RESUMO
BACKGROUND: The prognostic factors for patients with full-thickness rotator cuff tears (RCTs) include tear size, muscle atrophy and fatty infiltration. However, the influence of early coexisting degenerative changes on RCT outcomes is unappreciated. The purpose of this study was to calculate the impact that pre-existing partial glenohumeral cartilaginous changes have on patients undergoing arthroscopic RCT repair. METHODS: A study of 54 patients undergoing arthroscopic RCT repair was undertaken. The presence of co-existing patches of glenohumeral degenerative cartilaginous changes and RCT size was recorded at surgery. Pre- and postoperative outcomes were assessed using traditional (Oxford Shoulder Score [OSS], 5-level EuroQol-5D [EQ-5D-5L] questionnaire and EuroQol visual analog scale [EQ-VAS]) and patient-centric re-formatted prisms. Outcomes were assessed as an entire dataset, and sub-group analysis was performed according to the grade of co-existing arthritis and tear size. RESULTS: Significant improvements (p<0.05) in clinical outcomes were recognized when assessed using either the traditional or reformatted prisms (average % improvements in OSS, EQ-5D-5L and EQ-VAS were 47%, 33% and 43%, respectively; average improvements in pain, function, and psychological well-being were 48%, 33% and, 29%, respectively). Positive gain was noted in all sub-groups of arthritic grading and tear size. CONCLUSIONS: Good clinical outcomes can be achieved following RCT repair even in the presence of local partial degenerative cartilage changes and advancing tear size. These benefits are patient-centered but require RCT repairability.
RESUMO
SLC4A11 is a H+/NH3/water transport protein, of corneal endothelial cells. SLC4A11 mutations cause congenital hereditary endothelial dystrophy and some cases of Fuchs endothelial corneal dystrophy. To probe SLC4A11's roles, we compared gene expression in RNA from corneas of 17-week-old slc4a11-/- (n = 3) and slc4a11+/+ mice (n = 3) and subjected to RNA sequencing. mRNA levels for a subset of genes were also assessed by quantitative real-time reverse transcription PCR (qRT RT-PCR). Cornea expressed 13,173 genes, which were rank-ordered for their abundance. In slc4a11-/- corneas, 100 genes had significantly altered expression. Abundant slc14a1 expression, encoding the urea transporter UT-A, suggests a significant role in the cornea. The set of genes with altered expression was subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, revealing that alterations clustered into extracellular region, cytoskeleton, cell adhesion and plasma membrane functions. Gene expression changes further clustered into classes (with decreasing numbers of genes): cell fate and development, extracellular matrix and cell adhesion, cytoskeleton, ion homeostasis and energy metabolism. Together these gene changes confirm earlier suggestions of a role of SLC4A11 in ion homeostasis, energy metabolism, cell adhesion, and reveal an unrecognized SLC4A11 role in cytoskeletal organization.
Assuntos
Proteínas de Transporte de Ânions/genética , Córnea/fisiologia , Expressão Gênica/genética , Simportadores/genética , Animais , Adesão Celular/genética , Membrana Celular/genética , Células Endoteliais/fisiologia , Endotélio Corneano/fisiologia , Células Epiteliais/fisiologia , Matriz Extracelular/genética , Regulação da Expressão Gênica/genética , Transporte de Íons/genética , Masculino , Camundongos , Mutação/genéticaRESUMO
Objective: This study provides independent examination of the validity of the Barkley Deficits of Executive Functioning Scale-Children and Adolescents (BDEFS-CA) in a sample of children diagnosed with ADHD (n = 50) and typically developing controls (n = 50). Method: Parents of participants completed the BDEFS-CA and the Conners 3 rating scales. Validity of BDEFS-CA was examined using a confirmatory factor analysis, correlational analyses with Conners 3 ratings, and receiver operating characteristic (ROC) curve analysis of diagnostic accuracy. Results: Findings support the construct, concurrent, and discriminant validity of the BDEFS-CA in a mixed sample. Conclusion: Findings provide independent examination of the validity of the BDEFS-CA as a measure of executive dysfunction and a screening tool for ADHD.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade , Adolescente , Transtorno do Deficit de Atenção com Hiperatividade/diagnóstico , Criança , Função Executiva , Análise Fatorial , Humanos , Programas de Rastreamento , Pais , Escalas de Graduação PsiquiátricaRESUMO
