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1.
Cell Rep Med ; 3(9): 100748, 2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-36130483

RESUMO

Endometriosis is a chronic pain condition affecting 1 in 10 women. There is an unmet need for better medical treatments for endometriosis. We spotlight trials of a single preparation combined HRT-GnRH antagonist (Relugolix) by Giudice et al.,1 for endometriosis-associated pain.


Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Endometriose , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/tratamento farmacológico , Endometriose/tratamento farmacológico , Feminino , Hormônio Liberador de Gonadotropina/uso terapêutico , Antagonistas de Hormônios/efeitos adversos , Humanos , Dor/induzido quimicamente
2.
Eur J Obstet Gynecol Reprod Biol ; 257: 144-148, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33388182

RESUMO

OBJECTIVE: To explore the risk of melanoma in women with endometriosis. STUDY DESIGN: A retrospective cohort study using Scottish national population-based data was conducted. The study comprised 281,937 women with nearly 5 million person years (4,923,628) of follow up from 1981 to 2010. 17,834 women with a new surgical diagnosis of endometriosis were compared with 83,303 women with no evidence of endometriosis at laparoscopy, 162,966 women who underwent laparoscopic sterilisation and 17,834 age-matched women from the general population to determine the risk of melanoma. Cox proportional hazards regression was used to calculate crude and adjusted Hazards ratios with 95 % Confidence Intervals. RESULTS: Women with endometriosis had a significantly higher risk of melanoma when compared to women with no evidence of endometriosis at laparoscopy (HR 1.59, 95 % CI 1.19-2.13), women who had undergone laparoscopic sterilisation (HR 1.82, 95 % CI 1.39-2.40) and age-matched women from the general population (HR 1.63, 95 % CI 1.08-2.45). CONCLUSION: A diagnosis of endometriosis was associated with an increased risk of developing melanoma compared to those without endometriosis. These findings highlight the need for further research to explore shared pathways in the pathogenesis of the two conditions. It is important to acknowledge that the absolute increase in the risk of melanoma in women with endometriosis remains low, which should be considered when counselling women.


Assuntos
Endometriose , Melanoma , Estudos de Coortes , Endometriose/complicações , Endometriose/epidemiologia , Feminino , Humanos , Melanoma/epidemiologia , Melanoma/etiologia , Estudos Retrospectivos , Fatores de Risco , Escócia/epidemiologia
3.
BJOG ; 125(1): 64-72, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28952173

RESUMO

OBJECTIVE: To evaluate the long-term risk of further gynaecological surgery and cancer in women with endometriosis. DESIGN: Cohort study. SETTING: Scotland. PARTICIPANTS: 281 937 women with nearly 5 million person years (4 923 628) of follow up from 1981 to 2010. METHODS: In this national population-based study we compared 17 834 women with a new surgical diagnosis of endometriosis with 83 303 women with no evidence of endometriosis at laparoscopy, 162 966 women who underwent laparoscopic sterilisation, and 17 834 age-matched women from the general population. Cox proportional hazards regression was used to calculate crude and adjusted hazard ratios with 95% confidence intervals. MAIN OUTCOME MEASURES: Risk of further gynaecological surgery, number and type of repeat surgery and time to repeat surgery from the diagnosis of endometriosis. Cancer outcomes included subsequent risk of all cancer, gynaecological and non-gynaecological cancers. RESULTS: Women with endometriosis had a significantly higher risk of further surgery when compared with women with no evidence of endometriosis at laparoscopy [hazard ratio (HR) 1.69, 95% (confidence interval) CI 1.65-1.73], women who had undergone laparoscopic sterilisation (HR 3.30, 95% CI 3.23-3.37) and age-matched women from the general population (HR 5.95, 95% CI 5.71-6.20). They also have an increased risk of ovarian cancer when compared with general population counterparts (HR 1.77, 95% CI 1.08-2.89) or those with laparoscopic sterilisation (HR 1.75, 95% CI 1.2-2.45). CONCLUSION: Women with surgically diagnosed endometriosis face an increased risk of multiple surgery. They have a higher chance of developing ovarian cancer in comparison with the general population and women with laparoscopic sterilisation. TWEETABLE ABSTRACT: Women with endometriosis face an increased risk of recurrent surgery and developing ovarian cancer.


