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Cell Death & Disease Feb 2022Isoform-specific functions of Numb in the development of cancers, especially in the initiation of epithelial-to-mesenchymal transition (EMT) remains controversial. We...
Isoform-specific functions of Numb in the development of cancers, especially in the initiation of epithelial-to-mesenchymal transition (EMT) remains controversial. We study the specific function of Numb-PRRL isoform in activated EMT of pancreatic ductal adenocarcinoma (PC), which is distinguished from our previous studies that only focused on the total Numb protein. Numb-PRRL isoform was specifically overexpressed and silenced in PC cells combining with TGF-β1 and EGF stimulus. We systematically explored the potential effect of Numb-PRRL in the activated EMT of PC in vitro and in vivo. The total Numb protein was overexpressed in the normal pancreatic duct and well-differentiated PC by IHC. However, Numb-PRRS isoform but not Numb-PRRL showed dominant expression in PC tissues. Numb-PRRL overexpression promoted TGF-β1-induced EMT in PANC-1 and Miapaca-2 cells. TGF-β1-induced EMT-like cell morphology, cell invasion, and migration were enhanced in Numb-PRRL overexpressing groups following the increase of N-cadherin, Vimentin, Smad2/3, Snail1, Snail2, and cleaved-Notch1 and the decrease of E-cadherin. Numb-PRRL overexpression activated TGFβ1-Smad2/3-Snail1 signaling was significantly reversed by the Notch1 inhibitor RO4929097. Conversely, Numb-PRRL silencing inhibited EGF-induced EMT in AsPC-1 and BxPC-3 cells following the activation of EGFR-ERK/MAPK signaling via phosphorylating EGFR at tyrosine 1045. In vivo, Numb-PRRL overexpression or silencing promoted or inhibited subcutaneous tumor size and distant liver metastases via regulating EMT and Snail signaling, respectively. Numb-PRRL promotes TGF-β1- and EGF-induced EMT in PC by regulating TGF-β1-Smad2/3-Snail and EGF-induced EGFR-ERK/MAPK signaling.
Topics: Cell Line, Tumor; Cell Movement; Epidermal Growth Factor; Epithelial-Mesenchymal Transition; ErbB Receptors; Humans; Membrane Proteins; Nerve Tissue Proteins; Pancreatic Neoplasms; Transforming Growth Factor beta1
PubMed: 35197444
DOI: 10.1038/s41419-022-04609-y -
BioMed Research International 2018TRIM24 (Tripartite Motif Containing 24) is a recently identified oncogene overexpressed in various cancers. However, the molecular mechanism of TRIM24 in the progression...
TRIM24 (Tripartite Motif Containing 24) is a recently identified oncogene overexpressed in various cancers. However, the molecular mechanism of TRIM24 in the progression of head and neck squamous cell carcinoma (HNSCC) remains ambiguous. In the present study, we analyzed the expression pattern of TRIM24 in 100 HNSCC tissues and found that TRIM24 was overexpressed in 43/100 HNSCC cases. Significant association was found between TRIM24 overexpression and tumor-node-metastasis (TNM) stage ( = 0.0034) and T stage ( = 0.0048). Furthermore, we overexpressed and knocked down TRIM24 in Detroit 562 and FaDu cell lines, respectively. TRIM24 overexpression promoted proliferation, colony formation, and invasion, while TRIM24 depletion inhibited proliferation, colony formation, and invasion. Further studies showed that TRIM24 facilitated cell cycle transition and upregulated cyclin D1 and p-Rb. In addition, we found that GLUT3, a key protein involved in regulating glucose metabolism, was altered in HNSCC cells overexpressing TRIM24. We demonstrated that TRIM24 overexpression increased glucose uptake ATP production. Overexpression of TRIM24 increases cell sensitivity to glucose deprivation in Detroit cells. Depleting TRIM24 in FaDu cells demonstrated the opposite results. We also showed that TRIM24 could bind to the promoter region of cyclin D1. In conclusion, TRIM24 is upregulated in HNSCC and promotes HNSCC cell growth and invasion through modulation of cell cycle, glucose metabolism, and GLUT3, making TRIM24 a potential oncoprotein in HNSCC.
