RESUMO
We recently suggested that oral malodor production involves two steps: (i) deglycosylation of glycoproteins and (ii) proteolysis and amino acid utilization of the protein core to yield volatiles such as volatile sulfide compounds (VSCs). Our aim was to test the hypothesis that ß-galactosidase activity and VSC production occur in distinct areas of the biofilm by two different bacterial populations. Biofilms were grown anaerobically for seven days with or without antibiotics (i.e. vancomycin and metronidazole). Biofilms were stained for ß-galactosidase activity and VSC production and studied using confocal laser scanning microscopy. Results showed that ß-galactosidase activity occurs in the outer layers and disappears following vancomycin addition, whereas VSC production occurs deeper within the biofilm and disappears following metronidazole application. These findings suggest that ß-galactosidase activity is produced mainly by Gram-positive oral bacteria at the outer portion of the biofilm, and VSC production occurs in the deeper layers by Gram-negative oral bacteria.
RESUMO
OBJECTIVE: Pseudohypoparathyroidism type Ia (PHP-Ia) is a hereditary disorder characterized by resistance to multiple hormones that work via cAMP such as PTH and TSH, accompanied by typical skeletal features including short stature and brachydactyly, termed Albright hereditary osteodystrophy (AHO). In affected kindreds, some members may have AHO but not hormone resistance; they are termed as pseudopseudohypoparathyroidism (PPHP). The molecular basis for the disorder is heterozygous inactivating mutation of the Gsalpha gene. In affected families, subjects with both PHP-Ia and PPHP have the same Gsalpha mutations. The skeletal features common to PPHP and PHP-Ia are presumably caused by tissue-specific Gsalpha haploinsufficiency. Other features that distinguish between PPHP and PHP-Ia, such as the multihormone resistance, are presumably caused by tissue-specific paternal imprinting of Gsalpha. This suggests that major differences in phenotype between PHP-Ia and PPHP point to specific tissues with Gsalpha imprinting. One such major difference may be cognitive function in PHP-Ia and PPHP. DESIGN: Description of a large family with PHP-Ia and PPHP. PATIENTS: Eleven affected subjects with PHP-Ia or PPHP in one family. MEASUREMENTS: Cognitive impairment (CI) was defined by a history of developmental delay, learning disability and the Wechsler intelligence scale. RESULTS: CI occurred only in the five PHP-Ia but not in the six PPHP subjects. Hypothyroidism which occurred in all PHP-Ia subjects was apparently not the cause of CI as it was mild, and was treated promptly. Analysis of additional Israeli cases, and the published cases from the literature, all with documented Gsalpha mutations, revealed that CI is prevalent in PHP-Ia [60 of 77 subjects (79%)] but not in PPHP [3 of 30 subjects (10%)] (P < 1 x 10(-6)). CONCLUSION: We suggest that Gsalpha is imprinted in the brain.
Assuntos
Encéfalo/metabolismo , Transtornos Cognitivos/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Impressão Genômica/genética , Pseudo-Hipoparatireoidismo/genética , Pseudopseudo-Hipoparatireoidismo/genética , Adolescente , Adulto , Criança , Pré-Escolar , Transtornos Cognitivos/etiologia , Feminino , Displasia Fibrosa Poliostótica/genética , Heterozigoto , Humanos , Lactente , Masculino , Mutação , Pseudo-Hipoparatireoidismo/fisiopatologia , Pseudopseudo-Hipoparatireoidismo/fisiopatologia , Adulto JovemRESUMO
BACKGROUND: WNK [With No K (lysine)] kinases are essential for regulation of blood pressure and potassium homeostasis. WNK4 expression was recently found not only in the distal nephron but also in chloride-transporting epithelia. To establish a physiological role for this distribution we studied patients with familial hyperkalaemia and hypertension (FHH), [pseudohypoaldosteronism type II (PHAII)], which is caused by mutations in WNK4. DESIGN: Measurement of nasal potential difference (NPD) and sweat electrolytes were performed in controls, in six subjects with FHH and ten subjects with cystic fibrosis (CF). RESULTS: Basal NPD was higher in FHH compared with controls (n = 20): 22.8 +/- 5.7 vs. 16.2 +/- 5.3 mV, respectively (P = 0.014). Maximal response to amiloride was also higher in FHH compared with controls: 14.8 +/- 3.5 vs. 10.0 +/- 4.8 mV, respectively (P = 0.03). In CF these values were 42.9 +/- 9.3 and 29.9 +/- 7.4 mV, respectively. The kinetics of the amiloride effect were faster in FHH, and as first reported here also in CF, compared with controls. At 30 s, amiloride-inhibitable residual PD in FHH was 50 +/- 30 vs. 81 +/- 9% in controls (P = 0.0003) and 56 +/- 7% in CF. The response to chloride-free and isoproterenol solutions, which determines chloride transport activity, was similar in FHH compared with controls [16.0 +/- 8.6 vs. 10.4 +/- 5.9 mV (P = 0.08)]. Sweat conductivity in FHH was 49.7 +/- 7.3 vs. 38.2 +/- 8.1 mmol (NaCl eq) L-1 in 16 controls (P = 0.007) and 94.0 +/- 19.3 in CF. CONCLUSIONS: Mutant WNK4 increases Na+ transport in airways, and therefore it is regulated by wild-type WNK4. This may be caused by a regulation of ENaC or a K+ channel.
