1.Continued surprises in the cytochrome c biogenesis story.
Elizabeth B SAWYER ; Paul D BARKER
Protein & Cell 2012;3(6):405-409
Cytochromes c covalently bind their heme prosthetic groups through thioether bonds between the vinyl groups of the heme and the thiols of a CXXCH motif within the protein. In Gram-negative bacteria, this process is catalyzed by the Ccm (cytochrome c maturation) proteins, also called System I. The Ccm proteins are found in the bacterial inner membrane, but some (CcmE, CcmG, CcmH, and CcmI) also have soluble functional domains on the periplasmic face of the membrane. Elucidation of the mechanisms involved in the transport and relay of heme and the apocytochrome from the bacterial cytosol into the periplasm, and their subsequent reaction, has proved challenging due to the fact that most of the proteins involved are membrane-associated, but recent progress in understanding some key components has thrown up some surprises. In this Review, we discuss advances in our understanding of this process arising from a substrate's point of view and from recent structural information about individual components.
Cytochromes c
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chemistry
;
metabolism
;
Models, Biological
2.Enhanced heterologous expression of the cytochrome c from uncultured anaerobic methanotrophic archaea.
Lingwei CUI ; Xiaojun FAN ; Yanning ZHENG
Chinese Journal of Biotechnology 2022;38(1):226-237
Cytochrome c is a type of heme proteins that are widely distributed in living organisms. It consists of heme and apocytochrome c, and has potential applications in bioelectronics, biomedicine and pollutant degradation. However, heterologous overexpression of cytochrome c is still challenging. To date, expression of the cytochrome c from uncultured anaerobic methanotrophic archaea has not been reported, and nothing is known about the function of this cytochrome c. A his tagged cytochrome c was successfully expressed in E. coli by introducing a thrombin at the N-terminus of CytC4 and co-expressing CcmABCDEFGH, which is responsible for the maturation of cytochrome c. Shewanella oneidensis, which naturally has enzymes for cytochrome c maturation, was then used as a host to further increase the expression of CytC4. Indeed, a significantly higher expression of CytC4 was achieved in S. oneidensis when compared with in E. coli. The successful heterologous overexpression of CytC4 will facilitate the exploitation of its physiological functions and biotechnological applications.
Anaerobiosis
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Archaea/metabolism*
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Cytochromes c/metabolism*
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Escherichia coli/metabolism*
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Heme/metabolism*
3.Effects of experimental varicocele on mitochondria calcium and cytochrome C of the epididymis in adolescent rats.
Xiaoru MA ; Shuqiu WANG ; Yuexin WANG ; Shuxiang WANG ; Weiqun WANG ; Jianhua ZHANG ; Yongyi LI
National Journal of Andrology 2004;10(6):420-422
OBJECTIVETo investigate the effects of experimental varicocele on mitochondria calcium and cytochrome C of the epididymal cells in adolescent rats.
METHODSForty male adult Wistar rats were divided into two groups randomly: varicocele group (VG) and sham operation group (SOG) by partial ligation or exposure of the left renal vein. Bilateral epididymides were removed after ten
RESULTSThe content of mitochondria weeks. Mitochondria calcium and cytochrome C levels of the epididymal cells were detected. calcium decreased (P < 0.001 ) while that of cytochrome C increased (P < 0.05) markedly in the experimental group compared with SOG.
CONCLUSIONCalcium dyshomeostasis and mitochondrial damage of the epididymal cells caused by varicocele may play an important role in leading to subfertility.
Animals ; Calcium ; metabolism ; Cytochromes c ; analysis ; Epididymis ; metabolism ; Infertility, Male ; etiology ; Male ; Mitochondria ; metabolism ; Rats ; Rats, Wistar ; Varicocele ; metabolism
4.Apoptosis of jurkat cells induced by ursolic acid and its mechanism.
