15 results on '"Tanjing Song"'
Search Results
2. Efficient and reversible Cas13d-mediated knockdown with an all-in-one lentivirus-vector
- Author
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Suli Lv, Xuefeng Zhao, Xianyun Ma, Qingli Zou, Neng Li, Yingying Yan, Lidong Sun, and Tanjing Song
- Subjects
Histology ,Biomedical Engineering ,Bioengineering ,Biotechnology - Abstract
Type VI CRISPR effector Cas13d from Ruminococcus flavefaciens XPD3002 (RfxCas13d) is an RNA-guided RNA endonuclease. RfxCas13d has been harnessed to knockdown gene expression with high specificity in various systems including mammalian cells. While inducible knockdown is advantageous over constitutive knockdown in many scenarios, current inducible systems of RfxCas13d express CRISPR RNA and Cas13d separately. Such systems could be cumbersome to handle and may hamper the application of RfxCas13d in some scenarios. Here, we design an all-in-one Cas13d lentivirus vector which renders efficient and inducible knockdown in a doxycycline dosage-dependent manner. Furthermore, we find that Cas13d has a short half-life in mammalian cells. As a result, knockdown can be promptly reversed after doxycycline withdrawal. This vector is particularly useful for applications involving indispensable genes and/or in cells hard to transduce.
- Published
- 2022
3. Versatile functions of RNA m6A machinery on chromatin
- Author
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Tanjing Song, Suli Lv, Neng Li, Xuefeng Zhao, Xianyun Ma, Yingying Yan, Weixia Wang, and Lidong Sun
- Subjects
DNA Repair ,Genetics ,RNA ,Cell Biology ,General Medicine ,RNA, Messenger ,RNA Processing, Post-Transcriptional ,Molecular Biology ,Chromatin - Abstract
m6A, a conserved and abundant modification on RNA, regulates RNA processing and function. RNA m6A machinery, including writers, erasers, and readers of m6A, is indispensable for m6A installation and function. Intriguingly, recent studies have revealed that m6A machinery can be recruited to chromatin by pleiotropic factors, including nascent RNA, transcription factors, regulatory RNA, histone modifications, and epigenetic machinery. Consequently, recruitment of m6A machinery can directly regulate chromatin biology, such as transcription, DNA damage repair, and DNA recombination beyond installation of m6A on nascent mRNA. Here, we discuss recent evidence showing that m6A machinery is targeted to chromatin and the direct biological consequences along with the underlying mechanisms.
- Published
- 2021
4. Interaction between p53 N terminus and core domain regulates specific and nonspecific DNA binding
- Author
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Tanjing Song, Fan He, Wade Borcherds, Lihong Chen, Xi Wei, Jiandong Chen, Gary W. Daughdrill, and Mousumi Das
- Subjects
Multidisciplinary ,Chemistry ,Proto-Oncogene Proteins c-mdm2 ,Promoter ,DNA ,DNA-binding domain ,DNA-binding protein ,Cell Line ,Chromatin ,Cell biology ,chemistry.chemical_compound ,Transactivation ,PNAS Plus ,Protein Domains ,Escherichia coli ,Nucleic acid ,Humans ,RNA Processing, Post-Transcriptional ,Tumor Suppressor Protein p53 ,Protein Binding ,P53 binding - Abstract
The p53 tumor suppressor is a sequence-specific DNA binding protein that activates gene transcription to regulate cell survival and proliferation. Dynamic control of p53 degradation and DNA binding in response to stress signals are critical for tumor suppression. The p53 N terminus (NT) contains two transactivation domains (TAD1 and TAD2), a proline-rich region (PRR), and multiple phosphorylation sites. Previous work revealed the p53 NT reduced DNA binding in vitro. Here, we show that TAD2 and the PRR inhibit DNA binding by directly interacting with the sequence-specific DNA binding domain (DBD). NMR spectroscopy revealed that TAD2 and the PRR interact with the DBD at or near the DNA binding surface, possibly acting as a nucleic acid mimetic to competitively block DNA binding. In vitro and in vivo DNA binding analyses showed that the NT reduced p53 DNA binding affinity but improved the ability of p53 to distinguish between specific and nonspecific sequences. MDMX inhibits p53 binding to specific target promoters but stimulates binding to nonspecific chromatin sites. The results suggest that the p53 NT regulates the affinity and specificity of DNA binding by the DBD. The p53 NT-interacting proteins and posttranslational modifications may regulate DNA binding, partly by modulating the NT-DBD interaction.
