3β) inhibitors prevent CypD phosphorylation, yet the phosphorylation site(s) was (were) not identified26. Indeed, the kinase GSK3β has been shown to mediate mPTP opening by acting as a final integrator of multiple signaling27. Moreover, we and others demonstrated that GSK3β inhibition was required for pre/postconditioning mediated resistance to ischemia-reperfusion (I/R) injury and mPTP opening30. Therefore, we decided to take advantage of in silico analysis of potential CypD phosphorylation sites for GSK3β to reveal a new CypD phosphorylation site crucial for mPTP sensitivity.Significant progress has been made regarding the upstream regulation of the mPTP, particularly the post-translational modification of CypD and how it modifies pore sensitivityBy combining genetic manipulations with both in vitro and in vivo mouse heart models, we demonstrated that the extent of myocardial damage is associated with the CypD phosphorylation on its S191 residue, thereby inducing its binding to OSCP, ROS production and the subsequent mPTP opening.33 have been suggested to alter mPTP regulation. We and others have previously reported that inhibition of GSK3β abolishes mPTP opening35 and that GSK3β can phosphorylate CypD in vitro26. To investigate whether CypD may act as a potential substrate for GSK3β, we performed CypD-in silico analysis for putative GSK3β phosphorylation sites (S/T-x-x-x-S/T)36. In silico sequence analysis performed by NetPhos3.1 revealed 10 consensus phosphorylation sites on residues S38, S39, S40, S41, S42, S43, S119, S123, S186 and S191 , suggesting that serines S38 to S43 may act as a negative regulator of the phosphorylation status of CypD. More interestingly, when S191 was mutated to alanine, the CypD phosphorylation was significantly reduced, averaging 0.44 ± 0.09% vs. 3.63 ± 1.62% in the WT group (p = 0.0481)  cleavage zone and to avoid any neighboring serine phosphorylation, we have grouped them under a single mutant S38–43A. CypD-FLAG-tagged mutant proteins were then overexpressed in HEK cells. After FLAG immunoprecipitation, our results showed that mutants S119A, S123A and S186A did not significantly modify the global serine phosphorylation level of CypD, averaging, respectively, 1.20 ± 0.62%, 3.10 ± 0.98% and 4.26 ± 1.66% vs. 3.63 ± 1.62% in the WT group (81) Fig. , suggestp < 0.05)  . These data suggest that throughout the putative serine residues identified in silico, only the phosphoresistant mutant S191A afforded a similar protection as loss of CypD in the regulation of the mPTP opening.We next tested whether identified mutations could alter mPTP opening. To this end, CRISPR/Cas9 was used to knock out endogenous CypD in HEK cells and subsequently rescued with either WT or mutated CypD  Fig. . In S119WT) Fig. . While tp < 0.05)  . In these conditions, our results showed that the mutant S191E not only reversed the mPTP protection induced by the phosphoresistant S191A mutant (05) Fig. , but als05) Fig. . Altoget37. To further define the mechanism of how CypD regulates mPTP opening, we then investigated whether S191 residue plays a role in CypD binding with the mPTP. It has been recently reported that CypD could bind to the OSCP subunit of the ATP synthase, a proposed pore constituent38. We here hypothesized that CypD phosphorylation on its residue S191 induces its translocation and binding to the OSCP to favor mPTP opening. To test our hypothesis, we used three different strategies to measure the effect of CypD phosphorylation on the interaction of CypD with the pore. First, we designed a FRET system in which OSCP was tagged with Clover GFP , whereas the phosphomimetic mutant S191E enhanced the FRET ratio up to 2.44 ± 0.94 (p < 0.05 vs. WT)  . These data suggest that the phosphorylation event of CypD at serine 191 regulates the binding of CypD to the OSCP subunit of the ATP synthase, which is known to sensitize mPTP opening39.It was recently demonstrated that CypD not only regulates the mPTP but also regulates the dynamic assembly of mitochondrial ATP synthaseGFP Fig. . DownstrGFP Fig. . Our resWT) Fig. . These rWT) Fig. , showingWT) Fig. , whereas05) Fig. . To furtp < 0.05, Supplementary Fig. Interestingly, in basal condition, although oxidative phosphorylation did not seem to be different in WT, S191A, and S191E mutants    Fig. . Moreove05) Fig. . Altogetp < 0.05 vs. S191E), these interactions were significantly increased in injured S191E mutant cells after starvation. Altogether, these data demonstrate that CypD phosphorylation at S191 enhances binding of CypD to the OSCP, alteration of mitochondrial function, and subsequent cell death, suggesting that residue S191 is important in this regulation.It is worth noticing that the protein interaction of CypD with OSCP was positively correlated with the level of cell death. Indeed, as shown in the Fig. p = ns, Supplementary Fig. p = ns, Supplementary Fig. Lastly, we tested whether the phosphorylation level of CypD regulates in vivo I/R injury