-
The Journal of Biological Chemistry Nov 2021Zinc deficiency has been linked to human diseases, including cancer. MDMX, a crucial zinc-containing negative regulator of p53, has been found to be amplified or...
Zinc deficiency has been linked to human diseases, including cancer. MDMX, a crucial zinc-containing negative regulator of p53, has been found to be amplified or overexpressed in various cancers and implicated in the cancer initiation and progression. We report here that zinc depletion by the ion chelator TPEN or Chelex resin results in MDMX protein degradation in a ubiquitination-independent and 20S proteasome-dependent manner. Restoration of zinc led to recovery of cellular levels of MDMX. Further, TPEN treatment inhibits growth of the MCF-7 breast cancer cell line, which is partially rescued by overexpression of MDMX. Moreover, in a mass-spectrometry-based proteomics analysis, we identified TRPM7, a zinc-permeable ion channel, as a novel MDMX-interacting protein. TRPM7 stabilizes and induces the appearance of faster migrating species of MDMX on SDS-PAGE. Depletion of TRPM7 attenuates, while TRPM7 overexpression facilitates, the recovery of MDMX levels upon adding back zinc to TPEN-treated cells. Importantly, we found that TRPM7 inhibition, like TPEN treatment, decreases breast cancer cell MCF-7 proliferation and migration. The inhibitory effect on cell migration upon TRPM7 inhibition is also partially rescued by overexpression of MDMX. Together, our data indicate that TRPM7 regulates cellular levels of MDMX in part by modulating the intracellular Zn concentration to promote tumorigenesis.
Topics: Animals; Cell Cycle Proteins; Humans; MCF-7 Cells; Mice; Mice, Knockout; PC-3 Cells; Protein Serine-Threonine Kinases; Proteolysis; Proto-Oncogene Proteins; TRPM Cation Channels; Zinc
PubMed: 34627839
DOI: 10.1016/j.jbc.2021.101292 -
International Journal of Molecular... Oct 2021Androgen receptor (AR) is a ligand-mediated transcription factor that belongs to the superfamily of steroid receptors. AR is overexpressed in most glioblastomas and is a...
Androgen receptor (AR) is a ligand-mediated transcription factor that belongs to the superfamily of steroid receptors. AR is overexpressed in most glioblastomas and is a potential therapeutic target. In prostate and breast cancers, AR activation can be achieved also by a ligand-independent signaling through receptor tyrosine kinases such as epidermal growth factor receptor (EGFR). Considering its major role in glioblastoma, we explored whether EGFR is involved in AR signaling in this tumor. Analysis of mRNA expression in 28 glioblastoma samples with quantitative real-time reverse-transcription polymerase chain reaction revealed a positive and significant correlation between AR and EGFR mRNA expression levels (R = 0.47, = 0.0092), which was validated by The Cancer Genome Atlas dataset ( = 671) analysis (R = 0.3, = 0.00006). Using Western blotting and immunofluorescence staining, we showed that the transduced overexpression of EGFR or its variant EGFRvIII in the U87MG cells induced AR protein overexpression and nuclear translocation and Protein kinase B (AKT) S473 and AR S210/213 phosphorylation. The EGFR kinase inhibitor afatinib and the AKT inhibitor MK2206 reduced AR nuclear translocation. Afatinib diminished AKT phosphorylation at 30 min and 6 h in the EGFR- and EGFRvIII-overexpressing cells, respectively, and decreased AR phosphorylation in EGFR-overexpressing cells at 4 h. Afatinib or MK2206 combination therapy with the AR antagonist enzalutamide in the EGFR and EGFRvIII-overexpressing cells had synergistic efficacy. Our findings suggest that EGFR signaling is involved in AR activation in glioblastoma and buttresses the concept of combining an EGFR signaling inhibitor with AR antagonists as a potential glioblastoma treatment.
Topics: Afatinib; Androgen Receptor Antagonists; Benzamides; Brain Neoplasms; Cell Line, Tumor; ErbB Receptors; Gene Expression Regulation; Glioblastoma; Humans; Ligands; Nitriles; Phenylthiohydantoin; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Receptors, Androgen; Signal Transduction
PubMed: 34681618
DOI: 10.3390/ijms222010954 -
PLoS Genetics Aug 2023Kar4p, the yeast homolog of the mammalian methyltransferase subunit METTL14, is required for efficient mRNA m6A methylation, which regulates meiotic entry. Kar4p is also...
