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1.
Frontiers of Medicine ; (4): 252-263, 2021.
Article in English | WPRIM | ID: wpr-880970

ABSTRACT

An unexpected observation among the COVID-19 pandemic is that smokers constituted only 1.4%-18.5% of hospitalized adults, calling for an urgent investigation to determine the role of smoking in SARS-CoV-2 infection. Here, we show that cigarette smoke extract (CSE) and carcinogen benzo(a)pyrene (BaP) increase ACE2 mRNA but trigger ACE2 protein catabolism. BaP induces an aryl hydrocarbon receptor (AhR)-dependent upregulation of the ubiquitin E3 ligase Skp2 for ACE2 ubiquitination. ACE2 in lung tissues of non-smokers is higher than in smokers, consistent with the findings that tobacco carcinogens downregulate ACE2 in mice. Tobacco carcinogens inhibit SARS-CoV-2 spike protein pseudovirions infection of the cells. Given that tobacco smoke accounts for 8 million deaths including 2.1 million cancer deaths annually and Skp2 is an oncoprotein, tobacco use should not be recommended and cessation plan should be prepared for smokers in COVID-19 pandemic.


Subject(s)
Adult , Animals , COVID-19 , Epithelial Cells , Humans , Lung , Mice , Pandemics , Peptidyl-Dipeptidase A , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Ubiquitin-Protein Ligases/genetics
2.
Article in Chinese | WPRIM | ID: wpr-880151

ABSTRACT

OBJECTIVE@#To investigate the effect of the tripartite motif containing 31 (TRIM31) gene silencing on the proliferation and apoptosis of multiple myeloma cells and its possible mechanism.@*METHODS@#The normal bone marrow plasma cells (nPCs) were selected as control, and the mRNA and protein expression levels of TRIM31 in human multiple myeloma cell lines (U266, RPMI-8226, NCI-H929 and KMS-11) were detected by RT-qPCR and Western blot. Recombinant lentivirol vector containing shRNA-TRIM31 and its negative control were used to infect U266 cells respectively, and the mRNA expression level of TRIM31 in infected cells was detected by RT-qPCR. Then cell proliferation, colony forming and apoptosis were analyzed by CCK-8, soft agar assay, and flow cytometry, respectively. The protein expression levels of TRIM31, cleaved-caspase-3, BCL-2, Bax, p-Akt (Ser473), Akt and PI3K (p110α) were evaluated by Western blot. In addition, the PI3K/Akt signaling pathway-specific inhibitor LY294002 and TRIM31-shRNA lentivirus were used to interfere with U266 cells, and the cell proliferation, apoptosis, and protein expression of p-Akt (Ser473) and Akt were detected by CCK-8, flow cytometry and Western blot, respectively.@*RESULTS@#Compared with nPCs, the expression levels of TRIM31 mRNA and protein in U266, RPMI-8226, NCI-H929 and KMS-11 cells were significantly increased (P<0.001), especially in U266 cells. After lentivirus infection, the levels of TRIM31 mRNA and protein in U266 cells were significantly decreased (P<0.001). TRIM31 silencing significantly inhibited the proliferation of U266 cells (P<0.05), attenuated the ability of cell cloning, improved cell apoptosis, up-regulated the protein expressions of cleaved-caspase-3 and Bas as well as down-regulated expressions of BCL-2, p-Akt (Ser473) and PI3K (p110α). There was no significant effect on Akt protein. Intervention of LY294002 significantly enhanced the inhibition on cell proliferation and the promotion on apoptosis mediated by TRIM31 gene silencing in U266 cells.@*CONCLUSION@#TRIM31 gene silencing can inhibit U266 cell proliferation and promote its apoptosis, which may be closely related to inhibition of PI3K/Akt signaling pathway.


