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PloS One 2015Mitochondrial DNA (mtDNA) copy number decreases in animal and human heart failure (HF), yet its role in cardiomyocytes remains to be elucidated. Thus, we investigated...
BACKGROUND
Mitochondrial DNA (mtDNA) copy number decreases in animal and human heart failure (HF), yet its role in cardiomyocytes remains to be elucidated. Thus, we investigated the cardioprotective function of increased mtDNA copy number resulting from the overexpression of human transcription factor A of mitochondria (TFAM) or Twinkle helicase in volume overload (VO)-induced HF.
METHODS AND RESULTS
Two strains of transgenic (TG) mice, one overexpressing TFAM and the other overexpressing Twinkle helicase, exhibit an approximately 2-fold equivalent increase in mtDNA copy number in heart. These TG mice display similar attenuations in eccentric hypertrophy and improved cardiac function compared to wild-type (WT) mice without any deterioration of mitochondrial enzymatic activities in response to VO, which was accompanied by a reduction in matrix-metalloproteinase (MMP) activity and reactive oxygen species after 8 weeks of VO. Moreover, acute VO-induced MMP-2 and MMP-9 upregulation was also suppressed at 24 h in both TG mice. In isolated rat cardiomyocytes, mitochondrial reactive oxygen species (mitoROS) upregulated MMP-2 and MMP-9 expression, and human TFAM (hTFAM) overexpression suppressed mitoROS and their upregulation. Additionally, mitoROS were equally suppressed in H9c2 rat cardiomyoblasts that overexpress hTFAM or rat Twinkle, both of which exhibit increased mtDNA copy number. Furthermore, mitoROS and mitochondrial protein oxidation from both TG mice were suppressed compared to WT mice.
CONCLUSIONS
The overexpression of TFAM or Twinkle results in increased mtDNA copy number and facilitates cardioprotection associated with limited mitochondrial oxidative stress. Our findings suggest that increasing mtDNA copy number could be a useful therapeutic strategy to target mitoROS in HF.
Topics: Animals; Cell Line; DNA Helicases; DNA, Mitochondrial; DNA-Binding Proteins; Gene Dosage; Heart Failure; Humans; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; Mitochondria; Mitochondrial Proteins; Myocytes, Cardiac; Oxidative Stress; Rats; Reactive Oxygen Species; Transcription Factors
PubMed: 25822152
DOI: 10.1371/journal.pone.0119687 -
Molecular and Cellular Biology 2023Hsp104 propagates the yeast prion [], the infectious form of Sup35, by severing the prion seeds, but when Hsp104 is overexpressed, it cures [] in a process that is not...
Hsp104 propagates the yeast prion [], the infectious form of Sup35, by severing the prion seeds, but when Hsp104 is overexpressed, it cures [] in a process that is not yet understood but may be caused by trimming, which removes monomers from the ends of the amyloid fibers. This curing was shown to depend on both the N-terminal domain of Hsp104 and the expression level of various members of the Hsp70 family, which raises the question as to whether these effects of Hsp70 are due to it binding to the Hsp70 binding site that was identified in the N-terminal domain of Hsp104, a site not involved in prion propagation. Investigating this question, we now find, first, that mutating this site prevents both the curing of [] by Hsp104 overexpression and the trimming activity of Hsp104. Second, we find that depending on the specific member of the Hsp70 family binding to the N-terminal domain of Hsp104, both trimming and the curing caused by Hsp104 overexpression are either increased or decreased in parallel. Therefore, the binding of Hsp70 to the N-terminal domain of Hsp104 regulates both the rate of [] trimming by Hsp104 and the rate of [] curing by Hsp104 overexpression.
Topics: Heat-Shock Proteins; Saccharomyces cerevisiae Proteins; HSP70 Heat-Shock Proteins; Saccharomyces cerevisiae; Prions; Peptide Termination Factors
PubMed: 37099734
DOI: 10.1080/10985549.2023.2198181 -
Microbial Cell Factories Mar 2017Oxidized glutathione (GSSG) is the preferred form for industrial mass production of glutathione due to its high stability compared with reduced glutathione (GSH). In our...
BACKGROUND
Oxidized glutathione (GSSG) is the preferred form for industrial mass production of glutathione due to its high stability compared with reduced glutathione (GSH). In our previous study, over-expression of the mitochondrial thiol oxidase ERV1 gene was the most effective for high GSSG production in Saccharomyces cerevisiae cells among three types of different thiol oxidase genes.
RESULTS
We improved Erv1 enzyme activity for oxidation of GSH and revealed that S32 and N34 residues are critical for the oxidation. Five engineered Erv1 variant proteins containing S32 and/or N34 replacements exhibited 1.7- to 2.4-fold higher in vitro GSH oxidation activity than that of parental Erv1, whereas the oxidation activities of these variants for γ-glutamylcysteine were comparable. According to three-dimensional structures of Erv1 and protein stability assays, S32 and N34 residues interact with nearby residues through hydrogen bonding and greatly contribute to protein stability. These results suggest that increased flexibility by amino acid replacements around the active center decrease inhibitory effects on GSH oxidation. Over-expressions of mutant genes coding these Erv1 variants also increased GSSG and consequently total glutathione production in S. cerevisiae cells. Over-expression of the ERV1 gene was the most effective for GSSG production in S. cerevisiae cells among the parent and other mutant genes, and it increased GSSG production about 1.5-fold compared to that of the parental ERV1 gene.
CONCLUSIONS
This is the first study demonstrating the pivotal effects of S32 and N34 residues to high GSH oxidation activity of Erv1. Furthermore, in vivo validity of Erv1 variants containing these S32 and N34 replacements were also demonstrated. This study indicates potentials of Erv1 for high GSSG production.
Topics: Dipeptides; Fermentation; Glutathione; Metabolic Engineering; Mitochondria; Mitochondrial Proteins; Models, Molecular; Mutation; Oxidation-Reduction; Oxidoreductases Acting on Sulfur Group Donors; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 28298220
DOI: 10.1186/s12934-017-0658-0 -
Genes & Development Jan 1997Yeast core telomeric heterochromatin can silence adjacent genes and requires RAP1, SIR2, SIR3, and SIR4 and histones H3 and H4 for this telomere position effect. SIR3...
Yeast core telomeric heterochromatin can silence adjacent genes and requires RAP1, SIR2, SIR3, and SIR4 and histones H3 and H4 for this telomere position effect. SIR3 overproduction can extend the silenced domain. We examine here the nature of these multiprotein complexes. SIR2 and SIR4 were immunoprecipitated from whole-cell extracts. In addition, using formaldehyde cross-linking we have mapped SIR2, SIR4, and RAP1 along telomeric chromatin before and after SIR3 overexpression. Our data demonstrate that SIR2 and SIR4 interact in a protein complex and that SIR2, SIR3, SIR4, and RAP1 map to the same sites along telomeric heterochromatin in wild-type cells. However, when overexpressed, SIR3 spreads along the chromosome and its interactions are dominant to those of SIR4 and especially SIR2, whose detection is decreased in extended heterochromatin. RAP1 binding at the core region is unaffected by SIR3 overproduction and RAP1 shows no evidence of spreading. Thus, we propose that the structure of core telomeric heterochromatin differs from that extended by SIR3.
Topics: Blotting, Western; Cell Extracts; Cross-Linking Reagents; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Fungal Proteins; GTP-Binding Proteins; Gene Expression Regulation, Fungal; Heterochromatin; Histone Deacetylases; Histones; Models, Genetic; Mutation; Precipitin Tests; Protein Binding; Saccharomyces cerevisiae; Silent Information Regulator Proteins, Saccharomyces cerevisiae; Sirtuin 2; Sirtuins; Telomere; Trans-Activators; rap GTP-Binding Proteins
PubMed: 9000052
DOI: 10.1101/gad.11.1.83 -
Scientific Reports Nov 2016The PIM family of serine/threonine kinases has three highly conserved isoforms (PIM1, PIM2 and PIM3). PIM proteins are regulated through transcription and stability by...
The PIM family of serine/threonine kinases has three highly conserved isoforms (PIM1, PIM2 and PIM3). PIM proteins are regulated through transcription and stability by JAK/STAT pathways and are overexpressed in hematological malignancies and solid tumors. The PIM kinases possess weak oncogenic abilities, but enhance other genes or chemical carcinogens to induce tumors. We generated conditional transgenic mice that overexpress PIM1 or PIM2 in male reproductive organs and analyzed their contribution to tumorigenesis. We found an increase in alterations of sexual organs and hyperplasia in the transgenic mice correlating with inflammation. We also found that PIM1/2 are overexpressed in a subset of human male germ cells and prostate tumors correlating with inflammatory features and stem cell markers. Our data suggest that PIM1/2 kinase overexpression is a common feature of male reproductive organs tumors, which provoke tissue alterations and a large inflammatory response that may act synergistically during the process of tumorigenesis. There is also a correlation with markers of cancer stem cells, which may contribute to the therapy resistance found in tumors overexpressing PIM kinases.
Topics: Animals; Biomarkers, Tumor; Humans; Male; Mice; Mice, Transgenic; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-pim-1
PubMed: 27901106
DOI: 10.1038/srep38079 -
FEBS Letters Dec 2006We searched for F-box proteins that might be related to the mechanism that protects Saccharomyces cerevisiae against the toxic effects of methylmercury. We found that...
We searched for F-box proteins that might be related to the mechanism that protects Saccharomyces cerevisiae against the toxic effects of methylmercury. We found that overexpression of Hrt3 and of Ylr224w rendered yeast cells resistant to methylmercury. Yeast cells that overexpressed Hrt3 and Ylr224w were barely resistant to methylmercury in the presence of a proteasome inhibitor. Our results suggest the existence of some protein(s) that enhances the toxicity of methylmercury in yeast cells and, also, that overexpression of Hrt3 or Ylr224w can confer resistance to methylmercury by enhancing the polyubiquitination of this protein(s) and its degradation in proteasomes.
Topics: Drug Resistance, Fungal; F-Box Motifs; F-Box Proteins; Methylmercury Compounds; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; Saccharomyces cerevisiae; Sensitivity and Specificity
PubMed: 17141224
DOI: 10.1016/j.febslet.2006.11.045 -
The Journal of Neuroscience : the... Jan 2020Neurofibrillary tangles likely cause neurodegeneration in Alzheimer's disease (AD). We demonstrate that the CX3CL1 C-terminal domain can upregulate neurogenesis, which...
Neurofibrillary tangles likely cause neurodegeneration in Alzheimer's disease (AD). We demonstrate that the CX3CL1 C-terminal domain can upregulate neurogenesis, which may ameliorate neurodegeneration. Here we generated transgenic (Tg-CX3CL1) mice by overexpressing CX3CL1 in neurons. Tg-CX3CL1 mice exhibit enhanced neurogenesis in both subgranular and subventricular zones. This enhanced neurogenesis correlates well with elevated expression of TGF-β2 and TGF-β3, and activation of their downstream signaling molecule Smad2. Intriguingly, the enhanced adult neurogenesis was mitigated when Smad2 expression was deleted in neurons, supporting a role for the CX3CL1-TGF-β2/3-Smad2 pathway in the control of adult neurogenesis. When Tg-CX3CL1 mice were crossed with Alzheimer's PS19 mice, which overexpress a tau P301S mutation and exhibit age-dependent neurofibrillary tangles and neurodegeneration, overexpressed CX3CL1 in both male and female mice was sufficient to rescue the neurodegeneration, increase survival time, and improve cognitive function. Hence, we provide evidence that CX3CL1 is a strong activator of adult neurogenesis, and that it reduces neuronal loss and improves cognitive function in AD. This study will be the first to demonstrate that enhanced neurogenesis by overexpressed CX3CL1 is mitigated by disruption of Smad2 signaling and is independent of its interaction with CX3CR1. Overexpression of CX3CL1 lengthens the life span of PS19 tau mice by enhancing adult neurogenesis while having minimal effect on tau pathology. Enhancing neuronal CX3CL1, mainly the C-terminal fragment, is a therapeutic strategy for blocking or reversing neuronal loss in Alzheimer's disease or related neurodegenerative disease patients.
Topics: Alzheimer Disease; Animals; Chemokine CX3CL1; Disease Models, Animal; Female; Male; Mice, Transgenic; Neurogenesis; Neurons; Smad2 Protein; Spatial Memory; tau Proteins
PubMed: 31822518
DOI: 10.1523/JNEUROSCI.1333-19.2019 -
The Journal of Cell Biology Dec 2005Keratins 8 and 18 (K8/18) are major constituents of Mallory bodies (MBs), which are hepatocyte cytoplasmic inclusions seen in several liver diseases. K18-null but not...
Keratins 8 and 18 (K8/18) are major constituents of Mallory bodies (MBs), which are hepatocyte cytoplasmic inclusions seen in several liver diseases. K18-null but not K8-null or heterozygous mice form MBs, which indicates that K8 is important for MB formation. Early stages in MB genesis include K8/18 hyperphosphorylation and overexpression. We used transgenic mice that overexpress K8, K18, or K8/18 to test the importance of K8 and/or K18 in MB formation. MBs were induced by feeding 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Livers of young K8 or K8/K18 overexpressors had no histological abnormalities despite increased keratin protein and phosphorylation. In aging mice, only K8-overexpressing livers spontaneously developed small "pre-MB" aggregates. Only K8-overexpressing young mice are highly susceptible to MB formation after short-term DDC feeding. Thus, the K8 to K18 ratio, rather than K8/18 overexpression by itself, plays an essential role in MB formation. K8 overexpression is sufficient to form pre-MB and primes animals to accumulate MBs upon DDC challenge, which may help explain MB formation in human liver diseases.
Topics: Animals; Gene Expression Regulation; Hepatocytes; Humans; Inclusion Bodies; Keratin-18; Keratin-8; Keratins; Liver; Mice; Mice, Transgenic; Microscopy, Fluorescence; Models, Biological; Proteins; RNA, Messenger
PubMed: 16365160
DOI: 10.1083/jcb.200507093 -
The Journal of Cell Biology Jun 1998beta-Catenin and plakoglobin are homologous proteins that function in cell adhesion by linking cadherins to the cytoskeleton and in signaling by transactivation together... (Comparative Study)
Comparative Study
beta-Catenin and plakoglobin are homologous proteins that function in cell adhesion by linking cadherins to the cytoskeleton and in signaling by transactivation together with lymphoid-enhancing binding/T cell (LEF/TCF) transcription factors. Here we compared the nuclear translocation and transactivation abilities of beta-catenin and plakoglobin in mammalian cells. Overexpression of each of the two proteins in MDCK cells resulted in nuclear translocation and formation of nuclear aggregates. The beta-catenin-containing nuclear structures also contained LEF-1 and vinculin, while plakoglobin was inefficient in recruiting these molecules, suggesting that its interaction with LEF-1 and vinculin is significantly weaker. Moreover, transfection of LEF-1 translocated endogenous beta-catenin, but not plakoglobin to the nucleus. Chimeras consisting of Gal4 DNA-binding domain and the transactivation domains of either plakoglobin or beta-catenin were equally potent in transactivating a Gal4-responsive reporter, whereas activation of LEF-1- responsive transcription was significantly higher with beta-catenin. Overexpression of wild-type plakoglobin or mutant beta-catenin lacking the transactivation domain induced accumulation of the endogenous beta-catenin in the nucleus and LEF-1-responsive transactivation. It is further shown that the constitutive beta-catenin-dependent transactivation in SW480 colon carcinoma cells and its nuclear localization can be inhibited by overexpressing N-cadherin or alpha-catenin. The results indicate that (a) plakoglobin and beta-catenin differ in their nuclear translocation and complexing with LEF-1 and vinculin; (b) LEF-1-dependent transactivation is preferentially driven by beta-catenin; and (c) the cytoplasmic partners of beta-catenin, cadherin and alpha-catenin, can sequester it to the cytoplasm and inhibit its transcriptional activity.
Topics: 3T3 Cells; Animals; Cadherins; Cell Line; Cell Nucleus; Cysteine Endopeptidases; Cytoskeletal Proteins; DNA-Binding Proteins; Desmoplakins; Dogs; Gene Expression; Humans; Lymphoid Enhancer-Binding Factor 1; Mice; Multienzyme Complexes; Proteasome Endopeptidase Complex; Saccharomyces cerevisiae Proteins; Trans-Activators; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Tumor Cells, Cultured; Ubiquitins; Vinculin; alpha Catenin; beta Catenin; gamma Catenin
PubMed: 9628899
DOI: 10.1083/jcb.141.6.1433 -
Journal of Virology Jan 2016DNAJC14, a heat shock protein 40 (Hsp40) cochaperone, assists with Hsp70-mediated protein folding. Overexpressed DNAJC14 is targeted to sites of yellow fever virus (YFV)...
UNLABELLED
DNAJC14, a heat shock protein 40 (Hsp40) cochaperone, assists with Hsp70-mediated protein folding. Overexpressed DNAJC14 is targeted to sites of yellow fever virus (YFV) replication complex (RC) formation, where it interacts with viral nonstructural (NS) proteins and inhibits viral RNA replication. How RCs are assembled and the roles of chaperones in this coordinated process are largely unknown. We hypothesized that chaperones are diverted from their normal cellular protein quality control function to play similar roles during viral infection. Here, we show that DNAJC14 overexpression affects YFV polyprotein processing and alters RC assembly. We monitored YFV NS2A-5 polyprotein processing by the viral NS2B-3 protease in DNAJC14-overexpressing cells. Notably, DNAJC14 mutants that did not inhibit YFV replication had minimal effects on polyprotein processing, while overexpressed wild-type DNAJC14 affected the NS3/4A and NS4A/2K cleavage sites, resulting in altered NS3-to-NS3-4A ratios. This suggests that DNAJC14's folding activity normally modulates NS3/4A/2K cleavage events to liberate appropriate levels of NS3 and NS4A and promote RC formation. We introduced amino acid substitutions at the NS3/4A site to alter the levels of the NS3 and NS4A products and examined their effects on YFV replication. Residues with reduced cleavage efficiency did not support viral RNA replication, and only revertant viruses with a restored wild-type arginine or lysine residue at the NS3/4A site were obtained. We conclude that DNAJC14 inhibition of RC formation upon DNAJC14 overexpression is likely due to chaperone dysregulation and that YFV probably utilizes DNAJC14's cochaperone function to modulate processing at the NS3/4A site as a mechanism ensuring virus replication.
IMPORTANCE
Flaviviruses are single-stranded RNA viruses that cause a wide range of illnesses. Upon host cell entry, the viral genome is translated on endoplasmic reticulum (ER) membranes to produce a single polyprotein, which is cleaved by host and viral proteases to generate viral proteins required for genome replication and virion production. Several studies suggest a role for molecular chaperones during these processes. While the details of chaperone roles have been elusive, in this report we show that overexpression of the ER-resident cochaperone DNAJC14 affects YFV polyprotein processing at the NS3/4A site. This work reveals that DNAJC14 modulation of NS3/4A site processing is an important mechanism to ensure virus replication. Our work highlights the importance of finely regulating flavivirus polyprotein processing. In addition, it suggests future studies to address similarities and/or differences among flaviviruses and to interrogate the precise mechanisms employed for polyprotein processing, a critical step that can ultimately be targeted for novel drug development.
Topics: Cell Line; Fetal Proteins; Host-Pathogen Interactions; Humans; Molecular Chaperones; Protein Folding; Proteolysis; Viral Nonstructural Proteins; Virus Replication; Yellow fever virus
PubMed: 26739057
DOI: 10.1128/JVI.03077-15