Kar4p, the yeast homolog of the mammalian methyltransferase subunit METTL14, is required for efficient mRNA m6A methylation, which regulates meiotic entry. Kar4p is also required for a second seemingly non-catalytic function during meiosis. Overexpression of the early meiotic transcription factor, IME1, can bypass the requirement for Kar4p in meiotic entry but the additional overexpression of the translational regulator, RIM4, is required to permit sporulation in kar4Δ/Δ. Using microarray analysis and RNA sequencing, we sought to determine the impact of removing Kar4p and consequently mRNA methylation on the early meiotic transcriptome in a strain background (S288c) that is sensitive to the loss of early meiotic regulators. We found that kar4Δ/Δ mutants have a largely wild type transcriptional profile with the exception of two groups of genes that show delayed and reduced expression: (1) a set of Ime1p-dependent early genes as well as IME1, and (2) a set of late genes dependent on the mid-meiotic transcription factor, Ndt80p. The early gene expression defect is likely the result of the loss of mRNA methylation and is rescued by overexpressing IME1, but the late defect is only suppressed by overexpression of both IME1 and RIM4. The requirement for RIM4 led us to predict that the non-catalytic function of Kar4p, like methyltransferase complex orthologs in other systems, may function at the level of translation. Mass spectrometry analysis identified several genes involved in meiotic recombination with strongly reduced protein levels, but with little to no reduction in transcript levels in kar4Δ/Δ after IME1 overexpression. The low levels of these proteins were rescued by overexpression of RIM4 and IME1, but not by the overexpression of IME1 alone. These data expand our understanding of the role of Kar4p in regulating meiosis and provide key insights into a potential mechanism of Kar4p's later meiotic function that is independent of mRNA methylation.
Topics: Animals; Cytoplasm; DNA-Binding Proteins; Gene Expression; Meiosis; Methyltransferases; RNA, Messenger; Saccharomyces cerevisiae Proteins; Transcription Factors; Gene Expression Regulation, Fungal
PubMed: 37639444
DOI: 10.1371/journal.pgen.1010898 -
Biochimica Et Biophysica Acta.... Aug 2020The Ser/Thr protein phosphatase Ppz1 from Saccharomyces cerevisiae is the best characterized member of a family of enzymes only found in fungi. Ppz1 is regulated in vivo...
The Ser/Thr protein phosphatase Ppz1 from Saccharomyces cerevisiae is the best characterized member of a family of enzymes only found in fungi. Ppz1 is regulated in vivo by two inhibitory subunits, Hal3 and Vhs3, which are moonlighting proteins also involved in the decarboxylation of the 4-phosphopantothenoylcysteine (PPC) intermediate required for coenzyme A biosynthesis. It has been reported that, when overexpressed, Ppz1 is the most toxic protein in yeast. However, the reasons for such toxicity have not been elucidated. Here we show that the detrimental effect of excessive Ppz1 expression is due to an increase in its phosphatase activity and not to a plausible down-titration of the PPC decarboxylase components. We have identified several genes encoding ribosomal proteins and ribosome assembly factors as mild high-copy suppressors of the toxic Ppz1 effect. Ppz1 binds to ribosomes engaged in translation and copurifies with diverse ribosomal proteins and translation factors. Ppz1 overexpression results in Gcn2-dependent increased phosphorylation of eIF2α at Ser-51. Consistently, deletion of GCN2 partially suppresses the growth defect of a Ppz1 overexpressing strain. We propose that the deleterious effects of Ppz1 overexpression are in part due to alteration in normal protein synthesis.
Topics: Carboxy-Lyases; Galactokinase; Gene Expression Regulation, Fungal; Phosphoprotein Phosphatases; Phosphorylation; Protein Serine-Threonine Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Saccharomycetales; Transcriptome
PubMed: 32339526
DOI: 10.1016/j.bbamcr.2020.118727 -
Clinical and Experimental Medicine Dec 2023Heat shock protein B6 (HSPB6) plays a certain role in the formation of several cancers, whereas its effect on osteosarcoma remains unclear. In this study, the effect of...