Subject(s)
Apoptosis , Cell Line, Tumor , Cell Proliferation , Gene Silencing , Humans , Multiple Myeloma , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Tripartite Motif Proteins/genetics , Ubiquitin-Protein Ligases/genetics
3.
Article in Chinese | WPRIM | ID: wpr-888362

ABSTRACT

OBJECTIVE@#To screen proteins interacting with ring finger protein 216(RNF216) through yeast two hybrid experiment, and further clarify the role of RNF216 in the pathogenesis of gonadotropin-releasing hormone deficiency.@*METHODS@#A recombinant expression vector pGBKT7-RNF216 was constructed and transformed into yeast Y2HGold, which was hybridized with a human cDNA library in order to screen proteins interacting with RNF216. The interaction was verified in yeast Y2HGold.@*RESULTS@#A recombinant expression vector pGBKT7-RNF216 was successfully constructed and expressed in yeast Y2HGold. Filamin B (FLNB) was identified by yeast two hybrid experiment, and their interaction was verified in yeast Y2HGold.@*CONCLUSION@#An interaction between FLNB and RNF216 was identified through yeast two hybrid experiment. RNF216 may affect the proliferation and migration of GnRH neurons by regulating FLNB or FLNB/FLNA heterodimers.


Subject(s)
Gene Library , Gonadotropin-Releasing Hormone/genetics , Humans , Proteins , Two-Hybrid System Techniques , Ubiquitin-Protein Ligases/genetics
4.
Article in Chinese | WPRIM | ID: wpr-879538

ABSTRACT

OBJECTIVE@#To explore the genetic basis for a patient with intellectual disability.@*METHODS@#Whole exome sequencing and Sanger sequencing were carried out for the patient. The result was verified in her family.@*RESULTS@#DNA sequencing revealed that the patient has carried a heterozygous nonsense c.40C>T (p.Arg14X) variant of the TRIP12 gene, which was de novo in origin. The variant was unrecorded in the Human Gene Mutation Database. Based on the American College of Medical Genetics and Genomics standards and guidelines, the variant was predicted to be pathogenic (PVS1+ PS2+ PP3).@*CONCLUSION@#The patient was diagnosed with autosomal dominant intellectual disability due to heterozygous c.40C>T variant of the TRIP12 gene.


Subject(s)
Carrier Proteins/genetics , Codon, Nonsense , Female , Heterozygote , Humans , Intellectual Disability/genetics , Ubiquitin-Protein Ligases/genetics , Whole Exome Sequencing
5.
Article in Chinese | WPRIM | ID: wpr-880798

ABSTRACT

OBJECTIVE@#To evaluate the effect of rosmarinic acid (RA) on mitophagy and hypertrophy of cardiomyocytes exposed to high glucose (HG).@*METHODS@#Rat cardiomyocytes (H9c2) exposed to HG (25 mmol/L) were treated with 50 μmol/L RA or with both RA treatment and Parkin siRNA transfection, with the cells cultured in normal glucose (5.5 mmol/L) and HG as the controls. The expressions of PINK1, Parkin and LC3II/LC3I in the cells were detected by Western blotting. The formation of mitochondrial autophagosomes was observed by transmission electron microscope. Flow cytometry was employed to detect the level of reactive oxygen species (ROS) and apoptotic rate of the cells. The activities of respiratory chain complex enzymes were measured by spectrophotometry. Fluorescence enzyme labeling and @*RESULTS@#RA treatment significantly increased the expression levels of PINK1, Parkin and LC3-II/I (@*CONCLUSIONS@#RA can protect rat cardiomyocytes against oxidative stress injury and cardiomyocyte hypertrophy induced by HG by activating Parkin-mediated mitophagy.


Subject(s)
Animals , Cinnamates , Depsides , Glucose , Hypertrophy , Mitophagy , Myocytes, Cardiac , Protein Kinases , Rats , Reactive Oxygen Species , Ubiquitin-Protein Ligases/genetics
6.
Biol. Res ; 53: 09, 2020. graf
Article in English | LILACS | ID: biblio-1100915