using a mouse model. To rule out a possible effect of the adenovirus deliverance and/or infection process on the infarct size by itself, a pilot study using GFP adenovirus was used as a positive infection control. As shown in the Supplementary Fig. p = ns among groups) and the CypD protein rescue level was also comparable among groups (p = ns)     Fig. . It is wWT) Fig. . ConversWT) Fig. , but alsThe present study suggests that the CypD phosphorylation at S191 controls cell death and the extent of myocardial infarction in vivo, via the modulation of the mPTP opening which is dependent of the binding state of CypD with OSCP, the mitochondrial electron transport chain coupling and the ROS generation.33. Based on publications suggesting that GSK3β/CypD complex plays a pivotal role in the regulation of the mPTP43, and although it has been reported that the consensus sequence S/T-X-X-X-S/T is neither essential nor sufficient to guide GSK3β-dependent phosphorylation44, our in silico analysis shows that CypD presents no less than ten potential target sites for GSK3β. Even if the role of GSK3β on CypD has been recently questioned45, these results are in line with a previous report showing that GSK3β can directly phosphorylate CypD at Ser/Thr in vitro26. We assume that these preferential domain-specific kinase-substrate relationships can be used only to distinguish cognate kinase-substrate pairs from all other non-cognate combinations and that further studies are required to provide the proof that CypD is a bona fide substrate for GSK3β.Although the pore structure is still debated, the mechanism through which CypD regulates mPTP opening is the subject of intensive studies. Post-translational modifications of CypD have been shown to modulate pore opening26. The study by Parks et al. addressed the question why the hearts from global MCU-KO mice are not protected from ischemic injury by investigating whether adaptive alterations occur in cell death signaling pathways. Their results show that MCU-KO mitochondria exhibit an increase in phosphorylation of CypD-S42 which decreases mPTP calcium sensitivity thus allowing activation of mPTP in the absence of an MCU-mediated increase in matrix calcium. Using a phosphoresistant S42A strategy, they conclude that the phosphorylation of CypD at S42 seems to regulate mPTP. However, in the present study, in silico results show that CypD can be also potentially phosphorylable on neighboring serines 38, 39, 40, 41, and 43. Since making a targeted mutation into a residue-long flanking regions is known to involve a risk for the kinase to phosphorylate neighboring serines +1, 2, 3 or 446, their mutant S42A has an high risk to have a phosphorylation compensation which was not discussed. For this reason, we chose to regroup all serines in a single mutant S38–43A, avoiding any possible phosphorylation compensation in neighboring serines. Our results show that the S38–43A mutant significantly increases the global phosphorylation level of CypD associated with a significant decrease of the CRC as compared to the WT group. Conversely to Parks′ results, these results might suggest that the multiple mutations of serines could potentiate the phosphorylation of CypD and the over-sensitization of the mPTP opening. Altogether, these results suggest that the domain S38–43, located just after the MTS and before the cyclophilin-isomerase-domain, seems to play a role in the phosphorylation process of CypD and the activation of the mPTP opening. But the mechanism by which this domain regulates mPTP opening by phosphorylation, modification of the protein addressing and/or the modification of the isomerase activity requires additional work.In this context, we show that the global phosphorylation level of CypD is dependent on the phosphorylation of some in silico identified serines and that the phosphorylation event of CypD seems to control the mPTP opening. Our results are in line with recent reports showing that the phosphorylation of CypD could be associated with increased mPTP opening47, these results suggest that even if other serines are functionally actives (including S42), S191 seems to plays a preponderant role on the regulation of mPTP opening through the phosphorylation event of CypD26.On the other hand, our results show that only the phosphoresistant mutant S191A is able to completely abolish the phosphorylation of CypD, and confers resistance of mPTP opening against calcium. Since cardioprotection requires inhibition of multiples kinases49 and oxidative phosphorylation50. Our results add to this by showing that the phosphorylation event of CypD at S191 regulates mitochondrial function, such as OXPHOS and ROS production. Indeed, while protected phosphoresistant S191A cells exhibit improved mitochondrial respiration associated with a reduction of ROS production, phosphomimetic S191E cells are more sensitive to cell death stress due to an alteration of oxidative phosphorylation and consecutive ROS