RESUMO
Parathyroid hormone (PTH) stimulates bone formation in both animals and humans, and the expression of a number of genes has been implicated in the mediation of this effect. To discover new bone factors that initiate and support this phenomenon, we used differential display reverse transcription polymerase chain reaction (DDRT-PCR) and screened for genes, which are differentially expressed in osteoblast-enriched femoral metaphyseal primary spongiosa of young male rats after a single subcutaneous (s.c.) injection of hPTH (1-38) (8 microg/100 g). We found and cloned one full-length cDNA, which encodes a putative 348 amino acid protein. Sequence analysis of this protein demonstrates a 98, 93.7, and 82.5% identity with mouse, human, and chicken ubiquitin-specific protease UBP41, respectively. Northern blot analysis confirmed that a 3.8-4 kb UBP41 mRNA transcript was rapidly increased 1 h after acute hPTH (1-38) exposure in both metaphyseal (6- to 8-fold) and diaphyseal (3-fold) bone, but returned to control levels by 24 h after exposure. In contrast, continuous exposure to hPTH (1-38), resulted in a rapid and sustained elevation of UBP41 mRNA. PTH (1-31), which stimulates intracellular cAMP, and PTHrP (1-34) both induced UBP41 mRNA expression; whereas PTH analogs (3-34) and (7-34), that do not stimulate cAMP, had no effect on UBP41 expression. UBP41 mRNA expression was also rapidly induced 1 h after injection of PGE2, but returned to the control level by 6 to 24 h. In vitro, UBP41 mRNA is expressed in primary osteoblasts (metaphyseal and diaphyseal derived) and in the osteoblast-like cell lines UMR106, ROS17/2.8, and BALC. PTH (1-38) treatment induced UPB41 expression (3.6- to 13-fold) in both primary cultures of osteoblasts and in UMR106 cells. Further analysis in UMR 106 cells demonstrated that PGE2, forskolin and dibutyryl cAMP increased UBP41 mRNA expression 4-, 4.5-, and 2.4-fold, respectively. Tissue distribution analysis of UBP41 mRNA detected transcripts in brain, heart, skeletal muscle, kidney, liver, and testis. Together, these results demonstrate that UBP41, an ubiquitin-specific protease, is selectively upregulated in bone by the osteotropic agents PTH, PTHrP, and PGE2, possibly via the PKA/cAMP pathway. We speculate that the rapid induction of UBP41 in response to these physiological regulators contributes to the mechanism by which either the structure, activity, half-life or localization of essential proteins are modified to maintain bone homeostasis.
Assuntos
Osso e Ossos/efeitos dos fármacos , Endopeptidases/biossíntese , Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/farmacologia , Ubiquitina/metabolismo , Animais , Northern Blotting , Osso e Ossos/metabolismo , Células Cultivadas , Primers do DNA/química , Endopeptidases/genética , Fêmur/metabolismo , Perfilação da Expressão Gênica , Biblioteca Gênica , Masculino , Osteoblastos/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Ubiquitina Tiolesterase , Regulação para CimaRESUMO
PTH stimulates bone formation in animals and humans, and the expressions of a number of genes have been implicated in the mediation of this effect. To discover new bone factors that initiate and support this phenomenon we used differential display RT-PCR and screened for genes that are selectively expressed in osteoblast-enriched femoral metaphyseal primary spongiosa of young male rats after a single s.c. injection of human PTH-(1-38) (8 microg/100 g). We show that one of the messenger RNAs that is up-regulated in bone is ADAMTS-1, a new member of the ADAM (A disintegrin and metalloprotease) gene family containing thrombospondin type I motifs. ADAMTS-1 consists of multiple domains common to ADAM family of proteins, including pro-, metalloprotease-like, and disintegrin-like