ABSTRACT

BACKGROUND: Osteoarthritis (OA) is one of the most common rheumatic diseases of which clinical symptoms includes swelling, synovitis and inflammatory pain, affect patients' daily life. It was reported that non-coding RNAs play vital roles in OA. However, the regulation mechanism of ncRNA in OA pathogenesis has not been fully elucidated. METHODS: The expression of SNHG7, miR-34a-5p and SYVN1 was detected using qRT-PCR in tissues, serum and cells. The protein expression of SYVN1, PCNA, cleavage-caspase 3, beclinl and LC3 were measured using western blot. The RNA immunoprecipitation (RIP), RNA pulldown, and luciferase reporter assays were used to verify the relationship between SNHG7, miR-34a-5p and SYVN1. The MTT and flow cytometry assay was performed to detected cell proliferation and cell apoptosis respectively. RESULTS: In this study, SNHG7 and SYVN1 expression were down-regulated, but miR-34a-5p was up-regulated in OA tissues and IL-1P treated cells compared with normal tissues and chondrocyte. Functional investigation revealed that up-regulated SNHG7 or down-regulated miR-34a-5p could promote cell proliferation and inhibit cell apoptosis and autophagy in OA cells. More than that, RIP, pulldown and luciferase reporter assay was applied to determine that miR-34a-5p was a target miRNA of SNHG7 and SYVN1 was a target mRNA of miR-34-5p. Rescue experiments showed that overexpression of miR-34a reversed high expression of SNHG7-mediated suppression of apoptosis and autophagy as well as promotion of proliferation, while its knockdown inhibited cell apoptosis and autophagy and promoted cell proliferation which could be impaired by silencing SYVN1. In addition, SNHG7 regulated SYVN1 through sponging miR-34a-5p. CONCLUSION: SNHG7 sponged miR-34a-5p to affect cell proliferation, apoptosis and autophagy through targeting SYVN1 which provides a novel sight into the pathogenesis of OA.


Subject(s)
Humans , Osteoarthritis/metabolism , Autophagy/physiology , Apoptosis/physiology , MicroRNAs/metabolism , Ubiquitin-Protein Ligases/metabolism , RNA, Long Noncoding/metabolism , Osteoarthritis/genetics , Autophagy/genetics , Enzyme-Linked Immunosorbent Assay , Down-Regulation , Up-Regulation , Blotting, Western , Apoptosis/genetics , MicroRNAs/genetics , Ubiquitin-Protein Ligases/genetics , Cell Proliferation , Real-Time Polymerase Chain Reaction , RNA, Long Noncoding/genetics
8.
Indian J Med Ethics ; 2014 Jan-Mar; 11(1): 34-35
Article in English | IMSEAR | ID: sea-153521

ABSTRACT

The decision of the actress, Angelina Jolie (AJ), to undergo preventive risk-reducing bilateral mastectomy has elicited extreme responses, in support and against. We will discuss whether her decision was justified and if there are other options available to women. AJ, who is 38 years old, inherited the BRCA 1 gene. Because of the lack of randomised trials, there is controversy about the overall benefit that various risk-reduction strategies offer carriers of the BRCA 1, but some of the strategies offer a clear benefit. The decision to opt for mastectomy must be driven by the patient's choice, evidence on the balance of the risks and benefits, the quality of life after surgery and issues relating to body image.


Subject(s)
Adult , Body Image , Breast Neoplasms/genetics , Breast Neoplasms/prevention & control , Choice Behavior , Female , Genes, BRCA1/genetics , Genetic Predisposition to Disease , Humans , Mastectomy , Quality of Life , Ubiquitin-Protein Ligases/genetics
9.
Gut and Liver ; : 487-494, 2014.
Article in English | WPRIM | ID: wpr-108133

ABSTRACT

BACKGROUND/AIMS: To investigate the differential expression of RING finger (RNF) proteins in Barrett esophagus (BE) and esophageal adenocarcinoma (EAC). METHODS: The differential expression of RNFs in normal esophagus (NE), BE, and EAC was screened using microarray assay. Real-time quantitative polymerase chain reaction (PCR), tissue microarray assay, and Western blot analysis were independently performed to detect the mRNA and protein expression of screened RNFs. RESULTS: The expression of nine RNFs in the BE or EAC was 2-fold higher than those in NE. Among these proteins, the RNF32 and RNF121 expression in BE was 20.3-fold and 16.4-fold higher, respectively, than that in NE, and the expression of RNF24, RNF130, RNF141, RNF139, RNF11, RNF14, and RNF159 was upregulated more than 2-fold compared with NE. The expression of nine RNFs was not only upregulated in the EAC but was also positively related to the RNF expression in BE. The PCR results also indicated increased expression of these RNFs in BE and EAC compared to NE. Furthermore, the mRNA expression of all RNFs, except for RNF141 in EAC, was dramatically higher than those in the BE. Similar results were also obtained from the Western blot analysis. CONCLUSIONS: A total of nine RNFs play critical roles in the progression of BE to EAC.