production. Thus, our result are in line with previous studies showing that the Ca2+ overload induced by the extensive ROS generation causes necrosis through the enhancement of the permeability of the mitochondrial membrane and mPTP opening52. Altogether, our results suggest that CypD phosphorylation at S191 not only regulates mPTP opening but also seems to be a crucial regulator of ROS production, underlining the key role of the triangle CypD-phosphorylation/ROS-production/mPTP-opening in cell death pathway, especially during ischemia-reperfusion in the heart in vivo52.Growing data indicate that CypD, in addition to its role in mPTP, may play a pivotal role in regulating overall cell metabolism. Studies including genetic or pharmacological inhibition of CypD revealed its contribution to the regulation of respiratory functionIn this context, the most significant finding to emerge from this study is that the extent of myocardial damage is dependent on the CypD phosphorylation event on its S191 residue. To our knowledge, this is the first in vivo research study to have demonstrated that the phosphorylation of CypD on a precise serine residue is involved in acute myocardial infarction. Our results show not only that the infarct size is dependent of the CypD-S191 phosphorylation, but also that phosphorylation inhibition of this same serine significantly reduces the infarct size, providing new insight in cardioprotection. Moreover, although we did not observe any heart rate alteration or any arrhythmic event after the CypD rescue in our acute mouse model (data not shown), these potential mechanisms underlying S191 phosphorylation of CypD are of interest for future LV remodeling studies.54, PiC55 and the FOF1-ATP synthase subunit OSCP50 and thus stimulate mPTP induction. Our data further show that the CypD/OSCP interaction is positively correlated with the sensitization of mPTP opening and that the phosphorylation event of CypD at S191 regulates the binding of the CypD with the mPTP.Regarding possible mechanisms for the modulation of mitochondrial function and subsequent cell death in our model, in addition to post-translational modifications, it has been demonstrated that interaction with proteins in the matrix and IMM can modulate the CypD activity and mPTP opening. Numerous studies provided evidence that CypD can interact with other proteins including ANT56. Such mechanisms remain to be investigated in-depth in future studies. However, although we notice a difference of CRC in our mutants, the slope of mitochondrial calcium uptake is similar in all groups ups Fig.  suggestiIn summary, our work identifies S191 of CypD as a key regulator of ROS production, mPTP opening and cell death. As illustrated in Fig. Material and methods are detailed in the Supplemental methods and figure legends. The data, analytic methods, and study materials will be/have been made available to other researchers for purposes of reproducing the results or replicating the procedure.Experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals , and were approved by local institutional animal research committees′ #19896–201903212127912 and #901I.57. Following starvation, cells were collected and resuspended in PBS or respiration buffer before analysis.HEK cells were serum starved in serum-free high-glucose DMEM in the presence of pyruvate for 18 h8 PFU), which was injected in 10–12 different sites of the LV wall to cover the maximum area of the area at risk58. The chest cavity was closed, and the mice were allowed to recover for 1 week prior to I/R surgery. Mice underwent I/R surgery as previously described29 (Supplementary Fig. Mice were randomized to receive 25 µl of an adenovirus solution (CypD mutants at 5 × 10t-test. For results with 2 variables, a two-way ANOVA followed by a Tukey′s post hoc test was performed. Statistical significance was set at a threshold of p ≤ 0.05. No data/animals were excluded from the study. The data were computed using GraphPad Prism 6.1 software.Data for multiple experiments were quantified and expressed as mean ± SD where indicated. Differences in means among multiple groups were analyzed using one-way ANOVA with a Bonferroni’s or Tukey’s post hoc test to determine significance between groups. For results with two groups, normal distribution of values was verified using the Kolmogorov–Smirnov test and the differences between groups were then determined using a two-tailed paired students Supplmental methods and figure legendsSupplemental TablesSupplemental Figure 1Supplemental Figure 2Supplemental Figure 3Supplemental Figure 4Supplemental Figure 5