- Published
- 2019
5. DOT1L O-GlcNAcylation promotes its protein stability and MLL-fusion leukemia cell proliferation
- Author
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Qingli Zou, Lidong Sun, Suli Lv, Haigang Liu, Yingying Yan, Borui Tang, Tanjing Song, Xuefeng Zhao, Xianyun Ma, and Neng Li
- Subjects
MLL ,Methyltransferase ,QH301-705.5 ,Histone lysine methylation ,Acylation ,Ubiquitin-Protein Ligases ,histone ,N-Acetylglucosaminyltransferases ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Histones ,O-GlcNAcylation ,Ubiquitin ,Histone methylation ,Humans ,RNA, Small Interfering ,glucose ,Biology (General) ,Myeloid Ecotropic Viral Integration Site 1 Protein ,Cell Proliferation ,Homeodomain Proteins ,Leukemia ,biology ,Protein Stability ,Chemistry ,Ubiquitination ,Hexosamines ,Histone-Lysine N-Methyltransferase ,DOT1L ,Methylation ,Cell biology ,Ubiquitin ligase ,Histone ,Mutagenesis, Site-Directed ,biology.protein ,RNA Interference ,methylation ,Myeloid-Lymphoid Leukemia Protein - Abstract
Summary: Histone lysine methylation functions at the interface of the extracellular environment and intracellular gene expression. DOT1L is a versatile histone H3K79 methyltransferase with a prominent role in MLL-fusion leukemia, yet little is known about how DOT1L responds to extracellular stimuli. Here, we report that DOT1L protein stability is regulated by the extracellular glucose level through the hexosamine biosynthetic pathway (HBP). Mechanistically, DOT1L is O-GlcNAcylated at evolutionarily conserved S1511 in its C terminus. We identify UBE3C as a DOT1L E3 ubiquitin ligase promoting DOT1L degradation whose interaction with DOT1L is susceptible to O-GlcNAcylation. Consequently, HBP enhances H3K79 methylation and expression of critical DOT1L target genes such as HOXA9/MEIS1, promoting cell proliferation in MLL-fusion leukemia. Inhibiting HBP or O-GlcNAc transferase (OGT) increases cellular sensitivity to DOT1L inhibitor. Overall, our work uncovers O-GlcNAcylation and UBE3C as critical determinants of DOT1L protein abundance, revealing a mechanism by which glucose metabolism affects malignancy progression through histone methylation.
- Published
- 2021
6. Mutant p53 Sequestration of the MDM2 Acidic Domain Inhibits E3 Ligase Activity
- Author
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Qian Cheng, Leixiang Yang, Jiandong Chen, Tanjing Song, and Lihong Chen
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Ubiquitin-Protein Ligases ,Mutant ,Mutation, Missense ,03 medical and health sciences ,0302 clinical medicine ,Ubiquitin ,Cell Line, Tumor ,Humans ,Missense mutation ,Avidity ,neoplasms ,Molecular Biology ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Binding Sites ,biology ,Ubiquitination ,Nuclear Proteins ,Proto-Oncogene Proteins c-mdm2 ,Cell Biology ,Cell biology ,Ubiquitin ligase ,enzymes and coenzymes (carbohydrates) ,Enzyme ,chemistry ,030220 oncology & carcinogenesis ,Domain (ring theory) ,biology.protein ,Mdm2 ,Tumor Suppressor Protein p53 ,Research Article ,Protein Binding - Abstract
Missense p53 mutants often accumulate in tumors and drive progression through gain of function. MDM2 efficiently degrades wild-type p53 but fails to degrade mutant p53 in tumor cells. Previous studies revealed that mutant p53 inhibits MDM2 autoubiquitination, suggesting that the interaction inhibits MDM2 E3 activity. Recent work showed that MDM2 E3 activity is stimulated by intramolecular interaction between the RING and acidic domains. Here, we show that in the mutant p53-MDM2 complex, the mutant p53 core domain binds to the MDM2 acidic domain with significantly higher avidity than wild-type p53. The mutant p53-MDM2 complex is deficient in catalyzing ubiquitin release from the activated E2 conjugating enzyme. An MDM2 construct with extra copies of the acidic domain is resistant to inhibition by mutant p53 and efficiently promotes mutant p53 ubiquitination and degradation. The results suggest that mutant p53 interferes with the intramolecular autoactivation mechanism of MDM2, contributing to reduced ubiquitination and increased accumulation in tumor cells.