Over 200 million people worldwide are exposed to the human carcinogen, arsenic, in contaminated drinking water. In laboratory animals, arsenic and the essential trace element, selenium, can undergo mutual detoxification through the formation of the seleno-bis(S-glutathionyl) arsinium ion [(GS)2AsSe]-, which undergoes biliary and fecal elimination. [(GS)2AsSe]-, formed in animal red blood cells (RBCs), sequesters arsenic and selenium, and slows the distribution of both compounds to peripheral tissues susceptible to toxic effects. In human RBCs, the influence of arsenic on selenium accumulation, and vice versa, is largely unknown. The study aims were to characterize arsenite (AsIII) and selenite (SeIV) uptake by human RBCs, to determine if SeIV and AsIII increase the respective accumulation of the other in human RBCs, and ultimately to determine if this occurs through the formation and sequestration of [(GS)2AsSe]-. 75SeIV accumulation was temperature and Cl--dependent, inhibited by 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS) (IC50 1 ± 0.2 µM), and approached saturation at 30 µM, suggesting uptake is mediated by the erythrocyte anion-exchanger 1 (AE1 or Band 3, gene SLC4A1). HEK293 cells overexpressing AE1 showed concentration-dependent 75SeIV uptake. 73AsIII uptake by human RBCs was temperature-dependent, partly reduced by aquaglyceroporin 3 inhibitors, and not saturated. AsIII increased 75SeIV accumulation (in the presence of albumin) and SeIV increased 73AsIII accumulation in human RBCs. Near-edge X-ray absorption spectroscopy revealed the formation of [(GS)2AsSe]- in human RBCs exposed to both AsIII and SeIV. The sequestration of [(GS)2AsSe]- in human RBCs potentially slows arsenic distribution to susceptible tissues and could reduce arsenic-induced disease.
Assuntos
Arsenitos/sangue , Eritrócitos/metabolismo , Glutationa/sangue , Ácido Selenioso/sangue , Arsenitos/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Relação Dose-Resposta a Droga , Eritrócitos/efeitos dos fármacos , Células HEK293 , Humanos , Ácido Selenioso/farmacologia , Espectroscopia por Absorção de Raios X/métodosRESUMO
Background: A quarter of the population present at least once a year with a musculoskeletal disorder. Primary hip osteoarthritis is a high-volume condition with significant clinical need and population-level costs. There remains much variation in patient outcomes and care delivery costs for this condition. Aims: The study aimed to gauge if pathway redesign based on the principles of value-based healthcare (VBHC) could increase value. The aim was to calculate the value of treatment for primary hip osteoarthritis through measuring outcomes that matter to patients, as well as the costs of delivering them. Additionally it aimed to compare two care pathways to identify which elements may better promote the delivery of high-value clinical care. Methods: Two care models were evaluated: the first being a traditional model with multiple entry points and without pathway standardisation, and the second an intentionally designed standardised multidisciplinary pathway. Mandated National Health Service patient-reported outcomes were assessed but were restructured into a patient-centred format to assess the impact on pain, function and psychological outcomes. Patient-level pathway economic evaluation was performed. Using these data, outcomes were mapped against cost to calculate value. Results: There were no significant differences in clinical outcomes between the two models. The intentionally designed model delivered better value care, having lower pathway costs. This model produced a small but inconsistent positive financial margin. Conclusions: Intentionally designed, integrated elective services offer an opportunity to develop and evaluate VBHC models. Analysis of two care pathways from a VBHC perspective demonstrated that an intentionally designed pathway had higher value. The higher value pathway maximised the benefits of having physiotherapists and orthopaedic surgeons working side by side. Developing and measuring patient-orientated outcomes and performing accurate economic evaluation are the key to understanding and achieving better value care.