Assuntos
Endometriose/epidemiologia , Procedimentos Cirúrgicos em Ginecologia/estatística & dados numéricos , Neoplasias/epidemiologia , Adulto , Endometriose/cirurgia , Feminino , Seguimentos , Humanos , Laparoscopia/estatística & dados numéricos , Masculino , Prognóstico , Reoperação/estatística & dados numéricos , Fatores de Risco , Escócia/epidemiologia , Cirurgia de Second-Look/estatística & dados numéricos , Fatores Socioeconômicos , Esterilização Reprodutiva/estatística & dados numéricos
4.
BJOG ; 124(3): 444-452, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26887349

RESUMO

OBJECTIVE: To determine pregnancy outcomes in women with endometriosis. DESIGN: A national population based cohort study using record linkage. SETTING: Scotland. PARTICIPANTS: A cohort of 14 655 women followed up over a 30-year period (1981-2010). METHODS: In a nationwide Scottish study, we compared pregnancy outcomes in 5375 women with surgically confirmed endometriosis with outcomes in 8710 women without endometriosis who were pregnant during the same time period. Data were analysed using univariable and multivariable logistic regression after adjusting for confounding factors. MAIN OUTCOME MEASURES: Outcome measures evaluated included miscarriage, ectopic pregnancy, stillbirths and other pregnancy complications such as hypertensive disorders of pregnancy, antepartum and postpartum haemorrhage, operative delivery and preterm births. The outcomes were presented as adjusted odds ratios (OR) with 95% confidence intervals (CI). RESULTS: On multivariable analysis, after adjusting for age, parity, socio-economic status and year of delivery, women with endometriosis when compared to women without endometriosis, had a significantly higher risk of early pregnancy complications with adjusted OR (95% CI) of 1.76 (1.44, 2.15) and 2.70 (1.09, 6.72) for miscarriage and ectopic pregnancy, respectively. A previous diagnosis of endometriosis was associated with a significantly increased risk of [adjusted OR (95% CI)] placenta praevia [2.24 (1.52, 3.31)], unexplained antepartum haemorrhage [1.67 (1.39, 2.00)], postpartum haemorrhage [1.30 (1.61, 1.46)] and preterm births [1.26 (1.07, 1.49)] in pregnancies progressing beyond 24 weeks. CONCLUSION: Endometriosis predisposes women to an increased risk of early pregnancy loss and later pregnancy complications. TWEETABLE ABSTRACT: Endometriosis predisposes women to an increased risk of early pregnancy loss and later pregnancy complications.


Assuntos
Endometriose/complicações , Complicações na Gravidez/epidemiologia , Resultado da Gravidez/epidemiologia , Adulto , Estudos de Coortes , Feminino , Humanos , Recém-Nascido , Gravidez , Escócia/epidemiologia , Adulto Jovem
6.
BJOG ; 117(3): 245-57, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19943827

RESUMO

BACKGROUND: Threatened miscarriage is a common complication in the first trimester of pregnancy and is often associated with anxiety regarding pregnancy outcome. OBJECTIVE: We undertook a systematic review to explore the effects of threatened miscarriage in the first trimester on maternal and perinatal outcomes. SEARCH STRATEGY: An electronic literature search using MEDLINE and EMBASE, and bibliographies of retrieved primary articles. No language restrictions were applied. SELECTION CRITERIA: All studies analysing outcomes of first-trimester bleeding where viability was confirmed on ultrasound or the pregnancy continued beyond viability. DATA COLLECTION AND ANALYSIS: Two review authors independently selected studies and extracted data on study characteristics, quality and accuracy. Meta-analysis was performed using Review Manager software. MAIN OUTCOME MEASURES: The outcome was broadly categorised into maternal and perinatal outcomes. The chief maternal outcomes included pre-eclampsia/eclampsia or pregnancy-induced hypertension, antepartum haemorrhage, preterm prelabour rupture of membranes (PPROM) and mode of delivery. The perinatal outcomes evaluated were preterm delivery, low birthweight, intrauterine growth restriction, perinatal mortality, indicators of perinatal morbidity (Apgar scores and neonatal unit admission) and presence of congenital anomalies. MAIN RESULTS: Fourteen studies met the inclusion criteria. Women with threatened miscarriage had a significantly higher incidence of antepartum haemorrhage due to placenta praevia [odds ratio (OR) 1.62, 95% CI 1.19, 2.22] or antepartum haemorrhage of unknown origin (OR 2.47, 95% CI 1.52, 4.02) when compared with those without first-trimester bleeding. They were more likely to experience PPROM (OR 1.78, 95% CI 1.28, 2.48), preterm delivery (OR 2.05, 95% CI 1.76, 2.4) and to have babies with intrauterine growth restriction (OR 1.54, 95% CI 1.18, 2.00). First-trimester bleeding was associated with significantly higher rates of perinatal mortality (OR 2.15, 95% CI 1.41, 3.27) and low-birthweight babies (OR 1.83, 95% CI 1.48, 2.28). AUTHORS' CONCLUSIONS: Threatened miscarriage in the first trimester is associated with increased incidence of adverse maternal and perinatal outcome.


Assuntos
Ameaça de Aborto/epidemiologia , Resultado da Gravidez/epidemiologia , Ameaça de Aborto/diagnóstico , Feminino , Ruptura Prematura de Membranas Fetais/epidemiologia , Humanos , Hipertensão Induzida pela Gravidez/epidemiologia , Recém-Nascido , Mortalidade Perinatal , Pré-Eclâmpsia/epidemiologia , Gravidez , Primeiro Trimestre da Gravidez , Prognóstico , Hemorragia Uterina/epidemiologia
7.
Biochemistry ; 40(13): 3796-803, 2001 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-11300759

RESUMO

Binding of ATP to the catalytic domain of myosin induces a local conformational change which is believed to cause a major rotation of an 8.5 nm alpha-helix that is stabilized by the regulatory and essential light chains. Here we attempt to follow this rotation by measuring the mobility and orientation of a fluorescent probe attached near the C- or N-terminus of essential light chain 1 (LC1). Cysteine 178 of wild-type LC1, or Cys engineered near the N-terminus of mutant LC1, was labeled with tetramethylrhodamine and exchanged into skeletal subfragment-1 (S1) or into striated muscle fibers. In the absence of ATP, the fluorescence anisotropy (r) and the rotational correlation time (rho) of S1 reconstituted with LC1 labeled near the C-terminus were 0.195 and 66.6 ns, respectively. In the presence of ATP, r and rho increased to 0.233 and 233 ns, indicating considerable immobilization of the probe. A related parameter indicating the degree of order of cross-bridges in muscle fibers, Deltar, was small in rigor fibers (-0.009) and increased in relaxed fibers (0.030). For S1 reconstituted with LC1 labeled near the N-terminus, the steady-state anisotropy was 0.168 in rigor, and increased to 0.223 in relaxed state. In fibers, the difference in rigor was large (Deltar = 0.080), because of binding to the thin filaments, and decreased to 0.037 in relaxed fibers. These results suggest that before the power stroke, in the presence of ATP or its products of hydrolysis, the termini of LC1 are immobilized and ordered, and after the stroke, they become more mobile and partially disordered. The results are consistent with crystallographic structures that show that the level of putative stabilizing interactions of LC1 with the heavy chain of S1 in the transition state is reduced as the regulatory domain rotates to its post-power stroke position.


Assuntos
Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Galinhas , Polarização de Fluorescência , Corantes Fluorescentes/metabolismo , Microscopia Eletrônica , Proteínas Motores Moleculares/ultraestrutura , Dados de Sequência Molecular , Cadeias Leves de Miosina/ultraestrutura , Subfragmentos de Miosina/metabolismo , Subfragmentos de Miosina/ultraestrutura , Fragmentos de Peptídeos/ultraestrutura , Ligação Proteica , Coelhos
8.
Biochemistry ; 38(8): 2480-5, 1999 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-10029542

RESUMO

Skeletal myosin has two isoforms of the essential light chain (ELC), called LC1 and LC3, which differ only in their N-terminal amino acid sequence. The LC1 has 41 additional residues containing seven pairs of Ala-Pro, which form an elongated structure, and two pairs of lysines located near the N-terminus. When myosin subfragment-1 (S1) binds to actin, these lysines may interact with the C-terminus of actin and be responsible for the isoform specific properties of myosin. Here we employ cross-linking to identify the LC1 residues that are in contact with actin. S1 was reconstituted with various LC1 mutants and reacted with the zero-length cross-linker 1-ethyl-3-[3-dimethyl-aminopropyl]-carbodiimide (EDC). Cross-linking occurred only when actin was in molar excess over S1. Wild-type LC1 could be cross-linked through the terminal alpha-NH2 group, as well as via the two pairs of lysines. In a mutant ELC, where the lysines were deleted but two arginines were introduced near the N-terminus, the light chain could still be cross-linked via the terminal alpha-NH2 group. When the charge was reduced in the N-terminal region while retaining the Ala-Pro rich region, the mutant could not be cross-linked. These results suggest that as long as the N-terminus contains charged residues and an Ala-Pro rich extension, the binding between LC1 and actin can occur.


Assuntos
Actinas/metabolismo , Músculo Esquelético/metabolismo , Cadeias Leves de Miosina/metabolismo , Fragmentos de Peptídeos/metabolismo , Actinas/fisiologia , Alanina/genética , Alanina/fisiologia , Sequência de Aminoácidos , Animais , Ácido Aspártico/genética , Ácido Aspártico/fisiologia , Carbodi-Imidas/metabolismo , Galinhas , Reagentes de Ligações Cruzadas/metabolismo , Ácido Glutâmico/genética , Ácido Glutâmico/fisiologia , Lisina/genética , Lisina/fisiologia , Dados de Sequência Molecular , Músculo Esquelético/química , Mutagênese Sítio-Dirigida , Cadeias Leves de Miosina/genética , Fragmentos de Peptídeos/genética , Prolina/genética , Prolina/fisiologia , Deleção de Sequência
9.
J Biol Chem ; 273(28): 17671-9, 1998 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-9651364

RESUMO

The regulatory domain (RD), or neck region of the myosin head, consists of two classes of light chains that stabilize an alpha-helical segment of the heavy chain. RD from chicken skeletal muscle myosin was prepared in Escherichia coli by coexpression of a 9-kDa heavy chain fragment with the essential light chain. Recombinant regulatory light chain (RLC), wild type or mutant, was added separately to reconstitute the complex. The affinity of RD for divalent cations was determined by measuring the change in fluorescence of a pair of heavy chain tryptophans upon addition of calcium or magnesium. The complex bound divalent cations with high affinity, similar to the association constants determined for native myosin. The intrinsic fluorescence of the tryptophans could be used as a donor to measure the fluorescence resonance energy transfer distance to a single labeled cysteine engineered at position 2 on RLC. Dansylated Cys2 could also serve as a donor by preparing RLC with a second cysteine at position 79 which was labeled with an acceptor probe. These fluorescence resonance energy transfer distances (24-30 A), together with a previous measurement between Cys2 and Cys155 (Wolff-Long, V. L., Tao, T., and Lowey, S. (1995) J. Biol. Chem. 270, 31111-31118) suggest a location for the NH2 terminus of RLC that appears to preclude a direct interaction between the phosphorylatable serine and specific residues in the COOH-terminal domain.


Assuntos
Músculo Esquelético/metabolismo , Cadeias Leves de Miosina/metabolismo , Animais , Cátions Bivalentes , Embrião de Galinha , Corantes Fluorescentes , Sondas Moleculares , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/isolamento & purificação , Naftalenossulfonatos , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência , Triptofano/química
10.
J Mol Biol ; 279(2): 387-402, 1998 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-9642045

RESUMO

Changes in the orientation of the myosin regulatory light chain (RLC) in single muscle fibres were measured using polarised fluorescence from acetamidotetramethylrhodamine (ATR). Mutants of chicken skeletal RLC containing single cysteine residues at positions 2, 73, 94, 126 and 155 were labelled with either the 5 or 6-isomer of iodo-ATR, giving ten different probes. The labelled RLCs were exchanged into demembranated fibres from rabbit psoas muscle without significant effect on active force generation. Fluorescence polarisation measurements showed that nine out of the ten probe dipoles were more perpendicular to the fibre axis in the absence of ATP (in rigor) than in either relaxation or active contraction. The orientational distribution of the RLC region of the myosin head in active contraction is closer to the relaxed than to the rigor orientation, and is not equivalent to a linear combination of the relaxed and rigor orientations. Rapid length steps were applied to the fibres to synchronise the motions of myosin heads attached to actin. In active contraction the fluorescence polarisation changed both during the step, indicating elastic distortion of the RLC region of the myosin head, and during the subsequent rapid force recovery that is thought to signal the working stroke. The peak change in fluorescence polarisation produced by an active release of 5 nm per half sarcomere indicates an axial tilt of less than 5 degrees for all ten probes, if all the myosin heads in the fibre respond to the length step. This tilting was towards the rigor orientation for all ten probes, and could be explained by 14% of the heads moving to the rigor orientation. An active stretch tilted the heads away from the rigor conformation by a similar extent.


Assuntos
Corantes Fluorescentes , Contração Muscular/fisiologia , Músculo Esquelético/metabolismo , Cadeias Leves de Miosina/metabolismo , Animais , Sítios de Ligação , Galinhas , Cisteína/química , Polarização de Fluorescência , Corantes Fluorescentes/química , Técnicas In Vitro , Mutagênese Sítio-Dirigida , Cadeias Leves de Miosina/química , Cadeias Leves de Miosina/genética , Conformação Proteica , Coelhos , Rodaminas
11.
J Biol Chem ; 268(31): 23162-7, 1993 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-8226834

RESUMO

Multiple disulfide bonds form in recombinant myosin light chain-2 mutants that contain an engineered cysteine at positions 2, 73, or 94, in addition to the endogenous cysteines at residues 126 and 155 (Saraswat, L.D., and Lowey, S. (1991) J. Biol. Chem. 266, 1977). By replacing one of the native cysteines with an alanine, mutants with a single pair of thiols were created: Cys2/Cys155, Cys2/Cys126, Cys73/Cys155 and Cys94/Cys155. Oxidation of these mutants resulted in a fast migrating band on nonreducing SDS gels, which was attributed to an intramolecular disulfide bond. To determine if disulfide formation could also occur when the light chains (LC) are bound to the myosin heavy chains, LCs were added to myosin which had been depleted of its native LC2 by an immunoadsorbent. When the reconstituted myosin was reacted with 5,5'-dithiobis(2-nitrobenzoic acid) in the absence of divalent cations, intramolecular disulfide bonds formed in the mutant and wild type LCs, but the LCs did not remain bound to the myosin heavy chains. Addition of magnesium ions prevented LC dissociation, but intramolecular disulfide bonds no longer formed. Instead, mutants containing cysteines in the NH2-terminal domain formed intermolecular disulfide bonds between the two heads of myosin. The ability to cross-link the heads demonstrates the existence of close head/head interactions in the myosin molecule, a feature that may be essential for regulation.


Assuntos
Dissulfetos/química , Miosinas/química , Animais , Sequência de Bases , Cátions Bivalentes , Galinhas , Cisteína/química , Ácido Ditionitrobenzoico/química , Técnicas In Vitro , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Miosinas/genética , Oligodesoxirribonucleotídeos/química , Oxirredução , Relação Estrutura-Atividade
12.
J Biol Chem ; 267(29): 21112-8, 1992 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-1400421

RESUMO

The NH2- and COOH-terminal regions of the regulatory light chain (LC2) have been mapped to the head/rod junction by immunoelectron microscopy, using monoclonal and anti-fluorescyl antibodies as probes. In order to map the entire length of the light chain, we have engineered and purified mutants that contain a single cysteine residue at positions 2, 73, 94, 126, or 155. The single cysteine residues were labeled with either 5-iodoacetamido fluorescein or N-ethylmaleimide-biotin. We observed that the reactivity of Cys126 is far greater than that of Cys155, confirming that cysteine 126 is the fast-reacting thiol in wild-type light chain. The labeled light chains were exchanged into myosin stripped of its native LC2 by immunoaffinity chromatography, and the reconstituted myosin was reacted with anti-fluorescein antibody or avidin prior to rotary shadowing for electron microscopy. The position of the antibody or avidin was found to be near the head/rod junction for all mutants. These mapping studies, together with our finding that cysteines widely separated in the primary sequence can form multiple disulfide bonds (Saraswat, L.D., and Lowey, S. (1991) J. Biol. Chem. 226, 19777-19785), support a model for LC2 as a flexible, globular molecule localized mainly in the vicinity of the head/rod junction of myosin.


Assuntos
Cisteína , Miosinas/genética , Sequência de Aminoácidos , Animais , Complexo Antígeno-Anticorpo , Sequência de Bases , Galinhas , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Microscopia Eletrônica , Dados de Sequência Molecular , Músculos/fisiologia , Mutagênese Sítio-Dirigida , Miosinas/isolamento & purificação , Miosinas/ultraestrutura , Oligodesoxirribonucleotídeos , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/ultraestrutura
13.
J Biol Chem ; 266(29): 19777-85, 1991 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-1918082

RESUMO

Site-directed mutagenesis has been used to insert cysteine residues at specific locations in the myosin light chain 2 (LC2) sequence. The aim was to modify these cysteines with one or more spectroscopic probes and to reconstitute myosin with labeled light chains for structural studies. Native LC2 has two endogenous cysteine residues at positions 126 and 155; a third sulfhydryl was added by replacing either Pro2, Ser73, or Pro94 with cysteine. By oxidizing the endogenous cysteines to an intramolecular disulfide bond (Katoh, T., and Lowey, S., (1989) J. Cell Biol. 109, 1549), it was expected that the new cysteine could be selectively labeled with a fluorescent probe. This proved more difficult to accomplish than anticipated due to the formation of secondary disulfide bonds between the newly engineered cysteines and the native ones. Nevertheless, the unpaired cysteines were labeled with 5-(iodoacetamido)fluorescein, and singly labeled species were purified by ion-exchange chromatography. Chymotryptic digestion of the light chains, followed by high performance liquid chromatography separation of the peptides, led to the identification of the fluorescein-labeled cysteines. After light chain exchange into myosin, the position of the thiols was mapped by antifluorescyl antibodies in the electron microscope. Rotary-shadowed images showed the antibody bound at the head/rod junction of myosin for all the mutants. These mapping studies, together with the finding that widely separated cysteines can form multiple disulfide bonds, support a model for LC2 as a flexible, globular molecule that resembles other Ca/Mg-binding proteins in tertiary structure.


Assuntos
Cisteína/metabolismo , Mutação , Miosinas/genética , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Sequência de Bases , Galinhas , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Fluoresceína , Fluoresceínas , Corantes Fluorescentes , Engenharia Genética , Microscopia Eletrônica , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Miosinas/imunologia , Miosinas/ultraestrutura , Oxirredução , Plasmídeos , Conformação Proteica
14.
J Biol Chem ; 263(34): 18247-52, 1988 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-2848031

RESUMO

Photoaffinity labeling with 8-azidoadenosine 3':5'-monophosphate is a highly selective method for probing the cAMP-binding sites of the regulatory subunits of cAMP-dependent protein kinase and for identifying specific residues that are in close proximity to the cAMP-binding sites. The cAMP-binding site of a mutant RI-subunit has been characterized here and contrasted to the native RI-subunit. This mutant RI-subunit was generated by oligonucleotide-directed muta-genesis and lacks the entire second cAMP-binding domain which includes both of the residues, Trp260 and Tyr371, that are photolabeled in the native RI-subunit. The mutant RI-subunit, nevertheless, is photoaffinity-labeled with high efficiency, and the residue covalently modified was identified as Tyr244. The position of Tyr244 based on a computer graphic model of cAMP-binding site A is proposed and correlated with the presumed locations of Tyr371 and Trp260 in the native R-subunit. Photoaffinity labeling also can be used to detect functional cAMP-binding sites following electrophoretic transfer of the denatured protein to nitrocellulose. Labeling of the immobilized protein on nitrocellulose required a functional cAMP-binding site A that can be photoaffinity-labeled in solution based on the following criteria. 1) The type I R-subunit is photolabeled, whereas the type II R-subunit is not. A primary feature which distinguishes these two R-subunits is that the RI-subunit is photolabeled at both sites A and B, whereas covalent modification of the RII-subunit occurs only at site B. 2) The truncated mutant of the RI-subunit which lacks the entire second cAMP-binding domain can be photolabeled on nitrocellulose. 3) A mutant RI-subunit which can no longer be photolabeled in site B is still photolabeled on nitrocellulose. 4) A mutation which abolished cAMP binding to site A also abolished photoaffinity labeling after transfer to nitrocellulose.


Assuntos
Marcadores de Afinidade/metabolismo , Azidas/metabolismo , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , Proteínas Quinases/metabolismo , Animais , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Cinética , Substâncias Macromoleculares , Músculos/enzimologia , Miocárdio/enzimologia , Fragmentos de Peptídeos/isolamento & purificação , Ligação Proteica , Conformação Proteica , Suínos , Triptofano
15.
J Biol Chem ; 263(34): 18241-6, 1988 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-2848030

RESUMO

The regulatory subunit of cAMP-dependent protein kinase has a well-defined domain structure, and recombinant DNA techniques have been used to define further the functional properties that are associated with each domain. Our initial question was to define the minimal structural unit that is required for forming a stable complex with the catalytic subunit that will still bind and hence be dissociated by cAMP. To answer these questions, the entire second cAMP-binding domain was deleted using oligonucleotide-directed mutagenesis to introduce a premature stop codon at Trp260. This mutation results in the expression of a stable protein with an Mr of 38,000 based on polyacrylamide gel electrophoresis. The resulting mutant protein is a dimer; and like the native R-subunit, the two protomers of the dimer are cross-linked by disulfide bonds at the amino terminus. The mutant R-subunit binds 1 mol of cAMP/monomer based on equilibrium dialysis. The Kd(cAMP) was 25 nM, which is slightly higher than the Kd(cAMP) for the native R-subunit. The removal of the second cAMP domain does not prevent aggregation with the catalytic subunit, and the inactive holoenzyme complex that is formed in the absence of cAMP can still be dissociated and consequently activated by cAMP. In conjunction with previous results based on limited proteolysis, it is concluded that the region extending from Arg94 to Lys259 constitutes a structural unit that will be sufficient to interact with the catalytic subunit in a cAMP-dependent manner.


Assuntos
Deleção Cromossômica , Mutação , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Códon , AMP Cíclico/metabolismo , Genes , Substâncias Macromoleculares , Dados de Sequência Molecular , Peso Molecular , Miocárdio/enzimologia , Ligação Proteica , Proteínas Quinases/genética , Suínos
16.
FASEB J ; 2(11): 2677-85, 1988 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-3294077

RESUMO

Protein kinases represent a diverse family of enzymes that play critical roles in regulation. The simplest and best-understood biochemically is the catalytic (C) subunit of cAMP-dependent protein kinase, which can serve as a framework for the entire family. The amino-terminal portion of the C subunit constitutes a nucleotide binding site based on affinity labeling, labeling of lysines, and a conserved triad of glycines. The region beyond this nucleotide fold also contains essential residues. Modification of Asp 184 with a hydrophobic carbodiimide leads to inactivation, and this residue may function as a general base in catalysis. Despite the diversity of the kinase family, all share a homologous catalytic core, and the residues essential for nucleotide binding or catalysis in the C subunit are invariant in every protein kinase. Affinity labeling and intersubunit cross-linking have localized a portion of the peptide binding site, and this region is variable in the kinase family. The crystal structure of the C subunit also is being solved. The C subunit is maintained in its inactive state by forming a holoenzyme complex with an inhibitory regulatory (R) subunit. This R subunit has a well-defined domain structure that includes two tandem cAMP binding domains at the carboxy-terminus, each of which is homologous to the catabolite gene activator protein in Escherichia coli. Affinity labeling with 8N3 cAMP has identified residues that are in close proximity to the cAMP binding sites and is consistent with models of the cAMP binding sites based on the coordinates of the CAP crystal structure. An expression vector was constructed for the RI subunit and several mutations have been introduced. These mutations address 1) the major site of photoaffinity labeling, 2) a conserved arginine in the cAMP binding site, and 3) the consequences of deleting the entire second cAMP binding domain.


Assuntos
Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Animais , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica
17.
J Biol Chem ; 263(20): 9668-73, 1988 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-2898473

RESUMO

Each regulatory subunit of cAMP-dependent protein kinase has two tandem cAMP-binding sites, A and B, at the carboxyl terminus. Based on sequence homologies with the cAMP-binding domain of the Escherichia coli catabolite gene activator protein, a model has been constructed for each cAMP-binding domain. Two of the conserved features of each cAMP-binding site are an arginine and a glutamic acid which interact with the negatively charged phosphate and with the 2'-OH on the ribose ring, respectively. In the type I regulatory subunit, this arginine in cAMP binding site A is Arg-209. Recombinant DNA techniques have been used to change this arginine to a lysine. The resulting protein binds cAMP with a high affinity and associates with the catalytic subunit to form holoenzyme. The mutant holoenzyme also is activated by cAMP. However, the mutant R-subunit binds only 1 mol of cAMP/R-monomer. Photoaffinity labeling confirmed that the mutant R-subunit has only one functional cAMP-binding site. In contrast to the native R-subunit which is labeled at Trp-260 and Tyr-371 by 8-N3cAMP, the mutant R-subunit is convalently modified at a single site, Tyr-371, which correlates with a functional cAMP-binding site B. The lack of functional cAMP-binding site A also was confirmed by activating the mutant holoenzyme with analogs of cAMP which have a high specificity for either site A or site B. 8-NH2-methyl cAMP which preferentially binds to site B was similar to cAMP in its ability to activate both mutant and wild type holoenzyme whereas N6-monobutyryl cAMP, a site A-specific analog, was a very poor activator of the mutant holoenzyme. The results support the conclusions that 1) Arg-209 is essential for cAMP binding to site A and 2) cAMP binding to domain A is not essential for dissociation of the mutant holoenzyme.


Assuntos
AMP Cíclico/metabolismo , Proteínas Quinases/metabolismo , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/metabolismo , Marcadores de Afinidade , Sequência de Aminoácidos , Animais , Arginina/metabolismo , Azidas/metabolismo , Sítios de Ligação , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , DNA Recombinante , Ativação Enzimática/efeitos dos fármacos , Escherichia coli/genética , Glutamatos/metabolismo , Ácido Glutâmico , Lisina , Dados de Sequência Molecular , Mutação , Hibridização de Ácido Nucleico , Fosfatos/metabolismo , Fotoquímica , Proteínas Quinases/genética , Suínos
18.
Biochemistry ; 27(5): 1570-6, 1988 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-2835094

RESUMO

The regulatory (R) subunit of cAMP-dependent protein kinase I has been expressed in Escherichia coli, and oligonucleotide-directed mutagenesis was initiated in order to better understand structural changes that are induced as a consequence of cAMP-binding. Photoaffinity labeling of the type I holoenzyme with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) leads to the covalent modification of two residues, Trp-260 and Tyr-371 [Bubis, J., & Taylor, S.S. (1987) Biochemistry 26, 3478-3486]. The site that was targeted for mutagenesis was Tyr-371. The intention was to establish whether the interactions between the tyrosine ring and the adenine ring of cAMP are primarily hydrophobic in nature or whether the hydroxyl group is critical for cAMP binding and/or for inducing conformational changes. A single base change converted Tyr-371 to Phe. This yielded an R subunit that reassociated with the catalytic subunit to form holoenzyme and bound 2 mol of cAMP/mol of R monomer. The cAMP binding properties of the holoenzyme that was formed with this mutant R subunit, however, were altered: (a) the apparent Kd(cAMP) was shifted from 16 to 60 nM; (b) Scatchard plots showed no cooperativity between the cAMP binding sites in the mutant in contrast to the positive cooperativity that is observed for the wild-type holoenzyme; (c) the Hill coefficient of 1.6 for the wild-type holoenzyme was reduced to 0.99. The Ka's for activation by cAMP were altered in the mutant holoenzyme in a manner that was proportional to the shift in Kd(cAMP).(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Proteínas de Transporte/genética , Peptídeos e Proteínas de Sinalização Intracelular , Tirosina , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/metabolismo , AMP Cíclico/metabolismo , Cinética , Modelos Moleculares , Músculos/enzimologia , Mutação , Miocárdio/enzimologia , Ligação Proteica , Conformação Proteica , Suínos
20.
J Biol Chem ; 261(24): 11091-6, 1986 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-3525560

RESUMO

An expression vector has been constructed for the type I regulatory subunit of cAMP-dependent protein kinase. A cDNA clone for the bovine RI-subunit has been inserted into pUC7. When Escherichia coli JM105 was transformed with this plasmid, R-subunit was expressed in amounts that approached 4 mg/liter. The expressed protein was visualized in total cell extracts by photolabeling with 8-azidoadenosine 3':5'-mono[32P]phosphate following transfer from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose. Expression of R-subunit was independent of isopropyl-beta-D-thiogalactopyranoside. R-subunit accumulated in large amounts only in the stationary phase of growth, and the addition of isopropyl-beta-D-thiogalactopyranoside during the log phase of growth actually blocked the accumulation of R-subunit. Maximum expression (20 mg/liter) was achieved when E. coli 222 was transformed with the RI-containing plasmid. E. coli 222 is a strain that contains two mutations; it is cya- and also has a mutation in the catabolite gene activator protein (crp) that enables the protein to bind to DNA in the absence of cAMP. The expressed RI-subunit was a soluble, dimeric protein, and no significant proteolysis was apparent in the cell extract. The purified RI-subunit bound 2 mol of cAMP/mol of R monomer, reassociated with C-subunit to form holoenzyme, and migrated as a dimer on sodium dodecyl sulfate-polyacrylamide gels in the absence of reducing agents. The expressed protein was also susceptible to limited proteolysis, yielding a monomeric cAMP-binding fragment having a molecular weight of 35,000. In all of these properties, the expressed protein was indistinguishable from RI purified from bovine tissue even though the R-subunit expressed in E. coli represents a fusion protein that contains 10 additional amino acids at the amino terminus that are provided by the lac Z' gene of the vector. This NH2-terminal sequence was confirmed by amino acid sequencing.


Assuntos
Escherichia coli/enzimologia , Proteínas Quinases/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Marcadores de Afinidade , Animais , Azidas/metabolismo , Bovinos , Clonagem Molecular , DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica , Substâncias Macromoleculares , Fotoquímica , Plasmídeos
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