They unveiled distinct conformations, formations of short helices and interactions of the loop with neighboring glycans that could potentially regulate the accessibility of the cleavage site to proteases and its processing. In most conformations, this loop protrudes from the spike, thus representing an attractive SARS-CoV-2 specific therapeutic target.At the end of 2019, a new highly virulent coronavirus known under the name SARS-CoV-2 emerged as a human pathogen. One key feature of SARS-CoV-2 is the presence of an enigmatic insertion in the spike glycoprotein gene representing a novel multibasic S1/S2 protease cleavage site. The proteolytic cleavage of the spike at this site is essential for viral entry into host cells. However, it has been systematically abrogated in structural studies in order to stabilize the spike in the prefusion state. In this study, multi-microsecond molecular dynamics simulations and  A new s1.1The entry of coronaviruses into host cells is mediated by the spike glycoprotein (S protein) , which iS proteins can be divided into several conserved domains and motifs a. The N-1.2At the beginning of 2020, structures of the SARS-CoV-2 S protein ectodomain trimer were published  providin1.3in vitro experiments reports that this tribasic site is not only recognized and cleaved by furin  also contains a related dibasic cleavage site e. Proteo22.1Two 10-µs molecular dynamics (MD) simulations of the SARS-CoV-2 S protein under physiological conditions  were carried out by D. E. Shaw Research on their Anton2 supercomputers. These simulations are freely available and can be downloaded from the D. E. Shaw Research website . The clo2.2A principal component analysis (PCA) of the molecular dynamics simulations was performed using ProDy . The k-m2.3The missing loop containing the tribasic protease cleavage site was modeled using the remodel procedure in Rosetta . The pro33.1In CoVs, two conserved beta-strands form an anti-parallel beta-sheet connected by a loop, which contains the S1/S2 protease cleavage site d. In ordIn order to isolate representative conformations, a k-means clustering was carried out in the eigenspace defined by the first three principal components, which account for 81% of the total variance. The optimal number of clusters (k = 8) was determined using the Silhouette method . During ab initio modeling procedure of Rosetta , which includes experimentally verified furin cleavage sites, it appears that a proline residue at position P5, i.e., the 5th residue prior to the furin cleavage site, is rare and appears in only 5 out of 132 sequences . Since proline is unable to adopt several main chain conformations in proteins, it imposes strong conformational restraints on the peptide chain. It is therefore often found in turns, which force the peptide chain to change directions and separate secondary structures. This is supported by the ab initio modeling, where the proline is found at the N-terminus of short helices in several models . GlycanMD-21452 .The presence of alanine (A684) at position P2, i.e., the 2nd residue prior to the furin cleavage site, is also unusual in a furin cleavage site and appears in only 5 out of 132 sequences in the FurinDB . This po3.3From a functional point of view, the insertion of a multibasic protease cleavage site at S1/S2 in SARS-CoV-2 is an important new feature, which may account for its increased virulence. The highly pathogenic avian influenza (AI) viruses are known to have evolved from low-pathogenic AI viruses . While lAn additional feature that will influence the protease cleavage efficiency at the S1/S2 site of CoVs, is the length of the loop containing this site, which is flanked by two conserved beta-strands d. Recent4ab initio modeling showed that the loop containing this cleavage site protrudes from the S protein surface, making it accessible to proteases. The neighboring N-linked glycans might, however, modulate accessibility of the protease cleavage site. The ab initio modeling also indicated that the loop might be moderately structured forming short helices close to the cleavage site. The impact of the nature, length, structure and dynamics of this loop on protease cleavage efficiency, and ultimately, the overall pathogenicity of CoVs remains, however, an open question that warrants immediate detailed analysis due to the current pandemic crisis.The novel multibasic S1/S2 protease cleavage site is an important new feature of SARS-CoV-2 and represents an attractive therapeutic target, since viral entry could be reduced or inhibited by blocking the proteolytic cleavage event. Our analyses of molecular dynamic simulations and Thomas Lemmin: Conceptualization, Methodology, Software, Data curation, Formal analysis, Investigation, Resources, Visualization, Writing - original draft, Writing - review & editing, Funding acquisition. David Kalbermatter: Formal analysis, Investigation, Visualization, Writing - review & editing. Daniel Harder: Formal analysis, Investigation, Writing - review & editing. Philippe Plattet: Conceptualization, Writing - original draft, Writing - review & editing, Funding acquisition. Dimitrios Fotiadis: Conceptualization, Writing - original draft, Writing - review & editing, Supervision, Project administration, Funding acquisition.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.