Assuntos
Artroplastia de Quadril/economia , Osteoartrite do Quadril/cirurgia , Qualidade da Assistência à Saúde/normas , Idoso , Artroplastia de Quadril/estatística & dados numéricos , Estudos de Coortes , Custos e Análise de Custo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite do Quadril/economia , Qualidade da Assistência à Saúde/estatística & dados numéricos , Estudos Retrospectivos , Medicina Estatal/organização & administração , Medicina Estatal/estatística & dados numéricosRESUMO
SLC4A11 mutations cause cases of congenital hereditary endothelial dystrophy (CHED), Harboyan syndrome (HS), and Fuchs endothelial corneal dystrophy (FECD). Defective water reabsorption from corneal stroma by corneal endothelial cells (CECs) leads to these corneal dystrophies. SLC4A11, in the CEC basolateral membrane, facilitates transmembrane movement of H2 O, NH3 , and H+ -equivalents. Some SLC4A11 disease mutants have impaired folding, leading to a failure to move to the cell surface, which in some cases can be corrected by the drug, glafenine. To identify SLC4A11 mutants that are targets for folding-correction therapy, we examined 54 SLC4A11 missense mutants. Cell-surface trafficking was assessed on immunoblots, by the level of mature, high molecular weight, cell surface-associated form, and using a bioluminescence resonance energy transfer assay. Low level of cell surface trafficking was found in four out of 18 (20%) of FECD mutants, 19/ out of 31 (61%) of CHED mutants, and three out of five (60%) of HS mutants. Amongst ER-retained mutants, 16 showed increased plasma membrane trafficking when grown at 30°C, suggesting that their defect has potential for rescue. CHED-causing point mutations mostly resulted in folding defects, whereas the majority of FECD missense mutations did not affect trafficking, implying functional impairment. We identified mutations that make patients candidates for folding correction of their corneal dystrophy.
Assuntos
Proteínas de Transporte de Ânions/genética , Antiporters/genética , Distrofia Endotelial de Fuchs/genética , Mutação de Sentido Incorreto/genética , Medicina de Precisão , Sequência de Aminoácidos , Animais , Proteínas de Transporte de Ânions/química , Antiporters/química , Membrana Celular/metabolismo , Temperatura Baixa , Cães , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Modelos Moleculares , FenótipoRESUMO
Lacking protein synthesis machinery and organelles necessary for autophagy or apoptosis, aged red blood cells (RBCs) are marked by circulating auto-antibodies for macrophage-mediated clearance. The antigen recognized by these auto-antibodies is the major protein of the RBC membrane, Band 3. To ensure regulation and specificity in clearance, the molecular "clock" must mark senescent cells in a way that differentiates them from younger cells, to prevent premature clearance. Predominant models of Band 3 senescence signaling are reviewed, and merits are discussed in light of the recently published crystal structure of the Band 3 membrane domain. © 2017 IUBMB Life, 70(1):32-40, 2018.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/química , Autoanticorpos/química , Autoantígenos/química , Epitopos/química , Eritrócitos/química , Proteínas Opsonizantes/química , Proteína 1 de Troca de Ânion do Eritrócito/sangue , Autoanticorpos/sangue , Autoantígenos/sangue , Sítios de Ligação de Anticorpos , Senescência Celular , Epitopos/sangue , Eritrócitos/citologia , Eritrócitos/imunologia , Humanos , Transporte de Íons , Macrófagos/imunologia , Proteínas Opsonizantes/sangue , Fagocitose/fisiologia , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Transdução de Sinais , Fatores de TempoRESUMO
The crystal structure of the dimeric membrane domain of human Band 3(1), the red cell chloride/bicarbonate anion exchanger 1 (AE1, SLC4A1), provides a structural context for over four decades of studies into this historic and important membrane glycoprotein. In this review, we highlight the key structural features responsible for anion binding and translocation and have integrated the following topological markers within the Band 3 structure: blood group antigens, N-glycosylation site, protease cleavage sites, inhibitor and chemical labeling sites, and the results of scanning cysteine and N-glycosylation mutagenesis. Locations of mutations linked to human disease, including those responsible for Southeast Asian ovalocytosis, hereditary stomatocytosis, hereditary spherocytosis, and distal renal tubular acidosis, provide molecular insights into their effect on Band 3 folding. Finally, molecular dynamics simulations of phosphatidylcholine self-assembled around Band 3 provide a view of this membrane protein within a lipid bilayer.