domains. However, unlike other ADAMs, ADAMTS-1 does not possess a transmembrane or cytoplasmic domain and is a secreted protein. Northern blot analysis confirmed that ADAMTS-1 was up-regulated in both metaphyseal (14- to 35-fold) and diaphyseal (4.2-fold) bone 1 h after PTH-(1-38) injection and returned to control levels by 24 h. We also analyzed the regulation of ADAMTS-1 in response to various PTH/PTH-related peptide (PTHrP) analogs and found that PTH-(1-31) and PTHrP-(1-34), which activate the protein kinase A (PKA) pathway, induce ADAMTS-1 expression 1 h after injection, whereas PTH-(3-34) and PTH-(7-34), which do not activate the PKA pathway, did not regulate expression. To investigate the effect of other osteotropic agents, we analyzed ADAMTS-1 expression after a single dose of PGE2 (6 mg/kg) and found that it was up-regulated 1 h after injection and returned to control levels by 6 h. In vitro ADAMTS-1 is expressed in primary osteoblasts and osteoblastic cell lines, but was not detectable in osteoclasts generated from macrophage colony-stimulating factor/receptor activator of NF-kappaB ligand/transforming growth factor-beta1-treated bone marrow cells. Treatment of UMR 106 osteosarcoma cells with PTH, PGE2, forskolin, or (Bu)2cAMP increased ADAMTS-1 expression 7-, 4-, 5-, and 5-fold, respectively. Also, in vitro treatment with 1alpha,25-dihydroxyvitamin D3 increased ADAMTS-1 expression 3-fold. Tissue distribution analysis showed that ADAMTS-1 is expressed at high levels in many tissues, including the heart, lung, liver, skeletal muscle, and kidney. Taken together, these results demonstrate that ADAMTS-1 is specifically up-regulated in bone and osteoblasts by the osteotropic agents PTH, PTHrP, and PGE2 possibly via the cAMP/PKA pathway. We speculate that the rapid and transient increase in ADAMTS-1 expression may contribute to some of the effects of PTH on bone turnover.
Assuntos
Osso e Ossos/enzimologia , Desintegrinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Metaloendopeptidases/genética , Hormônio Paratireóideo/farmacologia , Proteínas ADAM , Proteína ADAMTS1 , Animais , Calcitriol/farmacologia , Bovinos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dinoprostona/farmacologia , Desintegrinas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fêmur , Humanos , Cinética , Masculino , Metaloendopeptidases/metabolismo , Especificidade de Órgãos , Osteoblastos/enzimologia , Osteoclastos/enzimologia , Osteossarcoma/enzimologia , Proteína Relacionada ao Hormônio Paratireóideo , Fragmentos de Peptídeos/farmacologia , Proteínas/farmacologia , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
We have screened a subtracted cDNA library in order to identify differentially expressed genes in omental adipose tissue of human patients with Type 2 diabetes. One clone (#1738) showed a marked reduction in omental adipose tissue from patients with Type 2 diabetes. Sequencing and BLAST analysis revealed clone #1738 was the adipocyte-specific secreted protein gene apM1 (synonyms ACRP30, AdipoQ, GBP28). Consistent with the murine orthologue, apM1 mRNA was expressed in cultured human adipocytes and not in preadipocytes. Using RT-PCR we confirmed that apM1 mRNA levels were significantly reduced in omental adipose tissue of obese patients with Type 2 diabetes compared with lean and obese normoglycemic subjects. Although less pronounced, apM1 mRNA levels were reduced in subcutaneous adipose tissue of Type 2 diabetic patients. Whereas the biological function of apM1 is presently unknown, the tissue specific expression, structural similarities to TNFalpha, and the dysregulated expression observed in obese Type 2 diabetic patients suggest that this factor may play a role in the pathogenesis of insulin resistance and Type 2 diabetes.
