58 results on '"Ying, Li"'
Search Results
2. CD38 produces nicotinic acid adenosine dinucleotide phosphate in the lysosome
- Author
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Qi Wen Deng, Ya Jie Chen, Cheng Fang, Ting Li, Yun Nan Hou, Ying Li, Hon Cheung Lee, Yong Juan Zhao, and Guan Jie Xu
- Subjects
0301 basic medicine ,030102 biochemistry & molecular biology ,Chemistry ,media_common.quotation_subject ,hemic and immune systems ,Cell Biology ,Endocytosis ,Biochemistry ,Endolysosome ,Cell biology ,03 medical and health sciences ,Enzyme activator ,030104 developmental biology ,medicine.anatomical_structure ,immune system diseases ,hemic and lymphatic diseases ,Lysosome ,Second messenger system ,medicine ,Internalization ,Molecular Biology ,Intracellular ,media_common ,Calcium signaling - Abstract
Nicotinic acid adenosine dinucleotide phosphate (NAADP) is a Ca2+-mobilizing second messenger that regulates a wide range of biological activities. However, the mechanism of its biogenesis remains controversial. CD38 is the only enzyme known to catalyze NAADP synthesis from NADP and nicotinic acid. CD38-mediated catalysis requires an acidic pH, suggesting that NAADP may be produced in acidic endolysosomes, but this hypothesis is untested. In this study, using human cell lines, we specifically directed CD38 to the endolysosomal system and assessed cellular NAADP production. First, we found that nanobodies targeting various epitopes on the C-terminal domain of CD38 could bind to cell surface–localized CD38 and induce its endocytosis. We also found that CD38 internalization occurred via a clathrin-dependent pathway, delivered CD38 to the endolysosome, and elevated intracellular NAADP levels. We also created a CD38 variant for lysosome-specific expression, which not only withstood the degradative environment in the lysosome, but was also much more active than WT CD38 in elevating cellular NAADP levels. Supplementing CD38-expressing cells with nicotinic acid substantially increased cellular NAADP levels. These results demonstrate that endolysosomal CD38 can produce NAADP in human cells. They further suggest that CD38's compartmentalization to the lysosome may allow for its regulation via substrate access, rather than enzyme activation, thereby providing a reliable mechanism for regulating cellular NAADP production.
- Published
- 2018
3. TFEB insufficiency promotes cardiac hypertrophy by blocking autophagic degradation of GATA4
- Author
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Rui Song, Han Lei, Wanwen Cheng, Ling Ling Yao, Li Feng, Jie Liu, and Ying Li
- Subjects
Autophagosome ,TF, transcription factor ,LVIDd, diastolic LV internal dimension ,CQ, chloroquine ,ATG7, autophagy-related protein 7 ,mTORC1 ,HF, heart failure ,Biochemistry ,Muscle hypertrophy ,Mice ,RFP, red fluorescent protein ,AAV, adeno-associated virus ,Medicine ,Myocytes, Cardiac ,TFEB, transcription factor EB ,MOI, multiplicity of infection ,FS, fractional shortening ,Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ,BW, body weight ,BafA1, bafilomycin A1 ,IP, immunoprecipitation ,mTORC1, mechanistic target of rapamycin kinase complex 1 ,NPPB, natriuretic peptide precursor B ,Cell biology ,LVIDs, systolic LV internal dimension ,cardiovascular system ,β-MHC, myosin heavy chain-β ,TAC, transverse aortic constriction ,hypertrophy ,Research Article ,autophagy ,NRVM, neonatal rat ventricular myocyte ,WGA, wheat germ agglutinin ,Cardiomegaly ,NIH, the National Institutes of Health ,heart ,MYH7B, myosin heavy chain 7B ,ALP, autophagosome–lysosome pathway ,GATA4 ,Downregulation and upregulation ,Animals ,Humans ,EF, ejection fraction ,PO, pressure overload ,Autophagy-Related Protein 7 ,ANP, atrial natriuretic peptide ,LV, left ventricular ,Molecular Biology ,PGC1α, peroxisome proliferator–activated receptor-gamma coactivator-1α ,KD, knockdown ,Pressure overload ,TFEB ,PE, phenylephrine ,autophagosome–lysosome pathway ,business.industry ,Autophagy ,Cell Biology ,NPPA, natriuretic peptide precursor A ,GATA4 Transcription Factor ,LC3, light chain 3 ,Proteolysis ,business - Abstract
Autophagosome–lysosome pathway (ALP) insufficiency has been suggested to play a critical role in the pathogenesis of cardiac hypertrophy. However, the mechanisms underlying ALP insufficiency remain largely unknown, and strategies to specifically manipulate ALP insufficiency for treating cardiac hypertrophy are lacking. Transcription factor EB (TFEB), as a master regulator of ALP, regulates the generation and function of autophagosomes and lysosomes. We found that TFEB was significantly decreased, whereas autophagosome markers were increased in phenylephrine (PE)-induced and transverse aortic constriction–induced cardiomyocyte hypertrophy and failing hearts from patients with dilated cardiomyopathy. Knocking down TFEB induced ALP insufficiency, as indicated by increased autophagosome markers, decreased light chain 3II flux, and cardiomyocyte hypertrophy manifested through increased levels of atrial natriuretic peptide and β-myosin heavy chain and enlarged cell size. The effects of TFEB knockdown were abolished by promoting autophagy. TFEB overexpression improved autophagic flux and attenuated PE-stimulated cardiomyocyte hypertrophy and transverse aortic constriction–induced hypertrophic remodeling, fibrosis, and cardiac dysfunction. Curcumin analog compound C1, a specific TFEB activator, similarly attenuated PE-induced ALP insufficiency and cardiomyocyte hypertrophy. TFEB knockdown increased the accumulation of GATA4, a transcription factor for several genes causing cardiac hypertrophy by blocking autophagic degradation of GATA4, whereas knocking down GATA4 attenuated TFEB downregulation–induced cardiomyocyte hypertrophy. Both TFEB overexpression and C1 promoted GATA4 autophagic degradation and alleviated PE-induced cardiomyocyte hypertrophy. In conclusion, TFEB downregulation plays a vital role in the development of pressure overload–induced cardiac hypertrophy by causing ALP insufficiency and blocking autophagic degradation. Activation of TFEB represents a potential therapeutic strategy for treating cardiac hypertrophy.
- Published
- 2021
4. Correction: CD38 produces nicotinic acid adenine dinucleotide phosphate in the lysosome
- Author
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Yun Nan Hou, Qi Wen Deng, Yong Juan Zhao, Cheng Fang, Ying Li, Hon Cheung Lee, Ting Li, Ya Jie Chen, and Guan Jie Xu
- Subjects
Vasodilator Agents ,CD38 ,Niacin ,Biochemistry ,chemistry.chemical_compound ,immune system diseases ,hemic and lymphatic diseases ,Lysosome ,medicine ,Humans ,Calcium Signaling ,Molecular Biology ,Membrane Glycoproteins ,Nicotinic acid adenine dinucleotide phosphate ,hemic and immune systems ,Cell Biology ,Single-Domain Antibodies ,ADP-ribosyl Cyclase 1 ,Endocytosis ,HEK293 Cells ,medicine.anatomical_structure ,chemistry ,Additions and Corrections ,Calcium ,Lysosomes ,NADP ,HeLa Cells - Abstract
Nicotinic acid adenosine dinucleotide phosphate (NAADP) is a Ca(2+)-mobilizing second messenger that regulates a wide range of biological activities. However, the mechanism of its biogenesis remains controversial. CD38 is the only enzyme known to catalyze NAADP synthesis from NADP and nicotinic acid. CD38-mediated catalysis requires an acidic pH, suggesting that NAADP may be produced in acidic endolysosomes, but this hypothesis is untested. In this study, using human cell lines, we specifically directed CD38 to the endolysosomal system and assessed cellular NAADP production. First, we found that nanobodies targeting various epitopes on the C-terminal domain of CD38 could bind to cell surface–localized CD38 and induce its endocytosis. We also found that CD38 internalization occurred via a clathrin-dependent pathway, delivered CD38 to the endolysosome, and elevated intracellular NAADP levels. We also created a CD38 variant for lysosome-specific expression, which not only withstood the degradative environment in the lysosome, but was also much more active than WT CD38 in elevating cellular NAADP levels. Supplementing CD38-expressing cells with nicotinic acid substantially increased cellular NAADP levels. These results demonstrate that endolysosomal CD38 can produce NAADP in human cells. They further suggest that CD38's compartmentalization to the lysosome may allow for its regulation via substrate access, rather than enzyme activation, thereby providing a reliable mechanism for regulating cellular NAADP production.
- Published
- 2019
5. Dihydroceramides: From Bit Players to Lead Actors
- Author
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Monowarul Mobin Siddique, Scott A. Summers, Ying Li, Bhagirath Chaurasia, and Vincent A. Kaddai
- Subjects
Cell signaling ,Autophagy ,Neurodegenerative Diseases ,Minireviews ,Cell Biology ,Biology ,Ceramides ,Biochemistry ,Sphingolipid ,Sphingomyelins ,Sphingolipid synthesis ,Apoptosis ,Neoplasms ,Reperfusion Injury ,Diabetes Mellitus ,Animals ,Humans ,lipids (amino acids, peptides, and proteins) ,Sphingomyelin ,Molecular Biology ,Glucosylceramides ,Cell Proliferation - Abstract
Sphingolipid synthesis involves a highly conserved biosynthetic pathway that produces fundamental precursors of complex sphingolipids. The final reaction involves the insertion of a double bond into dihydroceramides to generate the more abundant ceramides, which are converted to sphingomyelins and glucosylceramides/gangliosides by the addition of polar head groups. Although ceramides have long been known to mediate cellular stress responses, the dihydroceramides that are transiently produced during de novo sphingolipid synthesis were deemed inert. Evidence published in the last few years suggests that these dihydroceramides accumulate to a far greater extent in tissues than previously thought. Moreover, they have biological functions that are distinct and non-overlapping with those of the more prevalent ceramides. Roles are being uncovered in autophagy, hypoxia, and cellular proliferation, and the lipids are now implicated in the etiology, treatment, and/or diagnosis of diabetes, cancer, ischemia/reperfusion injury, and neurodegenerative diseases. This minireview summarizes recent findings on this emerging class of bioactive lipids.
- Published
- 2015
6. MicroRNA-137 Is a Novel Hypoxia-responsive MicroRNA That Inhibits Mitophagy via Regulation of Two Mitophagy Receptors FUNDC1 and NIX
- Author
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Sijie Wang, Longxuan Li, Wen Li, Ying Li, Sen-Fang Sui, Weili Tian, Liangqing Zhang, Haixia Zhuang, Yusen Chen, He-ge Chen, Bin Zhao, Wenxian Wu, Du Feng, Zhe Hu, Yinqin Chen, and Xingli Zhang
- Subjects
Genetic Vectors ,Biology ,Mitochondrion ,Biochemistry ,Parkin ,Mitochondrial Proteins ,Mice ,Phagosomes ,microRNA ,Mitophagy ,Autophagy ,Animals ,Humans ,Receptor ,3' Untranslated Regions ,Molecular Biology ,Regulation of gene expression ,HEK 293 cells ,Membrane Proteins ,Cell Biology ,Fibroblasts ,Cell Hypoxia ,Mitochondria ,Cell biology ,Mice, Inbred C57BL ,MicroRNAs ,HEK293 Cells ,Gene Expression Regulation ,RNA ,HeLa Cells - Abstract
Mitophagy receptors mediate the selective recognition and targeting of damaged mitochondria by autophagosomes. The mechanism for the regulation of these receptors remains unknown. Here, we demonstrated that a novel hypoxia-responsive microRNA, microRNA-137 (miR-137), markedly inhibits mitochondrial degradation by autophagy without affecting global autophagy. miR-137 targets the expression of two mitophagy receptors NIX and FUNDC1. Impaired mitophagy in response to hypoxia caused by miR-137 is reversed by re-expression of FUNDC1 and NIX expression vectors lacking the miR-137 recognition sites at their 3' UTR. Conversely, miR-137 also suppresses the mitophagy induced by fundc1 (CDS+3'UTR) but not fundc1 (CDS) overexpression. Finally, we found that miR-137 inhibits mitophagy by reducing the expression of the mitophagy receptor thereby leads to inadequate interaction between mitophagy receptor and LC3. Our results demonstrated the regulatory role of miRNA to mitophagy receptors and revealed a novel link between miR-137 and mitophagy.