- Published
- 2019
7. Regulation of SirT1-Nucleomethylin Binding by rRNA Coordinates Ribosome Biogenesis with Nutrient Availability
- Author
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Jiandong Chen, Neha Kabra, Edward Seto, Leixiang Yang, Tanjing Song, Hong Zheng, Lihong Chen, and John M. Koomen
- Subjects
Blotting, Western ,Molecular Sequence Data ,Gene Expression ,Ribosome biogenesis ,Biology ,Ribosome ,Sirtuin 1 ,23S ribosomal RNA ,Transcription (biology) ,Cell Line, Tumor ,RNA, Ribosomal, 28S ,Humans ,Amino Acid Sequence ,Molecular Biology ,Ribosomal DNA ,Sequence Homology, Amino Acid ,Reverse Transcriptase Polymerase Chain Reaction ,RNA, Ribosomal, 5S ,Nuclear Proteins ,RNA-Binding Proteins ,RNA ,Methyltransferases ,Articles ,Cell Biology ,Ribosomal RNA ,Molecular biology ,RRNA transcription ,RNA, Ribosomal, 5.8S ,Cell biology ,Glucose ,RNA, Ribosomal ,RNA Interference ,Ribosomes ,HeLa Cells ,Protein Binding - Abstract
Nucleomethylin (NML), a novel nucleolar protein, is important for mediating the assembly of the energy-dependent nucleolar silencing complex (eNoSC), which also contains SirT1 and SUV39H1. eNoSC represses rRNA transcription during nutrient deprivation, thus reducing energy expenditure and improving cell survival. We found that NML is an RNA binding protein that copurifies with 5S, 5.8S, and 28S rRNA. The SirT1 and RNA binding regions on NML showed partial overlap, and the NML-SirT1 interaction was competitively inhibited by rRNA. Nutrient deprivation triggered downregulation of rRNA transcription, reduced the level of NML-associated rRNA, and stimulated NML-SirT1 binding. Assembly of eNoSC facilitated repression of pre-rRNA transcription. These results suggest that nascent rRNA generates a positive-feedback signal by suppressing the assembly of eNoSC and protecting active ribosomal DNA units from heterochromatin formation. This RNA-mediated mechanism enables the eNoSC to amplify the effects of upstream nutrient-responsive regulators.
- Published
- 2013
8. Secondary interaction between MDMX and p53 core domain inhibits p53 DNA binding
- Author
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Jiandong Chen, Lihong Chen, Shaofang Wu, Gary W. Daughdrill, Wade Borcherds, Gao Ming, Xi Wei, and Tanjing Song
- Subjects
0301 basic medicine ,MDMX ,Protein domain ,Cell Cycle Proteins ,Plasma protein binding ,03 medical and health sciences ,Protein Domains ,Ubiquitin ,Cell Line, Tumor ,Neoplasms ,Proto-Oncogene Proteins ,Protein Interaction Mapping ,Humans ,Binding site ,Binding Sites ,Multidisciplinary ,biology ,Nuclear Proteins ,DNA, Neoplasm ,DNA-binding domain ,Molecular biology ,Cell biology ,Ubiquitin ligase ,030104 developmental biology ,PNAS Plus ,biology.protein ,Mdm2 ,Tumor Suppressor Protein p53 ,Protein Binding - Abstract
The MDMX oncoprotein is an important regulator of tumor suppressor p53 activity during embryonic development. Despite sequence homology to the ubiquitin E3 ligase MDM2, MDMX depletion activates p53 without significant increase in p53 level, implicating a degradation-independent mechanism. We present evidence that MDMX inhibits the sequence-specific DNA binding activity of p53. This function requires the cooperation between MDMX and CK1α, and phosphorylation of S289 on MDMX. Depletion of MDMX or CK1α increases p53 DNA binding without stabilization of p53. A proteolytic fragment release assay revealed that in the MDMX-p53 complex, the MDMX acidic domain and RING domain interact stably with the p53 DNA binding domain. These interactions are referred to as secondary interactions because they only occur after the canonical-specific binding between the MDMX and p53 N termini, but exhibit significant binding stability in the mature complex. CK1α cooperates with MDMX to inhibit p53 DNA binding by further stabilizing the MDMX acidic domain and p53 core domain interaction. These results suggest that secondary intermolecular interaction is important in p53 regulation by MDMX, which may represent a common phenomenon in complexes containing multidomain proteins.