Assuntos
Desequilíbrio Ácido-Base/sangue , Acidose Tubular Renal/sangue , Anemia Hemolítica Congênita/sangue , Proteína 1 de Troca de Ânion do Eritrócito/química , Eliptocitose Hereditária/sangue , Erros Inatos do Metabolismo/sangue , Esferocitose Hereditária/sangue , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Desequilíbrio Ácido-Base/genética , Desequilíbrio Ácido-Base/patologia , Acidose Tubular Renal/genética , Acidose Tubular Renal/patologia , Anemia Hemolítica Congênita/genética , Anemia Hemolítica Congênita/patologia , Proteína 1 de Troca de Ânion do Eritrócito/antagonistas & inibidores , Proteína 1 de Troca de Ânion do Eritrócito/genética , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Bicarbonatos/metabolismo , Eliptocitose Hereditária/genética , Eliptocitose Hereditária/patologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Eritrócitos/patologia , Eritrócitos Anormais/patologia , Expressão Gênica , Glicosilação , Humanos , Ligantes , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/patologia , Mutação , Ligação Proteica , Esferocitose Hereditária/genética , Esferocitose Hereditária/patologiaRESUMO
Bicarbonate (HCO3(-)) has a central place in human physiology as the waste product of mitochondrial energy production and for its role in pH buffering throughout the body. Because bicarbonate is impermeable to membranes, bicarbonate transport proteins are necessary to enable control of bicarbonate levels across membranes. In humans, 14 bicarbonate transport proteins, members of the SLC4 and SLC26 families, function by differing transport mechanisms. In addition, some anion channels and ZIP metal transporters contribute to bicarbonate movement across membranes. Defective bicarbonate transport leads to diseases, including systemic acidosis, brain dysfunction, kidney stones, and hypertension. Altered expression levels of bicarbonate transporters in patients with breast, colon, and lung cancer suggest an important role of these transporters in cancer.
Assuntos
Bicarbonatos/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Modelos Moleculares , Neoplasias/metabolismo , Filogenia , Simportadores de Sódio-Bicarbonato/metabolismo , Animais , Dióxido de Carbono/metabolismo , Antiportadores de Cloreto-Bicarbonato/genética , Humanos , Camundongos , Simportadores de Sódio-Bicarbonato/genéticaRESUMO
BACKGROUND: Carbonic anhydrase enzymes (CA) catalyze the reversible hydration of carbon dioxide to bicarbonate in mammalian cells. Trans-membrane transport of CA-produced bicarbonate contributes significantly to cellular pH regulation. A body of evidence implicates pH-regulatory processes in the hypertrophic growth pathway characteristic of hearts as they fail. In particular, Na+/H+ exchange (NHE) activation is pro-hypertrophic and CA activity activates NHE. Recently Cardrase (6-ethoxyzolamide), a CA inhibitor, was found to prevent and revert agonist-stimulated cardiac hypertrophy (CH) in cultured cardiomyocytes. Our goal thus was to determine whether hypertrophied human hearts have altered expression of CA isoforms. METHODS: We measured CA expression in hypertrophied human hearts to begin to examine the role of carbonic anhydrase in progression of human heart failure. Ventricular biopsies were obtained from patients undergoing cardiac surgery (CS, n = 14), or heart transplantation (HT, n = 13). CS patients presented mild/moderate concentric left ventricular hypertrophy and normal right ventricles, with preserved ventricular function; ejection fractions were ~60%. Conversely, HT patients with failing hearts presented CH or ventricular dilation accompanied by ventricular dysfunction and EF values of 20%. Non-hypertrophic, non-dilated ventricular samples served as controls. RESULTS: Expression of atrial and brain natriuretic peptide (ANP and BNP) were markers of CH. Hypertrophic ventricles presented increased expression of CAII, CAIV, ANP, and BNP, mRNA levels, which increased in failing hearts, measured by quantitative real-time PCR. CAII, CAIV, and ANP protein expression also increased approximately two-fold in hypertrophic/dilated ventricles. CONCLUSIONS: These results, combined with in vitro data that CA inhibition prevents and reverts CH, suggest that increased carbonic anhydrase expression is a prognostic molecular marker of cardiac hypertrophy.