- Published
- 2014
7. Histone Deacetylase Inhibition Promotes Osteoblast Maturation by Altering the Histone H4 Epigenome and Reduces Akt Phosphorylation
- Author
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Jared M. Evans, Ying Li, Meghan E. McGee-Lawrence, Amel Dudakovic, Andre J. van Wijnen, Jennifer J. Westendorf, and Sumit Middha
- Subjects
Chromatin Immunoprecipitation ,Sodium-Hydrogen Exchangers ,Cellular differentiation ,FOXO1 ,Biology ,Hydroxamic Acids ,Biochemistry ,Histone Deacetylases ,Cell Line ,Epigenesis, Genetic ,Histones ,Histone H4 ,Mice ,Axin Protein ,medicine ,Animals ,Insulin ,Gene Regulation ,RNA, Messenger ,Phosphorylation ,Promoter Regions, Genetic ,Molecular Biology ,Protein kinase B ,Vorinostat ,Genome ,Osteoblasts ,Gene Expression Profiling ,Reproducibility of Results ,Acetylation ,Cell Differentiation ,Osteoblast ,Cell Biology ,Epigenome ,Phosphoproteins ,Histone Deacetylase Inhibitors ,medicine.anatomical_structure ,Cancer research ,Histone deacetylase ,Proto-Oncogene Proteins c-akt ,Signal Transduction ,medicine.drug - Abstract
Bone has remarkable regenerative capacity, but this ability diminishes during aging. Histone deacetylase inhibitors (HDIs) promote terminal osteoblast differentiation and extracellular matrix production in culture. The epigenetic events altered by HDIs in osteoblasts may hold clues for the development of new anabolic treatments for osteoporosis and other conditions of low bone mass. To assess how HDIs affect the epigenome of committed osteoblasts, MC3T3 cells were treated with suberoylanilide hydroxamic acid (SAHA) and subjected to microarray gene expression profiling and high-throughput ChIP-Seq analysis. As expected, SAHA induced differentiation and matrix calcification of osteoblasts in vitro. ChIP-Seq analysis revealed that SAHA increased histone H4 acetylation genome-wide and in differentially regulated genes, except for the 500 bp upstream of transcriptional start sites. Pathway analysis indicated that SAHA increased the expression of insulin signaling modulators, including Slc9a3r1. SAHA decreased phosphorylation of insulin receptor β, Akt, and the Akt substrate FoxO1, resulting in FoxO1 stabilization. Thus, SAHA induces genome-wide H4 acetylation and modulates the insulin/Akt/FoxO1 signaling axis, whereas it promotes terminal osteoblast differentiation in vitro.
- Published
- 2013
8. Progesterone Receptor A Stability Is Mediated by Glycogen Synthase Kinase-3β in the Brca1-deficient Mammary Gland
- Author
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Pang-Hung Hsu, Yoon Kyung Kim, Sou-Ying Lee, Ying Li, Eva Y.-H. P. Lee, and Shaohui Wang
- Subjects
Progesterone receptor A ,endocrine system diseases ,Mammary gland ,Mutation, Missense ,Breast Neoplasms ,Mammary Neoplasms, Animal ,Protein degradation ,Biology ,Biochemistry ,Glycogen Synthase Kinase 3 ,Mice ,Mammary Glands, Animal ,GSK-3 ,Cell Line, Tumor ,Progesterone receptor ,medicine ,Animals ,Humans ,Phosphorylation ,Mammary Glands, Human ,skin and connective tissue diseases ,Receptor ,Molecular Biology ,Mice, Knockout ,Glycogen Synthase Kinase 3 beta ,BRCA1 Protein ,Protein Stability ,Kinase ,Molecular Bases of Disease ,Cell Biology ,medicine.anatomical_structure ,Cancer research ,Female ,Receptors, Progesterone - Abstract
Germ line mutations of the BRCA1 gene increase the risk of breast and ovarian cancer, but the basis of this tissue-specific tumor predisposition is not fully understood. Previously, we reported that the progesterone receptors are stabilized in Brca1-deficient mammary epithelial cells, and treating with anti-progesterone delays mammary tumorigenesis in Brca1/p53 conditional knock-out mice, suggesting that the progesterone has a critical role in breast carcinogenesis. To further explore how the stability of progesterone receptor is modulated, here, we have found that glycogen synthase kinase (GSK)-3β phosphorylation of progesterone receptor-A (PR-A) facilitates its ubiquitination. GSK-3β-mediated phosphorylation of serine 390 in PR-A regulates its subsequent ubiquitination and protein stability. Expression of PR-AS390A mutant in the human breast epithelial cells, MCF-10A, results in enhanced proliferation and formation of aberrant acini structure in the three-dimensional culture. Consistently, reduction of phosphorylation of serine 390 of PR-A and GSK-3β activity is observed in the Brca1-deficient mammary gland. Taken together, these results provide important aspects of tissue specificity of BRCA1-mediated suppression of breast carcinogenesis. Background: Stabilization of progesterone receptors contributes to mammary tumorigenesis in Brca1-deficient mice. Results: Deficiency in phosphorylation on serine 390 of progesterone receptor A by GSK-3β enhances the receptor stability. Conclusion: Stabilization of progesterone receptor A is mediated by GSK-3β kinase in the Brca1-deficient mammary gland. Significance: This finding provides a novel insight of how tumor suppression of Brca1 is mediated by PR-A.
- Published
- 2013
9. Virus-triggered Ubiquitination of TRAF3/6 by cIAP1/2 Is Essential for Induction of Interferon-β (IFN-β) and Cellular Antiviral Response
- Author
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Jie Yan, Chengwen Teng, Ying Li, Shu Li, Qi Li, Ai-Ping Mao, Bo Zhong, and Hong-Bing Shu
- Subjects
TRAF3 ,Cytoplasm ,Ubiquitin-Protein Ligases ,viruses ,Biology ,Antiviral Agents ,Biochemistry ,Inhibitor of Apoptosis Proteins ,Cytosol ,Ubiquitin ,Interferon ,RNA interference ,medicine ,Humans ,Molecular Biology ,Transcription factor ,RNA, Double-Stranded ,TNF Receptor-Associated Factor 6 ,Gene knockdown ,TNF Receptor-Associated Factor 3 ,NF-kappa B ,Interferon-beta ,Cell Biology ,Molecular biology ,Baculoviral IAP Repeat-Containing 3 Protein ,Cell biology ,biology.protein ,RNA Interference ,Signal transduction ,IRF3 ,Signal Transduction ,medicine.drug - Abstract
Viral infection causes activation of transcription factors NF-kappaB and IRF3, which collaborate to induce type I interferons (IFNs) and cellular antiviral response. Here we show that knockdown of the E3 ubiquitin ligases cIAP1 and cIAP2 markedly inhibited virus-triggered activation of IRF3 and NF-kappaB as well as IFN-beta induction. Knockdown of cIAP1 and cIAP2 also inhibited cytoplasmic dsRNA-triggered cellular antiviral response. Endogenous coimmunoprecipitation experiments indicated that viral infection caused recruitment of cIAP1 and cIAP2 to TRAF3, TRAF6, and VISA. Furthermore, we demonstrated that cIAP1- and cIAP2-mediated virus-triggered ubiquitination of TRAF3 and TRAF6. These findings suggest that virus-triggered ubiquitination of TRAF3 and TRAF6 by cIAP1 and cIAP2 is essential for type I IFN induction and cellular antiviral response.
- Published
- 2010
10. Molecular Basis of Cav2.3 Calcium Channels in Rat Nociceptive Neurons
- Author
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Chul-Kyu Park, Richard J. Miller, Joong Soo Kim, Hai Ying Li, Seong-Hae Park, Seog Bae Oh, Arnaud Monteil, Zhi Fang, Hyun Yeong Kim, and Sung Jun Jung
- Subjects
Gene isoform ,P-type calcium channel ,Cell ,Pain ,TRPV Cation Channels ,Sensory system ,Calcium Channels, R-Type ,Biochemistry ,Article ,Trigeminal ganglion ,medicine ,Animals ,Pain Management ,Protein Isoforms ,Neurons, Afferent ,Cation Transport Proteins ,Molecular Biology ,Voltage-dependent calcium channel ,Reverse Transcriptase Polymerase Chain Reaction ,Chemistry ,T-type calcium channel ,Nociceptors ,Cell Biology ,Anatomy ,Reverse transcriptase ,Rats ,Cell biology ,medicine.anatomical_structure ,Trigeminal Ganglion ,Biomarkers - Abstract
Ca(v)2.3 calcium channels play an important role in pain transmission in peripheral sensory neurons. Six Ca(v)2.3 isoforms resulting from different combinations of three inserts (inserts I and II in the II-III loop and insert III in the carboxyl-terminal region) have been identified in different mammalian tissues. To date, however, Ca(v)2.3 isoforms unique to primary sensory neurons have not been identified. In this study, we determined Ca(v)2.3 isoforms expressed in the rat trigeminal ganglion neurons. Whole tissue reverse transcription (RT)-PCR analyses revealed that only two isoforms, Ca(v)2.3a and Ca(v)2.3e, are present in TG neurons. Using single cell RT-PCR, we found that Ca(v)2.3e is the major isoform, whereas Ca(v)2.3e expression is highly restricted to small (16 mum) isolectin B4-negative and tyrosine kinase A-positive neurons. Ca(v)2.3e was also preferentially detected in neurons expressing the nociceptive marker, transient receptor potential vanilloid 1. Single cell RT-PCR following calcium imaging and whole-cell patch clamp recordings provided evidence of an association between an R-type calcium channel component and Ca(v)2.3e expression. Our results suggest that Ca(v)2.3e in sensory neurons may be a potential target for the treatment of pain.
- Published
- 2007
11. Functional Expression of Thermo-transient Receptor Potential Channels in Dental Primary Afferent Neurons
- Author
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Sung Joong Lee, Joong Soo Kim, Zhi Fang, Kyungpyo Park, Se-Young Choi, Mi-Sun Kim, Hai Ying Li, Seog Bae Oh, Sung Jun Jung, and Chul-Kyu Park
- Subjects
Agonist ,medicine.drug_class ,TRPV1 ,Icilin ,Cell Biology ,Anatomy ,Biochemistry ,Calcium in biology ,Transient receptor potential channel ,chemistry.chemical_compound ,chemistry ,Capsaicin ,TRPA1 Cation Channel ,medicine ,TRPM8 ,Molecular Biology ,Neuroscience - Abstract
Temperature signaling can be initiated by members of transient receptor potential family (thermo-TRP) channels. Hot and cold substances applied to teeth usually elicit pain sensation. This study investigated the expression of thermo-TRP channels in dental primary afferent neurons of the rat identified by retrograde labeling with a fluorescent dye in maxillary molars. Single cell reverse transcription-PCR and immunohistochemistry revealed expression of TRPV1, TRPM8, and TRPA1 in subsets of such neurons. Capsaicin (a TRPV1 agonist), menthol (a TRPM8 agonist), and icilin (a TRPM8 and TRPA1 agonist) increased intracellular calcium and evoked cationic currents in subsets of neurons, as did the appropriate temperature changes (>43 degrees
- Published
- 2006
12. Role of ATP Hydrolysis in the Antirecombinase Function of Saccharomyces cerevisiae Srs2 Protein
- Author
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Margaret A. Macris, Lumir Krejci, Patrick Sung, Hannah L. Klein, Stephen Van Komen, Jana Villemain, Ying Li, and Thomas E. Ellenberger
- Subjects
Recombination, Genetic ,Saccharomyces cerevisiae Proteins ,DNA repair ,Hydrolysis ,FLP-FRT recombination ,Mutant ,DNA Helicases ,RAD51 ,Helicase ,DNA ,Saccharomyces cerevisiae ,Cell Biology ,Biology ,Biochemistry ,Rad52 DNA Repair and Recombination Protein ,DNA-Binding Proteins ,Recombinases ,Adenosine Triphosphate ,Mutation ,biology.protein ,Homologous recombination ,Molecular Biology ,Replication protein A ,Sgs1 - Abstract
Mutants of the Saccharomyces cerevisiae SRS2 gene are hyperrecombinogenic and sensitive to genotoxic agents, and they exhibit a synthetic lethality with mutations that compromise DNA repair or other chromosomal processes. In addition, srs2 mutants fail to adapt or recover from DNA damage checkpoint-imposed G2/M arrest. These phenotypic consequences of ablating SRS2 function are effectively overcome by deleting genes of the RAD52 epistasis group that promote homologous recombination, implicating an untimely recombination as the underlying cause of the srs2 mutant phenotypes. TheSRS2-encodedproteinhasasingle-stranded (ss) DNA-dependent ATPase activity, a DNA helicase activity, and an ability to disassemble the Rad51-ssDNA nucleoprotein filament, which is the key catalytic intermediate in Rad51-mediated recombination reactions. To address the role of ATP hydrolysis in Srs2 protein function, we have constructed two mutant variants that are altered in the Walker type A sequence involved in the binding and hydrolysis of ATP. The srs2 K41A and srs2 K41R mutant proteins are both devoid of ATPase and helicase activities and the ability to displace Rad51 from ssDNA. Accordingly, yeast strains harboring these srs2 mutations are hyperrecombinogenic and sensitive to methylmethane sulfonate, and they become inviable upon introducing either the sgs1Delta or rad54Delta mutation. These results highlight the importance of the ATP hydrolysisfueled DNA motor activity in SRS2 functions.