Assuntos
Tecido Adiposo/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus/genética , Peptídeos e Proteínas de Sinalização Intercelular , Obesidade/genética , Proteínas/genética , Transcrição Gênica , Adiponectina , Adulto , Glicemia/análise , Índice de Massa Corporal , Colágeno/genética , Diabetes Mellitus/sangue , Diabetes Mellitus/metabolismo , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Jejum , Biblioteca Gênica , Humanos , Insulina/sangue , Pessoa de Meia-Idade , Obesidade/sangue , Obesidade/metabolismo , Omento , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele , MagrezaRESUMO
The initial steps involved in mediating the transduction of PTH signal via its G protein-coupled receptors are well understood and occur through the activation of cAMP and phospholipase C pathways. However, the cellular and molecular mechanisms for subsequent receptor desensitization are less well understood. Recently, a new family of GTPase activating proteins known as regulators of G protein signaling (RGS), has been implicated in desensitization of several G protein-coupled ligand-induced processes. At present, it is not known whether any of the RGS proteins play a role in PTH signaling. Using the differential display method, we screened for genes that are selectively expressed after a single s.c. injection of human PTH (1-38) (8 microg/100 g) in osteoblast-enriched femoral metaphyseal spongiosa of young male rats (3-4 weeks old). We found and cloned one full-length complementary DNA that encodes a 211-amino acid RGS protein and shares 97% sequence identity with mouse and human RGS2. Based on sequence similarity, we have designated this clone as rat RGS2. Northern blot analysis confirmed that the expression of RGS2 messenger RNA (mRNA) is rapidly and transiently increased by human PTH (1-38) in both metaphyseal (4-to 5-fold) and diaphyseal (2- to 3-fold) bone, as well as in cultured osteoblast cultures (2- to 37-fold). In vitro, forskolin and dibutyryl cAMP similarly elevated RGS2 mRNA. In vivo, PTH analog (1-31) [which stimulates intracellular cAMP accumulation, PTHrP (1-34), and prostaglandin E2] induced RGS2 mRNA expression; whereas PTH analogs (3-34) and (7-34), which do not stimulate cAMP production, had no effect on expression. In tissue distribution analysis, RGS2 is widely expressed and was detected in all tissues examined (heart, spleen, liver, skeletal muscle, kidney, and testis), with significant expression in two nonclassical PTH-sensitive tissues: the brain, and the heart. After PTH injection, RGS2 mRNA expression was induced in rat bone but not in any of the other tissues examined. These findings demonstrate that RGS2 is regulated by PTH, prostaglandin E2, and PTHrP and that regulation by PTH in bone occurs via the cAMP pathway. Additionally, these results suggest the exciting possibility that increased RGS2 expression in osteoblasts may be one of the early events influencing PTH signaling.
Assuntos
Comunicação Autócrina/fisiologia , Osso e Ossos/fisiologia , Hormônio Paratireóideo/fisiologia , Proteínas RGS/biossíntese , Sequência de Aminoácidos , Animais , Northern Blotting , Osso e Ossos/metabolismo , Células Cultivadas , DNA Complementar/biossíntese , DNA Complementar/genética , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Osteoblastos/metabolismo , Poli A/isolamento & purificação , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Regulação para CimaRESUMO
The benzothiophene LY329146 reverses the drug resistance phenotype in multidrug resistance protein (MRP1)-overexpressing cells when dosed in combination with MRP1-associated oncolytics doxorubicin and vincristine. Additionally, LY329146 inhibited MRP1-mediated uptake of the MRP1 substrate LTC4 into membrane vesicles prepared from MRP1-overexpressing cells.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Resistência a Múltiplos Medicamentos/genética , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Antineoplásicos/farmacologia , Transporte Biológico , Membrana Celular/metabolismo , Células HL-60 , Células HeLa , Humanos , Leucotrieno C4/antagonistas & inibidores , Leucotrieno C4/metabolismo , Proteínas Associadas à Resistência a Múltiplos MedicamentosRESUMO
Compounds were synthesized where oxygen in the ethoxypiperidine region of raloxifene is replaced with carbon, sulfur, or nitrogen linkages. Thia- and aza-substituted compounds were prepared by novel methodology. The compounds were evaluated in vitro as selective estrogen receptor modulators (SERMs). Calculations suggested the compounds exhibit an ER-alpha binding affinity/conformational energy relationship.
Assuntos
Carbono/química , Oxigênio/química , Piperidinas/química , Piperidinas/farmacologia , Receptores de Estrogênio/efeitos dos fármacos , Ligação Competitiva , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cristalografia por Raios X , Humanos , Piperidinas/metabolismo , Cloridrato de Raloxifeno , Receptores de Estrogênio/metabolismo , Relação Estrutura-AtividadeRESUMO
The substituted triphenylethylene antiestrogen clomiphene (CLO) prevents cancellous bone loss in ovariectomized (OVX'd) rats. However, CLO is a mixture of two stereoisomers, enclomiphene (ENC) and zuclomiphene (ZUC), which have distinctly different activities on reproductive tissues and tumor cells. The purpose of the present dose response study was to determine the effects of ENC and ZUC on nonreproductive estrogen target tissues. These studies were performed in 7-month-old female rats with moderate cancellous osteopenia that was established by ovariectomizing rats 1 month before initiating treatment. OVX resulted in increases in body weight, serum cholesterol, endocortical resorption, and indices of cancellous bone turnover, as well as decreases in uterine weight, uterine epithelial cell height, bone mineral density, bone strength, and cancellous bone area. Estrogen treatment for 3 months restored body weight, uterine histology, dynamic bone measurements, and osteoblast and osteoclast surfaces in OVX'd rats to the levels found in the age-matched sham-operated rats. In contrast, estrogen only partially restored cancellous bone volume and uterine weight, and it reduced serum cholesterol to subnormal values. CLO was a weak estrogen agonist on uterine measurements and a much more potent agonist on body weight, serum cholesterol, and dynamic bone measurements. CLO increased trabecular thickness in osteopenic rats and was the most effective treatment in improving cancellous bone volume and architecture. ZUC was a potent estrogen agonist on all tissues investigated and had dose-dependent effects. In contrast, ENC had dose-dependent effects on most measurements similar to CLO and decreased the uterotrophic effects of ZUC. It is concluded that ENC antagonizes the estrogenic effects of ZUC on the uterus but that the beneficial effects of CLO on nonreproductive tissues in OVX'd rats is conferred by both isomers. Furthermore, the combined actions of the two isomers on bone volume and architecture were more beneficial than either isomer given alone.