Bin WU ; Xu WANG ; Hui-Han WANG ; Hong-Tao WANG ; Wei YANG ; Zuo-Fei CHI ; Zhuo-Gang LIU
Journal of Experimental Hematology 2010;18(1):61-66
The aim of this study was to investigate the apoptosis effect of Jurkat cells induced by ursolic acid (UA) and its molecular mechanism so as to provide the theoretical basis for treatment of hematological malignancies by using UA. The cytotoxic effect of different concentration UA on Jurkat cells and inhibitory effect of caspase-9 inhibitor on cytotoxicity of UA were assayed by using WST-8 method; the Jurkat cells treated with 20 or 40 micromol/L UA for 2 or 4 hours were collected and were stained by Annexin/PI, then the apoptosis rate of Jurkat cells was detected by flow cytometry; the Jurkat cells in logarithmic growth phase were collected after treatment with different concentrations of UA for different times, the cell protein was extracted, then the activation of caspase-9, -3 and cytochrome C as well as phosphorylation level of Akt were determined by Western blot. The results indicated that the cytotoxic effect of UA on Jurkat cells was significant. UA induced apoptosis of Jurkat cells. Caspase-9, caspase-3 and cytochrome C were activated, and the phosphorylation of Akt was inhibited in the Jurkat cell apoptosis process induced by UA. It is concluded that the UA shows significant cytotoxic effect on Jurkat cells, UA can induce apoptosis of Jurkat cells through the mitochondria pathway. The mechanism may be associated with the inhibition of Akt phosphorylation.
Apoptosis
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drug effects
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Caspase 3
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metabolism
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Caspase 9
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metabolism
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Cytochromes c
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metabolism
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Humans
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Jurkat Cells
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Triterpenes
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pharmacology
5.Effect of rhIGF-1 on cytochrome C and caspase-3 expression in neonatal rats with hypoxic-ischemic brain damage in vivo.
Ming-Yan HEI ; Wei-Qing HUANG ; Fu-Rong LIU
Chinese Journal of Contemporary Pediatrics 2010;12(12):963-966
OBJECTIVETo study the effect of rhIGF-1on the mRNA and protein expression of cytochrome C (Cyt-C) and caspase-3 in neonatal rats with hypoxic-ischemic brain damage (HIBD).
METHODSNinety neonatal Sprague-Dawley rats were randomly divided into three groups: normal control, HIBD, and HIBD+rhIGF-1 (rhIGF-1 was given intraperitoneally right after HI). Rat HIBD model was prepared according the Rice-Vannucci method. RT-PCR and Western blot methods were used to measure the mRNA and protein expression of Cyt-C and caspase-3 24, 48 and 72 hrs after HI (n=10 each time point).
RESULTSAt all time points, both Cyt-C mRNA and caspase-3 mRNA expression levels in the HIBD group increased compared with those in the normal control group, and those in the HIBD+rhIGF-1 group also increased compared with that in the normal control group but decreased compared with that in the HIBD group. There were statistical significances among the three groups (P<0.01). At all time points, the changes of both Cyt-C and caspase-3 protein expression in the three groups were similar to those of the mRNA expression: both Cyt-C and caspase-3 protein expression levels increased in the HIBD group compared with those in the normal control group, and those in the HIBD+rhIGF-1 group also increased compared with those in the normal control group but decreased compared with those in the HIBD group. There were statistical significances among the three groups (P<0.01).Pearson correlation analysis showed that mRNA and protein expression of Cyt-C were positively correlated to casapse-3 mRNA and protein expression in the HIBD and the HIBD+rhIGF-1 groups.
CONCLUSIONSrhIGF-1 may inhibit the Cyt-C release and caspase-3 expression, and thus provides neuroprotection against HIBD in neonatal rats.
Animals ; Animals, Newborn ; Brain ; metabolism ; Caspase 3 ; metabolism ; Cytochromes c ; Hypoxia-Ischemia, Brain ; metabolism ; Rats ; Rats, Sprague-Dawley
6.Impacts of electroacupuncture on neurological function and protein expressions of apoptosis-related Cyt-C and Caspase-9 in rats with traumatic brain injury.
Ting GU ; Xin WANG ; Huan YANG ; Xue-Ni SHE ; Kun-Huang-Yu CHEN ; Tao WU ; Qi YANG ; Qiang YANG ; Rui-Hui WANG
Chinese Acupuncture & Moxibustion 2020;40(7):749-755
OBJECTIVE:
To observe the impacts of electroacupuncture (EA) on neurological function, the pathological morphology in brain tissue, apoptosis level and the protein expressions of apoptosis-related cytochrome C (Cyt-C) and cysteine aspartic acid protease-9 (Caspase-9) in the rats with traumatic brain injury (TBI) and explore the potential mechanism of EA in treatment of TBI.