Subject(s)
Adenocarcinoma/enzymology , Adult , Aged , Barrett Esophagus/enzymology , Carrier Proteins/genetics , Disease Progression , Esophageal Neoplasms/enzymology , Female , Gene Expression Profiling , Humans , Intracellular Signaling Peptides and Proteins/genetics , Male , Middle Aged , Proteins/genetics , RING Finger Domains , Receptors, Cell Surface/genetics , Ubiquitin-Protein Ligases/genetics
10.
Rio de Janeiro; s.n; 2013. 145 p. ilus, tab.
Thesis in Portuguese | LILACS | ID: lil-716900

ABSTRACT

A doença de Parkinson (DP) é uma das desordens neurodegenerativas mais comuns associada ao envelhecimento, alcançando 2% aos 70 anos. É uma doença caracterizada pela degeneração progressiva de neurônios dopaminérgicos nigrais nos gânglios basais e pela presença de inclusões protéicas citoplasmáticas denominadas corpúsculos e neuritos de Lewy nos neurônios sobreviventes. A etiologia da DP é pouco conhecida, sendo considerada, na maioria dos casos, idiopática. Conhecimentos alcançados nos últimos 15 anos sobre a base genética da DP demonstram, claramente, que os fatores genéticos desempenham um importante papel na etiologia desta desordem. Neste trabalho, rastreamos mutações nos genes que codificam proteínas participantes de vias metabólicas mitocondriais (Parkin, PINK1 e DJ-1) em 136 pacientes brasileiros com manifestação precoce da DP, através do sequenciamento automático e da técnica de MLPA. Avaliamos a presença de variantes de sequência por meio do sequenciamento dos exons 1 a 12 do gene Parkin e dos exons 1 a 8 do gene PINK1. Em Parkin foram identificadas três mutações patogênicas ou potencialmente patogênicas, ambas em heterozigose: p.T240M, p.437L e p.S145N. Em PINK1 não encontramos variantes de ponto patogênicas. Através da técnica de MLPA investigamos alterações de dosagem nos genes Parkin, PINK1 e DJ-1. Identificamos cinco alterações no gene Parkin em quatro pacientes: uma duplicação heterozigota do exon 4 no paciente PAR2256, uma deleção heterozigota do exon 4 no probando PAR2099, uma deleção homozigota do exon 4 na paciente PAR3380 e um probando heterozigoto composto (PAR2396) com duas alterações, uma duplicação do exon 3 e uma deleção dos exons 5 e 6. No gene PINK1 identificamos uma deleção heterozigota do exon 1, que nunca foi descrita na literatura, em um paciente (PAR2083). Não encontramos alteração quantitativa no gene DJ-1. Neste estudo obtivemos uma frequência total de mutações patogênicas (pontuais e de dosagem) nos genes estudados ...


Parkinson's disease (PD) is one of the most common neurodegenerative disorders associated with aging, reaching 2% at age 70. It is a disease characterized by progressive degeneration of nigra dopaminergic neurons in the basal ganglia and the presence of cytoplasmic protein inclusions known as Lewy bodies and neurites in surviving neurons. The etiology of PD is poorly understood, being considered, in most cases, idiopathic. Knowledge achieved in the last 15 years about the genetic basis of PD clearly shows that genetic factors play an important role in the etiology of this disorder. In this study, we screened mutations in genes that encode proteins participating in mitochondrial metabolic pathways (Parkin, PINK1 and DJ-1) in 136 Brazilian patients with early onset PD, through automatic sequencing and MLPA technique. We evaluated the presence of sequence variants by means of sequencing of exons 1 to 12 of Parkin gene and exons 1 to 8 of PINK1 gene. In Parkin gene were identified three pathogenic or potentially pathogenic mutations, both in heterozygous state: p.T240M, p.437L e p.S145N. In PINK1 gene we did not find pathogenic point mutations. Through the MLPA technique we investigated dosage changes in Parkin, PINK1 and DJ-1 genes. We identified five exon rearrangements in Parkin gene in four patients: a heterozygous duplication of exon 4 in patient PAR2256, a heterozygous deletion of exon 4 in proband PAR2099, a homozygous deletion of exon 4 in patient PAR3380 and a compound heterozygote (PAR2396) with two changes, a duplication of exon 3 and a deletion of exons 5 and 6. In PINK1 gene we identified a heterozygous deletion of exon 1, which has never been described in literature, in one patient (PAR2083). We found no quantitative change in DJ-1 gene. In this study, we obtained an overall frequency of pathogenic mutations (sequence and dosage) in the genes studied of 7.3%, being 6.6% in Parkin gene and 0.7% in PINK1 gene