Assuntos
Osso e Ossos/efeitos dos fármacos , Osso e Ossos/fisiologia , Clomifeno/farmacologia , Enclomifeno , Estrogênios/fisiologia , Fármacos para a Fertilidade Feminina/farmacologia , Animais , Doenças Ósseas Metabólicas/etiologia , Doenças Ósseas Metabólicas/prevenção & controle , Colesterol/sangue , Feminino , Ovariectomia , Ratos , Ratos Sprague-Dawley , Útero/efeitos dos fármacos , Útero/fisiologiaRESUMO
The 2-arylbenzothiophene raloxifene, 1, is a selective estrogen receptor modulator (SERM) which is currently under clinical evaluation for the prevention and treatment of postmenopausal osteoporosis. In vivo structure-activity relationships and molecular modeling studies have indicated that the orientation of the basic amine-containing side chain of 1, relative to the stilbene plane, is an important discriminating factor for the maintenance of tissue selectivity. We have constructed a series of analogues of 1 in which this side chain is held in an orientation which is orthogonal to the stilbene plane, similar to the low-energy conformation predicted for raloxifene. Herein, we report on the synthesis of these compounds and on their activity in a series of in vitro and in vivo biological assays reflective of the SERM profile. In particular, we describe their ability to (1) bind the estrogen receptor, (2) antagonize estrogen-stimulated proliferation of MCF-7 cells in vitro, (3) stimulate TGF-beta3 gene expression in cell culture, (4) inhibit the uterine effects of ethynyl estradiol in immature rats, and (5) potently reduce serum cholesterol and protect against osteopenia in ovariectomized (OVX) rats without estrogen-like stimulation of uterine tissue. These data demonstrate that one of these compounds, LY357489,4, is among the most potent SERMs described to date with in vivo efficacy on bone and cholesterol metabolism in OVX rats at doses as low as 0.01 mg/kg/d.
Assuntos
Antagonistas de Estrogênios/síntese química , Naftoquinonas/síntese química , Piperidinas/farmacologia , Receptores de Estrogênio/antagonistas & inibidores , Animais , Densidade Óssea/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Colesterol/sangue , Antagonistas de Estrogênios/química , Antagonistas de Estrogênios/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Modelos Moleculares , Conformação Molecular , Naftoquinonas/química , Naftoquinonas/metabolismo , Naftoquinonas/farmacologia , Tamanho do Órgão/efeitos dos fármacos , Ovariectomia , Piperidinas/química , Piperidinas/metabolismo , Cloridrato de Raloxifeno , Ratos , Ratos Sprague-Dawley , Receptores de Estrogênio/metabolismo , Relação Estrutura-Atividade , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genética , Células Tumorais Cultivadas , Útero/efeitos dos fármacos , Útero/metabolismoRESUMO
The skeleton has the ability to alter its mass, geometry, and strength in response to mechanical stress. In order to elucidate the molecular mechanisms underlying this phenomenon, differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) was used to analyze gene expression in endocortical bone of mature female rats. Female Sprague-Dawley rats, approximately 8 months old, received either a sham or bending load using a four-point loading apparatus on the right tibia. RNA was collected at 1 h and 24 h after load was applied, reverse-transcribed into cDNA, and used in DDRT-PCR. Parallel display of samples from sham and loaded bones on a sequencing gel showed several regulated bands. Further analysis of seven of these bands allowed us to isolate two genes that are regulated in response to a loading stimulus. Nucleotide analysis showed that one of the differentially expressed bands shares 99% sequence identity with rat osteopontin (OPN), a noncollagenous bone matrix protein. Northern blot analysis confirms that OPN mRNA expression is increased by nearly 4-fold, at 6 h and 24 h after loading. The second band shares 90% homology with mouse myeloperoxidase (MPO), a bactericidal enzyme found primarily in neutrophils and monocytes. Semiquantitative PCR confirms that MPO expression is decreased 4- to 10-fold, at 1 h and 24 h after loading. Tissue distribution analysis confirmed MPO expression in bone but not in other tissues examined. In vitro analysis showed that MPO expression was not detectable in total RNA from UMR 106 osteoblastic cells or in confluent primary cultures of osteoblasts derived from either rat primary spongiosa or diaphyseal marrow. Database analysis suggests that MPO is expressed by osteocytes. These findings reinforce the association of OPN expression to bone turnover and describes for the first time, decreased expression of MPO during load-induced bone formation. These results suggest a role for both OPN and MPO expression in bone cell function.