- Published
- 2016
9. BMP signaling pathway is required for commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage
- Author
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Hai-Yan Huang, Tanjing Song, Xi Li, Qi Qun Tang, Mei Liu, M. Daniel Lane, Ling Ling Hu, and Qun He
- Subjects
Pluripotent Stem Cells ,medicine.medical_specialty ,animal structures ,Bone Morphogenetic Protein 2 ,Smad Proteins ,Lysyl oxidase ,Bone Morphogenetic Protein 4 ,SMAD ,Biology ,Bone Morphogenetic Protein Receptors, Type II ,Bone morphogenetic protein ,p38 Mitogen-Activated Protein Kinases ,Protein-Lysine 6-Oxidase ,Mice ,chemistry.chemical_compound ,Internal medicine ,Adipocyte ,Adipocytes ,medicine ,Animals ,Cell Lineage ,Induced pluripotent stem cell ,BMP signaling pathway ,Bone Morphogenetic Protein Receptors, Type I ,Cells, Cultured ,Multidisciplinary ,Cell Differentiation ,Biological Sciences ,BMPR2 ,Cell biology ,Endocrinology ,chemistry ,embryonic structures ,Stem cell ,Signal Transduction - Abstract
Obesity is accompanied by an increase in both adipocyte number and size. The increase in adipocyte number is the result of recruitment to the adipocyte lineage of pluripotent stem cells present in the vascular stroma of adipose tissue. These pluripotent cells have the potential to undergo commitment and then differentiate into adipocytes, as well as myocytes, osteocytes, and chondrocytes. In this article, we show that both bone morphogenetic protein (BMP)2 and BMP4 can induce commitment of C3H10T1/2 pluripotent stem cells into adipocytes. After treatment of C3H10T1/2 stem cells with these BMPs during proliferation followed by exposure to differentiation inducers at growth arrest, nearly all cells enter the adipose development pathway, express specific adipocyte markers, and acquire the adipocyte phenotype. Overexpression of constitutively active BMP receptor (CA)-BMPr1A or CA-BMPr1B induces commitment in the absence of BMP2/4, whereas overexpression of a dominant-negative receptor dominant-negative-BMPr1A suppresses commitment induced by BMP. Also, knockdown of the expression of Smad4 (coregulator in the BMP/Smad signaling pathway) with RNAi disrupts commitment by the BMPs. However, knockdown of expression of p38 MAPK (an intermediary in the BMP/MAPK signaling pathway) with RNAi had little effect on BMP-induced commitment. Together, these findings indicate that the BMP/Smad signaling pathway has a dominant role in adipocyte lineage determination. Proteomic analysis identified lysyl oxidase (LOX), a bona fide downstream target gene of the BMP signaling pathway. Expression of LOX is induced by BMP2/4 during adipocyte lineage commitment, and knockdown of its expression disrupts the commitment process.
