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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 -
The Journal of Biological Chemistry Nov 2018Alzheimer's disease mouse models that overexpress amyloid precursor protein (APP) and presenilin 1 (PS1) form β-amyloid (Aβ) plaques, a hallmark Alzheimer's disease...
Alzheimer's disease mouse models that overexpress amyloid precursor protein (APP) and presenilin 1 (PS1) form β-amyloid (Aβ) plaques, a hallmark Alzheimer's disease lesion. It has been assumed that the neuroinflammation, synaptic dysfunction, neurodegeneration, and cognitive impairment observed in these mice are caused by cerebral Aβ accumulation. However, it is also possible that accumulation of the overexpressed transmembrane proteins APP and PS1 in the endoplasmic reticulum (ER) triggers chronic ER stress and activation of the unfolded protein response (UPR). The 5XFAD mouse, a widely used amyloid pathology model, overexpresses APP and PS1, displays aggressive amyloid pathology, and has been reported to exhibit ER stress. To systematically evaluate whether 5XFAD mice have increased ER stress, here we used biochemical approaches to assess a comprehensive panel of UPR markers. We report that APP and PS1 levels are 1.8- and 1.5-fold, respectively, of those in 5XFAD compared with nontransgenic brains, indicating that transgenes are not massively overexpressed in 5XFAD mice. Using immunoblotting, we quantified UPR protein levels in nontransgenic, 5XFAD, and 5XFAD;BACE1 mice at 4, 6, and 9 months of age. Importantly, we did not observe elevation of the ER stress markers p-eIF2α, ATF4, CHOP, p-IRE1α, or BiP at any age in 5XFAD or 5XFAD;BACE1 compared with nontransgenic mice. Despite lacking Aβ generation, 5XFAD;BACE1 mice still expressed APP and PS1 transgenes, indicating that their overexpression does not cause ER stress. These results reveal the absence of ER stress in 5XFAD mice, suggesting that artifactual phenotypes associated with overexpression-induced ER stress are not a concern in this model.
Topics: Alzheimer Disease; Amyloid Precursor Protein Secretases; Amyloid beta-Protein Precursor; Animals; Aspartic Acid Endopeptidases; Disease Models, Animal; Endoplasmic Reticulum Stress; Female; Humans; Male; Mice; Mice, Transgenic; Mutation; Presenilin-1; Unfolded Protein Response
PubMed: 30315100
DOI: 10.1074/jbc.RA118.005769 -
Nucleic Acids Research Apr 2022Restricting the localization of CENP-A (Cse4 in Saccharomyces cerevisiae) to centromeres prevents chromosomal instability (CIN). Mislocalization of overexpressed CENP-A...
Restricting the localization of CENP-A (Cse4 in Saccharomyces cerevisiae) to centromeres prevents chromosomal instability (CIN). Mislocalization of overexpressed CENP-A to non-centromeric chromatin contributes to CIN in budding and fission yeasts, flies, and humans. Overexpression and mislocalization of CENP-A is observed in cancers and is associated with increased invasiveness. Mechanisms that remove mislocalized CENP-A and target it for degradation have not been defined. Here, we report that Cdc48 and its cofactors Ufd1 and Npl4 facilitate the removal of mislocalized Cse4 from non-centromeric chromatin. Defects in removal of mislocalized Cse4 contribute to lethality of overexpressed Cse4 in cdc48,ufd1 andnpl4 mutants. High levels of polyubiquitinated Cse4 and mislocalization of Cse4 are observed in cdc48-3, ufd1-2 and npl4-1mutants even under normal physiological conditions, thereby defining polyubiquitinated Cse4 as the substrate of the ubiquitin directed segregase Cdc48Ufd1/Npl4. Accordingly, Npl4, the ubiquitin binding receptor, associates with mislocalized Cse4, and this interaction is dependent on Psh1-mediated polyubiquitination of Cse4. In summary, we provide the first evidence for a mechanism that facilitates the removal of polyubiquitinated and mislocalized Cse4 from non-centromeric chromatin. Given the conservation of Cdc48Ufd1/Npl4 in humans, it is likely that defects in such pathways may contribute to CIN in human cancers.