Assuntos
Hiperpotassemia/fisiopatologia , Hipertensão/fisiopatologia , Proteínas Serina-Treonina Quinases/fisiologia , Sódio/metabolismo , Fibrose Cística/fisiopatologia , Transporte de ÍonsRESUMO
Molecular imaging techniques allow visualization of specific gene products and their physiological processes in living tissues. In this study, we present a new approach for molecular imaging of endogenous tyrosine kinase receptor activity. Met and its ligand hepatocyte growth factor scatter factor (HGF/SF), which mediate mitogenicity, tumorigenicity, and angiogenesis, were used as a model. HGF/SF and Met play a significant role in the pathogenesis and biology of a wide variety of human epithelial cancers and, therefore, may serve as potential targets for cancer prognosis and therapy. We have shown previously that in vitro activation of Met by HGF/SF increases oxygen consumption. In this study, we demonstrate that Met activation in vivo by HGF/SF alters the hemodynamics of normal and malignant Met-expressing tissues. Tumor-bearing BALB/C mice were i.v. injected with HGF/SF and imaged using magnetic resonance imaging (MRI) and Doppler ultrasound. Organs and tumors expressing high levels of Met showed the most substantial alteration in blood oxygenation levels as measured by blood oxygenation level depended (BOLD)-MRI. No significant alteration was observed in tumors or organs that does not express Met. In the liver, which expresses high levels of Met, MRI signal alteration of about 60% was observed. In the kidneys, signal alteration was approximately 30%, and no change was observed in muscles. The extent of MRI signal alteration was also in correlation with HGF/SF doses. Injection of 7 and 170 ng/g body weight resulted in signal alteration of 5% and 30%, respectively, in tumors. Doppler ultrasound measurements demonstrated that these MRI changes are at least partially attributable to altered blood flow. These hemodynamic alterations, measured by MRI and Doppler ultrasound, were used in this study for the molecular imaging of Met activity in vivo. This novel molecular imaging technique may be used for in vivo diagnosis, prognosis, and therapy of Met-expressing tumors.