Subject(s)
Humans , Parkinson Disease/genetics , Mutation/genetics , DNA Mutational Analysis , Exons/genetics , Gene Duplication , Mitochondria/genetics , Point Mutation , Intracellular Signaling Peptides and Proteins/genetics , Oncogene Proteins/genetics , Protein Kinases/genetics , Nucleic Acid Amplification Techniques/methods , Ubiquitin-Protein Ligases/genetics
11.
Article in English | WPRIM | ID: wpr-122574

ABSTRACT

Angelman syndrome (AS) is a severe neurobehavioural disorder caused by failure of expression of the maternal copy of the imprinted domain located on 15q11-q13. There are different mechanisms leading to AS: maternal microdeletion, uniparental disomy, defects in a putative imprinting centre, mutations of the E3 ubiquitin protein ligase (UBE3A) gene. However, some of suspected cases of AS are still scored negative to all the latter mutations. Recently, it has been shown that a proportion of negative cases bear large deletions overlapping one or more exons of the UBE3A gene. These deletions are difficult to detect by conventional gene-scanning methods due to the masking effect by the non-deleted allele. In this study, we have used for the first time multiplex ligation-dependent probe amplification (MLPA) and comparative multiplex dosage analysis (CMDA) to search for large deletions affecting the UBE3A gene. Using this approach, we identified a novel causative deletion involving exon 8 in an affected sibling. Based on our results, we propose the use of MLPA as a fast, accurate and inexpensive test to detect large deletions in the UBE3A gene in a small but significant percentage of AS patients.


Subject(s)
Angelman Syndrome/genetics , Child , Female , Gene Deletion , Gene Dosage , Genetic Testing , Humans , Male , Ubiquitin-Protein Ligases/genetics
12.
Braz. j. med. biol. res ; 42(12): 1128-1137, Dec. 2009. tab, ilus
Article in English | LILACS | ID: lil-532305

ABSTRACT

Epithelial intercellular cohesion, mainly mediated by E-cadherin (CDH1) expression and function, may be deregulated during cancer cell invasion of adjacent tissues and lymphatic and vascular channels. CDH1 expression is down-modulated in invasive lobular breast carcinomas but its regulation in invasive ductal carcinomas (IDC) is less clear. CDH1 expression is repressed by transcription factors such as Snail (SNAI1) and its product is degraded after Hakai ubiquitination. We compared CDH1, SNAI1 and HAKAI mRNA expression in IDC and paired adjacent normal breast tissue and evaluated its relation with node metastasis and circulating tumor cells. Matched tumor/peritumoral and blood samples were collected from 30 patients with early IDC. Epithelial cells from each compartment (tumor/peritumoral) were recovered by an immunomagnetic method and gene expression was determined by real time RT-PCR. There were no differences in CDH1, SNAI1 and HAKAI mRNA expression between tumor and corresponding peritumoral samples and no differential tumoral gene expression according to nodal involvement. Another 30 patients with a long-term follow-up (at least 5 years) and a differential prognosis (good or poor, as defined by breast cancer death) had E-cadherin and Snail protein detected by immunohistochemistry in tumor samples. In this group, E-cadherin-positive expression, but not Snail, may be associated with a better prognosis. This is the first report simultaneously analyzing CDH1, SNAI1 and HAKAI mRNA expression in matched tumor and peritumoral samples from patients with IDC. However, no clear pattern of their expression could distinguish the invasive tumor compartment from its adjacent normal tissue.


Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Middle Aged , Breast Neoplasms/metabolism , Cadherins/metabolism , Carcinoma, Ductal, Breast/metabolism , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cadherins/genetics , Carcinoma, Ductal, Breast/genetics , Carcinoma, Ductal, Breast/pathology , Epithelial Cells/chemistry , Gene Expression Regulation, Neoplastic , Immunohistochemistry , Lymphatic Metastasis , Neoplasm Invasiveness , Neoplasm Staging , Prognosis , Reverse Transcriptase Polymerase Chain Reaction , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription Factors/genetics , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Ubiquitin-Protein Ligases/genetics
13.
Arq. neuropsiquiatr ; 67(1): 7-11, Mar. 2009. tab
Article in English | LILACS | ID: lil-509099

ABSTRACT

OBJECTIVE: To describe clinical and neuroimaging (SPECT) characteristics of Brazilian patients with Parkinson's disease (PD) and mutations in PARK2 or PARK8 genes. METHOD: A total of 119 patients meeting clinical criteria for PD were evaluated. RESULTS: Of all patients studied, 13 had mutations in either PARK2 (n=9) or PARK8 genes (n=4). No statistically significant differences in clinical characteristics in both groups were seen. SPECT with [99mTc] TRODAT-1 showed significant differences between patient and control and the most remarkable difference was between PARK2 and control. CONCLUSION: The study found a frequency of mutation of 10.1 percent and it was most commonly seen in women. These patients had long disease course and high rates of dyskinesia after L-DOPA use. PARK8 patients did not have a relevant family history of PD.


OBJETIVO: Descrever as características clínicas e de neuroimagem (SPECT) de pacientes brasileiros com doença de Parkinson e mutações PARK2 e PARK8. MÉTODO: Foram avaliados 119 pacientes com critérios clínicos para a doença de Parkinson. RESULTADO: Entre os pacientes avaliados foram encontrados 13 pacientes com mutação nos genes PARK2 (n=9) ou PARK8 (n=4). Não houve diferença significativa na avaliação das características clínicas entre os dois grupos. Os resultados de SPECT mostraram diferenças significativas quanto ao potencial de ligação do [99mTc] TRODAT-1 SPECT entre pacientes vs. controle, sendo a diferença mais pronunciada entre PARK2 e controle. CONCLUSÃO: A freqüência de mutação encontrada foi 10,1 por cento, sendo mais comum em mulheres. Estes pacientes apresentavam longo tempo de doença e alta prevalência de discinesias associadas ao uso da levodopa. Nossos pacientes com PARK8 não apresentaram uma história familiar relevante de doença de Parkinson.


Subject(s)
Adult , Female , Humans , Male , Middle Aged , Mutation , Parkinson Disease/genetics , Protein-Serine-Threonine Kinases/genetics , Ubiquitin-Protein Ligases/genetics , Age of Onset , Brazil/epidemiology , Environment , Gene Frequency , Parkinson Disease/epidemiology , Parkinson Disease , Sex Factors , Tomography, Emission-Computed, Single-Photon
14.
Article in English | WPRIM | ID: wpr-34744

ABSTRACT

Fe65 has been characterized as an adaptor protein, originally identified as an expressed sequence tag (EST) corresponding to an mRNA expressed at high levels in the rat brain. It contains one WW domain and two phosphotyrosine interaction/phosphotyrosine binding domains (PID1/PID2). As the neuronal precursor cell expressed developmentally down regulated 4-2 (Nedd4-2) has a putative WW domain binding motif (72PPLP75) in the N-terminal domain, we hypothesized that Fe65 associates with Nedd4-2 through a WW domain interaction, which has the characteristics of E3 ubiquitin-protein ligase. In this paper, we present evidence for the interaction between Fe65 WW domain and Nedd4-2 through its specific motif, using a pull down approach and co-immunoprecipitation. Additionally, the co-localization of Fe65 and Nedd4-2 were observed via confocal microscopy. Co-localization of Fe65 and Nedd4-2 was disrupted by either the mutation of Fe65 WW domain or its putative binding motif of Nedd4-2. When the ubiquitin assay was performed, the interaction of Nedd4-2 (wt) with Fe65 is required for the cell apoptosis and the ubiquitylation of Fe65. We also observed that the ubiquitylation of Fe65 (wt) was augmented depending on Nedd4-2 expression levels, whereas the Fe65 WW domain mutant (W243KP245K) or the Nedd4-2 AL mutant (72PPLP75 was changed to 72APLA75) was under-ubiquitinated significantly. Thus, our observations implicated that the protein-protein interaction between the WW domain of Fe65 and the putative binding motif of Nedd4-2 down-regulates Fe65 protein stability and subcellular localization through its ubiquitylation, to contribute cell apoptosis.


Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Cell Line , Down-Regulation , Endosomal Sorting Complexes Required for Transport/genetics , Gene Expression Regulation, Developmental , Humans , Immunoprecipitation , Microscopy, Confocal , Mutation , Protein Interaction Mapping , Protein Structure, Tertiary/physiology , Transfection , Ubiquitin-Protein Ligases/genetics , Ubiquitination
15.
Braz. j. med. biol. res ; 40(3): 293-299, Mar. 2007. tab, ilus
Article in English | LILACS | ID: lil-441764

ABSTRACT

Almost identical polyglutamine-containing proteins with unknown structures have been found in human, mouse and rat genomes (GenBank AJ277365, AF525300, AY879229). We infer that an identical new gene (RING) finger domain of real interest is located in each C-terminal segment. A three-dimensional (3-D) model was generated by remote homology modeling and the functional implications are discussed. The model consists of 65 residues from terminal position 707 to 772 of the human protein with a total length of 796 residues. The 3-D model predicts a ubiquitin-protein ligase (E3) as a binding site for ubiquitin-conjugating enzyme (E2). Both enzymes are part of the ubiquitin pathway to label unwanted proteins for subsequent enzymatic degradation. The molecular contact specificities are suggested for both the substrate recognition and the residues at the possible E2-binding surface. The predicted structure, of a ubiquitin-protein ligase (E3, enzyme class number 6.3.2.19, CATH code 3.30.40.10.4) may contribute to explain the process of ubiquitination. The 3-D model supports the idea of a C3HC4-RING finger with a partially new pattern. The putative E2-binding site is formed by a shallow hydrophobic groove on the surface adjacent to the helix and one zinc finger (L722, C739, P740, P741, R744). Solvent-exposed hydrophobic amino acids lie around both zinc fingers (I717, L722, F738, or P765, L766, V767, V733, P734). The 3-D structure was deposited in the protein databank theoretical model repository (2B9G, RCSB Protein Data Bank, NJ).


Subject(s)
Humans , Animals , Mice , Rats , Peptides/metabolism , Ubiquitin-Protein Ligases/metabolism , Zinc Fingers/genetics , Imaging, Three-Dimensional , Models, Genetic , Protein Structure, Tertiary , Peptides/chemistry , Peptides/genetics , Sequence Alignment , Ubiquitin-Protein Ligases/genetics
16.
Article in English | WPRIM | ID: wpr-115058

ABSTRACT

PURPOSE: The purpose of this study was to identify differences in signal transduction gene expression between normal and diabetic keratocytes stimulated with interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha). METHODS: Normal and diabetic keratocytes were primarily cultured and treated with 20 ng/ml IL-1alpha and TNF-alpha for 6 h. cDNA was hybridized to an oligonucleotide microarray. Genes identified by the microarray were further evaluated by real-time PCR. RESULTS: Diabetic keratocytes over-expressed components of the MAPK and Notch pathways, and under-expressed components of the insulin, calcium, and TGF-beta pathways. Cytokine treated diabetic keratocytes differentially expressed components of the TGF-beta and MAPK pathways. After IL-1alpha and TNF-alpha treatment, nine genes were under-expressed, falling in the insulin, TGF-beta, and Toll-like receptor pathways. Real-time PCR showed a significant decrease in the IL-6 and TGF-beta2 genes and a significant increase in the Ppm1a gene. CONCLUSIONS: There were some differences in gene expression between normal and diabetic keratocytes related to signal transduction pathways, such as the insulin, MAPK, calcium, and TGF-beta pathways. In addition, IL-1alpha and TNF-alpha stimulating the insulin, TGF-beta, and Toll-like receptor signaling pathways may have different effects in diabetic keratocytes.