Topics: Centromere; Centromere Protein A; Chromatin; Chromosomal Proteins, Non-Histone; DNA-Binding Proteins; Histones; Humans; Nucleocytoplasmic Transport Proteins; Proteolysis; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin; Valosin Containing Protein; Vesicular Transport Proteins
PubMed: 35234920
DOI: 10.1093/nar/gkac135 -
Yakugaku Zasshi : Journal of the... Mar 2007To elucidate the mechanism of toxicity of methylmercury (MeHg), we searched for factors that determine the sensitivity of yeast cells to MeHg and found that... (Review)
Review
To elucidate the mechanism of toxicity of methylmercury (MeHg), we searched for factors that determine the sensitivity of yeast cells to MeHg and found that overexpression of Cdc34 or Rad23, both proteins related to the ubiquitin-proteasome (UP) system, induces resistance to MeHg toxicity. The acquisition of resistance to MeHg in Cdc34-overexpressing yeast cells requires the ubiquitin-conjugating activity of Cdc34 and the proteolytic activity of proteasomes. Therefore, it seems likely that certain as-yet-unidentified proteins that increase MeHg toxicity might exist in cells and that the toxicity of MeHg might be reduced by the enhanced degradation of such proteins through the UP system when Cdc34 is overexpressed. Unlike Cdc34, Rad23 suppresses the degradation of ubiquitinated proteins by proteasomes. This activity of Rad23 might be involved in the acquisition of resistance to MeHg toxicity when Rad23 is overexpressed. Overexpression of Rad23 might induce resistance to MeHg by suppressing the degradation of proteins that reduce the MeHg toxicity. Moreover, when we overexpressed Cdc34 in normal and Rad23-defective yeasts, resistance to MeHg was enhanced to almost the same extent in both lines of yeast cells. Thus it is possible that the binding of Rad23 to ubiquitinated proteins might be regulated by a mechanism that involves the recognition of substrate proteins and that the functions of Rad23 might not affect the protein-degradation system in which Cdc34 is involved. Many proteins that reduce or enhance MeHg toxicity and are ubiquitinated might exist in cells. The UP system and related proteins might determine the extent of MeHg toxicity by regulating the cellular concentrations of these various proteins.
Topics: Anaphase-Promoting Complex-Cyclosome; DNA Repair Enzymes; DNA-Binding Proteins; Drug Resistance, Fungal; F-Box Proteins; Gene Expression; Methylmercury Compounds; Proteasome Endopeptidase Complex; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligase Complexes
PubMed: 17329932
DOI: 10.1248/yakushi.127.463 -
Journal of the National Cancer Institute Jan 2020Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA-binding protein that is aberrantly expressed in cancers. We and others have previously shown that reduced...
BACKGROUND
Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA-binding protein that is aberrantly expressed in cancers. We and others have previously shown that reduced hnRNP K expression downmodulates tumor-suppressive programs. However, overexpression of hnRNP K is the more commonly observed clinical phenomenon, yet its functional consequences and clinical significance remain unknown.
METHODS
Clinical implications of hnRNP K overexpression were examined through immunohistochemistry on samples from patients with diffuse large B-cell lymphoma who did not harbor MYC alterations (n = 75). A novel transgenic mouse model that overexpresses hnRNP K specifically in B cells was generated to directly examine the role of hnRNP K overexpression in mice (three transgenic lines). Molecular consequences of hnRNP K overexpression were determined through proteomics, formaldehyde-RNA-immunoprecipitation sequencing, and biochemical assays. Therapeutic response to BET-bromodomain inhibition in the context of hnRNP K overexpression was evaluated in vitro and in vivo (n = 3 per group). All statistical tests were two-sided.
RESULTS
hnRNP K is overexpressed in diffuse large B-cell lymphoma patients without MYC genomic alterations. This overexpression is associated with dismal overall survival and progression-free survival (P < .001). Overexpression of hnRNP K in transgenic mice resulted in the development of lymphomas and reduced survival (P < .001 for all transgenic lines; Line 171[n = 30]: hazard ratio [HR] = 64.23, 95% confidence interval [CI] = 26.1 to 158.0; Line 173 [n = 31]: HR = 25.27, 95% CI = 10.3 to 62.1; Line 177 [n = 25]: HR = 119.5, 95% CI = 42.7 to 334.2, compared with wild-type mice). Clinical samples, mouse models, global screening assays, and biochemical studies revealed that hnRNP K's oncogenic potential stems from its ability to posttranscriptionally and translationally regulate MYC. Consequently, Hnrnpk overexpression renders cells sensitive to BET-bromodomain-inhibition in both in vitro and transplantation models, which represents a strategy for mitigating hnRNP K-mediated c-Myc activation in patients.