- Published
- 2004
13. Stability of Homologue of Slimb F-box Protein Is Regulated by Availability of Its Substrate
- Author
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Serge Y. Fuchs, Zhen-Qiang Pan, Ying Li, and Stefan Gazdoiu
- Subjects
Proteasome Endopeptidase Complex ,IκB kinase ,Biochemistry ,NEDD8 ,F-box protein ,Cell Line ,Substrate Specificity ,SCF complex ,Ubiquitin ,Multienzyme Complexes ,Humans ,Molecular Biology ,DNA Primers ,Base Sequence ,biology ,Hydrolysis ,Proteins ,Cell Biology ,Ubiquitin ligase ,Cysteine Endopeptidases ,IκBα ,Mutation ,biology.protein ,Cullin ,DNA Damage - Abstract
The homologue of Slimb (HOS) F-box protein is a receptor of the Skp1-Cullin1-F-box protein (SCF(HOS)) E3 ubiquitin ligase, which mediates ubiquitination and degradation of beta-catenin and the inhibitor of NFkappaB, IkappaB. We found that HOS itself is an unstable protein that undergoes ubiquitination and degradation in a 26 S proteasome-dependent manner. A HOS mutant lacking the F-box that is deficient in binding to the core SCF components underwent ubiquitination less efficiently and was more stable than the wild type protein. Furthermore, ubiquitination and degradation of HOS was impaired in ts41 cells, in which the activities of Cullin-based ligases were decreased because the NEDD8 pathway was abrogated. Whereas HOS was directly ubiquitinated within the SCF(HOS) complex in vitro, the addition of phosphorylated IkappaBalpha inhibited this ubiquitination. Increasing cellular levels of HOS substrate (phosphorylated IkappaBalpha) by activating IkappaB kinase inhibited HOS ubiquitination and led to stabilization of HOS, indicating that interaction between HOS and its substrate might protect HOS from proteolysis. Taken together, our data suggest that proteolysis of HOS depends on its interaction with active components of the SCF complex and that HOS stability is regulated by a bound substrate. These findings may define a mechanism for maintaining activities of specific SCF complexes based on availability of a particular substrate.
- Published
- 2004
14. Effects of Hydrogen Peroxide upon Nicotinamide Nucleotide Metabolism in Escherichia coli
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Julia L. Brumaghim, Ernst S. Henle, Stuart Linn, and Ying Li
- Subjects
inorganic chemicals ,chemistry.chemical_classification ,biology ,Nicotinamide ,Stereochemistry ,Dehydrogenase ,Cell Biology ,Biochemistry ,chemistry.chemical_compound ,Enzyme ,Glycerol-3-phosphate dehydrogenase ,chemistry ,biology.protein ,Nucleotide ,NAD+ kinase ,Hydrogen peroxide ,Molecular Biology ,Peroxidase - Abstract
DNA is damaged in vivo by the Fenton reaction mediated by Fe2+ and cellular reductants such as NADH, which reduce Fe3+ to Fe2+ and allow the recycling of iron. To study the response of Escherichia coli to such cycling, the activities of several enzymes involved in nicotinamide nucleotide metabolism were measured following an H2O2 challenge. NADPH-dependent peroxidase, NADH/NADP+ transhydrogenase, and glucose-6-phosphate dehydrogenase were most strongly induced, increasing 2.5-3-fold. In addition, the cellular ratios of NADPH to NADH increased 6- or 92-fold 15 min after exposure to 0.5 or 5 mm H2O2, respectively. In vitro, NADH was oxidized by Fe3+ up to 16-fold faster than NADPH, despite their identical reduction potentials. To understand this rate difference, the interactions of Fe3+ and Ga3+ with NAD(P)H were examined by 1H, 13C, and 31P NMR spectroscopy. Association with NADH occurred primarily with adenine at N7 and the amino group, but for NADPH, strong metal interactions also occurred at the 2'-phosphate group. Interaction of M3+ (Fe3+ or Ga3+) with the adenine ring would bring it into close proximity to the redox-active nicotinamide ring in the folded form of NAD(P)H, but interaction of M3+ with the 2'-phosphate group would avoid this close contact. In addition, as determined by absorbance spectroscopy, the energy of the charge-transfer species was significantly higher for the Fe3+.NADPH complex than for the Fe3+.NADH complex. We therefore suggest that upon exposure to H2O2 the NADH pool is depleted, and NADPH, which is less reactive with Fe3+, functions as the major nicotinamide nucleotide reductant.
- Published
- 2003
15. The Amino Acid Residues Asparagine 354 and Isoleucine 372 of Human Farnesoid X Receptor Confer the Receptor with High Sensitivity to Chenodeoxycholate
- Author
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James M. Schaeffer, Jinghua Yu, Li Huang, Jisong Cui, Samuel D. Wright, Thomas S. Heard, Jane-L. Lo, and Ying Li
- Subjects
medicine.drug_class ,Receptors, Cytoplasmic and Nuclear ,Biology ,Chenodeoxycholic Acid ,Ligands ,Biochemistry ,Cell Line ,Mice ,Structure-Activity Relationship ,Transactivation ,medicine ,Animals ,Humans ,Amino Acid Sequence ,RNA, Messenger ,Isoleucine ,Receptor ,Chenodeoxycholate ,Molecular Biology ,ATP Binding Cassette Transporter, Subfamily B, Member 11 ,Binding Sites ,Bile acid ,Cell Biology ,G protein-coupled bile acid receptor ,Bile Salt Export Pump ,DNA-Binding Proteins ,Liver ,Nuclear receptor ,Mutagenesis, Site-Directed ,ATP-Binding Cassette Transporters ,Farnesoid X receptor ,Asparagine ,Transcription Factors - Abstract
The critical steps in bile acid metabolism have remarkable differences between humans and mice. It is known that human cholesterol 7 alpha-hydroxylase, the enzyme catalyzing the rate-limiting step of bile acid synthesis, is more sensitive to bile acid suppression. In addition, hepatic bile acid export in humans is more dependent on the bile salt export pump (BSEP). To explore the molecular basis for these species differences, we analyzed the function of the ligand-binding domain (LBD) of human and murine farnesoid X receptor (FXR), a nuclear receptor for bile acids. We observed a strong interspecies difference in bile acid-mediated FXR function; in the coactivator association assay, chenodeoxycholate (CDCA) activated human FXR-LBD with 10-fold higher affinity and 3-fold higher maximum response than murine FXR-LBD. Consistently, in HepG2 cells human FXR-LBD increased reporter expression more robustly in the presence of CDCA. The basis for these differences was investigated by preparing chimeric receptors and by site-directed mutagenesis. Remarkably, the double replacements of Lys(366) and Val(384) in murine FXR (corresponding to Asn(354) and Ile(372) in human FXR) with Asn(366) and Ile(384) explained the difference in both potency and maximum activation; compared with the wild-type murine FXR-LBD, the double mutant gained 8-fold affinity and more than 250% maximum response to CDCA in vitro. This mutant also increased reporter expression to an extent comparable with that of human FXR-LBD in HepG2 cells. These results demonstrate that Asn(354) and Ile(372) are critically important for FXR function and that murine FXR can be "humanized" by substituting with the two corresponding residues of human FXR. Consistent with the difference in FXR-LBD transactivation, CDCA induced endogenous expression of human BSEP by 10-12-fold and murine BSEP by 2-3-fold in primary hepatocytes. This study not only provides the identification of critical residues for FXR function but may also explain the species difference in bile acids/cholesterol metabolism.
- Published
- 2002
16. AKT/PKB Phosphorylation of p21Cip/WAF1 Enhances Protein Stability of p21Cip/WAF1 and Promotes Cell Survival
- Author
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Donald Dowbenko, Laurence A. Lasky, and Ying Li
- Subjects
Cyclin-Dependent Kinase Inhibitor p21 ,Cell Survival ,Molecular Sequence Data ,Cyclin A ,AKT1 ,Protein Serine-Threonine Kinases ,DEPTOR ,Biochemistry ,mTORC2 ,Cyclin D1 ,Cyclins ,Proto-Oncogene Proteins ,Tumor Cells, Cultured ,Humans ,Amino Acid Sequence ,Phosphorylation ,Molecular Biology ,Protein kinase B ,DNA Primers ,Base Sequence ,biology ,Chemistry ,Akt/PKB signaling pathway ,Cyclin-Dependent Kinase 4 ,Cell Biology ,Protein-Tyrosine Kinases ,Cell cycle ,Cyclin-Dependent Kinases ,Up-Regulation ,Cell biology ,biology.protein ,Cancer research ,Proto-Oncogene Proteins c-akt - Abstract
p21(Cip1/WAF1) (p21), a p53-inducible protein, is a critical regulator of cell cycle and cell survival. p21 binds to and inhibits both the DNA synthesis regulator proliferating cell nuclear antigen and cyclin A/E-CDK2 complexes. Recently, p21 has also been shown to be a positive regulator of cell cycle progression as p21 is necessary for the assembly and activation of cyclin D1-CDK4/6 complexes. Furthermore, elevated p21 protein levels have been observed in various aggressive tumors as well as linked to chemoresistance. Here we demonstrate that p21 is directly phosphorylated by AKT/PKB, a survival kinase that is hyperactivated in many late stage tumors. Two sites (Thr(145) and Ser(146)) in the carboxyl terminus of p21 are phosphorylated by AKT/PKB in vitro and in vivo. Phosphorylation of Thr(145) inhibits PCNA binding, whereas phosphorylation of Ser(146) significantly increases p21 protein stability. Glioblastoma cell lines with activated AKT/PKB show enhanced p21 stability, and they are more resistant to taxol-mediated toxicity. Finally, AKT/PKB controls the assembly of cyclin D1-CDK4 complexes through modulation of p21 and cyclin D1 levels. These data imply that enhanced levels of p21 in tumors are due, in part, to phosphorylation by activated AKT/PKB. Furthermore, they suggest that one mechanism of AKT/PKB regulation of tumor cell survival and/or proliferation is to stabilize p21 protein.
- Published
- 2002
17. Differential Gene Regulation in Human Versus Rodent Hepatocytes by Peroxisome Proliferator-activated Receptor (PPAR) α
- Author
-
Diane R. Umbenhauer, Joel P. Berger, Gaochao Zhou, Shiying Chen, David E. Moller, Jeffrey W. Lawrence, John G. DeLuca, and Ying Li
- Subjects
Regulation of gene expression ,chemistry.chemical_classification ,Messenger RNA ,medicine.medical_specialty ,Receptor expression ,Peroxisome Proliferation ,Peroxisome proliferator-activated receptor ,Cell Biology ,Transfection ,Biology ,Peroxisome ,Biochemistry ,Molecular biology ,Endocrinology ,chemistry ,Internal medicine ,medicine ,Receptor ,Molecular Biology - Abstract
We compared the ability of rat and human hepatocytes to respond to fenofibric acid and a novel potent phenylacetic acid peroxisome proliferator-activated receptor (PPAR) α agonist (compound 1). Fatty acyl-CoA oxidase (FACO) activity and mRNA were increased after treatment with either fenofibric acid or compound 1 in rat hepatocytes. In addition, apolipoprotein CIII mRNA was decreased by both fenofibric acid and compound 1 in rat hepatocytes. Both agonists decreased apolipoprotein CIII mRNA in human hepatocytes; however, very little change in FACO activity or mRNA was observed. Furthermore, other peroxisome proliferation (PP)-associated genes including peroxisomal 3-oxoacyl-CoA thiolase (THIO), peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), peroxisomal membrane protein-70 (PMP-70) were not regulated by PPARα agonists in human hepatocytes. Moreover, other genes that are regulated by PPARα ligands in human hepatocytes such as mitochondrial HMG-CoA synthase and carnitine palmitoyl transferase-1 (CPT-1) were also regulated in HepG2 cells by PPARα agonists. Several stably transfected HepG2 cell lines were established that overexpressed human PPARα to levels between 6- and 26-fold over normal human hepatocytes. These PPARα-overexpressing cells had higher basal mRNA levels of mitochondrial HMG-CoA synthase and CPT-1; however, basal FACO mRNA levels and other PP-associated genes including THIO, HD, or PMP-70 mRNA were not substantially affected. In addition, FACO, THIO, HD, and PMP-70 mRNA levels did not increase in response to PPARα agonist treatment in the PPARα-overexpressing cells, although mitochondrial HMG-CoA synthase and CPT-1 mRNAs were both induced. These results suggest that other factors besides PPARα levels determine the species-specific response of human and rat hepatocytes to the induction of PP.