Subject(s)
Animals , Apoptosis , Cells, Cultured , Cornea/drug effects , DNA/genetics , Diabetes Mellitus, Experimental/genetics , Gene Expression Profiling , Insulin/genetics , Interleukin-1alpha/pharmacology , Mitogen-Activated Protein Kinase Kinases/genetics , Nuclear Proteins/genetics , Oligonucleotide Array Sequence Analysis/methods , Phosphoric Monoester Hydrolases/genetics , Polymerase Chain Reaction , Prolactin/genetics , Rats , Rats, Long-Evans , Receptors, Notch/genetics , Signal Transduction/drug effects , Transforming Growth Factor beta/genetics , Tumor Necrosis Factor-alpha/pharmacology , Ubiquitin-Protein Ligases/genetics
17.
J Biosci ; 2005 Jun; 30(3): 329-37
Article in English | IMSEAR | ID: sea-111137

ABSTRACT

Heat induced differentiation of mouse embryonal carcinoma cells PCC4 has been reported earlier. We have further characterized the phenotype of the differentiated cells and by DD-RT-PCR identified several partial cDNAs that are differentially expressed during differentiation. Nucleotide homology search revealed that the genes corresponding to some of the up-regulated partial cDNAs are indeed part of differentiation pathway. 5'extension of an EST that has homology to one of the partial cDNAs led to the identification of mouse cullin4B. Cullin4B is coded by a separate gene and has a unique and longer amino-terminal end with a putative nuclear localization signal sequence (NLS). We have cloned, expressed and raised antibodies against the amino and carboxy-terminal halves of cullin4B. Immuno staining of differentiated PCC4 cells with N-terminal Cul4B antibody showed enhanced expression of Cul4B and its translocation into the nucleus upon differentiation. Transient transfection of a chimeric gene encoding the N-terminal part of Cul4B fused to green fluorescent protein into PCC4 cells revealed that the protein was localized in the nucleus confirming the functional significance of the putative NLS. Since cullins are involved in recognition of specific proteins for degradation, based on the evidence presented here, we hypothesize that cullin4B is probably involved in differentiation specific degradation/modification of nuclear proteins.


Subject(s)
Animals , Antibodies , Cell Differentiation/physiology , Cell Line, Tumor , Cloning, Molecular , Mice , Protein Sorting Signals , Ubiquitin-Protein Ligases/genetics , Up-Regulation
18.
Article in English | WPRIM | ID: wpr-634147

ABSTRACT

Mutations in the parkin gene have recently been identified in familial and isolated patients with early-onset Parkinson disease (PD) and that subregions between exon 2 and 4 of the parkin gene are hot spots of deletive mutations. To study the distribution of deletions in the parkin gene among variant subset patients with PD in China, and to explore the role of parkin gene in the pathogenesis of PD, 63 patients were divided into early onset and later onset groups. Exons 1-12 were amplified by PCR, templated by the genomic DNA of patients, and then the deletion distribution detected by agarose electrophoresis. Four patients were found to be carrier of exon deletions in 63 patients with PD. The location of the deletion was on exon 2 (1 case), exon 3 (2 cases) and exon 4 (1 case). All patients were belong to the group of early onset PD. The results showed that parkin gene deletion on exon 2, exon 3 and exon 4 found in Chinese population contributes partly to early onset PD.


Subject(s)
Exons/genetics , Gene Deletion , Parkinson Disease/genetics , Point Mutation , Ubiquitin-Protein Ligases/genetics
19.
Article in English | WPRIM | ID: wpr-634060

ABSTRACT

To investigate the distribution of possible novel mutations from parkin gene in variant subset of patients with Parkinson's disease (PD) in China and explore whether parkin gene plays an important role in the pathogenesis of PD, 70 patients were divided into early-onset group and late-onset group; 70 healthy subjects were included as controls. Genomic DNA from 70 normal controls and from those of PD patients were extracted from peripheral blood leukocytes by using standard procedures. Mutations of parkin gene (exon 1-12) in all the subjects were screened by PCR-single strand conformation polymorphism (SSCP), and further sequencing was performed in the samples with abnormal SSCP results, in order to confirm the mutation and its location. A new missense mutation Gly284Arg in a patient and 3 abnormal bands in SSCP electrophoresis from samples of another 3 patients were found. All the DNA variants were sourced from the samples of the patients with early-onset PD. It was concluded that Parkin point mutation also partially contributes to the development of early-onset Parkinson's disease in Chinese.


Subject(s)
DNA Mutational Analysis , Exons , Genotype , Parkinson Disease/genetics , Point Mutation , Polymorphism, Single-Stranded Conformational , Ubiquitin-Protein Ligases/biosynthesis , Ubiquitin-Protein Ligases/genetics
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