- Published
- 2009
10. Transcription factor YY1 promotes adipogenesis via inhibiting CHOP-10 expression
- Author
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Qi Qun Tang, Hai Yan Huang, Chun Gu Ma, Tanjing Song, Xi Li, Mei Liu, and Qun He
- Subjects
Serum ,Leucine zipper ,medicine.medical_treatment ,Biophysics ,Biology ,Biochemistry ,Mice ,chemistry.chemical_compound ,Transactivation ,3T3-L1 Cells ,Adipocyte ,medicine ,Animals ,Insulin-Like Growth Factor I ,RNA, Small Interfering ,Promoter Regions, Genetic ,Molecular Biology ,Transcription factor ,YY1 Transcription Factor ,Regulation of gene expression ,ATF3 ,Adipogenesis ,Growth factor ,Cell Biology ,Cell biology ,Gene Expression Regulation ,chemistry ,embryonic structures ,Cancer research ,Cattle ,biological phenomena, cell phenomena, and immunity ,Transcription Factor CHOP - Abstract
CHOP-10, a dominant-negative member of the C/EBP family of transcription factors, is initially expressed by growth-arrested preadipocytes and sequesters/inactivates C/EBPbeta through heterodimerization with its leucine zipper during 3T3-L1 preadipocyte differentiation. Our previous studies indicated that, FBS leads to the down-regulation of CHOP-10 expression after induction, and releasing C/EBPbeta from inhibitory constraint, allowing the transactivation of C/EBPalpha and PPARgamma genes, transcription factors required for terminal adipocyte differentiation. In the present study, we reported that FBS induced the expression of YY1, which bound to CHOP-10 promoter via two adjacent YY1-binding sites, suppressing its expression. The knock-down of YY1 expression with YY1 siRNA increased the expression of CHOP-10, inhibiting adipocyte differentiation. IGF-1, a growth factor present in greater concentration in FBS, independently induced the expression of YY1, and contributed to the down-regulation of CHOP-10 during the adipocyte differentiation program. Our studies suggested that YY1 can be a new adipocyte differentiation stimulator.
- Published
- 2008
11. Functional expression of the globular domain of human adiponectin in Pichia pastoris
- Author
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De Guo Liu, Qi Qun Tang, Hai Yan Huang, Tanjing Song, Xi Li, and Hong Lei Liu
- Subjects
Blood Glucose ,Genetic Vectors ,Immunoblotting ,Molecular Sequence Data ,Biophysics ,White adipose tissue ,Biochemistry ,Pichia ,Cell Line ,Diabetes Mellitus, Experimental ,Pichia pastoris ,law.invention ,Myoblasts ,Mice ,Transformation, Genetic ,Plasmid ,law ,Sequence Homology, Nucleic Acid ,Animals ,Humans ,Amino Acid Sequence ,Phosphorylation ,Codon ,Molecular Biology ,chemistry.chemical_classification ,Binding Sites ,Expression vector ,Base Sequence ,Dose-Response Relationship, Drug ,Adiponectin ,biology ,Fatty Acids ,Fatty acid ,Cell Biology ,biology.organism_classification ,Molecular biology ,Recombinant Proteins ,Yeast ,Diabetes Mellitus, Type 1 ,chemistry ,Recombinant DNA ,Acetyl-CoA Carboxylase - Abstract
Adiponectin is an adipokine that predominantly synthesized and secreted from adipocytes mainly in the white adipose tissue. Here, we report that we have successfully expressed human gAdiponectin (the globular domain of adiponectin) in the methylotrophic yeast Pichia pastoris after codon optimization and established the purification procedure. The human gAdiponectin gene was designed and synthesized by PCR according to the P. pastoris preferred codons, and then inserted into the P. pastoris pPIC9K expression vector. The plasmid was electroporated into the P. pastoris strain GS115 and only the G418 resistance colonies could produce the gAdiponectin. After fermentation and purification, we could get 1.2g of recombinant gAdiponectin (purity is approximately 95%) from a 24 L culture media. The recombinant gAdiponectin is fully functional as evidenced by induction the phosphorylation of ACC in differentiated C2C12 myotubes, significantly lowering the blood glucose level and accelerating the clearance of free fatty acid in animal models.