Assuntos
Adenocarcinoma/enzimologia , Neoplasias Mamárias Experimentais/enzimologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Adenocarcinoma/irrigação sanguínea , Animais , Ativação Enzimática , Feminino , Hemodinâmica/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Imageamento por Ressonância Magnética , Neoplasias Mamárias Experimentais/irrigação sanguínea , Camundongos , Camundongos Endogâmicos BALB C , Oxigênio/sangue , Ultrassonografia Doppler em CoresRESUMO
Activation of the Met tyrosine kinase growth factor receptor by its ligand HGF/SF has been shown to increase in vitro invasiveness in epithelial cell lines. To study the effect of Met-HGF/SF signaling in breast cancer cells, we transfected met, hgf/sf and dominant negative (DN) forms of met into the poorly differentiated metastatic murine mammary adenocarcinoma cell line DA3. These cells express moderate levels of endogenous Met, which is rapidly phosphorylated in response to HGF/SF treatment. Met+hgf/sf transfection results in significantly increased tumorigenic and metastatic activity in vivo accompanied by reduced tubule formation. DA3 cells transfected with DN forms of Met (DN-DA3) exhibit reduced Met phosphorylation following exposure to HGF/SF. Furthermore, as compared to the parental cells, the DN-DA3 cells exhibit diminished in vitro scattering and invasiveness, while in vivo they display greatly reduced tumorigenicity and spontaneous metastasis. Tumors emanating from DN-DA3 cells injected to BALB/C mice are highly differentiated and display extensive tubule formation. These results suggest that Met-HGF/SF signaling is a determining factor in the delicate balance between differentiation/tubule formation and tumorigenicity-metastasis. Oncogene (2000) 19, 2386 - 2397
Assuntos
Adenocarcinoma/patologia , Neoplasias Mamárias Experimentais/patologia , Metástase Neoplásica/genética , Proteínas Proto-Oncogênicas c-met/fisiologia , Adenocarcinoma/metabolismo , Animais , Divisão Celular , Genes Dominantes , Fator de Crescimento de Hepatócito/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microtúbulos , Transplante de Neoplasias , Fosforilação , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
The spitz gene encoding a TGF-alpha homolog, has been shown to affect a subset of developmental processes that are similar to those regulated by DER, the Drosophila EGF receptor homolog. This work demonstrates that Spitz triggers the DER signaling cascade. Addition of a secreted, but not the membrane-associated form of Spitz to S2 Drosophila cells expressing DER gives rise to a rapid tyrosine autophosphorylation of DER. Following autophosphorylation, DER associates with the Drk adapter protein. Consequently, activation of MAP kinase is observed. The profile of MAP kinase activation provides a quantitative assay for DER activation. A dose response between the levels of Spitz and MAP kinase activity was observed. The secreted Spitz protein was expressed in embryos to assess its biological activity. An alteration in cell fates was observed in the ventral ectoderm, such that lateral cells acquired the ventral-most fates. The result indicates that graded activation of the DER pathway may normally give rise to a repertoire of discrete cell fates in the ventral ectoderm. Spatially restricted processing of Spitz may be responsible for this graded activation. The Rhomboid (Rho) and Star proteins were suggested, on the basis of genetic interactions, to act as modulators of DER signaling. No alteration in DER autophosphorylation or the pattern of MAP kinase activation by secreted Spitz was observed when the Rho and Star proteins were coexpressed with DER in S2 cells. In embryos mutant for rho or Star the ventralizing effect of secreted Spitz is epistatic, suggesting that Rho and Star may normally facilitate processing of the Spitz precursor.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/embriologia , Ectoderma/fisiologia , Fator de Crescimento Epidérmico , Receptores ErbB/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Quinases , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Peptídeos de Invertebrados/fisiologia , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Epistasia Genética , Receptores ErbB/genética , Regulação da Expressão Gênica no Desenvolvimento , Hormônios de Inseto/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Receptores de Peptídeos de Invertebrados/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de SinaisRESUMO
The reverse transcriptase (RT) of equine infectious anemia virus (EIAV) shares sequence similarity with the RTs of other lentiviruses, particularly with the RTs of human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2, respectively), the causative agents of acquired immunodeficiency syndrome (AIDS). There is a 41-42% sequence identity between EIAV RT and both HIV RTs (which have 61% sequence identity to each other). We have compared the enzymic properties of EIAV RT with those of HIV-1 RT. Several aspects of the activities of EIAV RT differ from the corresponding activities of HIV-1 RT. There are significant differences in the inhibition of the DNA polymerase activities by the deoxynucleoside triphosphate analogs, 3'-azido-2,3'-dideoxythymidine triphosphate, dideoxyTTP and dideoxyGTP and by the nonnucleoside inhibitor, tetrahydroimidazo[4,5,1-jk-1,4]benzodiazepin-2-(1H)-one and thione; in the dependence of DNA polymerase and RNase H activities on pH; in the inhibition of the DNA polymerase activities by the thiol-specific reagent N-ethylmaleimide; in the specific DNA polymerase activity; in the inhibition of the ribonuclease H activity by the zinc chelator orthophenanthroline. However, there are several cases in which EIAV RT and HIV-1 RT are more similar than was previously found for HIV-1 RT and HIV-2 RT. These include the Km values for the DNA polymerase activities, the heat stability of the DNA polymerase functions and the specific activity of the RNase H function.