METHODS:
A total of 70 clean-grade SD mice were randomized into a blank group (8 rats), a sham-operation group (8 rats), a model group (27 rats) and an EA group (27 rats). In terms of interventions of 3, 7 and 14 days, 3 subgroups were divided in the model group and the EA group successively, 9 rats in each subgroup. The modified Feeney free-fall percussion method was adopted to establish TBI models of rats. In the sham-operation group, only the skull was exposed and drilled and no free-fall percussion was exerted. One day after modeling, EA was given in the rats of EA group at "Shuigou" (GV 26), "Baihui" (GV 20) and "Neiguan" (PC 6) and "Zusanli" (ST 36) on the affected side, with intermittent wave, 2 Hz in frequency, once daily, 10 min each time, for 3, 7 and 14 days successively. Separately, on the day 3, 7 and 14 of intervention, the modified neurological severity scale (mNSS) was used to evaluate the degree of neurological function injury in the rats, HE staining and Nissl staining were to observe the pathological and morphological changes in brain tissue, TUNEL method was to observe the level of apoptosis in brain tissue and immunohistochemistry (IHC) method and Western blot were to determine the protein expressions of Cyt-C and Caspase-9 in brain tissue.
RESULTS:
Compared with the sham-operation group, on the day 3, 7 and 14 of intervention, mNSS scores were increased obviously in the rats of the model group respectively (<0.01). Compared with the model group, on the day 3, 7 and 14 of intervention, mNSS scores were reduced in the rats of the EA group respectively (<0.05). On day 3 of intervention, in brain injury region of the rats in the model group and the EA group, gross tissue necrosis, nuclear fragmentation, consolidation and obvious vacuolar changes, reduced Nissl bodies and scattered arrangement were found. On day 7 and 14 of intervention, in the model group and the EA group, the new connective tissue filling and normal cells were visible and Nissl bodies increased. The overall repair and Nissl body quantity in the EA group were better than the model group. Compared with the sham-operation group, on day 3, 7 and 14 of intervention, the numbers of apoptotic cells were increased obviously in the model group (<0.01) and they were reduced in the EA group as compared with the model group (<0.05). Compared with the sham-operation group, on day 3, 7 and 14 of intervention, the protein expressions of Cyt-C and Caspase-9 in damaged brain tissue were all increased obviously in the model group (<0.01) and they were all reduced in the EA group as compared with the model group successively (<0.05).
CONCLUSION
Electroacupuncture remarkably improves the condition in the neurological function injury and reduces apoptosis degree in TBI model rats, which is likely related to the down-regulation of the protein expressions of Cyt-C and Caspase-9 in damaged brain tissue and further to bring the impacts on mitochondria mediated apoptosis process.
Animals
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Apoptosis
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Brain Injuries, Traumatic
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therapy
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Caspase 9
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metabolism
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Cytochromes c
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metabolism
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Electroacupuncture
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Random Allocation
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Rats
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Rats, Sprague-Dawley
7.Role of apoptosis in trichloroethylene induced irritant injury in the skin.
Chinese Journal of Industrial Hygiene and Occupational Diseases 2009;27(1):16-20
OBJECTIVETo investigate the expression of apoptosis and caspase-8, cyt c in the skin of the BALB/c mice exposed to trichloroethylene (TCE).
METHODS30 BALB/c mice were divided in random into the solvent control group, 10% TCE group, 20% TCE group, 40% TCE group, 80% TCE group and 100% TCE group. Apoptotic cells were detected by TUNEL and EM. The expressions of caspase-8 and cyt c were detected with immunohistochemical method.
RESULTSEM showed that the apoptosis of cells was found in the high dosage groups. The immunohistochemical results showed that there were significant differences in the apoptosis rate and the activity of cyt c between the different dosage groups. There was the significant difference in the apoptosis rate between the 40%, 80%, 100% TCE groups the control group (P < 0.01). There was the significant difference in the expression of cyt c between the 20%, 40%, 80%, 100% TCE groups [(2.60 +/- 0.54), (3.42 +/- 0.56), (5.81 +/- 1.30) and (6.00 +/- 0.70), respectively] and the control group (P < 0.01). The expressions of caspase-8 had no significant differences (P > 0.05).
CONCLUSIONApoptosis plays an important role in trichloroethylene induced irritant injury in skin and the apoptosis may be related with the mitochondrial injury.
Animals ; Apoptosis ; drug effects ; Caspase 8 ; metabolism ; Cytochromes c ; metabolism ; Female ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Skin ; drug effects ; metabolism ; pathology ; Trichloroethylene ; toxicity
8.Relationship between hypoxia-induced apoptosis and caspases-3 activation, intracellular calcium overload in cardiomyocytes.
Zhou ZHOU ; Xiao-hua WANG ; Guang-xu ZHU ; Zheng-ping YU
Chinese Journal of Applied Physiology 2005;21(1):10-14
AIMTo explore the effects of hypoxia on Caspases activation in cardiomyocyte and role of intracellular calcium in this event in cardiomyocytes.