- Published
- 2007
12. Lactacystin inhibits 3T3-L1 adipocyte differentiation through induction of CHOP-10 expression
- Author
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Qi Qun Tang, Hou Yan Song, Chun Gu Ma, Lin Jiang, Hong Lei Liu, Hai Yan Huang, Tanjing Song, De Guo Liu, Qun He, Jie Gen Chen, Xi Li, and Duan Ma
- Subjects
Lactacystin ,Biophysics ,Mitosis ,Peroxisome proliferator-activated receptor ,Biology ,Biochemistry ,Mice ,chemistry.chemical_compound ,Downregulation and upregulation ,Transcription (biology) ,3T3-L1 Cells ,Adipocyte ,Adipocytes ,medicine ,Animals ,Receptor ,Molecular Biology ,chemistry.chemical_classification ,CCAAT-Enhancer-Binding Protein-beta ,Cyclin-Dependent Kinase 2 ,Cell Differentiation ,DNA ,Cell Biology ,Phenotype ,Molecular biology ,Acetylcysteine ,Up-Regulation ,chemistry ,Proteasome inhibitor ,biological phenomena, cell phenomena, and immunity ,Transcription Factor CHOP ,Protein Binding ,medicine.drug - Abstract
Hormonal induction triggers a cascade leading to the expression of CCAAT/enhancer-binding protein(C/EBP)alpha and peroxisome proliferator-activated receptor (PPAR) gamma, C/EBPalpha, and PPARgamma turns on series of adipocyte genes that give rise to the adipocyte phenotype. Previous findings indicate that C/EBPbeta, a transcriptional activator of the C/EBPalpha and PPARgamma genes, is rapidly expressed after induction, but lacks DNA-binding activity and therefore cannot activate transcription of the C/EBPalpha and PPARgamma genes early in the differentiation program. Acquisition of DNA-binding activity of C/EBPbeta occurs when CHOP-10, a dominant-negative form of C/EBP family members, is down-regulated and becomes hyperphosphorylated as preadipocytes traverse the G1-S checkpoint of mitotic clonal expansion. Evidences are presented in this report that lactacystin, a proteasome inhibitor, up-regulated the CHOP-10 expression, blocked the DNA-binding activity of C/EBPbeta, and subsequently inhibited MCE as well as adipocyte differentiation.
- Published
- 2006
13. Identification of a peroxisome proliferator responsive element (PPRE)-like cis-element in mouse plasminogen activator inhibitor-1 gene promoter
- Author
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Duan Ma, Hong Lei Liu, Qi Qun Tang, Xi Li, Hou Yan Song, De Guo Liu, Chun Gu Ma, Hai Yan Huang, Tanjing Song, and Jie Gen Chen
- Subjects
Transcription, Genetic ,Biophysics ,Adipose tissue ,Biology ,Ligands ,Response Elements ,Biochemistry ,Mice ,chemistry.chemical_compound ,Transactivation ,3T3-L1 Cells ,Adipocyte ,Plasminogen Activator Inhibitor 1 ,Adipocytes ,Animals ,Electrophoretic mobility shift assay ,Promoter Regions, Genetic ,Receptor ,Molecular Biology ,Cell Differentiation ,Promoter ,3T3-L1 ,Cell Biology ,Molecular biology ,PPAR gamma ,chemistry ,Plasminogen activator inhibitor-1 ,Peroxisome Proliferators - Abstract
PAI-1 is expressed and secreted by adipose tissue which may mediate the pathogenesis of obesity-associated cardiovascular complications. Evidence is presented in this report that PAI-1 is not expressed by preadipocyte, but significantly induced during 3T3-L1 adipocyte differentiation and the PAI-1 expression correlates with the induction of peroxisome proliferator-activated receptor gamma (PPARgamma). A peroxisome proliferator responsive element (PPRE)-like cis-element (-206TCCCCCATGCCCT-194) is identified in the mouse PAI-1 gene promoter by electrophoretic mobility shift assay (EMSA) combined with transient transfection experiments; the PPRE-like cis-element forms a specific DNA-protein complex only with adipocyte nuclear extracts, not with preadipocyte nuclear extracts; the DNA-protein complex can be totally competed away by non-labeled consensus PPRE, and can be supershifted with PPARgamma antibody. Mutation of this PPRE-like cis-element can abolish the transactivation of mouse PAI-1 promoter mediated by PPARgamma. Specific PPARgamma ligand Pioglitazone can significantly induce the PAI-1 expression, and stimulate the secretion of PAI-1 into medium.