Assuntos
Vírus da Anemia Infecciosa Equina/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Antivirais/farmacologia , Benzodiazepinas/farmacologia , Catálise , DNA Polimerase Dirigida por DNA/metabolismo , Estabilidade Enzimática , Etilmaleimida/farmacologia , Transcriptase Reversa do HIV , Temperatura Alta , Concentração de Íons de Hidrogênio , Imidazóis/farmacologia , Fenantrolinas/farmacologia , Proteínas Recombinantes/metabolismo , Inibidores da Transcriptase Reversa , Ribonuclease H/metabolismoRESUMO
The reverse transcriptase of equine infectious anemia virus (EIAV) shows sequence similarity with the reverse transcriptases of other lentiviruses, particularly with those of human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2). We have constructed a plasmid that when introduced into E. coli induces the synthesis of substantial quantities of the nearly authentic EIAV reverse transcriptase. The viral and bacterially expressed reverse transcriptases are similar in their molecular weights. The bacterial expression clone was used to generate deletion mutants of the protein. Mutations in both amino and carboxyl terminal regions of the polypeptide strongly affect the DNA polymerase activity of the enzyme. Thus, EIAV reverse transcriptase resembles the reverse transcriptases of HIV-1 and HIV-2 and can serve as a suitable enzyme for studying the structure-function relationship in lentiviral reverse transcriptase.
Assuntos
Vírus da Anemia Infecciosa Equina/enzimologia , DNA Polimerase Dirigida por RNA/biossíntese , Clonagem Molecular , Análise Mutacional de DNA , DNA Polimerase Dirigida por DNA/metabolismo , Genes pol , Transcriptase Reversa do HIV , HIV-1/enzimologia , HIV-2/enzimologia , Vírus da Anemia Infecciosa Equina/genética , Cinética , Provírus/enzimologia , Provírus/genética , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Deleção de SequênciaRESUMO
We have studied the effects of four nonnucleoside inhibitors, including the novel natural product inhibitor calanolide A, on molecular chimeras containing complementary segments of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) reverse transcriptases (RTs). All four compounds specifically inhibited the DNA polymerase activity of HIV-1 RT but had no apparent effect on the RNase H activity of this enzyme or on the DNA polymerase or RNase H activity of HIV-2 RT. Three of these compounds showed the generally expected patterns of resistance and susceptibility with the various chimeric RTs. However, the inhibition patterns of the chimeric RTs by calanolide A provided evidence that there is a segment between residues 94 and 157 in HIV-1 RT that is critical for inhibition. However, the data also suggest that there may be a second segment located between amino acids 225 and 427 in HIV-1 RT that is also important for specifying susceptibility to the drug.
Assuntos
Antivirais/farmacologia , Cumarínicos/farmacologia , HIV-1/enzimologia , HIV-2/enzimologia , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Inibidores da Transcriptase Reversa , Transcriptase Reversa do HIV , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Mutagênese Insercional , Piranocumarinas , DNA Polimerase Dirigida por RNA/genética , Proteínas Recombinantes de Fusão/genéticaRESUMO
We have generated by site-directed mutagenesis plasmids that induce the synthesis of specific mutants of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). These recombinant mutants of HIV-1 RT, designed on the basis of our previous studies of HIV-1 and HIV-2 RTs, were analyzed for structure-function relationship by assessing their RNA-dependent and DNA-dependent DNA polymerase as well as the ribonuclease H activities. Three groups of mutants were studied. 1) We have investigated the importance of the only two sets of highly conserved double prolines found in the sequence of HIV-1 RT. The results indicate that the conversion of either one or both prolines (at positions 225 and 226) to threonines have no significant effect on all catalytic activities of the enzyme. The mutants in which prolines 419 and 420 were individually modified to threonines exhibit full activities, whereas the double proline 419/420 mutant lost most of its RNase H activity (although the DNA polymerase function was fully retained). 2) We have deleted phenylalanine 346 from HIV-1 RT, which is absent in wild type HIV-2 RT. This mutant of HIV-1 RT lost practically all catalytic activities. 3) A mutant of HIV-1 RT in which a cysteine residue substituted for alanine 446, was found to be slightly hyperactive for both DNA polymerase and RNase H activities.