- Published
- 2001
18. Heat Shock Protein 70 Inhibits Apoptosis Downstream of Cytochrome c Release and Upstream of Caspase-3 Activation
- Author
-
Jae-Seon Lee, Jong Il Kim, Jeong-Sun Seo, Chun-Ying Li, and Young Gyu Ko
- Subjects
Programmed cell death ,Hot Temperature ,Cell Survival ,Apoptosis ,Cytochrome c Group ,Caspase 3 ,DNA Fragmentation ,Biology ,Transfection ,Inhibitor of apoptosis ,Biochemistry ,Mice ,Cytosol ,Animals ,Humans ,HSP70 Heat-Shock Proteins ,Molecular Biology ,Cell Nucleus ,Cytochrome c ,U937 Cells ,Cell Biology ,Flow Cytometry ,Molecular biology ,Recombinant Proteins ,Protein Structure, Tertiary ,Hsp70 ,Enzyme Activation ,Liver ,Caspases ,Mutagenesis, Site-Directed ,biology.protein ,DNA fragmentation ,Electrophoresis, Polyacrylamide Gel ,Apoptosome ,Gene Deletion ,Protein Binding - Abstract
Heat shock protein 70 (HSP70) has been shown to act as an inhibitor of apoptosis. We have also observed an inhibitory effect of HSP70 on apoptotic cell death both in preheated U937 and stably transfected HSP70-overexpressing U937 (U937/HSP70) cells. However, the molecular mechanism whereby HSP70 prevents apoptosis still remains to be solved. To address this issue, we investigated the effect of HSP70 on apoptotic processes in an in vitro system. Caspase-3 cleavage and DNA fragmentation were detected in cytosolic fractions from normal cells upon addition of dATP, but not from preheated U937 or U937/hsp70 cells. Moreover, the addition of purified recombinant HSP70 to normal cytosolic fractions prevented caspase-3 cleavage and DNA fragmentation, suggesting that HSP70 prevents apoptosis upstream of caspase-3 processing. Because cytochrome c was still released from mitochondria into the cytosol by lethal heat shock despite prevention of caspase-3 activation and cell death in both preheated U937 and U937/hsp70 cells, it was evident that HSP70 acts downstream of cytochrome c release. Results obtained in vitro with purified deletion mutants of HSP70 showed that the carboxyl one-third region (from amino acids 438 to 641) including the peptide-binding domain and the carboxyl-terminal EEVD sequence was essential to prevent caspase-3 processing. From these results, we conclude that HSP70 acts as a strong suppressor of apoptosis acting downstream of cytochrome c release and upstream of caspase-3 activation.
- Published
- 2000
19. Analysis of PDZ Domain-Ligand Interactions Using Carboxyl-terminal Phage Display
- Author
-
M. Teresa Pisabarro, Germaine Fuh, Sachdev S. Sidhu, Clifford Quan, Laurence A. Lasky, and Ying Li
- Subjects
Models, Molecular ,Phage display ,Protein Conformation ,Molecular Sequence Data ,PDZ domain ,Nerve Tissue Proteins ,Biology ,Ligands ,Biochemistry ,Capsid ,Protein structure ,Peptide Library ,Humans ,Amino Acid Sequence ,Homology modeling ,Binding site ,Peptide library ,Molecular Biology ,Peptide sequence ,Conserved Sequence ,Binding selectivity ,Binding Sites ,Sequence Homology, Amino Acid ,Membrane Proteins ,Cell Biology ,Molecular biology ,Recombinant Proteins ,Kinetics ,Mutagenesis, Site-Directed ,Biophysics ,Capsid Proteins ,Sequence Alignment ,Bacteriophage M13 - Abstract
PDZ domains mediate protein-protein interactions at specialized subcellular sites, such as epithelial cell tight junctions and neuronal post-synaptic densities. Because most PDZ domains bind extreme carboxyl-terminal sequences, the phage display method has not been amenable to the study of PDZ domain binding specificities. For the first time, we demonstrate the functional display of a peptide library fused to the carboxyl terminus of the M13 major coat protein. We used this library to analyze carboxyl-terminal peptide recognition by two PDZ domains. For each PDZ domain, the library provided specific ligands with sub-micromolar binding affinities. Synthetic peptides and homology modeling were used to dissect and rationalize the binding interactions. Our results establish carboxyl-terminal phage display as a powerful new method for mapping PDZ domain binding specificity.
- Published
- 2000
20. Both Coactivator LXXLL Motif-dependent and -independent Interactions Are Required for Peroxisome Proliferator-activated Receptor γ (PPARγ) Function
- Author
-
Ralph T. Mosley, David E. Moller, Susan D. Aster, Shiying Chen, Gaochao Zhou, Bruce A. Johnson, Brian M. McKeever, and Ying Li
- Subjects
Models, Molecular ,Transcriptional Activation ,Peroxisome proliferator-activated receptor gamma ,Magnetic Resonance Spectroscopy ,Receptors, Cytoplasmic and Nuclear ,Peroxisome proliferator-activated receptor ,Biochemistry ,Protein Structure, Secondary ,Nuclear Receptor Coactivator 1 ,Coactivator ,Animals ,Humans ,Molecular Biology ,Histone Acetyltransferases ,chemistry.chemical_classification ,Binding Sites ,Nuclear Proteins ,Cell Biology ,Cell biology ,Nuclear receptor coactivator 1 ,chemistry ,Nuclear receptor ,COS Cells ,Mutation ,Nuclear receptor coactivator 3 ,Trans-Activators ,Nuclear receptor coactivator 2 ,PPARGC1B ,Protein Binding ,Transcription Factors - Abstract
Nuclear receptor activation is dependent on recruitment of coactivators, including CREB-binding protein (CBP/p300) and steroid receptor coactivator-1 (SRC-1). A three-dimensional NMR approach was used to probe the coactivator binding interface in the peroxisome proliferator-activated receptor gamma (PPARgamma) ligand binding domain (LBD). In the presence of a CBP peptide, peaks corresponding to 20 residues in helices 3, 4, 5, and 12 of the LBD were attenuated. Alanine mutants revealed that K301A, V315A, Y320A, L468A, and E471A were required for binding of both CBP and SRC-1 and for cell-based transcription. Several additional amino acids in helix 4 of the PPARgammaLBD were defective with respect to CBP recruitment, but retained relatively normal SRC-1 recruitment. Thus these amino acid residues may be important determinants of specificity for nuclear receptor LBD interactions with discrete coactivator molecules.
- Published
- 2000
21. Novel Peroxisome Proliferator-activated Receptor (PPAR) γ and PPARδ Ligands Produce Distinct Biological Effects
- Author
-
Bei Zhang, Gregory D. Berger, Chhabi Biswas, Robert W. Marquis, Joel Berger, Richard L. Tolman, Conrad Santini, Roy G. Smith, Ying Li, Gaochou Zhou, Margaret Wu, John Ventre, Nancy S. Hayes, David E. Moller, Thomas W. Doebber, Michael R. Tanen, Soumya P. Sahoo, Alex Elbrecht, Mark D. Leibowitz, Catherine A. Cullinan, and Ralph T. Mosley
- Subjects
Blood Glucose ,medicine.medical_specialty ,Protein Conformation ,medicine.drug_class ,Receptors, Cytoplasmic and Nuclear ,Peroxisome proliferator-activated receptor ,Biology ,Ligands ,Biochemistry ,PPAR agonist ,Cell Line ,Diabetes Mellitus, Experimental ,Mice ,Transactivation ,Mediator ,In vivo ,Internal medicine ,Adipocytes ,medicine ,Animals ,Humans ,Thiazolidinedione ,Receptor ,Molecular Biology ,chemistry.chemical_classification ,Cell Differentiation ,Cell Biology ,Endocrinology ,Nuclear receptor ,chemistry ,Transcription Factors - Abstract
The peroxisome proliferator-activated receptors (PPARs) include three receptor subtypes encoded by separate genes: PPARalpha, PPARdelta, and PPARgamma. PPARgamma has been implicated as a mediator of adipocyte differentiation and the mechanism by which thiazolidinedione drugs exert in vivo insulin sensitization. Here we characterized novel, non-thiazolidinedione agonists for PPARgamma and PPARdelta that were identified by radioligand binding assays. In transient transactivation assays these ligands were agonists of the receptors to which they bind. Protease protection studies showed that ligand binding produced specific alterations in receptor conformation. Both PPARgamma and PPARdelta directly interacted with a nuclear receptor co-activator (CREB-binding protein) in an agonist-dependent manner. Only the PPARgamma agonists were able to promote differentiation of 3T3-L1 preadipocytes. In diabetic db/db mice all PPARgamma agonists were orally active insulin-sensitizing agents producing reductions of elevated plasma glucose and triglyceride concentrations. In contrast, selective in vivo activation of PPARdelta did not significantly affect these parameters. In vivo PPARalpha activation with WY-14653 resulted in reductions in elevated triglyceride levels with minimal effect on hyperglycemia. We conclude that: 1) synthetic non-thiazolidinediones can serve as ligands of PPARgamma and PPARdelta; 2) ligand-dependent activation of PPARdelta involves an apparent conformational change and association of the receptor ligand binding domain with CREB-binding protein; 3) PPARgamma activation (but not PPARdelta or PPARalpha activation) is sufficient to potentiate preadipocyte differentiation; 4) non-thiazolidinedione PPARgamma agonists improve hyperglycemia and hypertriglyceridemia in vivo; 5) although PPARalpha activation is sufficient to affect triglyceride metabolism, PPARdelta activation does not appear to modulate glucose or triglyceride levels.
- Published
- 1999
22. Sites Important for PLCβ2 Activation by the G Protein βγ Subunit Map to the Sides of the β Propeller Structure
- Author
-
Mikhail P. Panchenko, Eva J. Neer, Tohru Kozasa, Kumkum Saxena, Temple F. Smith, Sara Charnecki, Alfred G. Gilman, Ying Li, and Pamela M. Sternweis
- Subjects
Chemistry ,G protein ,Stereochemistry ,Effector ,Protein subunit ,Beta sheet ,Cell Biology ,Biochemistry ,Adenylyl cyclase ,chemistry.chemical_compound ,Heterotrimeric G protein ,Helix ,Molecular Biology ,Ion channel - Abstract
The βγ subunits of the heterotrimeric GTP-binding proteins (G proteins) that couple heptahelical, plasma membrane-bound receptors to intracellular effector enzymes or ion channels directly regulate several types of effectors, including phospholipase Cβ and adenylyl cyclase. The β subunit is made up of two structurally different regions: an N-terminal α helix followed by a toroidal structure made up of 7 blades, each of which is a twisted β sheet composed of four anti-parallel β strands (Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995)Cell 83, 1047–1058; Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311–319). We have previously shown that sites for activation of PLCβ2, PLCβ3, and adenylyl cyclase II overlap on the “top” surface of the propeller, where Gα also binds (Li, Y., Sternweis, P. M., Charnecki, S., Smith, T. F., Gilman, A. G., Neer, E. J., and Kozasa, T. (1998)J. Biol. Chem. 273, 16265–16272). The present study was undertaken to identify the regions on the side of the torus that might be important for effector interactions. We made mutations in each of the outer β strands of the G protein β1 propeller, as well as mutations in the loops that connect the outer strands to the adjacent β strands. Our results suggest that activation of PLCβ2 involves residues in the outer strands of blades 2, 6, and 7 of the propeller. We tested three of the mutations that most severely affected PLCβ2 activity against two forms of adenylyl cyclase (ACI and ACII). Both inhibition of ACI and activation of ACII were unaffected by these mutations, suggesting that if ACI and ACII contact the outer strands, the sites of contact are different from those for PLCβ2. We propose that distinct sets of contacts along the sides of the propeller will define the specificity of the interaction of βγ with effectors.
- Published
- 1998
23. Fluid Shear Stress Activation of Focal Adhesion Kinase
- Author
-
Shila Jalali, Shu Chien, John Y.-J. Shyy, Song Li, Michael Kim, Tony Hunter, Ying-Li Hu, and David D. Schlaepfer
- Subjects
PTK2B ,MAP kinase kinase kinase ,Cyclin-dependent kinase 2 ,PTK2 ,Cell Biology ,Biology ,Mitogen-activated protein kinase kinase ,Biochemistry ,MAP2K7 ,Cell biology ,biology.protein ,ASK1 ,biological phenomena, cell phenomena, and immunity ,Kinase activity ,Molecular Biology - Abstract
Shear stress, the tangential component of hemodynamic forces, activates the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) signal transduction pathways in cultured vascular endothelial cells to induce the transcriptional activation of many immediate early genes. It appears that integrins, protein-tyrosine kinases, and the structural integrity of actin are important factors involved in these shear stress-induced responses. The underlying molecular events were investigated by the application of a shear stress of 12 dyn/cm2 on bovine aortic endothelial cells (BAEC). We found that such a shear stress increased the tyrosine phosphorylation and the kinase activity of focal adhesion kinase (FAK) and its association with growth factor receptor binding protein 2 (Grb2) in a rapid and transient manner, suggesting that FAK may be linked to these mitogen-activated protein kinase signaling pathways through a Grb2. Son of sevenless (Sos) complex. FAK(F397Y), which encodes a dominant negative mutant of FAK, attenuated the shear stress-induced kinase activity of Myc epitope-tagged ERK2 and hemagglutinin epitope-tagged JNK1. DeltamSos1, encoding a dominant negative mutant of Sos in which the guanine nucleotide exchange domain has been deleted, also attenuated shear stress activation of Myc-ERK2 and hemagglutinin-JNK1. Pretreating the confluent BAEC monolayers with a blocking type anti-vitronectin receptor monoclonal antibody had similar inhibitory effects in these shear stress-activated ERKs and JNKs. Confocal microscopic observation further demonstrated that FAK tended to cluster with vitronectin receptor near the abluminal side of the sheared BAEC. These results demonstrate that FAK signaling is critical in the shear stress-induced dual activation of ERK and JNK.