This study focuses on the feature extraction and classification of surface discharges of ice-covered insulator strings during process of alternating current flashover. The test specimen was the five units suspension ceramic insulators, which was artificially accreted with wet-grown ice in the cold-climate room of CIGELE. Based on the IEEE Standard 1783/2009, flashover experiments were conducted on iced insulators to measure the minimum flashover voltage (VMF) and record the propagating process of surface discharges to flashover by using a high-speed video camera. The gray-level co-occurrence matrix (GLCM) method has been used to extract four parameters of arc discharge images features that characterize different stages of flashover process. The parameters are angular second moment (ASM), contrast (CON), inverse difference moment (IDM) and entropy (ENT). These statistical parameters of GLCM can be extracted to reveal the underlying properties of ice flashover on the insulator surface from the quantitative perspective. The different values of these indicators are representative of the different stages in the process of arc discharge. Once the value of quantitative indicators  of surface discharges exceeds the threshold value, the higher flashover risk of iced insulators will appear. Hence, the proposed methods are helpful to understand and monitor surface discharge on iced outdoor insulator strings for preventing flashover accidents. From viewpoint of reducing the ice flashover accident caused by ice-covered insulators, many investigations have studied the properties of ice flashover process15. It is obtained that the icing flashover is a complex discharge phenomenon, which results from various parameters, such as the types of insulators, the shape of ice, the conductivity of the ice, amount of air gaps of iced insulator strings and atmospheric environment.Ice flashover experience has been recognized as one of serious accidents for power systems operating in most of atmospheric icing regions16. Wen et al. used ultraviolet (UV) imaging technology to observe the discharging phenomenon. The number of UV photons was regarded as the characteristic quantity to detect discharging, which clearly revealed a periodical corresponding relationship between the leakage current and the UV image during the icing flashover. Finally, the three-segment method of UV warning was proposed in the discharging process of ice-covered insulators18. Porkar et al. put forward the method of image charges to improve static and dynamic arc models. The improved arc model had a better correspondence with the test results than the previous model. It could be used as a powerful tool for reducing icing flashover accident of insulator strings19. For optimizing the method of insulator flashover, Taheri et al. established an empirical model by the electrical conductivity of the melted ice accumulation to determine the flashover voltage under icing conditions and investigated the influence of air gaps on the flashover stress of iced post insulator20. Liu et al. found that the arc on the long ice-covered insulators need more time up to the critical length and the development of arc will be easily effected by the decreasing of melted water conductivity and ice shedding than the short insulators21. Hao et al. researched the natural iced of in-service glass insulator string by image processing methods. Based on the GrabCut segmentation method, the algorithm of graphical shed spacing and graphical shed overhang are presented by recognizing the convexity defects of the contours of ice-covered insulators23.Li et al. analyzed the discharge images of ice-covered insulator during the flashover for the characteristic extraction. They use the image gray value and the arc length as the characteristic value for the flashover prediction24.These previous studies are helpful to improve the ability of judging the accident risk of insulator in power system. Nevertheless, these previous investigations have disadvantage dealing with the complexity phenomenon of ice accretion and indeterminacy performance of partial discharge arcs, so it is hard to exactly locate initiation and propagation of partial arcs discharge along surface of iced insulator strings, which influences the condition of discharge development and the significant factors of flashover experience28.Over the past decades, many researchers have investigated the discharge mechanism and performance evaluation of iced insulators to decrease the ice flashover accident. Ma et al. used the ultraviolet photons to reveal the discharge properties of iced insulators. They found that the ultraviolet photons could be regarded as features to effectively evaluate the reliability of ice-covered insulatorsUp to now, although the optical measurement can record strength and dynamic distribution of surface discharges, few investigations have been conducted on the feature extraction and analysis of visual images of arc discharge along the iced outdoor insulator strings. In this paper, the multiscale geometric analysis method is presented for the feature extraction and classification of arc discharges images of iced insulator strings during AC flashover process. Due to the indeterminacy of cold outdoor environment, the natural ice-covering flashover is complex. Thus, the artificial ice-covering flashover is different from the natural ice-covering flashover in some degree. However, the image processing method proposed in this paper can overcome these differences because of the process of ice-covering flashover can be characterized by image parameters which is only related to the pixel intensity, the homogeneity of the