Assuntos
HIV-1/enzimologia , Mutagênese Sítio-Dirigida , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Expressão Gênica , Plasmídeos , DNA Polimerase Dirigida por RNA/metabolismo , Ribonuclease H/metabolismoRESUMO
The reverse transcriptases (RTs) from human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2, respectively) are relatively highly related yet there are several significant differences in their catalytic activities. In an attempt to relate these functional dissimilarities to the differences in amino acid sequences, we have employed a novel approach of constructing chimeric molecules composed of complementary amino acid sequences derived from the two HIV RTs. These recombinant proteins were analyzed for their enzymatic activities and for their sensitivity to tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2[1H]-one and thione (TIBO), which selectively inhibits only HIV-1 RT. The active chimeric RTs were used to map the TIBO binding site on the HIV-1 RT molecules and to localize the putative sequences responsible for the high RNase H activity of HIV-1 RT relative to that of HIV-2 RT. The results suggest that TIBO interacts with amino acid residues located around residue 200 within the DNA polymerase domain of HIV-1 RT which shows a relatively low similarity to HIV-2 RT. The difference in the RNase H activity maps to a position in the DNA polymerase domain rather than to the RNase H domain. Out of the 12 chimeric RTs generated, four were either fully active or hyperactive, three others lost most of their catalytic activities, and the rest were totally inactive. The pattern of catalytic activities of these hybrid proteins can be explained by a model for the initial folding of HIV RTs, which entails the formation of three distinct and independently folded regions. Each region can be formed by amino acid sequences derived exclusively from either HIV-1 RT or HIV-2 RT.
Assuntos
HIV-1/enzimologia , HIV-2/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Sequência de Aminoácidos , Benzodiazepinas/metabolismo , Benzodiazepinas/farmacologia , Sítios de Ligação , Catálise , DNA Polimerase Dirigida por DNA/metabolismo , Transcriptase Reversa do HIV , Imidazóis/metabolismo , Imidazóis/farmacologia , Dados de Sequência Molecular , Plasmídeos , DNA Polimerase Dirigida por RNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Inibidores da Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
Chemical modification of HIV-1 and HIV-2 (human immunodeficiency virus, types 1 and 2) reverse transcriptases (RT) with three thiol reactive compounds selectively inhibits the RNase H function of the enzyme. HIV-1 RT has 2 cysteines (at positions 38 and 280); HIV-2 RT has 3 (38, 280, 445). Both of the cysteines in HIV-1 RT are in the polymerase domain. To investigate the role of the cysteines in the structure and function of the HIV RTs, we have converted each cysteine to serine and made combinations of the mutations. Since HIV-1 RT has alanine at position 445, we have also substituted alanine for serine at this position in HIV-2 RT. Neither of the single mutations in HIV-1 RT nor the double mutation mimics the effects of the chemical modification. The serine 280 mutation has little effect on either polymerase or RNase H; the serine 38 mutation affects both activities, as does the 38/280 double mutant. The 38 and 280 serine mutations in HIV-2 RT resemble the equivalent mutations in HIV-1 RT. Substitution of serine or alanine at position 445 (which lies in the RNase H domain) diminishes, but does not abolish, the RNase H activity of HIV-2 without affecting polymerase activity. The RNase H activity of a mutant HIV-1 RT with serine at position 280 is completely resistant to inactivation by the three thiol reactive compounds we tested, which demonstrates that cysteine 280 is the critical residue. We suggest that the reason the mutation (cysteine 280 to serine) does not mimic the chemical modification is because the chemical modification produces a greater change in the structure of the protein. We also suggest that position 280 lies at or near the important points of contact between the RNase H and polymerase domains, so that chemical modification of this position, which lies within the polymerase domain, distorts the RNase H domain.
Assuntos
Cisteína/genética , HIV-1/enzimologia , HIV-2/enzimologia , Mutação , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/enzimologia , Expressão Gênica , Transcriptase Reversa do HIV , DNA Polimerase Dirigida por RNA/metabolismo , Inibidores da Transcriptase Reversa , Ribonuclease H/antagonistas & inibidores , Ribonuclease H/metabolismo , Compostos de Sulfidrila/farmacologiaRESUMO
We have constructed a series of plasmids which, when introduced into Escherichia coli, induce the overexpression of soluble wild-type and mutated forms of the reverse transcriptases (RTs) from human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2, respectively). These proteins were analyzed previously for their RNA-dependent DNA polymerase (RDDP) and ribonuclease H (RNase H) activities. In the present study we assayed the different mutant RTs for their DNA-dependent DNA polymerase (DDDP) activity, employing an in situ polyacrylamide gel activity assay. The results indicate that both the RDDP and DDDP catalytic functions of HIV-1 RT mutants are affected similarly by mutations suggesting a high degree of overlap between the catalytic domains involved in both activities. Contrariwise, many of the HIV-2 RT mutants display no correlation between these two DNA polymerase activities, that is, the DDDP activity was not affected by the mutations introduced in the native enzyme in contrast to the RDDP activity. We were thus able to generate mutants of HIV-2 RT that unlike the wild-type RT, are capable of transcribing only DNA and not RNA. The disparity in mutational-catalytic relations between the two HIV-related RTs may reflect a possible difference in the structure and folding properties of the two proteins.