METHODSAfter hypoxia 0 min, 30 min, 1 h, 3 h, 6 h, 12 h, 24 h, apoptotic cell percentage was determined with Hoechst 33342 straining. Expressions of Caspases-3 mRNA and release of mitochondrial cytochrome c in primary culture of cardiomyocytes were determined by using RT-PCR and Western blotting respectively.
RESULTSElevation of Cyt c in cytosol was in accordance with the decline in mitochondrial Cyt c content. Significant increase in Cyt c in cytosol appeared at 12 h post hypoxia and peaked at 24 h while Cyt c in mitochondria could not be detected at 24 h post hypoxia. Hypoxia up-regulated Caspases-3 mRNA expressions beginning at 3 h post hypoxia. Intracellular calcium overload occurred earlier than release of mitochondrial Cyt c and the activation of Caspase-3 during the hypoxic insult. Inhibition of Caspase-3 activation and pretreatment with calcium chelator BAPTA/AM offered a marked protective effect on hypoxia induced cardiomyocyte apoptosis.
CONCLUSIONHypoxia can induce mitochondrion-dependent Caspase-3 activation in cardiomyocytes and therefore leads to cell apoptosis. Increase of intracellular Ca2+ plays an important role in the activation of Caspase-3 and the induction of apoptosis in cardiomyocytes.
Animals ; Apoptosis ; Calcium ; metabolism ; Caspase 3 ; metabolism ; Cell Hypoxia ; Cytochromes c ; metabolism ; Cytosol ; metabolism ; Male ; Mitochondria ; metabolism ; Myocytes, Cardiac ; cytology ; metabolism ; Rats ; Rats, Wistar
9.Expression and purification of recombinant human cytochrome C in Escherichia coli.
Huawei CAI ; Lin WAN ; Hao YANG ; Lihong CHEN ; Xiaofeng LU
Journal of Biomedical Engineering 2007;24(3):620-625
Cytochrome C plays important roles in electron transferring, oxidative stress and apoptosis. In this study, soluble cytochrome C was accumulated in cytoplasm of E. coli by utilizing the co-expression of human cytochrome c and yeast heme lyase from a single plasmid. After ultrasonic disruption of the bacteria, a lot of contaminated proteins were discarded by addition of 350 g/L ammonium sulfate into the supernatant. Then the recombinant human cytochrome C was purified to 80% homogeneity after two times cation exchange chromatography on SP-Sepharose Fast Flow. Yields of cytochrome C greater than 10 mg per liter culture were attained. This efficient system for producing human cytochrome C is helpful for us to understand the roles of this protein in biological processes and therapy of human diseases relevant to apoptosis and oxidative stress.
Cytochromes c
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biosynthesis
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genetics
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Escherichia coli
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genetics
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metabolism
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Genetic Vectors
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Humans
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Recombinant Proteins
;
biosynthesis
;
genetics
;
isolation & purification
10.Distinct Cellular Calcium Metabolism in Radiation-sensitive RKO Human Colorectal Cancer Cells.
Yun Tai KIM ; Soo Shin JO ; Young Jun PARK ; Myung Za LEE ; Chang Kook SUH
The Korean Journal of Physiology and Pharmacology 2014;18(6):509-516
Radiation therapy for variety of human solid tumors utilizes mechanism of cell death after DNA damage caused by radiation. In response to DNA damage, cytochrome c was released from mitochondria by activation of pro-apoptotic Bcl-2 family proteins, and then elicits massive Ca2+ release from the ER that lead to cell death. It was also suggested that irradiation may cause the deregulation of Ca2+ homeostasis and trigger programmed cell death and regulate death specific enzymes. Thus, in this study, we investigated how cellular Ca2+ metabolism in RKO cells, in comparison to radiation-resistant A549 cells, was altered by gamma (gamma)-irradiation. In irradiated RKO cells, Ca2+ influx via activation of NCX reverse mode was enhanced and a decline of [Ca2+]i via forward mode was accelerated. The amount of Ca2+ released from the ER in RKO cells by the activation of IP3 receptor was also enhanced by irradiation. An increase in [Ca2+]i via SOCI was enhanced in irradiated RKO cells, while that in A549 cells was depressed. These results suggest that gamma-irradiation elicits enhancement of cellular Ca2+ metabolism in radiation-sensitive RKO cells yielding programmed cell death.
Calcium*
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Cell Death
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Colorectal Neoplasms*
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Cytochromes c
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DNA Damage
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Homeostasis
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Humans
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Inositol 1,4,5-Trisphosphate Receptors
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Metabolism*
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Mitochondria