- Published
- 1997
24. Mutagenesis of the Regulatory Subunit (RIIβ) of cAMP-dependent Protein Kinase IIβ Reveals Hydrophobic Amino Acids That Are Essential for RIIβ Dimerization and/or Anchoring RIIβ to the Cytoskeleton
- Author
-
Ying Li and Charles S. Rubin
- Subjects
chemistry.chemical_classification ,endocrine system ,Protein subunit ,hemic and immune systems ,chemical and pharmacologic phenomena ,Cell Biology ,Plasma protein binding ,Biochemistry ,Cell biology ,A Kinase Anchor Proteins ,Amino acid ,chemistry ,Beta protein ,Binding site ,Protein kinase A ,Molecular Biology ,Peptide sequence - Abstract
In neurons cAMP-dependent protein kinase II beta (PKAII beta) is sequestered in the dendritic cytoskeleton because the regulatory subunit (RII beta) of the enzyme is tightly bound by A Kinase Anchor Proteins (AKAPs). The prototypic neuronal anchor protein AKAP75 has a COOH-terminal 22-residue RII beta binding (tethering) site. A key feature of the tethering site is that several amino acids with large aliphatic side chains mediate the high-affinity binding of RII beta. Mutagenesis, recombinant protein expression, and physicochemical characterization were used to investigate the structural basis for the homodimerization and AKAP75 binding activities of RII beta. Several crucial residues are located in an NH2-terminal region that encompasses amino acids 13-36. Substitution of Ala for Leu13 or Phe36 generates monomeric RII beta subunits that cannot bind AKAP75. The results are not due to general misfolding since mutant RII beta monomers bind cAMP and inhibit the catalytic subunit of PKAII beta with the same affinity and efficacy as wild-type RII beta dimers. Moreover, substitution of Ala for Leu12, Val20, Leu21, Phe31, Leu33, or Leu39 and replacement of Leu13 with Ile or Val did not impair the dimerization reaction. Evidently, large hydrophobic side chains of Leu13 and Phe36 play pivotal roles in stabilizing RII beta-RII beta interactions. A secondary consequence of destabilizing RII beta dimers is the loss of intracellular targeting/anchoring capacity because monomers fail to bind AKAP75. Other NH2-terminal residues directly modulate the affinity of RII beta dimers for the AKAP75 tethering site. Replacement of Val20-Leu21 with Ala-Ala produced a dimeric RII beta protein that binds AKAP75 approximately 4% as avidly as wild-type RII beta. It is possible that the aliphatic side chains of Val20 and Leu21 interact with the essential Leu and Ile residues in the AKAP75 tethering region.
- Published
- 1995
25. Regulation of alpha o expression by the 5'-flanking region of the alpha o gene
- Author
-
Eva J. Neer, Ying Li, and Richard M. Mortensen
- Subjects
Regulation of gene expression ,Heterotrimeric G protein ,G12/G13 alpha subunits ,5' flanking region ,CAAT box ,Alpha (ethology) ,Promoter ,Cell Biology ,Biology ,Molecular Biology ,Biochemistry ,Molecular biology ,G alpha subunit - Abstract
Many responses of cells to external signals require activation of the heterotrimeric G proteins. These responses depend on the type and amount of G proteins that are expressed. Each cell has a characteristic complement of G protein subunits. For example, the alpha o subunit is very abundant in neural tissues. Very little is known about the mechanisms that determine cellular levels of G proteins. In the present study, we have isolated a genomic clone for mouse alpha o gene and identified the promoter region. There are multiple transcription initiation sites located about 750 base pairs upstream of the translational start site. The promoter region is GC-rich and contains neither a TATA-box nor a CAAT box. Transient expression assays using a series of constructs containing various lengths of the 5'-flanking region of the alpha o promoter demonstrated that the region 300-700 base pairs upstream of the transcription initiation sites is responsible for the basic promoter activity. The relative activity of alpha o promoter is 8-12-fold higher in cells expressing alpha o than in cells lacking alpha o. The level of alpha o in cells may also be regulated at the level of protein translation because deletions in the 5'-noncoding region of alpha o gene increase reporter enzyme expression without a corresponding increase in reporter enzyme mRNA level. Our results suggest that both transcriptional and post-transcriptional mechanisms are involved in regulating the expression of alpha o in vivo. Transcriptional regulation probably is important for control of tissue-specific expression, while posttranscriptional mechanisms may be used to regulate the alpha o level in cells.
- Published
- 1994
26. Crystal structure at 1.9-A resolution of human immunodeficiency virus (HIV) II protease complexed with L-735,524, an orally bioavailable inhibitor of the HIV proteases
- Author
-
Chris Culberson, Dawn L. Hall, Lawrence Kuo, Jules A. Shafer, Paul L. Darke, Zhongguo Chen, Ying Li, and Elizabeth Chen
- Subjects
Proteases ,Protease ,biology ,Stereochemistry ,Chemistry ,medicine.medical_treatment ,Active site ,Cell Biology ,Biochemistry ,chemistry.chemical_compound ,Indinavir ,Amide ,Hydrolase ,medicine ,biology.protein ,HIV Protease Inhibitor ,Binding site ,Molecular Biology ,medicine.drug - Abstract
L-735,524 is a potent, orally bioavailable inhibitor of human immunodeficiency virus (HIV) protease currently in a Phase II clinical trial. We report here the three-dimensional structure of L-735,524 complexed to HIV-2 protease at 1.9-A resolution, as well as the structure of the native HIV-2 protease at 2.5-A resolution. The structure of HIV-2 protease is found to be essentially identical to that of HIV-1 protease. In the crystal lattice of the HIV-2 protease complexed with L-735,524, the inhibitor is chelated to the active site of the homodimeric enzyme in one orientation. This feature allows an unambiguous assignment of protein-ligand interactions from the electron density map. Both Fourier and difference Fourier maps reveal clearly the closure of the flap domains of the protease upon L-735,524 binding. Specific interactions between the enzyme and the inhibitor include the hydroxy group of the hydroxyaminopentane amide moiety of L-735,524 ligating to the carboxyl groups of the essential Asp-25 and Asp-25' enzymic residues and the amide oxygens of the inhibitor hydrogen bonding to the backbone amide nitrogen of Ile-50 and Ile-50' via an intervening water molecule. A second bridging water molecule is found between the amide nitrogen N2 of L-735,524 and the carboxyl oxygen of Asp-29'. Although other hydrogen bonds also add to binding, an equally significant contribution to affinity arises from hydrophobic interactions between the protease and the inhibitor throughout the pseudo-symmetric S1/S1', S2/S2', and S3/S3' regions of the enzyme. Except for its pyridine ring, all lipophilic moieties (t-butyl, indanyl, benzyl, and piperidyl) of L-735,524 are rigidly defined in the active site.
- Published
- 1994
27. Characterization of distinct tethering and intracellular targeting domains in AKAP75, a protein that links cAMP-dependent protein kinase II beta to the cytoskeleton
- Author
-
Charles S. Rubin, Ying Li, and S. B. Glantz
- Subjects
endocrine system ,Protein subunit ,Binding protein ,Cell Biology ,Biology ,Biochemistry ,A Kinase Anchor Proteins ,Cell biology ,Cytosol ,Protein kinase A ,Site-directed mutagenesis ,Beta (finance) ,Cytoskeleton ,Molecular Biology - Abstract
Cyclic AMP-dependent protein kinase II beta (PKAII beta) is the principal mediator of cAMP action in neurons. A Kinase Anchor Proteins (AKAPs) are enriched in forebrain neurons and have distinct high affinity binding domains for the regulatory subunit (RII beta) of PKAII beta and components of the dendritic cytoskeleton. The selective accumulation of AKAP.RII beta complexes near dendritic microtubules tethers PKAII beta in proximity with adenylate cyclase in the synaptic plasma membrane and cytoskeletal proteins that are substrates for the kinase, thereby creating intraneuronal target sites for signals carried by cAMP. We have characterized the targeting (anchoring) and tethering (RII beta binding) domains of a prototypic anchor protein AKAP75. Deletion of N-terminal residues 27-48 generated a truncated RII beta-binding protein that partitions equally between the cytosol and detergent-insoluble fractions of HEK293 cells. Further removal of a non-adjacent sequence (residues 77-91) produced a cytosolic protein with unimpaired RII beta binding activity. Thus, two noncontiguous domains mediate the intracellular localization of AKAP75. Boundaries for the RII beta tethering domain were mapped to residues 392-413 by scanning mutagenesis. Residues containing long aliphatic side chains are essential for the high affinity binding of RII beta by AKAP75. Contributions of hydrophobic amino acids to tethering activity also depend on the position of the residue in the sequence. Certain conservative mutations that should not alter significantly the overall hydrophobicity or helicity of the tethering region (e.g. replacement of Leu with Ala) diminish the RII beta binding activity of AKAP75.
- Published
- 1993
28. Tissue-specific expression and alternative mRNA processing of the mammalian acetylcholinesterase gene
- Author
-
Ying Li, Palmer Taylor, and Shelley Camp
- Subjects
Gene Expression ,Biology ,Biochemistry ,Mice ,Exon ,Exon trapping ,Species Specificity ,Gene expression ,Tumor Cells, Cultured ,Animals ,Humans ,Coding region ,Tissue Distribution ,RNA, Messenger ,Molecular Biology ,Gene ,Cells, Cultured ,Splice site mutation ,Alternative splicing ,DNA ,Exons ,Cell Biology ,Molecular biology ,Alternative Splicing ,RNA splicing ,Acetylcholinesterase - Abstract
This study examines the tissue specificity and the gene products arising from alternative mRNA processing of the mammalian acetylcholinesterase gene. By splicing either alternative exons 5 or 6 in the mouse and human genes directly to the invariant exons (exons 2, 3, and 4), we show that the acetylcholinesterase species expressed by transfected recombinant DNA have the properties expected for the respective enzyme forms found in tissue. Antisense mRNA derived from these cDNAs has been employed to examine differential splicing in various tissues. In most cells, the hydrophilic form of AChE encoded by the exon 4 to exon 6 splice to form the mRNA is the predominant species. However, splicing of exon 4 to exon 5, yielding a mRNA encoding the glycophospholipid-linked form of acetylcholinesterase, is seen primarily in erythroid and to a lesser extent in AtT-20 cells. Only small amounts of this mRNA species appear in some other cells in culture. A novel third mRNA species, which arises from an extension of exon 4 without splicing to a downstream exon, is seen in mouse erythroid but not in human erythroid cells. A cDNA encoding this species when expressed in COS cells gives rise to a unique hydrophilic, secreted form of acetylcholinesterase. Transfection of a human genomic clone into mouse erythroleukemia cells does not result in the appearance of a mRNA species with an extension of exon 4 as seen with the endogenous mouse gene. Hence, differential splicing between the mouse and human genes appears intrinsic to the coding sequence and is not dependent solely on specific factors in the mouse erythroleukemia cell.
- Published
- 1993
29. Regulation of human NAD(P)H:quinone oxidoreductase gene. Role of AP1 binding site contained within human antioxidant response element
- Author
-
Ying Li and Anil K. Jaiswal
- Subjects
Regulation of gene expression ,animal diseases ,Cell Biology ,Transfection ,Biology ,NAD(P)H Dehydrogenase (Quinone) ,Quinone oxidoreductase ,Biochemistry ,Molecular biology ,Chloramphenicol acetyltransferase ,Regulatory sequence ,Gene expression ,Binding site ,Molecular Biology - Abstract
Deletion mutagenesis and transfection studies into hepatic (mouse hepatoma (Hepa-1) and human hepatoblastoma (Hep-G2)) and nonhepatic (HeLa) cells indicated that high levels of expression of the human NAD(P)H:quinone oxidoreductase gene in tumor cells and its induction by beta-naphthoflavone and 3-(2)-tert-butyl-4-hydroxyanisole are mediated by human antioxidant response element (hARE) located in the region between -470 and -445. The hARE, when attached to the thymidine kinase promoter and transfected into several mammalian cells, expressed high levels of the chloramphenicol acetyltransferase gene that was inducible by beta-naphthoflavone and 3-(2)-tert-butyl-4-hydroxyanisole. Nucleotide sequence analysis of the hARE revealed the presence of a recognition site for binding to the AP1 protein. Mutation of the AP1 binding site located within the hARE resulted in the loss of expression and induction upon transfection into various cell types. Band shift and competition assays with hARE and nuclear extracts from control and beta-naphthoflavone-treated Hepa-1, Hep-G2 and HeLa cells indicated specific interaction of regulatory protein(s) to the hARE. The supershift assays using antibodies against specific proteins of the AP1 family identified Jun-D and c-Fos as two members in the hARE-protein complex observed in band shift assays.