Assuntos
Anidrases Carbônicas/genética , Insuficiência Cardíaca/genética , Hipertrofia Ventricular Esquerda/genética , Miocárdio/enzimologia , Idoso , Animais , Fator Natriurético Atrial/genética , Biópsia , Anidrase Carbônica II/genética , Anidrase Carbônica IV/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Marcadores Genéticos , Células HEK293 , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Hipertrofia Ventricular Esquerda/enzimologia , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Isoenzimas , Masculino , Camundongos , Pessoa de Meia-Idade , Miocárdio/patologia , Peptídeo Natriurético Encefálico/genética , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Volume Sistólico , Transfecção , Função Ventricular EsquerdaRESUMO
Pathological cardiac hypertrophy, the maladaptive remodelling of the myocardium, often progresses to heart failure. The sodium-proton exchanger (NHE1) and chloride-bicarbonate exchanger (AE3) have been implicated as important in the hypertrophic cascade. Carbonic anhydrase II (CAII) provides substrates for these transporters (protons and bicarbonate, respectively). CAII physically interacts with NHE1 and AE3, enhancing their respective ion transport activities by increasing the concentration of substrate at their transport sites. Earlier studies found that a broad-spectrum carbonic anhydrase inhibitor prevented cardiomyocyte hypertrophy (CH), suggesting that carbonic anhydrase is important in the development of hypertrophy. Here we investigated whether cytosolic CAII was the CA isoform involved in hypertrophy. Neonatal rat ventricular myocytes (NRVMs) were transduced with recombinant adenoviral constructs to over-express wild-type or catalytically inactive CAII (CAII-V143Y). Over-expression of wild-type CAII in NRVMs did not affect CH development. In contrast, CAII-V143Y over-expression suppressed the response to hypertrophic stimuli, suggesting that CAII-V143Y behaves in a dominant negative fashion over endogenous CAII to suppress hypertrophy. We also examined CAII-deficient (Car2) mice, whose hearts exhibit physiological hypertrophy without any decrease in cardiac function. Moreover, cardiomyocytes from Car2 mice do not respond to prohypertrophic stimulation. Together, these findings support a role of CAII in promoting CH.
Assuntos
Anidrase Carbônica II/metabolismo , Cardiomegalia/enzimologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Animais , Cardiomegalia/genética , Cardiomegalia/patologia , Antiportadores de Cloreto-Bicarbonato/genética , Antiportadores de Cloreto-Bicarbonato/metabolismo , Expressão Gênica/genética , Técnicas de Transferência de Genes , Ventrículos do Coração/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenilefrina/metabolismo , Ratos , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismoRESUMO
AE1, which exists in the erythrocyte plasma membrane as a noncovalent dimer, facilitates transmembrane Clâ»/HCO3â» exchange. Here a concatamer of AE1 (two AE1 monomers fused via a two-residue linker to form an intramolecular dimer) was designed to facilitate fluorescence resonance energy transfer (FRET) studies. The concatameric protein (AE1·AE1) was expressed at the plasma membrane at levels similar to that of wild-type AE1 and had Clâ»/HCO3â» exchange activity indistinguishable from that of wild-type AE1. Nondenaturing gel electrophoresis revealed that AE1·AE1 does not associate into higher-order oligomers when expressed in HEK293 cells and Xenopus laevis oocytes. The cysteine-less concatamer (AE1·AE1-Câ») enabled introduction of unique cysteine residues into the whole intramolecular dimer. AE1(Q434C)·AE1(Q434C)-Câ», with a single cysteine residue in each AE1 subunit, was labeled with the donor Alexa Fluor 488 C(5)-maleimide (AF) and the acceptor tetramethylrhodamine methanethiosulfonate (TMR-MTS). Energy transfer efficiency revealed that the distance between these residues in the AE1 dimer is 49 ± 5 Å. The 72% FRET efficiency observed between AE1(Q434C)·AE1-Câ» labeled with AF and the lipid bilayer labeled with 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate indicates that Q434 is less than 33 Å from the lipid bilayer. We thus provide two distance constraints for the position of Q434, which is located in extracellular loop 1, connecting the first two transmembrane segments of AE1.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/química , Transferência Ressonante de Energia de Fluorescência/métodos , Animais , Proteína 1 de Troca de Ânion do Eritrócito/genética , Linhagem Celular , Cisteína , Membrana Eritrocítica , Humanos , Proteínas de Membrana , Oócitos , Conformação Proteica , Engenharia de Proteínas , Multimerização Proteica , Xenopus laevisRESUMO