- Published
- 2006
14. Autoactivation of the MDM2 E3 ligase by intramolecular interaction
- Author
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Tanjing Song, Qian Cheng, Lihong Chen, and Jiandong Chen
- Subjects
Ubiquitin-Protein Ligases ,Biology ,Catalysis ,Cell Line ,DDB1 ,Protein structure ,Ubiquitin ,Humans ,Protein Interaction Domains and Motifs ,Binding site ,Phosphorylation ,Molecular Biology ,Acidic Region ,Binding Sites ,Proto-Oncogene Proteins c-mdm2 ,Cell Biology ,Articles ,Ubiquitin ligase ,Protein Structure, Tertiary ,Biochemistry ,biology.protein ,Biophysics ,Mdm2 ,Tumor Suppressor Protein p53 ,Binding domain ,DNA Damage ,Protein Binding - Abstract
The RING domain ubiquitin E3 ligase MDM2 is a key regulator of p53 degradation and a mediator of signals that stabilize p53. The current understanding of the mechanisms by which MDM2 posttranslational modifications and protein binding cause p53 stabilization remains incomplete. Here we present evidence that the MDM2 central acidic region is critical for activating RING domain E3 ligase activity. A 30-amino-acid minimal region of the acidic domain binds to the RING domain through intramolecular interactions and stimulates the catalytic function of the RING domain in promoting ubiquitin release from charged E2. The minimal activation sequence is also the binding site for the ARF tumor suppressor, which inhibits ubiquitination of p53. The acidic domain-RING domain intramolecular interaction is modulated by ATM-mediated phosphorylation near the RING domain or by binding of ARF. These results suggest that MDM2 phosphorylation and association with protein regulators share a mechanism in inhibiting the E3 ligase function and stabilizing p53 and suggest that targeting the MDM2 autoactivation mechanism may be useful for therapeutic modulation of p53 levels.
- Published
- 2014
15. Involvement of Cytoskeleton-associated Proteins in the Commitment of C3H10T1/2 Pluripotent Stem Cells to Adipocyte Lineage Induced by BMP2/4
- Author
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Pengyuan Yang, Qun He, Hai Yan Huang, Qi Qun Tang, Tanjing Song, Hao Jie Lu, Xi Li, Yi Ming Li, Ling Ling Hu, and Xia Sun
- Subjects
Pluripotent Stem Cells ,Proteomics ,Bone Morphogenetic Protein 2 ,Lysyl oxidase ,Bone Morphogenetic Protein 4 ,Biology ,Biochemistry ,Bone morphogenetic protein 2 ,Analytical Chemistry ,Protein-Lysine 6-Oxidase ,Mice ,chemistry.chemical_compound ,Adipocyte ,Adipocytes ,Biomarkers, Tumor ,Animals ,Cell Lineage ,Induced pluripotent stem cell ,Molecular Biology ,Extracellular Matrix Proteins ,Gene knockdown ,Research ,Mesenchymal stem cell ,Reproducibility of Results ,Tumor Protein, Translationally-Controlled 1 ,alpha-Crystallin B Chain ,Molecular biology ,Actins ,Up-Regulation ,Cell biology ,Cytoskeletal Proteins ,Bone morphogenetic protein 4 ,chemistry ,Gene Knockdown Techniques ,Signal transduction ,Signal Transduction - Abstract
The developmental pathway that gives rise to mature adipocytes involves two distinct stages: commitment and terminal differentiation. Although the important proteins/factors contributing to terminal adipocyte differentiation have been well defined, the proteins/factors in the commitment of mesenchymal stem cells to the adipocyte lineage cells have not. In this study, we applied proteomics analysis profiling to characterize differences between uncommitted C3H10T1/2 pluripotent stem cells and those that have been committed to the adipocyte lineage by BMP4 or BMP2 with the goal to identify such proteins/factors and to understand the molecular mechanisms that govern the earliest stages of adipocyte lineage commitment. Eight proteins were found to be up-regulated by BMP2, and 27 proteins were up-regulated by BMP4, whereas five unique proteins were up-regulated at least 10-fold by both BMP2/4, including three cytoskeleton-associated proteins (i.e. lysyl oxidase (LOX), translationally controlled tumor protein 1 (TPT1), and αB-crystallin). Western blotting further confirmed the induction of the expression of these cytoskeleton-associated proteins in the committed C3H10T1/2 induced by BMP2/4. Importantly, knockdown of LOX expression totally prevented the commitment, whereas knockdown of TPT1 and αB-crystallin expression partially inhibited the commitment. Several published reports suggest that cell shape can influence the differentiation of partially committed precursors of adipocytes, osteoblasts, and chondrocytes. We observed a dramatic change of cell shape during the commitment process, and we showed that knockdown of these cytoskeleton-associated proteins prevented the cell shape change and restored F-actin organization into stress fibers and inhibited the commitment to the adipocyte lineage. Our studies indicate that these differentially expressed cytoskeleton-associate proteins might determine the fate of mesenchymal stem cells to commit to the adipocyte lineage through cell shape regulation.
- Published
- 2011
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