Topics: Aged; Carcinoma, Squamous Cell; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Glucose; Head and Neck Neoplasms; Humans; Male; Middle Aged; Neoplasm Invasiveness; Neoplasm Proteins
PubMed: 29862279
DOI: 10.1155/2018/6142843 -
PloS One 2019The Acinetobacter baumannii BlsA photoreceptor has an N-terminal (NT) BLUF domain and a C-terminal (CT) amino acid sequence with no significant homology to characterized...
The Acinetobacter baumannii BlsA photoreceptor has an N-terminal (NT) BLUF domain and a C-terminal (CT) amino acid sequence with no significant homology to characterized bacterial proteins. In this study, we tested the biological role of specific residues located in these BlsA regions. Site-directed mutagenesis, surface motility assays at 24°C and protein overexpression showed that residues Y7, Q51 and W92 are essential for not only light-regulated motility, but also BlsA's solubility when overexpressed in a heterologous host. In contrast, residues A29 and F32, the latter representing a difference when compared with other BLUF-containing photoreceptors, do not play a major role in BlsA's biological functions. Analysis of the CT region showed that the deletion of the last five BlsA residues has no significant effect on the protein's light-sensing and motility regulatory functions, but the deletion of the last 14 residues as well as K144E and K145E substitutions significantly alter light-regulated motility responses. In contrast to the NT mutants, these CT derivatives were overexpressed and purified to homogeneity to demonstrate that although these mutations do not significantly affect flavin binding and photocycling, they do affect BlsA's photodynamic properties. Notably, these mutations map within a potential fifth α-helical component that could play a role in predicted interactions between regulatory partners and BlsA, which could function as a monomer according to gel filtration data. All these observations indicate that although BlsA shares common structural and functional properties with unrelated photoreceptors, it also exhibits unique features that make it a distinct BLUF photoreceptor.
Topics: Acinetobacter baumannii; Bacterial Proteins; Mutation; Protein Domains
PubMed: 31415622
DOI: 10.1371/journal.pone.0220918 -
Journal of Thrombosis and Haemostasis :... Aug 2016Essentials Epithelial cell apoptosis is critical in the pathogenesis of idiopathic pulmonary fibrosis. Protein S, a circulating anticoagulant, inhibited apoptosis of...
UNLABELLED
Essentials Epithelial cell apoptosis is critical in the pathogenesis of idiopathic pulmonary fibrosis. Protein S, a circulating anticoagulant, inhibited apoptosis of lung epithelial cells. Overexpression of protein S in lung cells reduced bleomycin-induced pulmonary fibrosis. Intranasal therapy with exogenous protein S ameliorated bleomycin-induced pulmonary fibrosis.
SUMMARY
Background Pulmonary fibrosis is the terminal stage of interstitial lung diseases, some of them being incurable and of unknown etiology. Apoptosis plays a critical role in lung fibrogenesis. Protein S is a plasma anticoagulant with potent antiapoptotic activity. The role of protein S in pulmonary fibrosis is unknown. Objectives To evaluate the clinical relevance of protein S and its protective role in pulmonary fibrosis. Methods and Results The circulating level of protein S was measured in patients with pulmonary fibrosis and controls by the use of enzyme immunoassays. Pulmonary fibrosis was induced with bleomycin in transgenic mice overexpressing human protein S and wild-type mice, and exogenous protein S or vehicle was administered to wild-type mice; fibrosis was then compared in both models. Patients with pulmonary fibrosis had reduced circulating levels of protein S as compared with controls. Inflammatory changes, the levels of profibrotic cytokines, fibrosis score, hydroxyproline content in the lungs and oxygen desaturation were significantly reduced in protein S-transgenic mice as compared with wild-type mice. Wild-type mice treated with exogenous protein S showed significant decreases in the levels of inflammatory and profibrotic markers and fibrosis in the lungs as compared with untreated control mice. After bleomycin infusion, mice overexpressing human protein S showed significantly low caspase-3 activity, enhanced expression of antiapoptotic molecules and enhanced Akt and Axl kinase phosphorylation as compared with wild-type counterparts. Protein S also inhibited apoptosis of alveolar epithelial cells in vitro. Conclusions These observations suggest clinical relevance and a protective role of protein S in pulmonary fibrosis.