Association of some plasma membrane bicarbonate transporters with carbonic anhydrase enzymes forms a bicarbonate transport metabolon to facilitate metabolic CO(2)-HCO(3)(-) conversions and coupled HCO(3)(-) transport. The transmembrane carbonic anhydrase, CAIX, with its extracellular catalytic site, is highly expressed in parietal and other cells of gastric mucosa, suggesting a role in acid secretion. We examined in transfected HEK293 cells the functional and physical interactions between CAIX and the parietal cell Cl(-)/HCO(3)(-) exchanger AE2 or the putative Cl(-)/HCO(3)(-) exchanger SLC26A7. Coexpression of CAIX increased AE2 transport activity by 28 +/- 7% and also activated transport mediated by AE1 and AE3 (32 +/- 10 and 37 +/- 9%, respectively). In contrast, despite a transport rate comparable to that of AE3, coexpressed CAIX did not alter transport associated with SLC26A7. The CAIX-associated increase of AE2 activity did not result from altered AE2 expression or cell surface processing. CAIX was coimmunoprecipitated with the coexpressed SLC4 polypeptides AE1, AE2, and AE3, but not with SLC26A7. GST pull-down assays with a series of domain-deleted forms of CAIX revealed that the catalytic domain of CAIX mediated interaction with AE2. AE2 and CAIX colocalized in human gastric mucosa, as indicated by coimmunofluorescence. This is the first example of a functional and physical interaction between a bicarbonate transporter and a transmembrane carbonic anhydrase. We conclude that CAIX can bind to some Cl(-)/HCO(3)(-) exchangers to form a bicarbonate transport metabolon.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Proteínas de Transporte de Ânions/metabolismo , Antígenos de Neoplasias/metabolismo , Antiporters/metabolismo , Bicarbonatos/metabolismo , Anidrases Carbônicas/metabolismo , Membrana Celular/metabolismo , Cloretos/metabolismo , Animais , Proteína 1 de Troca de Ânion do Eritrócito/genética , Proteínas de Transporte de Ânions/genética , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Antiporters/genética , Anidrase Carbônica IX , Anidrases Carbônicas/química , Anidrases Carbônicas/genética , Domínio Catalítico , Linhagem Celular , Membrana Celular/enzimologia , Mucosa Gástrica/enzimologia , Mucosa Gástrica/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Mutação , Células Parietais Gástricas/enzimologia , Células Parietais Gástricas/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Proteínas SLC4A , Fatores de Tempo , TransfecçãoRESUMO
Integral membrane proteins of the plasma membrane span from the inside to the outside of the cell. The primary structural element of integral membrane proteins is their topology: the pattern in which the protein traverses the membrane. A full description of topology, defining which parts of the protein face outside versus inside, goes a long way toward understanding the folding of these proteins. Many approaches have been established to define membrane protein topology. Here, we present the technique of scanning cysteine accessibility mutagenesis (SCAM). This approach uses the unique chemical reactivity of the cysteine sulfhydryl to probe membrane protein structure. Individual cysteine residues are introduced into the target protein by mutagenesis. The ability to chemically react these residues using sulfhydryl-directed reagents (either membrane permeant or impermeant) defines each site as either extracellular or intracellular, thus establishing topology of a location. This analysis performed on many sites in the protein will define the protein's topology.
Assuntos
Bioquímica/métodos , Cisteína/química , Proteínas de Membrana/química , Mutagênese Sítio-Dirigida/métodos , Células Cultivadas , Humanos , Rim/citologia , Dobramento de Proteína , Estrutura Terciária de Proteína , Reagentes de Sulfidrila/químicaRESUMO
Myocardial pH(i) recovery from intracellular alkalization results in part from the acid load (-J(H+)) carried by Cl(-)/HCO(3)(-) anion-exchangers (AE). Three AE isoforms, AE1, AE2 and AE3, have been identified in cardiac membranes, but the function of each isoform on pH(i) homeostasis is still under investigation. This work explored, by means of specific antibodies, the role of AE3 isoform in myocardial pH(i) regulation. We developed rabbit polyclonal antibodies against the extracellular "loops": one connecting the fifth to sixth and the other one the seventh to eighth transmembrane domains (loops 3 and 4, respectively) of AE3, and their effect on pH(i) regulation was studied in rat papillary muscles. The anti-AE3 loop 3 antibody decreased -J(H+) in response to myocardial alkalization (from a mean control value of 1.06+/-0.26 to 0.32+/-0.13 mmol/L/min, n=7, P<0.05) without affecting the baseline pH(i) (7.22+/-0.03 vs. 7.21+/-0.04). The anti-AE3 loop 4 antibody did not modify either pH(i) recovery or baseline pH(i). Under control conditions, endothelin-1 (ET-1) increased -J(H+) in response to myocardial alkalization from 1.30+/-0.18 to 2.01+/-0.33 mmol/L /min (n=5, P<0.05). This effect of ET-1 on -J(H+) was abolished by anti-AE3 loop 3 antibody. In addition, the MgATP-induced stimulation of AE activity was reduced by the anti-AE3 loop 3 antibody. These data support the key role of the AE3 isoform in myocardial pH(i) recovery from alkaline loads and also in the stimulatory effect of ET-1 on AE activity. To a lesser extent, it may also contribute to the effect of MgATP on pH(i).