Assuntos
DNA Viral/fisiologia , DNA Polimerase Dirigida por DNA/análise , HIV-1/enzimologia , HIV-2/enzimologia , RNA Viral/fisiologia , DNA Polimerase Dirigida por RNA/análise , Catálise , Análise Mutacional de DNA , DNA Polimerase Dirigida por DNA/genética , Escherichia coli/genética , Vetores Genéticos , Transcriptase Reversa do HIV , HIV-1/genética , HIV-2/genética , Plasmídeos , DNA Polimerase Dirigida por RNA/genéticaRESUMO
The enzyme reverse transcriptase (RT) is crucial in the early steps of the life cycle of retroviruses. We have expressed in bacteria the RTs from human immunodeficiency viruses (HIV) types 1 and 2 in order to study the structural-functional relationships of these two multifunctional enzymes that share a relatively high degree of amino acid sequence homology. For comparison purposes, we have analyzed several catalytic functions of both enzymes. The two HIV RTs show a high similarity in many aspects studied but exhibit profound differences in several other properties. For instance, the specific RNase H activity of HIV-2 RT is about 10 times lower than the corresponding activity of HIV-1 RT. There are also significant dissimilarities between some of the apparent Km values calculated for the DNA polymerizing functions of both enzymes. Furthermore, the heat stability of the DNA polymerizing activity of HIV-2 RT is about 15-fold higher than that of HIV-1 RT. On the other hand, the susceptibility of the RNase H activities of the two enzymes to heat inactivation was found to be similar. Other treatments also enable discrimination between the RNase H and DNA polymerizing catalytic properties of the two enzymes (although both reverse transcriptases respond similarily). Thus, the RNase H activity was inactivated by N-ethylmaleimide, suggesting the possible involvement of cysteine residues in performing this activity, whereas the DNA polymerizing functions of the two enzymes were fully resistant to this chemical modification. The zinc chelator 1,10-phenanthroline affected the DNA polymerase activities of both enzymes to a significantly higher extent than the RNase H activity. In all, the two HIV RTs were shown to be substantially different one from the other in several of their properties and also distinct from other RTs thus far studied.
Assuntos
HIV-1/enzimologia , HIV-2/enzimologia , DNA Polimerase Dirigida por RNA/metabolismo , Catálise , DNA Polimerase Dirigida por DNA/análise , Difosfatos/farmacologia , Endorribonucleases/análise , Escherichia coli/genética , Etilmaleimida/farmacologia , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Temperatura Alta , Concentração de Íons de Hidrogênio , Fenantrolinas/farmacologia , Fosfato de Piridoxal/farmacologia , DNA Polimerase Dirigida por RNA/genética , Inibidores da Transcriptase Reversa , Ribonuclease H , Rosa Bengala/farmacologiaRESUMO
We have constructed a series of plasmids that, when introduced into Escherichia coli, induce the expression of high levels of either wild-type or mutated forms of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). Mutant forms of RT that had been previously analyzed for their RNA-dependent DNA polymerase activity were tested for RNase H activity using an in situ polyacrylamide gel assay. Mutations affecting the RNase H are not clustered in a single region of the 66-kDa RT molecule. With only few exceptions, mutations that affect the RNase H activity also cause a substantial decrease in the DNA polymerase function. This suggests that, unlike the RT from murine leukemia virus (MuLV), it is difficult to genetically separate the catalytic domains responsible for the RNase H and DNA polymerase functions of HIV-1 RT. Those few mutations that differentially affect the RNase H and the polymerase activities of HIV-1 RT suggest that, as in MuLV, the polymerase domain is in the amino-terminus and the RNase H domain is in the carboxy-terminus. We have also generated chimeric molecules that are composed of sequences from the RT of HIV-1 and MuLV and these hybrid RTs were analyzed for their enzymatic properties. Two of these chimeric RTs possess RNase H activity but lack detectable DNA polymerase activity.