Topics: A549 Cells; Aged; Animals; Apoptosis; Bleomycin; Blood Proteins; Bronchoalveolar Lavage Fluid; Caspase 3; Epithelial Cells; Female; Fibrosis; Gene Expression Profiling; Humans; Idiopathic Pulmonary Fibrosis; Immunoenzyme Techniques; Inflammation; Lung; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Middle Aged; Phosphorylation; Protein S
PubMed: 27172994
DOI: 10.1111/jth.13362 -
Viruses Oct 2022Prions replicate by a self-templating mechanism. Infidelity in the process can lead to the emergence of new infectious structures, referred to as variants or strains....
Prions replicate by a self-templating mechanism. Infidelity in the process can lead to the emergence of new infectious structures, referred to as variants or strains. The question of whether prions are prone to mis-templating is not completely answered. Our previous experiments with 23 variants of the yeast [] prion do not support broad mutability. However, it became clear recently that the heat shock protein Hsp104 can restrict [] strain variation. This raises the possibility that many transmutable variants of the prion may have been mistaken as faithful-propagating simply because the mutant structure was too sturdy or too frail to take root in the wild-type cell. Here, I alter the strength of Hsp104 in yeast, overexpressing wild-type Hsp104 or expressing the hypo-active Hsp104 mutant, and check if the new environments enable the variants to mutate. Two variants hitherto thought of as faithful-propagating are discovered to generate different structures, which are stabilized with the hypo-active chaperone. In contrast, most transmutable variants discovered in cells overexpressing Hsp104 have been correctly identified as such previously in wild-type cells without the overexpression. The majority of transmutable variants only mis-template the structure of VH, VK, or VL, which are the most frequently observed variants and do not spontaneously mutate. There are four additional variants that never give rise to different structures in all cell conditions tested. Therefore, quite a few [] variants are faithful-propagating, and even the transmutable ones do not freely evolve but can only change to limited structural types.
Topics: Saccharomyces cerevisiae; Prions; Peptide Termination Factors; Saccharomyces cerevisiae Proteins; Heat-Shock Proteins
PubMed: 36366434
DOI: 10.3390/v14112337 -
Cancer Science Jan 2022CD28, one of the costimulatory molecules, has a pivotal role in T-cell activation, and its expression is strictly regulated in normal T cells. Gain-of-function genetic...
CD28, one of the costimulatory molecules, has a pivotal role in T-cell activation, and its expression is strictly regulated in normal T cells. Gain-of-function genetic alterations involving CD28 have been frequently observed in adult T-cell leukemia/lymphoma (ATLL). These abnormalities, such as CD28 fusions and copy number variations, may not only confer continuous, prolonged, and enhanced CD28 signaling to downstream pathways but also induce overexpression of the CD28 protein. In this study, 120 ATLL cases were examined by immunohistochemistry for CD28 and its ligands CD80 and CD86, and their expression on tumor cells was semiquantitatively evaluated. CD28 was overexpressed in 55 (46%) cases, and CD80 or CD86 (CD80/CD86) was infrequently overexpressed in 12 (11%). Compared with non-overexpressers, CD28 overexpressers showed a higher frequency of CD28 genetic alterations and had an increased number of CD80/CD86-positive non-neoplastic cells infiltrating tumor microenvironment. In the entire ATLL patient cohort, CD28 overexpressers showed a significantly poorer overall survival (OS) compared with non-overexpressers (P = .001). The same was true for a subgroup who were treated with multidrug regimens with or without mogamulizumab. CD28 overexpression had no prognostic impact in the group who received allogeneic hematopoietic stem cell transplantation. In the multivariate analysis for OS, CD28 overexpression was selected as an independent risk factor. These results suggest ATLL patients with CD28 overexpression have more aggressive clinical course and are more refractory to treatment with multidrug chemotherapy. CD28 overexpression appears to be a novel unfavorable prognostic marker in ATLL patients, and further prospective studies are warranted to establish its prognostic significance.