Assuntos
Alcalose/metabolismo , Antiporters/antagonistas & inibidores , Antiporters/metabolismo , Miocárdio/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Anticorpos Bloqueadores/farmacologia , Especificidade de Anticorpos , Antiporters/agonistas , Soluções Tampão , Reações Cruzadas , Endotelina-1/farmacologia , Glutationa/metabolismo , Concentração de Íons de Hidrogênio , Masculino , Membranas/efeitos dos fármacos , Membranas/metabolismo , Coelhos , Ratos , Ratos Wistar , Estimulação QuímicaRESUMO
A detailed inhibition study of five carbonic anhydrase (CA, EC 4.2.1.1) isozymes with carboxylates including aliphatic (formate, acetate), dicarboxylic (oxalate, malonate), hydroxy/keto acids (l-lactate, l-malate, pyruvate), tricarboxylic (citrate), or aromatic (benzoate, tetrafluorobenzoate) representatives, some of which are important intermediates in the Krebs cycle, is presented. The cytosolic isozyme hCA I was strongly activated by acetate, oxalate, pyruvate, l-lactate, and citrate (K(A) around 0.1 microM), whereas formate, malonate, malate, and benzoate were weaker activators (K(A) in the range 0.1-1mM). The cytosolic isozyme hCA II was weakly inhibited by all the investigated anions, with inhibition constants in the range of 0.03-24 mM. The membrane-associated isozyme hCA IV was the most sensitive to inhibition by carboxylates, showing a K(I) of 99 nM for citrate and oxalate, of 2.8 microM for malonate and of 14.5 microM for pyruvate among others. The mitochondrial isozyme hCA V was weakly inhibited by all these carboxylates (K(I)s in the range of 1.67-25.9 mM), with the best inhibitor being citrate (K(I) of 1.67 mM), whereas this is the most resistant CA isozyme to pyruvate inhibition (K(I) of 5.5mM), which may be another proof that CA V is the isozyme involved in the transfer of acetyl groups from the mitochondrion to the cytosol for the provision of substrate(s) for de novo lipogenesis. Furthermore, the relative resistance of CA V to inhibition by pyruvate may be an evolutionary adaptation of this mitochondrial isozyme to the presence of high concentrations of this anion within this organelle. The transmembrane, tumor-associated isozyme hCA IX was similar to isozyme II in its slight inhibition by all these anions (K(I) in the range of 1.12-7.42 mM), except acetate, lactate, and benzoate, which showed a K(I)>150 mM. The lactate insensitivity of CA IX also represents an interesting finding, since it is presumed that this isozyme evolved in such a way as to show a high catalytic activity in hypoxic tumors rich in lactate, and suggests a possible metabolon in which CA IX participates together with the monocarboxylate/H(+) co-transporter in dealing with the high amounts of lactate/H(+) present in tumors.
Assuntos
Inibidores da Anidrase Carbônica/química , Ácidos Carboxílicos/química , Antígenos de Neoplasias , Transporte Biológico , Anidrase Carbônica I/antagonistas & inibidores , Anidrase Carbônica II/antagonistas & inibidores , Anidrase Carbônica IV/antagonistas & inibidores , Anidrase Carbônica IX , Anidrase Carbônica V/antagonistas & inibidores , Anidrases Carbônicas , Citosol/metabolismo , Humanos , Isoenzimas/antagonistas & inibidores , Mitocôndrias/metabolismo , Relação Estrutura-AtividadeRESUMO
A series of positively charged sulfonamides were obtained by reaction of aminobenzolamide [5-(4-aminobenzenesulfonylamino)-1,3,4-thiadiazole-2-sulfonamide] with tri-/tetrasubstituted pyrilium salts possessing alkyl-, aryl- or combinations of alkyl and aryl groups at the pyridinium ring. The new compounds reported here were assayed for the inhibition of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes: the cytosolic hCA I and II, the membrane-anchored bCA IV, and the membrane-bound, tumor-associated isozyme hCA IX. They showed potent inhibitory activity against all investigated isozymes, although with different profiles. For CA I the new derivatives showed inhibition constants in the range of 3-12 nM, for CA II in the range of 0.20-5.96 nM, against CA IV in the range of 2.0-10.3 nM, and against CA IX in the range of 3-45 nM, respectively. These new compounds are membrane-impermeant due to their salt-like character. Some of these derivatives were also tested for their inhibitory activity against the Cl(-)/HCO(3)(-) anion exchanger AE1: two derivatives showed inhibitory activity in the low micromolar range, whereas one compound was inactive at these concentrations. The high affinity of these new derivatives for the tumor-associated isozyme CA IX and their membrane impermeability make this type of CA inhibitor interesting candidates for the selective inhibition of only the tumor-associated isozyme and not the cytosolic ones, for which they also show high potency. Furthermore, we prove here for the first time that the CA-AE metabolon can be inhibited by the same type of sulfonamide derivative.