- Published
- 1992
30. Cloning and expression of an intron-less gene for AKAP 75, an anchor protein for the regulatory subunit of cAMP-dependent protein kinase II beta
- Author
-
Charles S. Rubin, Ying Li, S. B. Glantz, A. H. Hirsch, and Young You
- Subjects
chemistry.chemical_classification ,endocrine system ,Protein subunit ,HEK 293 cells ,Cell Biology ,Transfection ,Biology ,Biochemistry ,Molecular biology ,Amino acid ,Gene product ,chemistry ,Cytoplasm ,Protein kinase A ,Molecular Biology ,Gene - Abstract
The A-Kinase Anchor Protein AKAP 75 (formerly designated bovine brain P75) is a particulate brain protein that avidly binds the regulatory subunit (RII beta) of cAMP-dependent protein kinase II beta (Bregman, D. B., Hirsch, A.H. and Rubin, C.S. (1991) J. Biol. Chem. 266, 7207-7213). The formation of stable AKAP 75.RII beta complexes provides a potential mechanism for targeting physiological signals carried by cAMP to specific effector sites within neurons and other brain cells. We have now cloned and characterized the AKAP 75 gene. Its coding sequence is novel and unexpectedly short (1284 base pairs) and contains no introns. When the AKAP 75 gene was transfected into HEK 293 cells, a new RII beta-binding protein with an apparent Mr of 75,000 accumulated. A high proportion (approximately 65%) of the AKAP 75 gene product was excluded from the cytoplasm and was recovered in the 40,000 x g pellet derived from disrupted transfected cells. In contrast, cells transfected with a construct encoding 249 amino acids from the central and C-terminal regions of AKAP 75 produced an RII beta-binding protein (apparent Mr = 45,000) that was exclusively cytosolic. AKAP 75 is a novel protein composed of only 428 amino acid residues (Mr = 47,878). A highly acidic C-terminal region mediates the binding of RII beta (and cAMP-dependent protein kinase II beta), whereas a positively charged N-terminal segment contains structural features that are essential for the association of AKAP 75 with the cytoskeleton and/or intracellular membranes.
- Published
- 1992
31. Gene structure of mammalian acetylcholinesterase. Alternative exons dictate tissue-specific expression
- Author
-
Shelley Camp, Palmer Taylor, Ying Li, D. Getman, and Tara L. Rachinsky
- Subjects
RNA Splicing ,Molecular Sequence Data ,Restriction Mapping ,Biology ,Exon shuffling ,Polymerase Chain Reaction ,Biochemistry ,Mice ,Exon ,Ribonucleases ,Exon trapping ,Sequence Homology, Nucleic Acid ,Tumor Cells, Cultured ,Animals ,Humans ,Amino Acid Sequence ,Molecular Biology ,Gene ,Mammals ,Splice site mutation ,Base Sequence ,Intron ,DNA ,Exons ,Cell Biology ,Blotting, Northern ,Molecular biology ,Introns ,Stop codon ,Organ Specificity ,Acetylcholinesterase ,Tandem exon duplication ,Sequence Alignment - Abstract
The genes encoding mouse and human acetylcholinesterases have been cloned from genomic and cosmid libraries. Restriction analysis and a comparison of sequence with the cDNAs have defined the exon-intron boundaries. In mammals, three invariant exons encode the signal peptide and the amino-terminal 535 amino acids common to all forms of the enzyme whereas alternative exon usage of the next exon accounts for the structural divergence in the carboxyl termini of the catalytic subunits. mRNA protection studies show that the cDNA encoding the hydrophilic catalytic subunits represents the dominant mRNA species in mammalian brain and muscle whereas divergent mRNA species are evident in cells of hematopoietic origin (bone marrow cells and a erythroleukemia cell line). Analyses of mRNA species in these cells and the genomic sequence have enabled us to define two alternative exons in addition to the one found in the cDNAs; they encode unique carboxyl-terminal sequences. One mRNA consists of a direct extension through the intervening sequence between the common exon and the 3' exon deduced from the cDNA. This sequence encodes a subunit lacking the cysteine critical to oligomer formation. Another mRNA results from a splice that encodes a stretch of hydrophobic amino acids immediately upstream of a stop codon. This exon, when spliced to the upstream invariant exons, should encode glycophospholipid-linked species of the enzyme. Homologous sequence, identity of exon-intron junctions, and identity of position of the stop codon are seen for this region in mouse and human. Polymerase chain reactions carried out across the expected intron region and mRNA protection studies show that this splice occurs in mouse bone marrow and erythroleukemia cells yielding the appropriate cDNA.
- Published
- 1991
32. Brain-selective Kinase 2 (BRSK2) Phosphorylation on PCTAIRE1 Negatively Regulates Glucose-stimulated Insulin Secretion in Pancreatic β-Cells
- Author
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Chen, Xin-Ya, primary, Gu, Xiu-Ting, additional, Saiyin, Hexige, additional, Wan, Bo, additional, Zhang, Yu-Jing, additional, Li, Jing, additional, Wang, Ying-Li, additional, Gao, Rui, additional, Wang, Yu-Fan, additional, Dong, Wei-Ping, additional, Najjar, Sonia M., additional, Zhang, Chen-Yu, additional, Ding, Han-Fei, additional, Liu, Jun O., additional, and Yu, Long, additional
- Published
- 2012
- Full Text
- View/download PDF
33. Dihydroceramides: From Bit Players to Lead Actors.
- Author
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Siddique, Monowarul Mobin, Ying Li, Chaurasia, Bhagirath, Kaddai, Vincent A., and Summers, Scott A.
- Subjects
- *
SPHINGOLIPIDS , *BIOSYNTHESIS , *CERAMIDES , *SPHINGOMYELIN , *GANGLIOSIDES , *DOUBLE bonds - Abstract
Sphingolipid synthesis involves a highly conserved biosynthetic pathway that produces fundamental precursors of complex sphingolipids. The final reaction involves the insertion of a double bond into dihydroceramides to generate the more abundant ceramides, which are converted to sphingomyelins and glucosylceramides/gangliosides by the addition of polar head groups. Although ceramides have long been known to mediate cellular stress responses, the dihydroceramides that are transiently produced during de novo sphingolipid synthesis were deemed inert. Evidence published in the last few years suggests that these dihydroceramides accumulate to a far greater extent in tissues than previously thought. Moreover, they have biological functions that are distinct and non-overlapping with those of the more prevalent ceramides. Roles are being uncovered in autophagy, hypoxia, and cellular proliferation, and the lipids are now implicated in the etiology, treatment, and/or diagnosis of diabetes, cancer, ischemia/ reperfusion injury, and neurodegenerative diseases. This minireview summarizes recent findings on this emerging class of bioactive lipids. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
34. Progesterone Receptor A Stability Is Mediated by Glycogen Synthase Kinase-3β in the Brca1-deficient Mammary Gland.
- Author
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Shaohui Wang, Ying Li, Pang-Hung Hsu, Sou-Ying Lee, Yoon Kim, and Lee, Eva Y.-H. P.
- Subjects
- *
PROGESTERONE receptors , *HORMONE receptors , *PROGESTATIONAL hormones , *MAMMARY glands , *GERM cells , *NEOPLASTIC cell transformation - Abstract
Germ line mutations of the BRCA1 gene increase the risk of breast and ovarian cancer, but the basis of this tissue-specific tumor predisposition is not fully understood. Previously, we reported that the progesterone receptors are stabilized in Brca1 -deficient mammary epithelial cells, and treating with anti-progesterone delays mammary tumorigenesis in Brca1/ p53 conditional knock-out mice, suggesting that the progesterone has a critical role in breast carcinogenesis. To further explore how the stability of progesterone receptor is modulated, here, we have found that glycogen synthase kinase (GSK)-3β phosphorylation of progesterone receptor-A (PR-A) facilitates its ubiquitination. GSK-3β-mediated phosphorylation of serine 390 in PR-A regulates its subsequent ubiquitination and protein stability. Expression of PR-AS390A mutant in the human breast epithelial cells, MCF-10A, results in enhanced proliferation and formation of aberrant acini structure in the three-dimensional culture. Consistently, reduction of phosphorylation of serine 390 of PR-A and GSK-3β activity is observed in the Brca1-deficient mammary gland. Taken together, these results provide important aspects of tissue specificity of BRCA1-mediated suppression of breast carcinogenesis. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
35. Fluid Shear Stress Activation of Focal Adhesion Kinase
- Author
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Li, Song, primary, Kim, Michael, additional, Hu, Ying-Li, additional, Jalali, Shila, additional, Schlaepfer, David D., additional, Hunter, Tony, additional, Chien, Shu, additional, and Shyy, John Y-J., additional
- Published
- 1997
- Full Text
- View/download PDF
36. Catalysis and pH Control by Membrane-associated Carbonic Anhydrase IX in MDA-MB-231 Breast Cancer Cells.
- Author
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Ying Li, Chingkuang Tu, Hai Wang, Silverman, David N., and Frost, Susan C.
- Subjects
- *
CANCER cells , *BREAST cancer , *CARBONIC anhydrase , *CATALYSIS , *LYASES , *MASS spectrometry , *HYPOXEMIA - Abstract
Carbonic anhydrase IX (CAIX) is a membrane-bound, tumorrelated enzyme whose expression is often considered a marker for hypoxia, an indicator of poor prognosis in the majority of cancer patients, and is associated with acidification ofthe tumor microenvironment. Here, we describe for the first time the catalytic properties of native CAIX in MDA-MB-231 breast cancer cells that exhibit hypoxia-inducible CAIX expression. Using "*O exchange measured by membrane inlet mass spectrometry, we determined catalytic activity in membrane ghosts and intact cells. Exofacial carbonic anhydrase activity increases with exposure to hypoxia, an activity which is suppressed by impermeant sulfonamide CA inhibitors. Inhibition by sulfonamide inhibitors is not sensitive to reoxygenation. CAIX activity in intact cells increases in response to reduced pH. Data from membrane ghosts show that the increase in activity at reduced pH is largely due to an increase in the dehydration reaction. In addition, the kinetic constants of CAIX in membrane ghosts are very similar to our previous measurements for purified, recombinant, truncated forms. Hence, the activity of CAIX is not affected by the proteoglycan extension or membrane environment. These activities were measured at a total concentration for all CO2 species at 25 mM and close to chemical equilibrium, conditions which approximate the physiological extracellular environment. Our data suggest that CAIX is particularly well suited to maintain the extracellular pH at a value that favors the survival fitness of tumor cells. [ABSTRACT FROM AUTHOR]
- Published
- 2011
- Full Text
- View/download PDF
37. Ectopic Expression of p73α, but Not p73β, Suppresses Myogenic Differentiation.
- Author
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Chun-Ying Li, Jiangyu Zhu, and Wang, Jean Y.J.
- Subjects
- *
MYOGENESIS , *CELL death , *CELLS , *MUSCLE cells , *GENETIC transformation , *NUCLEIC acids , *GENE expression , *GENETIC regulation , *ALKYLATING agents , *ANTINEOPLASTIC agents , *CISPLATIN - Abstract
The TRP73 gene, a member of the p53 family, encodes several variants through differential splicing and use of alternative promoters. At the N terminus, two different promoters generate the full-length and the ΔN isoforms, with or without the transactivating domain. At the C terminus, seven isoforms generated through alternative splicing have been cloned. Previous studies have demonstrated that ΔN-p73 interferes with p73-induced apoptosis. However, there has been no evidence for functional diversity of the C-terminal p73 variants. In this study, we found that p73α and p73β exerted differential effect on the differentiation of C2C12 myoblasts. Although p73β lacked any detectable effect on differentiation, p73α caused a substantial delay in the expression of muscle-specific genes. In co-transfection experiments p73α, but not p73β, attenuated the transcriptional activity of MyoD. Microarray-based gene profiling confirmed the protraction of MyoD-dependent gene expression in C2C12 cells stably expressing p73α. Notwithstanding the differential effect on differentiation, p73α and p73β showed similar activity in sensitizing C2C12 myoblasts to cisplatin-induced cell death. These results demonstrated a functional diversity between the two C-terminal variants of p73 and suggested that p73α can regulate cellular differentiation in addition to its role in stimulating cell death. [ABSTRACT FROM AUTHOR]
- Published
- 2005
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- View/download PDF
38. Stability of Homologue of Slimb F-box Protein Is Regulated by Availability of Its Substrate.
- Author
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Ying Li, Gazdoiu, Stefan, Pan, Zhen-Qiang, and Fuchs, Serge Y.