CONCLUSION
Our findings indicate that hnRNP K is a bona fide oncogene when overexpressed and represents a novel mechanism for c-Myc activation in the absence of MYC lesions.
Topics: Adult; Aged; Animals; Antineoplastic Agents; Cell Line, Tumor; Disease Models, Animal; Disease Susceptibility; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Heterogeneous-Nuclear Ribonucleoprotein K; Humans; Lymphoma, B-Cell; Male; Mice; Mice, Transgenic; Middle Aged; Neoplasm Staging; Phenotype; Protein Binding; Protein Interaction Domains and Motifs; RNA-Binding Proteins
PubMed: 31077320
DOI: 10.1093/jnci/djz078 -
Journal of Experimental Botany Feb 2019The activity of the protein kinase STN7, involved in phosphorylation of the light-harvesting complex II (LHCII) proteins, has been reported as being co-operatively...
The activity of the protein kinase STN7, involved in phosphorylation of the light-harvesting complex II (LHCII) proteins, has been reported as being co-operatively regulated by the redox state of the plastoquinone pool and the ferredoxin-thioredoxin (Trx) system. The present study aims to investigate the role of plastid Trxs in STN7 regulation and their impact on photosynthesis. For this purpose, tobacco plants overexpressing Trx f or m from the plastid genome were characterized, demonstrating that only Trx m overexpression was associated with a complete loss of LHCII phosphorylation that did not correlate with decreased STN7 levels. The absence of phosphorylation in Trx m-overexpressing plants impeded migration of LHCII from PSII to PSI, with the concomitant loss of PSI-LHCII complex formation. Consequently, the thylakoid ultrastructure was altered, showing reduced grana stacking. Moreover, the electron transport rate was negatively affected, showing an impact on energy-demanding processes such as the Rubisco maximum carboxylation capacity and ribulose 1,5-bisphosphate regeneration rate values, which caused a strong depletion in net photosynthetic rates. Finally, tobacco plants overexpressing a Trx m mutant lacking the reactive redox site showed equivalent physiological performance to the wild type, indicating that the overexpressed Trx m deactivates STN7 in a redox-dependent way.
Topics: Chloroplast Thioredoxins; Chloroplasts; Gene Expression Regulation, Plant; Oxidation-Reduction; Photosynthesis; Photosystem II Protein Complex; Plant Proteins; Protein Serine-Threonine Kinases; Nicotiana
PubMed: 30476130
DOI: 10.1093/jxb/ery415 -
Molecular and Biochemical Parasitology Sep 2020Altering amounts of a protein in a cell has become a crucial tool for understanding its function. In many organisms, including the protozoan parasite Trypanosoma brucei,...
Altering amounts of a protein in a cell has become a crucial tool for understanding its function. In many organisms, including the protozoan parasite Trypanosoma brucei, protein overexpression has been achieved by inserting a protein-coding sequence into an overexpression vector. Here, we have adapted the PCR only based system for tagging trypanosome proteins at their endogenous loci such that it in addition enables a tetracycline-inducible T7 RNA polymerase-mediated protein overexpression. Hence, this approach bypasses the need for molecular cloning, making it rapid and cost effective. We validated the approach for ten flagellum-associated proteins with molecular weights ranging from 40 to over 500 kDa. For a majority of the recombinant proteins a significant (3-50 fold) increase in the cellular amount was achieved upon induction of overexpression. Two of the largest proteins studied, the dynein heavy chains, were significantly overexpressed, while two were not. Our data suggest that this may reflect the extent of the T7 RNA polymerase processivity on the trypanosome genomic DNA. We further show that the overexpression is informative as to cellular functions of the studied proteins, and that these cultures can serve as an excellent source for purification of the overexpressed proteins. We believe that this rapid in locus overexpression system will become a valuable tool to interrogate cellular functions and biochemical activities of trypanosome proteins.
Topics: DNA-Directed RNA Polymerases; Dyneins; Gene Expression; Genes, Protozoan; Protozoan Proteins; Recombinant Proteins; Trypanosoma brucei brucei; Viral Proteins
PubMed: 32682799
DOI: 10.1016/j.molbiopara.2020.111300 -
Communications Biology Oct 2020High expression of centrosomal protein CEP55 has been correlated with clinico-pathological parameters across multiple human cancers. Despite significant in vitro studies...