discharge image and the local variations in gray level. In accordance with the IEEE Std. 1783/2009, all the experiments were conducted on the five-unit suspension ceramic insulators. The minimum flashover voltages were measured and dynamic discharge behaviors were recorded by using a high-speed video camera during the flashover process. Based on the image analysis of surface discharges, the quantitative indicators were proposed to reveal the discharge propagating characteristics of the flashover on iced insulator strings.Figure The tests were conducted in a designed artificial climate room with a length of 4.8 m, a wide of 2.8 m and a height of 3.5 m of CIGELE Laboratories. By using the proportional integral and differential system, the freezing devices can make the ambient temperature drops to -12 °C after the test setup was fixed. When making the insulators under the setting room temperature last about sixteen hours, the voltage of 75 kVrms (15 kVrms per unit) energized on insulators. Meanwhile, the water conductivity was set at 30 µS/cm. The spray device mainly consists of a water supplying system and a wind blowing equipment. Ice was formed from super cooled droplets produced by the former system through four oscillating nozzles, which can accrete ice in the form of a uniform ice thickness on the surface of insulators; The latter system produced the uniform air flow through a series of fans with a diffusing honeycomb panel. The wind speed was fixed at 3.3 m/s to blow on the windward side of insulators in the ice accumulation period and the up-to-down oscillating rate of nozzles was 16 rpm.Once the ice accretion was finished, then the flashover experiment was prepared and the duration of the preparation was approximately two minutes. After that, for reaching the estimated flashover voltage, the voltage was reapplied on insulators with a rate of 3.9 kV/s. Once flashover appears or no flashover for duration of 15 min, the applied voltage was diminished or increment by the settled voltage step of 3 kV for following experiment. The minimum flashover voltage (VMF) and the maximum withstand voltage (VMW) are determined when VMF and VMW differs by a voltage step of 3 kV, and when VMF is applied two flashovers in all experiments were produced. Simultaneously, the high-speed video camera which has 256 grey levels was selected visually for recording the performance of arc discharges by the rate of six thousand frames per second.Before icing flashover tests, the icing state of ice-covered insulators is shown in Fig. 30. Under the same condition of icing accumulation, the performance of ice accreted on insulator strings are identical, as shown in Fig. When the icing accumulation completed, the icing flashover test was carried out to obtain VMF with the value of 84 kVrms, as depicted in Fig. Initial stage of surface discharge: Generally, due to the insulators was energized at AC voltage, ice accumulated on insulator surface is not symmetrical. Moreover, most of ice accretion along the windward side of the insulators surface under the test icing environment, and the thermal energy of discharge generated, ice shedding, all of these led to the icing-free air gaps formed on the insulators. According to observations from Fig. 29. The leakage current generated heating energy on ice plane and also in the ambient air, which has a positive effect on the arcs re-ignition under the applied voltage. When the applied voltage near the minimum flashover voltage level, the electrical discharge sustaining stable burning in the air gaps. During AC pollution flashover process, the resistance of polluted surface is a vital factor in extinguishing and re-ignition of arcs discharge. Similarly, the surface resistance of iced insulators play a fundamental part in arc formation, re-ignition and propagation under the applied voltage. Arcs are unable to kept in a stationary location under the AC voltage, but instead develop and extinguish at each half-cycle of AC voltage. The arc root is generated in the air gaps that led to the non-uniformity distribution of current along the surface of iced insulators, which turn to effects the residual surface resistance. If this resistance is low, the arc current will be higher. Therefore, the arcs may develop across the air gaps directly from high-voltage electrode to the grounded line.Development stage of surface discharge: The discharge arc spread from ice layer to the bottom side of insulator strings after that it along the icing caps under the freezing conditionsStage of complete flashover: The partial arcs across the air gaps then move to the HV electrode near by, which induce the distribution of voltage to shift on the iced insulators. The stable and bright discharge arcs extend between the icicles and electrode. The local discharge arcs can spread along the ice plane with an increasing speed. The luminosity of discharge path show longer and bigger characteristics in Fig. Due to the high-speed video camera operated at the sampling rate of 6000 fps, there are a large number of repetitions of images at the same stage during the flashover process. In different flashover stages, there are obvious differences in flashover arc length, flashover arc position, continuity of flashover arc, and flashover arc area, and there will be dark-bright-darkness in each flashover stage during the experiment process. This article combines the various characteristics of the above flashover arc, divides the entire flashover process into different stages, and selects clear and obvious pictures in each stage as the research object of this article, so as to fully reveal the process from arc discharge to flashover. As