Heat shock protein B6 (HSPB6) plays a certain role in the formation of several cancers, whereas its effect on osteosarcoma remains unclear. In this study, the effect of HSPB6 on osteosarcoma was validated through numerous experiments. HSPB6 was down-regulated in osteosarcoma. As indicated by the result of CCK-8 and colony formation assays, HSPB6 overexpression was likely to inhibit the osteosarcoma cells proliferation, whereas the flow cytometry analysis suggested that apoptosis of osteosarcoma cells was increased after HSPB6 overexpression. Furthermore, transwell and wound healing assays suggested that when HSPB6 was overexpressed, osteosarcoma cells migration and invasion were declined. Moreover, the western blotting assay suggested that the protein level of p-ERK1/2 was down-regulated in osteosarcoma when HSPB6 was overexpressed. Besides, the effect of HSPB6 on osteosarcoma in vivo was examined. As indicated by the result, HSPB6 overexpression was likely to prevent osteosarcoma growth and lung metastasis in vivo. As revealed by the findings of this study, HSPB6 overexpression exerted anticancer effects in osteosarcoma through the ERK signaling pathway and HSPB6 may be suitable target for osteosarcoma molecular therapies.
Topics: Humans; Apoptosis; Bone Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; HSP20 Heat-Shock Proteins; MAP Kinase Signaling System; Osteosarcoma; Signal Transduction
PubMed: 37861934
DOI: 10.1007/s10238-023-01216-9 -
The Journal of Neuroscience : the... Nov 2020Decline of protein quality control in neurons contributes to age-related neurodegenerative disorders caused by misfolded proteins. 4E-BP1 is a key node in the regulation...
Decline of protein quality control in neurons contributes to age-related neurodegenerative disorders caused by misfolded proteins. 4E-BP1 is a key node in the regulation of protein synthesis, as activated 4E-BP1 represses global protein translation. Overexpression of 4E-BP1 mediates the benefits of dietary restriction and can counter metabolic stress, and 4E-BP1 disinhibition on mTORC1 repression may be neuroprotective; however, whether 4E-BP1 overexpression is neuroprotective in mammalian neurons is yet to be fully explored. To address this question, we generated 4E-BP1-overexpressing transgenic mice and confirmed marked reductions in protein translation in 4E-BP1-overexpressing primary neurons. After documenting that 4E-BP1-overexpressing neurons are resistant to proteotoxic stress elicited by brefeldin A treatment, we exposed primary neurons to three different Parkinson's disease (PD)-linked toxins (rotenone, maneb, or paraquat) and documented significant protection in neurons from newborn male and female transgenic mice. We observed 4E-BP1-dependent upregulation of genes encoding proteins that comprise the mitochondrial unfolded protein response, and noted 4E-BP1 overexpression required activation of the mitochondrial unfolded protein response for neuroprotection against rotenone toxicity. We also tested whether 4E-BP1 could prevent α-synuclein neurotoxicity by treating 4E-BP1-overexpressing primary neurons with α-synuclein preformed fibrils, and we observed marked reductions in α-synuclein aggregation and neurotoxicity, thus validating that 4E-BP1 is a powerful suppressor of PD-linked pathogenic insults. Our results indicate that increasing 4E-BP1 expression or enhancing 4E-BP1 activation can robustly induce the mitochondrial unfolded protein response and thus could be an appealing strategy for treating a variety of neurodegenerative diseases, including especially PD. In neurodegenerative disease, misfolded proteins accumulate and overwhelm normal systems of homeostasis and quality control. One mechanism for improving protein quality control is to reduce protein translation. Here we investigated whether neuronal overexpression of 4E-BP1, a key repressor of protein translation, can protect against misfolded protein stress and toxicities linked to Parkinson's disease, and found that 4E-BP1 overexpression prevented cell death in neurons treated with brefeldin A, rotenone, maneb, paraquat, or preformed fibrils of α-synuclein. When we sought the basis for 4E-BP1 neuroprotection, we discovered that 4E-BP1 activation promoted the mitochondrial unfolded protein response. Our findings highlight 4E-BP1 as a therapeutic target in neurodegenerative disease and underscore the importance of the mitochondrial unfolded protein response in neuroprotection against various insults.
Topics: Adaptor Proteins, Signal Transducing; Animals; Animals, Newborn; Brefeldin A; Cell Cycle Proteins; Female; Male; Mice; Mice, Transgenic; Mitochondria; Neurons; Parkinson Disease, Secondary; Primary Cell Culture; Protein Biosynthesis; Protein Synthesis Inhibitors; Protein Unfolding; Proteostasis Deficiencies; Rotenone; Uncoupling Agents; alpha-Synuclein
PubMed: 33046555
DOI: 10.1523/JNEUROSCI.0940-20.2020 -
Molecular Medicine (Cambridge, Mass.) Dec 2022Alzheimer's disease (AD) is the most common neurodegenerative disease worldwide but has no effective treatment. Amyloid beta (Aβ) protein, a primary risk factor for AD,...