Topics: Adult; Aged; Aged, 80 and over; B7-1 Antigen; B7-2 Antigen; CD28 Antigens; DNA Copy Number Variations; Female; Gene Expression Regulation, Neoplastic; Humans; Leukemia-Lymphoma, Adult T-Cell; Male; Middle Aged; Prognosis; Survival Analysis; Tumor Microenvironment; Up-Regulation
PubMed: 34738707
DOI: 10.1111/cas.15191 -
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 -
Investigative Ophthalmology & Visual... Feb 2012Neuroglobin (Ngb) is a vertebrate globin that is predominantly expressed in the retina and brain. To explore the role of Ngb in retinal neuroprotection during ischemia...
PURPOSE
Neuroglobin (Ngb) is a vertebrate globin that is predominantly expressed in the retina and brain. To explore the role of Ngb in retinal neuroprotection during ischemia reperfusion (IR), the authors examined the effect of Ngb overexpression in the retina in vivo by using Ngb-transgenic (Ngb-Tg) mice.
METHODS
Retinal IR was induced in Ngb overexpressing Ngb-Tg mice and wild type (WT) mice by cannulating the anterior chamber and transiently elevating the IOP for 60 minutes. After Day 7 of reperfusion, the authors evaluated Ngb mRNA and protein expression in nonischemic control as well as ischemic mice and its effect on retinal histology, mitochondrial oxidative stress, and apoptosis, using morphometry and immunohistochemistry, quantitative PCR analysis and Western blot techniques.
RESULTS
Ngb-Tg mice without ischemia overexpress Ngb mRNA 11.3-fold (SE ± 0.457, P < 0.05) higher than WT control mice, and this overexpression of Ngb protein was localized to the mitochondria of the ganglion cells, outer and inner plexiform layers, and photoreceptor inner segments. This overexpression of Ngb is associated with decreased mitochondrial DNA damage in Ngb-Tg mice with IR in comparison with WT. Ngb-Tg mice with IR also revealed significant preservation of retinal thickness, significantly less activated caspase 3 protein expression, and apoptosis in comparison with WT mice.
CONCLUSIONS
Neuroglobin overexpression plays a neuroprotective role against retinal ischemia reperfusion injury due to decreasing of mitochondrial oxidative stress-mediated apoptosis.
Topics: Animals; Apoptosis; Blotting, Western; Case-Control Studies; Caspase 3; Disease Models, Animal; Globins; Mice; Mice, Transgenic; Mitochondria; Nerve Tissue Proteins; Neuroglobin; Oxidative Stress; Real-Time Polymerase Chain Reaction; Reperfusion Injury; Retinal Diseases
PubMed: 22167093
DOI: 10.1167/iovs.11-7408 -
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 -
Biochimica Et Biophysica Acta Feb 2003Membrane transporter proteins play critical physiological roles in the cell and constitute 5-10% of prokaryotic and eukaryotic genomes. High-resolution structural... (Review)
Review
Membrane transporter proteins play critical physiological roles in the cell and constitute 5-10% of prokaryotic and eukaryotic genomes. High-resolution structural information is essential for understanding the functional mechanism of these proteins. A prerequisite for structural study is to overexpress such proteins in large quantities. In the last few years, over 20 bacterial membrane transporters were overexpressed at a level of 1 mg/l of culture or higher, most often in Escherichia coli. In this review, we analyzed those factors that affect the quantity and quality of the protein produced, and summarized recent progress in overexpression of membrane transporters from bacterial inner membrane. Rapid progress in genome sequencing provides opportunities for expressing several homologues and orthologues of the target protein simultaneously, while the availability of various expression vectors allows flexible experimental design. Careful optimization of cell culture conditions can drastically improve the expression level and homogeneity of the target protein. New sample preparation techniques for mass spectrometry of membrane proteins have enabled one to identity the rigid protein core, which can be subsequently overexpressed. Size-exclusion chromatography on HPLC has proven to be an efficient method in screening detergent, pH an other conditions required for maintaining the stability and monodispersity of the protein. Such high-quality preparations of membrane transporter proteins will probably lead to successful crystallization and structure determination of these proteins in the next few years.
Topics: Bacterial Proteins; Culture Media; Detergents; Escherichia coli Proteins; Genetic Vectors; Humans; Intracellular Membranes; Membrane Transport Proteins; Quality Control; Temperature
PubMed: 12586376
DOI: 10.1016/s0005-2736(02)00709-5