- Subjects
- *
LIGASES , *CELL receptors , *CELL adhesion molecules , *CELL adhesion , *UBIQUITIN , *BIOCHEMISTRY - Abstract
The homologue of Slimb (HOS) F-box protein is a receptor of the Skp1-Cullin1-F-box protein (SCFHOS) E3 ubiquitin ligase, which mediates ubiquitination and degradation of β-catenin and the inhibitor of NFκB, IκB. We found that HOS itself is an unstable protein that undergoes ubiquitination and degradation in a 26 S proteasome-dependent manner. A HOS mutant lacking the F-box that is deficient in binding to the core SCF components underwent ubiquitination less efficiently and was more stable than the wild type protein. Furthermore, ubiquitination and degradation of HOS was impaired in ts41 cells, in which the activities of Cullin-based ligases were decreased because the NEDD8 pathway was abrogated. Whereas HOS was directly ubiquitinated within the SCFHOS complex in vitro, the addition of phosphorylated IκBα inhibited this ubiquitination. Increasing cellular levels of HOS substrate (phosphorylated IκBα) by activating IκB kinase inhibited HOS ubiquitination and led to stabilization of HOS, indicating that interaction between HOS and its substrate might protect HOS from proteolysis. Taken together, our data suggest that proteolysis of HOS depends on its interaction with active components of the SCF complex and that HOS stability is regulated by a bound substrate. These findings may define a mechanism for maintaining activities of specific SCF complexes based on availability of a particular substrate. [ABSTRACT FROM AUTHOR]
- Published
- 2004
- Full Text
- View/download PDF
39. Effects of Hydrogen Peroxide upon Nicotinamide Nucleotide Metabolism in Escherichia coli.
- Author
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Brumaghim, Julia L., Ying Li, Henle, Ernst, and Linn, Stuart
- Subjects
- *
HYDROGEN peroxide , *NICOTINAMIDE , *ESCHERICHIA coli - Abstract
DNA is damaged in vivo by the Fenton reaction mediated by Fe[sup 2+] and cellular reductants such as NADH, which reduce Fe[sup 3+] to Fe[sup 2+] and allow the recycling of iron. To study the response of Escherichia coli to such cycling, the activities of several enzymes involved in nicotinamide nucleotide metabolism were measured following an H[sub 2]O[sub 2] challenge. NADPH-dependent peroxidase, NADH/NADP[sup +] transhydrogenase, and glucose-6phosphate dehydrogenase were most strongly induced, increasing 2.5-3-fold. In addition, the cellular ratios of NADPH to NADH increased 6- or 92-fold 15 min after exposure to 0.5 or 5 mM H[sub 2]O[sub 2], respectively. In vitro, NADH was oxidized by Fe[sup 3+] up to 16-fold faster than NADPH, despite their identical reduction potentials. To understand this rate difference, the interactions of Fe[sup 3+] and Ga[sup 3+] with NAD(P)H were examined by ¹H, [sup 13]C, and [sup 31]P NMR spectroscopy. Association with NADH occurred primarily with adenine at N7 and the amino group, but for NADPH, strong metal interactions also occurred at the 2'-phosphate group. Interaction of M[sup 3+] (Fe[sup 3+] or Ga[sup 3+]) with the adenine ring would bring it into close proximity to the redox-active nicotinamide ring in the folded form of NAD(P)H, but interaction of M[sup 3+] with the 2'-phosphate group would avoid this close contact. In addition, as determined by absorbance spectroscopy, the energy of the charge-transfer species was significantly higher for the Fe[sup 3+]·NADPH complex than for the Fe[sup 3+]·NADH complex. We therefore suggest that upon exposure to H[sub 2]O[sub 2] the NADH pool is depleted, and NADPH, which is less reactive with Fe[sup 3+], functions as the major nicotinamide nucleotide reductant. [ABSTRACT FROM AUTHOR]
- Published
- 2003
- Full Text
- View/download PDF
40. An Analog of Lophotoxin Reacts Covalently with Tyr190 in the α-Subunit of the Nicotinic Acetylcholine Receptor
- Author
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Ying Li, Paul Culver, Palmer Taylor, and Stewart N. Abramson
- Subjects
chemistry.chemical_classification ,Gel electrophoresis ,Protease ,Chemistry ,Stereochemistry ,medicine.medical_treatment ,Peptide ,Cell Biology ,Biochemistry ,Nicotinic acetylcholine receptor ,chemistry.chemical_compound ,Nicotinic agonist ,medicine ,Cyanogen bromide ,Molecular Biology ,G alpha subunit ,Acetylcholine receptor - Abstract
Lophotoxin and lophotoxin analog-1 are natural diterpenes from coral that inhibit nicotinic acetylcholine receptors by covalent reaction with the acetylcholine recognition sites on the alpha-subunits. Although both toxins contain potentially reactive epoxides and alpha,beta-unsaturated aldehydes, the mechanism of their covalent reaction with the receptor is not known. The role of the alpha,beta-unsaturated aldehyde in analog-1 was investigated by reduction of the aldehyde to an alcohol with [3H]NaBH4. The reduced [3H]analog-1 bound selectively and covalently to the alpha-subunit of the receptor. Covalent binding was inhibited by agonists and antagonists, but not by noncompetitive allosteric inhibitors. The apparent dissociation constant of the reduced [3H]analog-1 was approximately 1.5 x 10(-6) M. These results demonstrate that the alpha,beta-unsaturated aldehyde in analog-1 is not an absolute requirement for covalent reaction with the receptor. Receptors were treated with the reduced-[3H]analog-1, and the labeled alpha-subunits were isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and digested with staphylococcal V8 protease. A labeled 20-kDa V8 protease fragment was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high performance liquid chromatography and subjected to sequence analysis. A peptide beginning at Ser173 was identified, and the label appeared in the 18th step corresponding to Tyr190. This assignment was confirmed by digestion of the labeled 20-kDa V8 protease fragment with cyanogen bromide, followed by purification of the labeled cyanogen bromide peptide on reverse-phase high performance liquid chromatography. A peptide beginning at Lys179 was identified, and the label appeared in the 12th step, again corresponding to Tyr190. Tyr190 may react with the coral toxin by nucleophilic addition at one of the carbons associated with an epoxide, and may form part of the alkylammonium-binding subsite of the acetylcholine recognition site.
- Published
- 1989
41. Lophotoxin and related coral toxins covalently label the alpha-subunit of the nicotinic acetylcholine receptor
- Author
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Leslie Gutman, Paul Guest, Ying Li, Toni Kline, Palmer Taylor, Paul Culver, and Stewart N. Abramson
- Subjects
chemistry.chemical_classification ,Electric Organ ,Iodoacetic acid ,Macromolecular Substances ,Terpenes ,Chemistry ,Cell Membrane ,Peptide ,Borohydrides ,Cell Biology ,Receptors, Nicotinic ,Torpedo ,Biochemistry ,chemistry.chemical_compound ,Nicotinic acetylcholine receptor ,Cnidarian Venoms ,Nicotinic agonist ,Iodoacetamide ,Animals ,Binding site ,Oxidation-Reduction ,Molecular Biology ,Acetylcholine receptor ,G alpha subunit - Abstract
Lophotoxin and lophotoxin analog-1 are uncharged cyclic diterpenes obtained from gorgonian corals. They have been shown to block the function of nicotinic acetylcholine receptors. Inhibition results from blockade of the agonist recognition site and appears irreversible in that extensive washing does not restore receptor function. This study was undertaken to determine whether this apparently irreversible inhibition involves covalent labeling at a selective site and to further characterize this site directly. Incubation of membranes prepared from the electric organ of Torpedo californica with analog-1 followed by reduction with NaB3H4 resulted in the incorporation of radioactivity into several membrane proteins. The incorporation of radioactivity into the alpha-subunit of the receptor was blocked by prior incubation with agonists and antagonists. [3H]Lophotoxin and [3H]analog-1 were prepared by reduction with NaB3H4 and back-oxidation with CrO3. The radiolabeled coral toxins reacted selectively and covalently with the alpha-subunit of the receptor. Their binding was prevented by prior exposure to agonists and antagonists. In contrast to the site-directed alkylating agent 4-(N-maleimido)benzyltrimethylammonium iodide, prior reduction of the receptor was not required for covalent binding of 3H-labeled coral toxins. Selective reduction of Cys192 and Cys193 followed by alkylation with 4-(N-maleimido)benzyltrimethylammonium iodide blocked the binding of [3H]analog-1, whereas alkylation with iodoacetic acid or iodoacetamide did not. Thus, the binding site for the coral toxins does not overlap the binding surface near Cys192 and Cys193. Digestion of isolated labeled alpha-subunits with endoglycosidase H revealed that the polypeptide portion of the protein retained the covalently bound [3H]analog-1. Digestion with staphylococcal V8 protease revealed two major peptides of approximately 19 and 20 kDa, along with several smaller peptides. Only the 20-kDa peptide retained the covalently bound [3H]analog-1, localizing the site of covalent attachment between Ser173 and Glu335. The unique chemical structure and covalent reactivity of these gorgonian coral toxins will undoubtedly allow further insights into the structure of the agonist recognition site.
- Published
- 1988
42. Modulation of conformational equilibrium by phosphorylation underlies the activation of deubiquitinase A.
- Author
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Kabra, Ashish, Rumpa, Efsita, and Ying Li
- Subjects
- *
POST-translational modification , *UBIQUITINATION , *TYPE I interferons , *DEPHOSPHORYLATION , *PHOSPHORYLATION , *ENZYME kinetics , *NUCLEAR magnetic resonance spectroscopy , *EQUILIBRIUM - Abstract
Deubiquitinases deconjugate ubiquitin modifications from target proteins and are involved in many cellular processes in eukaryotes. The functions of deubiquitinases are regulated by post-translational modifications, mainly phosphorylation and ubiquitination. Post-translational modifications can result in subtle changes in structural and dynamic properties, which are difficult to identify but functionally important. In this work, we used NMR spectroscopy to characterize the conformational properties of the human deubiquitinase A (DUBA), a negative regulator of type I interferon. DUBA activity is regulated by phosphorylation at a single serine residue, Ser-177. We found that the catalytic rate constant of DUBA is enhanced by phosphorylation. By comparing NMR and enzyme kinetics data among different forms of DUBA with low and high activities, we concluded that a two-state equilibrium that was present only in phosphorylated DUBA is important for DUBA activity. Our results highlight the importance of defining conformational dynamics in understanding the mechanism of DUBA activation. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
43. CD38 produces nicotinic acid adenosine dinucleotide phosphate in the lysosome.
- Author
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Cheng Fang, Ting Li, Ying Li, Guan Jie Xu, Qi Wen Deng, Ya Jie Chen, Yun Nan Hou, Hon Cheung Lee, and Yong Juan Zhao
- Subjects
- *
CD38 antigen , *NICOTINIC acid adenine dinucleotide phosphate , *LYSOSOMES , *CATALYSIS , *ENZYME analysis - Abstract
Nicotinic acid adenosine dinucleotide phosphate (NAADP) is aCa2+-mobilizing second messenger that regulates a wide range of biological activities. However, the mechanism of its biogenesis remains controversial. CD38 is the only enzyme known to catalyze NAADP synthesis from NADP and nicotinic acid. CD38-mediated catalysis requires an acidic pH, suggesting that NAADP may be produced in acidic endolysosomes, but this hypothesis is untested. In this study, using human cell lines, we specifically directed CD38 to the endolysosomal system and assessed cellularNAADPproduction. First, we found that nanobodies targeting various epitopes on the C-terminal domain of CD38 could bind to cell surface-localized CD38 and induce its endocytosis. We also found that CD38 internalization occurred via a clathrin-dependent pathway, delivered CD38 to the endolysosome, and elevated intracellular NAADP levels. We also created a CD38 variant for lysosome-specific expression, which not only withstood the degradative environment in the lysosome, but was also much more active than WT CD38 in elevating cellular NAADP levels. Supplementing CD38-expressing cells with nicotinic acid substantially increased cellular NAADP levels. These results demonstrate that endolysosomal CD38 can produceNAADPin human cells. They further suggest that CD38's compartmentalization to the lysosome may allow for its regulation via substrate access, rather than enzyme activation, thereby providing a reliable mechanism for regulating cellular NAADP production. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
44. Identification and characterization of a novel hydroxylamine oxidase, DnfA, that catalyzes the oxidation of hydroxylamine to N2.