BACKGROUND
Alzheimer's disease (AD) is the most common neurodegenerative disease worldwide but has no effective treatment. Amyloid beta (Aβ) protein, a primary risk factor for AD, accumulates and aggregates in the brain of patients with AD. Paired immunoglobulin-like receptor B (PirB) has been identified as a receptor of Aβ and Aβ-PirB molecular interactions that cause synapse elimination and synaptic dysfunction. PirB deletion has been shown to suppress Aβ-induced synaptic dysfunction and behavioral deficits in AD model mice, implying that PirB mediates Aβ-induced AD pathology. Therefore, inhibiting the Aβ-PirB molecular interaction could be a successful approach for combating AD pathology. We previously showed that lateral olfactory tract usher substance (LOTUS) is an endogenous antagonist of type1 Nogo receptor and PirB and that LOTUS overexpression promotes neuronal regeneration following damage to the central nervous system, including spinal cord injury and ischemic stroke. Therefore, in this study, we investigated whether LOTUS inhibits Aβ-PirB interaction and Aβ-induced dendritic spine elimination.
METHODS
The inhibitory role of LOTUS against Aβ-PirB (or leukocyte immunoglobulin-like receptor subfamily B member 2: LilrB2) binding was assessed using a ligand-receptor binding assay in Cos7 cells overexpressing PirB and/or LOTUS. We assessed whether LOTUS inhibits Aβ-induced intracellular alterations and synaptotoxicity using immunoblots and spine imaging in a primary cultured hippocampal neuron.
RESULTS
We found that LOTUS inhibits the binding of Aβ to PirB overexpressed in Cos7 cells. In addition, we found that Aβ-induced dephosphorylation of cofilin and Aβ-induced decrease in post-synaptic density-95 expression were suppressed in cultured hippocampal neurons from LOTUS-overexpressing transgenic (LOTUS-tg) mice compared with that in wild-type mice. Moreover, primary cultured hippocampal neurons from LOTUS-tg mice improved the Aβ-induced decrease in dendritic spine density. Finally, we studied whether human LOTUS protein inhibits Aβ binding to LilrB2, a human homolog of PirB, and found that human LOTUS inhibited the binding of Aβ to LilrB2 in a similar manner.
CONCLUSIONS
This study implied that LOTUS improved Aβ-induced synapse elimination by suppressing Aβ-PirB interaction in rodents and inhibited Aβ-LilrB2 interaction in humans. Our findings revealed that LOTUS may be a promising therapeutic agent in counteracting Aβ-induced AD pathologies.
Topics: Animals; Humans; Mice; Alzheimer Disease; Amyloid beta-Peptides; Dendritic Spines; Immunoglobulins; Mice, Transgenic; Neurodegenerative Diseases; Receptors, Immunologic; Calcium-Binding Proteins
PubMed: 36510132
DOI: 10.1186/s10020-022-00581-7 -
CNS Neuroscience & Therapeutics Sep 2020Astrogliosis and glial scar formation following spinal cord injury (SCI) are viewed as major obstacles that hinder axonal regeneration and functional recovery....
INTRODUCTION
Astrogliosis and glial scar formation following spinal cord injury (SCI) are viewed as major obstacles that hinder axonal regeneration and functional recovery. Regulating the glial scar and axonal regeneration in the lesion site is important for treating SCI.
AIMS
Considering the important role of astrocyte in glial scar formation and subsequent axonal regeneration, we intended to investigate the effect of the transcription factors OCT4 and KLF4 on astrocyte and the underlying mechanism after spinal cord contusion injury in transgenic mice.
RESULTS
Western blotting, q-PCR, immunofluorescence, and functional evaluation suggested that glial fibrillary acidic protein (GFAP) expression decreased in the lesion area, the porosity of the scar increased, and remyelination enhanced. Mice overexpressing the transcription factors OCT4 and KLF4 had higher Basso Mouse Scale scores than did the control mice. Moreover, using immunofluorescence and Western blotting, we discovered that some astrocytes expressed nestin and sox2 protein, suggesting that these astrocytes were reprogrammed into neural stem cell-like cells. Furthermore, a cell scratch assay showed that the migration ability of the astrocytes was significantly inhibited in the presence of the transcription factors OCT4 and KLF4. In addition, we demonstrated that the Hippo/Yap pathway was activated after these two transcription factors overexpressed in astrocytes.