High expression of centrosomal protein CEP55 has been correlated with clinico-pathological parameters across multiple human cancers. Despite significant in vitro studies and association of aberrantly overexpressed CEP55 with worse prognosis, its causal role in vivo tumorigenesis remains elusive. Here, using a ubiquitously overexpressing transgenic mouse model, we show that Cep55 overexpression causes spontaneous tumorigenesis and accelerates Trp53 induced tumours in vivo. At the cellular level, using mouse embryonic fibroblasts (MEFs), we demonstrate that Cep55 overexpression induces proliferation advantage by modulating multiple cellular signalling networks including the hyperactivation of the Pi3k/Akt pathway. Notably, Cep55 overexpressing MEFs have a compromised Chk1-dependent S-phase checkpoint, causing increased replication speed and DNA damage, resulting in a prolonged aberrant mitotic division. Importantly, this phenotype was rescued by pharmacological inhibition of Pi3k/Akt or expression of mutant Chk1 (S280A) protein, which is insensitive to regulation by active Akt, in Cep55 overexpressing MEFs. Moreover, we report that Cep55 overexpression causes stabilized microtubules. Collectively, our data demonstrates causative effects of deregulated Cep55 on genome stability and tumorigenesis which have potential implications for tumour initiation and therapy development.
Topics: Animals; Biomarkers, Tumor; Biopsy; Cell Cycle Proteins; Cell Line; Cell Transformation, Neoplastic; Checkpoint Kinase 1; Disease Susceptibility; Fibroblasts; Gene Expression; Genomic Instability; Genotype; Immunohistochemistry; Karyotype; Lymph Nodes; Mice; Mice, Transgenic; Microtubules; Mitosis; Protein Stability; Proto-Oncogene Proteins c-akt; Signal Transduction; Stress, Physiological; Tumor Suppressor Protein p53
PubMed: 33087841
DOI: 10.1038/s42003-020-01304-6 -
International Journal of Oncology Jun 2016The contribution of CXCL12/CXCR4/CXCR7 axis to cancer progression has been increasingly recognized. However, its role in thyroid cancer development remains unclear. The...
The contribution of CXCL12/CXCR4/CXCR7 axis to cancer progression has been increasingly recognized. However, its role in thyroid cancer development remains unclear. The present study aimed to examine the expression and function of CXCL12 and its receptors in thyroid cancer. The expression of CXCL12/CXCR4/CXCR7 in human tissue specimens of papillary, follicular, medullary, and anaplastic thyroid carcinoma, follicular adenoma, Hashimoto's thyroiditis and nodular goiter were examined by immunohistochemistry using a tissue microarray. CXCR4 and CXCR7 were over-expressed in human thyroid cancer cells K1 by transduction of recombinant lentivirus. The effect of overexpression of CXCR4 and CXCR7 on K1 cell proliferation and invasion and the molecular mechanism underlying the effect were investigated. CXCL12 was exclusively expressed in papillary thyroid carcinoma tissue but absent in other types of thyroid malignancies and benign lesions. CXCR7 was widely expressed in the endothelial cells of all types of malignancy but only occasionally detected in benign lesions. CXCR4 was expressed in 62.5% of papillary thyroid carcinoma tissue specimens and in 30-40% of other types of malignancy, and it was either absent or weakly expressed in benign lesions. CXCL12 stimulated the invasion and migration of K1 cells overexpressing CXCR4, but did not affect K1 cells overexpressing CXCR7. K1 cell proliferation was not affected by overexpression of CXCR4 or CXCR7. Overexpression of CXCR4 in K1 cells significantly increased AKT and ERK phosphorylation and markedly induced the expression and activity of matrix metalloproteinase-2 (MMP‑2). Thus, CXCL12 may be an effective diagnostic marker for papillary thyroid carcinoma, and CXCL12/CXCR4/CXCR7 axis may contribute to thyroid cancer development by regulating cancer cell migration and invasion via AKT and ERK signaling and MMP-2 activation.
Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Chemokine CXCL12; Extracellular Signal-Regulated MAP Kinases; Humans; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Phosphorylation; Proto-Oncogene Proteins c-akt; Receptors, CXCR; Receptors, CXCR4; Thyroid Neoplasms; Up-Regulation
PubMed: 27082011
DOI: 10.3892/ijo.2016.3485