Assuntos
Osteogênese/fisiologia , Tíbia/metabolismo , Animais , Sequência de Bases , Northern Blotting , Desenvolvimento Ósseo/genética , Desenvolvimento Ósseo/fisiologia , Feminino , Expressão Gênica/genética , Regulação da Expressão Gênica , Dados de Sequência Molecular , Osteogênese/genética , Osteopontina , Peroxidase/genética , Reação em Cadeia da Polimerase , RNA/análise , RNA/genética , Ratos , Ratos Sprague-Dawley , Homologia de Sequência do Ácido Nucleico , Sialoglicoproteínas/genética , Estresse Mecânico , Tíbia/fisiopatologia , Fatores de Tempo , Distribuição TecidualRESUMO
Interaction of the estrogen receptor/ligand complex with a DNA estrogen response element is known to regulate gene transcription. In turn, specific conformations of the receptor-ligand complex have been postulated to influence unique subsets of estrogen-responsive genes resulting in differential modulation and, ultimately, tissue-selective outcomes. The estrogen receptor ligands raloxifene and tamoxifen have demonstrated such tissue-specific estrogen agonist/antagonist effects. Both agents antagonize the effects of estrogen on mammary tissue while mimicking the actions of estrogen on bone. However, tamoxifen induces significant stimulation of uterine tissue whereas raloxifene does not. We postulate that structural differences between raloxifene and tamoxifen may influence the conformations of their respective receptor/ligand complexes, thereby affecting which estrogen-responsive genes are modulated in various tissues. These structural differences are 4-fold: (A) the presence of phenolic hydroxyls, (B) different substituents on the basic amine, (C) incorporation of the stilbene moiety into a cyclic benzothiophene framework, and (D) the imposition of a carbonyl "hinge" between the basic amine-containing side chain and the olefin. A series of raloxifene analogs that separately exemplify each of these differences have been prepared and evaluated in a series of in vitro and in vivo assays. This strategy has resulted in the development of a pharmacophore model that attributes the differences in effects on the uterus between raloxifene and tamoxifen to a low-energy conformational preference imparting an orthogonal orientation of the basic side chain with respect to the stilbene plane. This three-dimensional array is dictated by a single carbon atom in the hinge region of raloxifene. These data indicate that differences in tissue selective actions among benzothiophene and triarylethylene estrogen receptor modulators can be ascribed to discrete ligand conformations.
Assuntos
Receptores de Estrogênio/metabolismo , Animais , Linhagem Celular , Congêneres do Estradiol/química , Congêneres do Estradiol/metabolismo , Congêneres do Estradiol/farmacologia , Antagonistas de Estrogênios/química , Antagonistas de Estrogênios/metabolismo , Antagonistas de Estrogênios/farmacologia , Feminino , Ligantes , Modelos Moleculares , Conformação Molecular , Piperidinas/química , Piperidinas/metabolismo , Piperidinas/farmacologia , Conformação Proteica , Cloridrato de Raloxifeno , Ratos , Ratos Sprague-Dawley , Receptores de Estrogênio/química , Receptores de Estrogênio/efeitos dos fármacos , Relação Estrutura-Atividade , Tamoxifeno/química , Tamoxifeno/metabolismo , Tamoxifeno/farmacologia , Termodinâmica , Distribuição Tecidual , Útero/anatomia & histologia , Útero/efeitos dos fármacos , Útero/metabolismoRESUMO
1H NMR assignments of the trans and cis isomers of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide were accomplished by two-dimensional NMR techniques. Conformational exchange between the cis and trans isomers was not detected in the two-dimensional exchange spectra (NOESY) until catalytic amounts of FK506-binding protein (FKbp) were added. The addition of FK506 to the enzyme-substrate solution inhibited the enzyme and removed the substrate exchange peaks.