shown in Fig. GRAY-level co-occurrence matrix (GLCM) with the related statistics is known as one of popular estimating method to reveal the image properties. The GLCM is defined as Eq. , which iAfter obtaining the GLCM of discharge images, the statistical parameters of GLCM are extracted to reveal the underlying properties of arc discharge from the quantitative perspective. The indicators are angular second moment (ASM), contrast (CON), inverse difference moment (IDM) and entropy (ENT). Each stage of arc discharges can be indicated by the four parameters which are extracted from the selected images through gray-level co-occurrence matrix (GLCM) method. The basic principle of the GLCM method is to analyze the pixel characteristics of the image. The influence of different ice thickness, wind speed and other factors on the flashover process is finally reflected in the change of image parameters. The GLCM method proposed to process the image parameters. The change of the image parameters will only affect the calculation result, not the calculation accuracy. Therefore, factors such as different ice thickness and wind speed will not affect the performance of the GLCM method. The model is only related to the image parameters, such as the pixel intensity, the homogeneity of the discharge image and the local variations in gray level. And this model can be applied to insulators with different geometric shapes and different surface contamination levels.ASM can be obtained as Eq.  to refleCON is calculated by Eq. , which i2), the far separate areas (i ≠ j) can have little influence on the IDM, which means the higher IDM value for local homogeneity and lower for the local inhomogeneity. Therefore, IDM is a measure of the appearance frequency for near neighboring pairings while CON is that of distant pairings.The definition of IDM is shown as Eq. . AlthougENT is defined by Eq. 5\documenThe variation of ASM of surface discharge images during AC flashover process is shown in Table 2 term, which can indicate the relative location of surface discharges along the ice-covered insulator string. If the discharge image shows a large value of this parameter, it can be obtained that the concentration of surface discharges is relatively localized in some specific spatial regions and the sustainable arcing discharge channel is being formed for the occurrence of ice flashover on the insulator surface. As shown in Table Table The variation of IDM of surface discharge images during the development of arc discharges to the flashover is shown in Table Table Figure The reason of the appearance of local arcs is the insulators are bridged by icicles that led to the nonuniform distribution of electric field. As stronger white arcs generated, they extend across the ice plane until reach the critical length with increasing the applied voltage.The melting water conductivity increases, which causes the increase of arc discharge intensity. The residual icing layer cannot withstand the applied voltage on it and flashover will take place on the surface ice.These four statistical parameters of GLCM can be extracted to reveal the discharge mechanism of ice flashover process. The different values of these indicators correspond well to the different stages of arc discharge process, which can be used as the risk value for monitoring iced insulators flashover. The proposed model is only related to the image parameters, such as the pixel intensity, the homogeneity of the discharge image and the local variations in gray level. Therefore, this method can be applied to the different insulator geometries and surface contamination levels.The higher ASM value is representative of the weak and random discharges, while the lower ASM value corresponds to the relatively stable discharge behaviors at the air gaps. When the value of ASM exceeds 0.9, the stage of arc discharge is in safe operating area (SOA). When the value of ASM is within 0.9–0.4, the stage of arc discharge is in attention operating area (AOA). When the value of ASM is less than 0.4, the stage of arc discharge is in dangerous operating area (DOA).The value of CON is a measure of the local variations in gray levels of the discharge image which can indicate the relative location of surface discharges along the ice-covered insulator string by analyzing, whereas the IDM is a measure of the appearance frequency for near neighboring pairings. When the value of CON is below 0.1, the stage of arc discharge is in safe operating area (SOA). When the value of CON is between 0.1 and 6.0, the stage of arc discharge is in attention operating area (AOA). When the value of CON exceeds 6.0, the stage of arc discharge is in dangerous operating area (DOA).When the value of IDM exceeds 0.9, the stage of arc discharge is in safe operating area (SOA). When the value of IDM is within 0.9–0.3, the stage of arc discharge is in attention operating area (AOA). When the value of IDM is below 0.3, the stage of arc discharge is in dangerous operating area (DOA).The value of ENT can be used to describe the randomness of the arc discharge images. The nonhomogeneous images have high ENT value, while homogeneous scenes have low ENT value. When the value of ENT is less than 0.2, the stage of arc discharge is in safe operating area (SOA). When the value of ENT is between 0.2 and 2.0, the stage of arc discharge is in attention operating area (AOA). When the value of ENT exceeds 2.0, the stage of arc discharge is in dangerous operating area (DOA).According to investigate the statistical indicators of GLCM, the arc discharge properties of icing flashover process are analyzed. The significant conclusions are obtained as follows: