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Scientific Reports Oct 2022Transgenic mice over-expressing human PRNP or murine Prnp transgenes on a mouse prion protein knockout background have made key contributions to the understanding of...
Transgenic mice over-expressing human PRNP or murine Prnp transgenes on a mouse prion protein knockout background have made key contributions to the understanding of human prion diseases and have provided the basis for many of the fundamental advances in prion biology, including the first report of synthetic mammalian prions. In this regard, the prion paradigm is increasingly guiding the exploration of seeded protein misfolding in the pathogenesis of other neurodegenerative diseases. Here we report that a well-established and widely used line of such mice (Tg20 or tga20), which overexpress wild-type mouse prion protein, exhibit spontaneous aggregation and accumulation of misfolded prion protein in a strongly age-dependent manner, which is accompanied by focal spongiosis and occasional neuronal loss. In some cases a clinical syndrome developed with phenotypic features that closely resemble those seen in prion disease. However, passage of brain homogenate from affected, aged mice failed to transmit this syndrome when inoculated intracerebrally into further recipient animals. We conclude that overexpression of the wild-type mouse prion protein can cause an age-dependent protein misfolding disorder or proteinopathy that is not associated with the production of an infectious agent but can produce a phenotype closely similar to authentic prion disease.
Topics: Animals; Brain Diseases; Humans; Mammals; Mice; Mice, Transgenic; Prion Diseases; Prion Proteins; Prions
PubMed: 36229637
DOI: 10.1038/s41598-022-21608-3 -
Biochimica Et Biophysica Acta.... Dec 2022In the endoplasmic reticulum (ER) membrane, transmembrane (TM) domain insertion occurs through the Sec61 channel with its auxiliary components, including Sec62. Sec62...
In the endoplasmic reticulum (ER) membrane, transmembrane (TM) domain insertion occurs through the Sec61 channel with its auxiliary components, including Sec62. Sec62 interacts with the Sec61 channel and is located on the front side of the Sec61 lateral gate, an entry site for TM domains to the lipid bilayer. Overexpression of Sec62 led to a growth defect in yeast, and we investigated its effects on protein translocation and membrane insertion by pulse labeling of Sec62 client proteins. Our data show that the insertion efficiency of marginally hydrophobic TM segments is reduced upon Sec62 overexpression. This result suggests a potential regulatory role of Sec62 as a gatekeeper of the lateral gate, thereby modulating the insertion threshold of TM segments.
Topics: Endoplasmic Reticulum; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Membrane Transport Proteins; Protein Transport; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 36122759
DOI: 10.1016/j.bbamem.2022.184051 -
International Journal of Molecular... Dec 2022Protein kinase CK2 plays an important role in cell survival and protects regulatory proteins from caspase-mediated degradation during apoptosis. The consensus sequence...
Protein kinase CK2 plays an important role in cell survival and protects regulatory proteins from caspase-mediated degradation during apoptosis. The consensus sequence of proteins phosphorylated by CK2 contains a cluster of acidic amino acids around the phosphorylation site. The poly-acidic sequence in yeast protein Asf1 is similar to the acidic loop in CK2β, which possesses a regulatory function. We observed that the overexpression of Asf1 in yeast cells influences cell growth. Experiments performed in vitro and in vivo indicate that yeast protein Asf1 inhibits protein kinase CK2. Our data suggest that each CK2 isoform might be regulated in a different way. Deletion of the amino or carboxyl end of Asf1 reveals that the acidic cluster close to the C-terminus is responsible for the activation or inhibition of CK2 activity.
Topics: Casein Kinase II; Phosphorylation; Saccharomyces cerevisiae; Fungal Proteins; Caspases; Cell Cycle Proteins; Molecular Chaperones; Saccharomyces cerevisiae Proteins
PubMed: 36555405
DOI: 10.3390/ijms232415764 -
Cell Reports Apr 2024Overexpression of Cyclin E1 perturbs DNA replication, resulting in DNA lesions and genomic instability. Consequently, Cyclin E1-overexpressing cancer cells increasingly...
Overexpression of Cyclin E1 perturbs DNA replication, resulting in DNA lesions and genomic instability. Consequently, Cyclin E1-overexpressing cancer cells increasingly rely on DNA repair, including RAD52-mediated break-induced replication during interphase. We show that not all DNA lesions induced by Cyclin E1 overexpression are resolved during interphase. While DNA lesions upon Cyclin E1 overexpression are induced in S phase, a significant fraction of these lesions is transmitted into mitosis. Cyclin E1 overexpression triggers mitotic DNA synthesis (MiDAS) in a RAD52-dependent fashion. Chemical or genetic inactivation of MiDAS enhances mitotic aberrations and persistent DNA damage. Mitosis-specific degradation of RAD52 prevents Cyclin E1-induced MiDAS and reduces the viability of Cyclin E1-overexpressing cells, underscoring the relevance of RAD52 during mitosis to maintain genomic integrity. Finally, analysis of breast cancer samples reveals a positive correlation between Cyclin E1 amplification and RAD52 expression. These findings demonstrate the importance of suppressing mitotic defects in Cyclin E1-overexpressing cells through RAD52.
Topics: Humans; Cyclin E; Genomic Instability; Rad52 DNA Repair and Recombination Protein; Mitosis; Oncogene Proteins; DNA Replication; Cell Line, Tumor; DNA Damage; DNA; Breast Neoplasms
PubMed: 38625790
DOI: 10.1016/j.celrep.2024.114116 -
The Journal of Physiology Sep 2021The maintenance of mitochondrial integrity is critical for skeletal muscle health. Mitochondrial dynamics play key roles in mitochondrial quality control; however, the...
KEY POINTS
The maintenance of mitochondrial integrity is critical for skeletal muscle health. Mitochondrial dynamics play key roles in mitochondrial quality control; however, the exact role that mitochondrial fission plays in the muscle ageing process remains unclear. Here we report that both Drp1 knockdown and Drp1 overexpression late in life in mice is detrimental to skeletal muscle function and mitochondrial health. Drp1 knockdown in 18-month-old mice resulted in severe skeletal muscle atrophy, mitochondrial dysfunction, muscle degeneration/regeneration, oxidative stress and impaired autophagy. Overexpressing Drp1 in 18-month-old mice resulted in mild skeletal muscle atrophy and decreased mitochondrial quality. Our data indicate that silencing or overexpressing Drp1 late in life is detrimental to skeletal muscle integrity. We conclude that modulating Drp1 expression is unlikely to be a viable approach to counter the muscle ageing process.
ABSTRACT
Sarcopenia, the ageing-related loss of skeletal muscle mass and function, is a debilitating process negatively impacting the quality of life of afflicted individuals. Although the mechanisms underlying sarcopenia are still only partly understood, impairments in mitochondrial dynamics, and specifically mitochondrial fission, have been proposed as an underlying mechanism. Importantly, conflicting data exist in the field and both excessive and insufficient mitochondrial fission were proposed to contribute to sarcopenia. In Drosophila melanogaster, enhancing mitochondrial fission in midlife through overexpression of dynamin-1-like protein (Drp1) extended lifespan and attenuated several key hallmarks of muscle ageing. Whether a similar outcome of Drp1 overexpression is observed in mammalian muscles remains unknown. In this study, we investigated the impact of knocking down and overexpressing Drp1 protein for 4 months in skeletal muscles of late middle-aged (18 months) mice using intra-muscular injections of adeno-associated viruses expressing shRNA targeting Drp1 or full Drp1 cDNA. We report that knocking down Drp1 expression late in life triggers severe muscle atrophy, mitochondrial dysfunctions, degeneration/regeneration, oxidative stress and impaired autophagy. Drp1 overexpression late in life triggered mild muscle atrophy and decreased mitochondrial quality. Taken altogether, our results indicate that both overexpression and silencing of Drp1 in late middle-aged mice negatively impact skeletal muscle mass and mitochondrial health. These data suggest that Drp1 content must remain within a narrow physiological range to preserve muscle and mitochondrial integrity during ageing. Altering Drp1 expression is therefore unlikely to be a viable target to counter sarcopenia.
Topics: Animals; Cytoskeletal Proteins; Drosophila melanogaster; Dynamins; GTP-Binding Proteins; Mice; Mitochondria; Mitochondrial Dynamics; Muscle, Skeletal; Quality of Life
PubMed: 34269418
DOI: 10.1113/JP281752 -
Genes May 2021Colorectal cancer (CRC) remains one of the leading causes of cancer-related death worldwide. The high mortality of CRC is related to its ability to metastasize to...
Colorectal cancer (CRC) remains one of the leading causes of cancer-related death worldwide. The high mortality of CRC is related to its ability to metastasize to distant organs. The kallikrein-related peptidase Kallikrein 6 (KLK6) is overexpressed in CRC and contributes to cancer cell invasion and metastasis. The goal of this study was to identify KLK6-associated markers for the CRC prognosis and treatment. Tumor Samples from the CRC patients with significantly elevated transcript levels were identified in the RNA-Seq data from Cancer Genome Atlas (TCGA) and their expression profiles were evaluated using Gene Ontology (GO), Phenotype and Reactome enrichment, and protein interaction methods. KLK6-high cases had a distinct spectrum of mutations in titin (), , , and genes. Differentially expressed genes (DEGs) found in the KLK6-overexpressing CRCs were associated with cell signaling, extracellular matrix organization, and cell communication regulatory pathways. The top KLK6-interaction partners were found to be the members of kallikrein family (KLK7, KLK8, KLK10), extracellular matrix associated proteins (keratins, integrins, small proline rich repeat, S100A families) and TGF-β, FOS, and Ser/Thr protein kinase signaling pathways. Expression of selected KLK6-associated genes was validated in a subset of paired normal and tumor CRC patient-derived organoid cultures. The performed analyses identified KLK6 itself and a set of genes, which are co-expressed with KLK6, as potential clinical biomarkers for the management of the CRC disease.
Topics: Adenomatous Polyposis Coli Protein; CA-125 Antigen; Colorectal Neoplasms; Connectin; Extracellular Matrix Proteins; Female; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Kallikreins; Male; Membrane Proteins; Proto-Oncogene Proteins p21(ras); Signal Transduction; Transcriptome; Tumor Cells, Cultured; Up-Regulation
PubMed: 34065672
DOI: 10.3390/genes12050749 -
The Journal of Biological Chemistry Nov 2020In , the Pif1 helicase functions in both nuclear and mitochondrial DNA replication and repair processes, preferentially unwinding RNA:DNA hybrids and resolving...
In , the Pif1 helicase functions in both nuclear and mitochondrial DNA replication and repair processes, preferentially unwinding RNA:DNA hybrids and resolving G-quadruplex structures. We sought to determine how the various activities of Pif1 are regulated Here, we report lysine acetylation of nuclear Pif1 and demonstrate that it influences both Pif1's cellular roles and core biochemical activities. Using Pif1 overexpression toxicity assays, we determined that the acetyltransferase NuA4 and deacetylase Rpd3 are primarily responsible for the dynamic acetylation of nuclear Pif1. MS analysis revealed that Pif1 was modified in several domains throughout the protein's sequence on the N terminus (Lys-118 and Lys-129), helicase domain (Lys-525, Lys-639, and Lys-725), and C terminus (Lys-800). Acetylation of Pif1 exacerbated its overexpression toxicity phenotype, which was alleviated upon deletion of its N terminus. Biochemical assays demonstrated that acetylation of Pif1 stimulated its helicase, ATPase, and DNA-binding activities, whereas maintaining its substrate preferences. Limited proteolysis assays indicate that acetylation of Pif1 induces a conformational change that may account for its altered enzymatic properties. We propose that acetylation is involved in regulating of Pif1 activities, influencing a multitude of DNA transactions vital to the maintenance of genome integrity.
Topics: Acetylation; Cell Nucleus; DNA Helicases; DNA, Fungal; Histone Acetyltransferases; Histone Deacetylases; Lysine; Mutagenesis, Site-Directed; Protein Domains; RNA, Fungal; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Substrate Specificity; Tandem Mass Spectrometry
PubMed: 32878983
DOI: 10.1074/jbc.RA120.015164 -
Journal of Carcinogenesis 2021Metastatic progression in triple-negative breast cancer (TNBC) patients occurs primarily because of nuclear reprogramming that includes chromatin remodeling and...
INTRODUCTION
Metastatic progression in triple-negative breast cancer (TNBC) patients occurs primarily because of nuclear reprogramming that includes chromatin remodeling and epigenetic modifications. The existing and most successful chemotherapies available for metastatic TNBC target nuclear proteins or damage DNA. The objectives here are to investigate an undescribed role for the molecular biology of nuclear angiopoietin-like protein 4 (ANGPTL4) and to characterize the effect of ectopic overexpression of ANGPTL4 in the metastatic biology of TNBC.
MATERIALS AND METHODS
Lentiviral-mediated transduction was used to overexpress ANGPTL4 in the TNBC cell line MD Anderson-metastatic breast cancer 231. The overexpression of ANGPTL4 was confirmed by western blot and ELISA. Subcellular fractionation, western blot, and immunofluorescence microscopy were used to characterize the intracellular localization of ANGPTL4. Mammosphere culture and the anchorage-independent growth assay analyzed the metastatic potential of the cell line. Xenograft assays assessed the effect of ANGPTL4 overexpression on TNBC metastases .
RESULTS
The ANGPTL4 overexpressing cell line formed larger mammospheres and anchorage-independent colonies and developed larger primary tumors, more liver metastases, and brain metastatic outgrowth in comparison to a cell line that expressed endogenous levels of ANGPTL4. ANGPTL4, aurora kinase A (AURKA), a mitotic kinase, and Tat-interacting protein p60 kDa (Tip60), a lysine acetyltransferase, associated with chromatin in the ANGPTL4 overexpressing cells but not in cells that expressed endogenous levels of ANGPTL4.
CONCLUSIONS
The ANGPTL4 overexpressing cell line showed and activities that suggest that nuclear ANGPTL4, AURKA, and Tip60 may cooperatively modulate TNBC metastases within chromatin-remodeling complexes or DNA-associated machinery.
PubMed: 34447288
DOI: 10.4103/jcar.JCar_20_20 -
The Journal of Nutrition Nov 2021Selenoprotein H (SELONOH), a member of the thioredoxin-like family proteins, is prioritized to degradation in selenium (Se) insufficiency. Recent studies implicate...
BACKGROUND
Selenoprotein H (SELONOH), a member of the thioredoxin-like family proteins, is prioritized to degradation in selenium (Se) insufficiency. Recent studies implicate protective roles of SELENOH in oxidative stress, cellular senescence, and intestinal tumorigenesis. Although the nonselenoprotein H0YE28 is suggested as shortened SELENOH according to genomic and proteomic data repositories, this variant has not been verified biochemically.
OBJECTIVES
We sought to identify SELENOH isoforms and explore the impact of Se flux on selenoprotein expression in SELENOH-overexpressing cells.
METHODS
A vector expressing a FLAG (the DYKDDDDK sequence) tag on the N-terminal end of wild-type SELENOH was constructed and transiently transfected into 293T cells incubated with graded concentrations of Na2SeO3 (0-200 nM). Cells were subjected to immunoprecipitation, LC-MS/MS protein analysis, immunoblotting, qRT-PCR, and senescence assays. Data were analyzed by 1-way or 2-way ANOVA.
RESULTS
Results of anti-FLAG immunoblotting showed that FLAG-SELENOH transfection increased (3.7-fold; P < 0.05) protein levels of the long, but not the short, SELENOH variants in the presence of Na2SeO3 (100 nM). By contrast, SELENOH mRNA levels were increased by 53-fold upon FLAG-SELENOH transfection but were comparable with or without supplemental Se (100 nM). LC-MS/MS analyses of anti-FLAG immunoprecipitates designated both anti-FLAG bands as SELENOH and co-identified three 60S ribosomal and 9 other proteins. Overexpression of FLAG-SELENOH 1) reduced glutathione peroxidase 1 and thioredoxin reductase 1 expression at the protein rather than the mRNA level in the absence but not presence of supplemental Se (100 nM; P < 0.05); 2) increased mRNA levels of 3 heat shock proteins (HSP27, HSP70-1A, and HSP70-1B; P < 0.05); and 3) reduced senescence induced by H2O2 (20 μM, 4 hours; P < 0.05).
CONCLUSIONS
These cellular studies demonstrate a Se-independent, shortened SELENOH variant and suggest competition of overexpressed FLAG-SELENOH with 2 other selenoproteins for the expression at the protein but not the mRNA level in Se insufficiency.
Topics: Chromatography, Liquid; DNA-Binding Proteins; Glutathione Peroxidase; HEK293 Cells; Humans; Hydrogen Peroxide; Protein Isoforms; Proteomics; RNA, Messenger; Selenium; Selenoproteins; Tandem Mass Spectrometry
PubMed: 34510207
DOI: 10.1093/jn/nxab290 -
Redox Biology Sep 2020NADPH oxidases produce reactive oxygen species that differ in localization, type and concentration. Within the Nox family only Nox4 produces HO which can directly...
NADPH oxidases produce reactive oxygen species that differ in localization, type and concentration. Within the Nox family only Nox4 produces HO which can directly oxidize cysteine residues. With this post-translational modification, activity, stability, localization and protein-protein interactions of the affected protein is altered. Nox4 controls differentiation, cellular homeostasis and prevents inflammation. Therefore, is likely that epigenetic mechanisms contribute to the effects of Nox4. One group of epigenetic modifiers are class IIa histone deacetylases (HDACs). We hypothesize that Nox4-derived HO oxidizes HDACs and analyzed whether HDACs can be differentially oxidized by Nox4. As an artificial system, we utilized HEK293 cells, overexpressing Nox4 in a tetracycline-inducible manner. HDAC4 was oxidized upon Nox4 overexpression. Additionally, Nox4 overexpression increased HDAC4 phosphorylation on Ser632. HO disrupted HDAC4/Mef2A complex, which de-represses Mef2A. In endothelial cells such as HUVECs and HMECs, overexpression of HDAC4 significantly reduced tube formation. Overexpression of a redox insensitive HDAC4 had no effect on endothelial tube formation. Treatment with HO, induction of Nox4 expression by treatment of the cells with TGFβ and co-overexpression of Nox4 not only induced phosphorylation of HDAC4, but also restored the repressive effect of HDAC4 for tube formation, while overexpression of a redox dead mutant of Nox4 did not. Taken together, Nox4 oxidizes HDAC4, increases its phosphorylation, and eventually ensures proper tube formation by endothelial cells.
Topics: Endothelial Cells; HEK293 Cells; Histone Deacetylases; Humans; Hydrogen Peroxide; NADPH Oxidase 4; NADPH Oxidases; Oxidation-Reduction; Reactive Oxygen Species; Repressor Proteins
PubMed: 32818796
DOI: 10.1016/j.redox.2020.101669