- Author
-
Meng-Ru Wu, Li-Li Miao, Ying Liu, Xin-Xin Qian, Ting-Ting Hou, Guo-Min Ai, Lu Yu, Lan Ma, Xi-Yan Gao, Ya-Ling Qin, Hai-Zhen Zhu, Lei Du, Sheng-Ying Li, Chang-Lin Tian, De-Feng Li, Zhi-Pei Liu, and Shuang-Jiang Liu
- Subjects
- *
HYDROXYLAMINE , *ELECTRON paramagnetic resonance , *OXIDATION-reduction reaction , *NITROGEN , *ENZYME kinetics , *OXIDATION , *NITROGEN removal (Water purification) - Abstract
Nitrogen (N2) gas in the atmosphere is partially replenished by microbial denitrification of ammonia. Recent study has shown that Alcaligenes ammonioxydans oxidizes ammonia to dinitrogen via a process featuring the intermediate hydroxylamine, termed "Dirammox" (direct ammonia oxidation). However, the unique biochemistry of this process remains unknown. Here, we report an enzyme involved in Dirammox that catalyzes the conversion of hydroxylamine to N2. We tested previously annotated proteins involved in redox reactions, DnfA, DnfB, and DnfC, to determine their ability to catalyze the oxidation of ammonia or hydroxylamine. Our results showed that none of these proteins bound to ammonia or catalyzed its oxidation; however, we did find DnfA bound to hydroxylamine. Further experiments demonstrated that, in the presence of NADH and FAD, DnfA catalyzed the conversion of 15N-labeled hydroxylamine to 15N2. This conversion did not happen under oxygen (O2)-free conditions. Thus, we concluded that DnfA encodes a hydroxylamine oxidase. We demonstrate that DnfA is not homologous to any known hydroxylamine oxidoreductases and contains a diiron center, which was shown to be involved in catalysis via electron paramagnetic resonance experiments. Furthermore, enzyme kinetics of DnfA were assayed, revealing a Km of 92.9 ± 3.0 μM for hydroxylamine and a kcat of 0.028 ± 0.001 s-1. Finally, we show that DnfA was localized in the cytoplasm and periplasm as well as in tubular membrane invaginations in HO-1 cells. To the best of our knowledge, we conclude that DnfA is the first enzyme discovered that catalyzes oxidation of hydroxylamine to N2. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
45. Histone Deacetylase Inhibition Promotes Osteoblast Maturation by Altering the Histone H4 Epigenome and Reduces Akt Phosphorylation.
- Author
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Dudakovic, Amel, Evans, Jared M., Ying Li, Middha, Sumit, McGee-Lawrence, Meghan E., van Wijnen, Andre J., and Westendorf, Jennifer J.
- Subjects
- *
HISTONE deacetylase inhibitors , *OSTEOBLASTS , *PHOSPHORYLATION , *EPIGENETICS , *REGENERATION (Biology) , *BONE regeneration - Abstract
Bone has remarkable regenerative capacity, but this ability diminishes during aging. Histone deacetylase inhibitors (HDIs) promote terminal osteoblast differentiation and extracellular matrix production in culture. The epigenetic events altered by HDIs in osteoblasts may hold clues for the development of new anabolic treatments for osteoporosis and other conditions of low bone mass. To assess how HDIs affect the epigenome of committed osteoblasts, MC3T3 cells were treated with suberoylanilide hydroxamic acid (SAHA) and subjected to microarray gene expression profiling and high-throughput ChIP-Seq analysis. As expected, SAHA induced differentiation and matrix calcification of osteoblasts in vitro. ChIP-Seq analysis revealed that SAHA increased histone H4 acetylation genome-wide and in differentially regulated genes, except for the 500 bp upstream of transcriptional start sites. Pathway analysis indicated that SAHA increased the expression of insulin signaling modulators, including Slc9a3r1.SAHAdecreased phosphorylation of insulin receptor β, Akt, and the Akt substrate FoxO1, resulting in FoxO1 stabilization. Thus, SAHA induces genome-wide H4 acetylation and modulates the insulin/Akt/FoxO1 signaling axis, whereas it promotes terminal osteoblast differentiation in vitro. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
46. Transmembrane Protein 214 (TMEM214) Mediates Endoplasmic Reticulum Stress-induced Caspase 4 Enzyme Activation and Apoptosis.
- Author
-
Chao Li, Jin Wei, Ying Li, Xiao He, Qian Zhou, Jie Yan, Jing Zhang, Ying Liu, Yu Liu, and Hong-Bing Shu
- Subjects
- *
MEMBRANE proteins , *APOPTOSIS , *PROTEIN folding , *ENDOPLASMIC reticulum , *ENZYME activation - Abstract
Endoplasmic reticulum (ER) stress caused by excessive aggregation of misfolded proteins induces apoptosis. Although ER stress-induced apoptosis has been implicated in many diseases, the detailed mechanisms are not well understood. Here, we identified human transmembrane protein 214 (TMEM214) as a critical mediator of ER stress-induced apoptosis. Overexpression of TMEM214 induced apoptosis, whereas knockdown of TMEM214 inhibited ER stress-induced apoptosis. TMEM214 was localized on the outer membrane of the ER and constitutively associated with procaspase 4, which was also critical for ER stressinduced apoptosis.TMEM214-inducedapoptosiswasabolishedby a dominant negative mutant of procaspase 4, whereas caspase 4-induced apoptosis was inhibited by knockdown of TMEM214. Furthermore, knockdown of TMEM214 inhibited the activation and cleavage of procaspase 4 by impairing its recruitment to the ER. Our findings suggest that TMEM214 is essential for ER stress-induced apoptosis by acting as an anchor for recruitment of procaspase 4 to the ER and its subsequent activation. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
47. The Extracellular cAMP-Adenosine Pathway Regulates Expression of Renal D1 Dopamine Receptors in Diabetic Rats.
- Author
-
Kuzhikandathil, Eldo V., Clark, Leslie, and Ying Li
- Subjects
- *
DOPAMINE receptors , *ADENOSINE monophosphate , *DIABETES , *LABORATORY rats , *KIDNEYS - Abstract
Activation of D1 dopamine receptors expressed in the kidneys promotes the excretion of sodium and regulates sodium levels during increases in dietary sodium intake. A decrease in the expression or function of D1 receptors results in increased sodium retention which can potentially lead to the development of hypertension. Studies have shown that in the absence of functional D1 receptors, in null mice, the systolic, diastolic, and mean arterial pressures are higher. Previous studies have shown that the expression and function of D1 receptors in the kidneys are decreased in animal models of diabetes. The mechanisms that down-regulate the expression of renal D1 receptor gene in diabetes are not well understood. Using primary renal cells and acutely isolated kidneys from the streptozotocin-induced rat diabetic model, we demonstrate that the renal D1 receptor expression is down-regulated by the extracellular cAMP-adenosine pathway in vitro and in vivo. In cultures of primary renal cells, a 3 mm, 60-h cAMP treatment down-regulated the expression of D1 receptors. In vivo, we determined that the plasma and urine cAMP levels as well as the expression of 5′-ectonucleotidase, tissue-nonspecific alkaline phosphatase, and adenosine A2a receptors are significantly increased in diabetic rats. Inhibitors of 5′-ectonucleotidase and tissue-nonspecific alkaline phosphatase, α,β-methyleneadenosine 5′-diphosphate, and levamisole, respectively, blocked the down-regulation of D1 receptors in the primary renal cells and in the kidney of diabetic animals. The results suggest that inhibitors of the extracellular cAMP-adenosine pathway reverse the down-regulation of renal D1 receptor in diabetes. [ABSTRACT FROM AUTHOR]
- Published
- 2011
- Full Text
- View/download PDF
48. Molecular Basis of Cav2.3 Calcium Channels in Rat Nociceptive Neurons.
- Author
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Zhi Fang, Chui-Kyu Park, Hai Ying Li, Hyun Yeong Kim, Seong-Hae Park, Sung Jun Jung, Joong Soo Kim, Monteil, Arnaud, Seog Bae Oh, and Miller, Richard J.
- Subjects
- *
NEURONS , *CALCIUM channels , *PAIN , *GANGLIONIC blocking agents , *CELLS - Abstract
Cav2.3 calcium channels play an important role in pain transmission in peripheral sensory neurons. Six Cav2.3 isoforms resulting from different combinations of three inserts (inserts I and II in the II-III loop and insert III in the carboxyl-terminal region) have been identified in different mammalian tissues. To date, however, Cav2.3 isoforms unique to primary sensory neurons have not been identified. In this study, we determined Cav2.3 isoforms expressed in the rat trigeminal ganglion neurons. Whole tissue reverse transcription (RT)-PCR analyses revealed that only two isoforms, Cav2.3a and Cav2.3e, are present in TG neurons. Using single cell RT-PCR, we found that Cav2.3e is the major isoform, whereas Cav2.3e expression is highly restricted to small (<16 µm) isolectin B4-negative and tyrosine kinase A-positive neurons. Cav2.3e was also preferentially detected in neurons expressing the nociceptive marker, transient receptor potential vanilloid 1. Single cell RT-PCR following calcium imaging and whole-cell patch clamp recordings provided evidence of an association between an R-type calcium channel component and Cav2.3e expression. Our results suggest that Cav2.3e in sensory neurons may be a potential target for the treatment of pain. [ABSTRACT FROM AUTHOR]
- Published
- 2007
- Full Text
- View/download PDF
49. Role of ATP Hydrolysis in the Antirecombinase Function of Saccharomyces cerevisiae Srs2 Protein.
- Author
-
Krejci, Lumir, Macris, Margaret, Ying Li, Van Komen, Stephen, Villemain, Jana, Ellenberger, Thomas, Klein, Hannah, and Sung, Patrick
- Subjects
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ADENOSINE triphosphate , *HYDROLYSIS , *SACCHAROMYCES cerevisiae , *GENETIC mutation , *DNA damage , *DNA repair , *NUCLEIC acids , *DNA - Abstract
Mutants of the Saccharomyces cerevisiae SRS2 gene are hyperrecombinogenic and sensitive to genotoxic agents, and they exhibit a synthetic lethality with mutations that compromise DNA repair or other chromosomal processes. In addition, srs2 mutants fail to adapt or recover from DNA damage checkpoint-imposed G2/M arrest. These phenotypic consequences of ablating SRS2 function are effectively overcome by deleting genes of the RAD52 epistasis group that promote homologous recombination, implicating an untimely recombination as the underlying cause of the srs2 mutant phenotypes. The SRS2-encoded protein has a single-stranded (ss) DNAdependent ATPase activity, a DNA helicase activity, and an ability to disassemble the Rad51-ssDNA nucleoprotein filament, which is the key catalytic intermediate in Rad51-mediated recombination reactions. To address the role of ATP hydrolysis in Srs2 protein function, we have constructed two mutant variants that are altered in the Walker type A sequence involved in the binding and hydrolysis of ATP. The srs2 K41A and srs2 K41R mutant proteins are both devoid of ATPase and helicase activities and the ability to displace Rad51 from ssDNA. Accordingly, yeast strains harboring these srs2 mutations are hyperrecombinogenic and sensitive to methylmethane sulfonate, and they become inviable upon introducing either the sgs1Δ or rad54Δ mutation. These results highlight the importance of the ATP hydrolysisfueled DNA motor activity in SRS2 functions. [ABSTRACT FROM AUTHOR]
- Published
- 2004
- Full Text
- View/download PDF
50. Toll-like receptor 4-induced ryanodine receptor 2 oxidation and sarcoplasmic reticulum Ca2+ leakage promote cardiac contractile dysfunction in sepsis.
- Author
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Jie Yang, Rui Zhang, Xin Jiang, Jingzhang Lv, Ying Li, Hongyu Ye, Wenjuan Liu, Gang Wang, Cuicui Zhang, Na Zheng, Ming Dong, Yan Wang, Peiya Chen, Santosh, Kumar, Yong Jiang, and Jie Liu
- Subjects
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RYANODINE receptors , *TOLL-like receptors , *OXIDATION , *SEPSIS , *CARDIAC contraction , *SARCOPLASMIC reticulum - Abstract
Studies suggest the potential role of a sarcoplasmic reticulum (SR) Ca2+ leak in cardiac contractile dysfunction in sepsis. However, direct supporting evidence is lacking, and the mechanisms underlying this SR leak are poorly understood. Here, we investigated the changes in cardiac Ca2+ handling and contraction in LPS-treated rat cardiomyocytes and a mouse model of polymicrobial sepsis produced by cecal ligation and puncture (CLP). LPS decreased the systolic Ca2+ transient and myocyte contraction as well as SR Ca2+ content. Meanwhile, LPS increased Ca2+ spark-mediated SR Ca2+leak. Preventing the SR leak with ryanodine receptor (RyR) blocker tetracaine restored SR load and increased myocyte contraction. Similar alterations in Ca2+ handling were observed in cardiomyocytes from CLP mice. Treatment with JTV-519, an anti-SR leak drug, restored Ca2+ handling and improved cardiac function. In the LPS-treated cardiomyocytes, mitochondrial reactive oxygen species and oxidative stress in RyR2 were increased, whereas the levels of the RyR2-associated FK506-binding protein 1B (FKBP12.6) were decreased. The Toll-like receptor 4 (TLR4)-specific inhibitor TAK-242 reduced the oxidative stress in LPS-treated cells, decreased the SR leak, and normalized Ca2+ handling and myocyte contraction. Consistently, TLR4 deletion significantly improved cardiac function and corrected abnormal Ca2+ handling in the CLP mice. This study provides evidence for the critical role of the SR Ca2+ leak in the development of septic cardiomyopathy and highlights the therapeutic potential of JTV-519 by preventing SR leak. Furthermore, it reveals that TLR4 activation-induced mitochondrial reactive oxygen species production and the resulting oxidative stress in RyR2 contribute to the SR Ca2+ leak. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
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