Assuntos
Isomerases de Aminoácido/metabolismo , Antibacterianos/metabolismo , Sequência de Aminoácidos , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Peptidilprolil Isomerase , Conformação Proteica , Proteínas Recombinantes , TacrolimoRESUMO
Incorporation of the DNA-cleaving moiety EDTA.Fe at discrete amino acid residues along a DNA-binding protein allows the positions of these residues relative to DNA bases, and hence the organization of the folded protein, to be mapped by high-resolution gel electrophoresis. A 52-residue protein, based on the sequence-specific DNA-binding domain of Hin recombinase (139-190), with EDTA at the NH2 terminus cleaves DNA at Hin recombination sites. The cleavage data for EDTA-Hin(139-190) reveal that the NH2 terminus of Hin(139-190) is bound in the minor groove of DNA near the symmetry axis of Hin-binding sites [Sluka, J. P., Horvath, S. J., Bruist, M. F., Simon, M. I., & Dervan, P. B. (1987) Science 238, 1129]. Six proteins, varying in length from 49 to 60 residues and corresponding to the DNA-binding domain of Hin recombinase, were synthesized by solid-phase methods: Hin(142-190), Hin(141-190), Hin(140-190), Hin(139-190), Hin(135-190), and Hin(131-190) were prepared with and without EDTA at the NH2 termini in order to test the relative importance of the residues Gly139-Arg140-Pro141-Arg142, located near the minor groove, for sequence-specific recognition at five imperfectly conserved 12-base-pair binding sites. Footprinting and affinity cleaving reveal that deletion of Gly139 results in a protein with affinity and specificity similar to those of Hin(139-190) but that deletion of Gly139-Arg140 affords a protein with altered affinities and sequence specificities for the five binding sites. It appears that Arg140 in the DNA-binding domain of Hin is important for recognition of the 5'-AAA-3' sequence in the minor groove of DNA. Our results indicate modular DNA and protein interactions with two adjacent DNA sites (major and minor grooves, respectively) bound on the same face of the helix by two separate parts of the protein.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Nucleotidiltransferases/metabolismo , DNA Bacteriano/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , DNA Bacteriano/ultraestrutura , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Salmonella typhimurium/enzimologia , Salmonella typhimurium/genética , Homologia de Sequência do Ácido NucleicoRESUMO
On the basis of sequence similarity with other known DNA-binding proteins, the DNA-binding domain of Hin recombinase, residues 139-190, is thought to bind DNA by a helix-turn-helix motif. Two models can be considered that differ in the orientation of the recognition helix in the major groove of DNA. One is based on the orientation of the recognition helix found in the 434 repressor (1-69) and lambda repressor-DNA cocrystals, and the other is based on the NMR studies of lac repressor headpiece. Cleavage by EDTA.Fe attached to a lysine side chain (Ser183----Lys183) near the COOH terminus of Hin(139-184) reveals that the putative recognition helix is oriented toward the center of the inverted repeats in a manner similar to that seen in the 434 and lambda repressor-DNA cocrystals.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Nucleotidiltransferases/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , DNA Bacteriano/genética , DNA Bacteriano/ultraestrutura , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Sequências Repetitivas de Ácido Nucleico , Proteínas Repressoras/metabolismo , Salmonella typhimurium/enzimologia , Salmonella typhimurium/genética , Proteínas Virais , Proteínas Virais Reguladoras e AcessóriasRESUMO
A synthetic 52-residue peptide based on the sequence-specific DNA-binding domain of Hin recombinase (139-190) has been equipped with ethylenediaminetetraacetic acid (EDTA) at the amino terminus. In the presence of Fe(II), this synthetic EDTA-peptide cleaves DNA at Hin recombination sites. The cleavage data reveal that the amino terminus of Hin(139-190) is bound in the minor groove of DNA near the symmetry axis of Hin recombination sites. This work demonstrates the construction of a hybrid peptide combining two functional domains: sequence-specific DNA binding and DNA cleavage.