CONCLUSIONS
In summary, these results suggest that overexpression of the transcription factors OCT4 and KLF4 could induce astrocyte reprogramming, which subsequently improves remyelination and functional recovery after SCI.
Topics: Animals; Animals, Newborn; Astrocytes; Cells, Cultured; Gene Expression; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Mice; Mice, Inbred C57BL; Mice, Transgenic; Octamer Transcription Factor-3; Recovery of Function; Spinal Cord Injuries; Transcription Factors
PubMed: 32449258
DOI: 10.1111/cns.13390 -
Molecular and Cellular Biochemistry Aug 2022Non-alcoholic steatohepatitis (NASH) is a rapidly growing liver disease. The chemoattractant chemerin is abundant in hepatocytes, and hepatocyte expressed prochemerin...
Non-alcoholic steatohepatitis (NASH) is a rapidly growing liver disease. The chemoattractant chemerin is abundant in hepatocytes, and hepatocyte expressed prochemerin protected from NASH. Prochemerin is inactive and different active isoforms have been described. Here, the effect of hepatocyte expressed muChem-156, a highly active murine chemerin isoform, was studied in the methionine-choline deficient dietary model of NASH. Mice overexpressing muChem-156 had higher hepatic chemerin protein. Serum chemerin levels and the capability of serum to activate the chemerin receptors was unchanged showing that the liver did not release active chemerin. Notably, activation of the chemerin receptors by hepatic vein blood did not increase in parallel to total chemerin protein in patients with liver cirrhosis. In experimental NASH, muChem-156 had no effect on liver lipids. Accordingly, overexpression of active chemerin in hepatocytes or treatment of hepatocytes with recombinant chemerin did not affect cellular triglyceride and cholesterol levels. Importantly, overexpression of muChem-156 in the murine liver did not change the hepatic expression of inflammatory and profibrotic genes. The downstream targets of chemerin such as p38 kinase were neither activated in the liver of muChem-156 producing mice nor in HepG2, Huh7 and Hepa1-6 cells overexpressing this isoform. Recombinant chemerin had no effect on global gene expression of primary human hepatocytes and hepatic stellate cells within 24 h of incubation. Phosphorylation of p38 kinase was, however, increased upon short-time incubation of HepG2 cells with chemerin. These findings show that muChem-156 overexpression in hepatocytes does not protect from liver steatosis and inflammation.
Topics: Animals; Chemokines; Disease Models, Animal; Hepatic Stellate Cells; Hepatocytes; Humans; Intercellular Signaling Peptides and Proteins; Liver; Mice; Non-alcoholic Fatty Liver Disease; Protein Isoforms
PubMed: 35449483
DOI: 10.1007/s11010-022-04430-3 -
Adipocyte Dec 2020Secreted frizzled-related protein (SFRP) 4 is an extracellular antagonist of Wnt signalling that regulates adipogenesis, and is highly in the visceral adipose tissue of...
Secreted frizzled-related protein (SFRP) 4 is an extracellular antagonist of Wnt signalling that regulates adipogenesis, and is highly in the visceral adipose tissue of obese individuals. However, it is still unclear how exactly SFRP4 regulates the secretion of adipokines in the adipose tissue , an event that is closely related to the pathogenesis of obesity and insulin resistance. In this study, we generated transgenic (Tg) mice overexpressing in the liver and investigated SFRP4 role in adipokine secretion in mice on a regular normal diet. In Tg mice, SFRP4 protein was overexpressed in the liver, as compared to wild-type littermates (non-Tg), and released into the blood. Moreover, the size of adipocytes was smaller in the visceral adipose tissue of Tg mice compared to controls. Additionally, overexpression affected the expression of genes related to adipocyte differentiation, causing the upregulation of adiponectin and glucose transporter 4, and the downregulation of CCAAT/enhancer-binding protein-β, in both visceral and subcutaneous adipose tissue. However, there was no difference in body weight or body composition between Tg and non-Tg mice. In summary, our data showed that overexpression altered adipocyte size and adipokine secretion, possibly affecting adipocyte differentiation, obesity, and glucose metabolism.
Topics: Adipocytes; Adipokines; Adipose Tissue, White; Animals; Cell Differentiation; Cell Size; Gene Expression; Gene Targeting; Humans; Intra-Abdominal Fat; Male; Mice; Mice, Transgenic; Proto-Oncogene Proteins
PubMed: 32657640
DOI: 10.1080/21623945.2020.1792614