Assuntos
Inibidores da Anidrase Carbônica/síntese química , Compostos de Piridínio/síntese química , Sulfonamidas/síntese química , Antígenos de Neoplasias/química , Anidrase Carbônica IX , Inibidores da Anidrase Carbônica/química , Inibidores da Anidrase Carbônica/metabolismo , Anidrases Carbônicas/química , Membrana Eritrocítica/metabolismo , Humanos , Técnicas In Vitro , Isoenzimas/antagonistas & inibidores , Permeabilidade , Compostos de Piridínio/química , Compostos de Piridínio/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/químicaRESUMO
AE1 facilitates Cl-/HCO3- exchange across the erythrocyte membrane. To identify residues involved in substrate selection and translocation, we prepared an array of single cysteine mutants in an otherwise cysteineless background. These mutants spanning the C-terminal portion of the AE1 membrane domain from Phe806-Cys885 were characterized for functional activity when expressed in human embryonic kidney 293 cells by measurement of changes of intracellular pH associated with bicarbonate transport. To identify residues involved in substrate translocation, transport activity was assessed for each mutant before and after treatment with the following sulfhydryl reagents: anionic para-chloromercuibenzenesulfonate; permeant (2-aminoethyl)methanethiosulfonate; and cationic [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET). Among the 80 mutants, only certain key residues in the Val849-Leu863 region were inhibited by the sulfhydryl reagent, consistent with direct involvement of these sites in anion transport. In the last two transmembrane segments, only mutants in the extracellular portion of the transmembrane segments could be inhibited by sulfhydryl reagent, suggesting that the outer portions line the translocation channel and the inner portions have some other role. Sensitivity to cationic MTSET and effects of Cl- identified the substrate charge filter as Ser852-Leu857.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Eritrócitos/metabolismo , Proteína 1 de Troca de Ânion do Eritrócito/genética , Ânions/metabolismo , Cisteína , Humanos , Mesilatos , Mutação , Transporte Proteico , Especificidade por SubstratoRESUMO
Human NBC3 is an electroneutral Na(+)/HCO(3)(-) cotransporter expressed in heart, skeletal muscle, and kidney in which it plays an important role in HCO(3)(-) metabolism. Cytosolic enzyme carbonic anhydrase II (CAII) catalyzes the reaction CO(2) + H(2)O left arrow over right arrow HCO(3)(-) + H(+) in many tissues. We investigated whether NBC3, like some Cl(-)/HCO(3)(-) exchange proteins, could bind CAII and whether PKA could regulate NBC3 activity through modulation of CAII binding. CAII bound the COOH-terminal domain of NBC3 (NBC3Ct) with K(d) = 101 nM; the interaction was stronger at acid pH. Cotransfection of HEK-293 cells with NBC3 and CAII recruited CAII to the plasma membrane. Mutagenesis of consensus CAII binding sites revealed that the D1135-D1136 region of NBC3 is essential for CAII/NBC3 interaction and for optimal function, because the NBC3 D1135N/D1136N retained only 29 +/- 22% of wild-type activity. Coexpression of the functionally dominant-negative CAII mutant V143Y with NBC3 or addition of 100 microM 8-bromoadenosine to NBC3 transfected cells reduced intracellular pH (pH(i)) recovery rate by 31 +/- 3, or 38 +/- 7%, respectively, relative to untreated NBC3 transfected cells. The effects were additive, together decreasing the pH(i) recovery rate by 69 +/- 12%, suggesting that PKA reduces transport activity by a mechanism independently of CAII. Measurements of PKA-dependent phosphorylation by mass spectroscopy and labeling with [gamma-(32)P]ATP showed that NBC3Ct was not a PKA substrate. These results demonstrate that NBC3 and CAII interact to maximize the HCO(3)(-) transport rate. Although PKA decreased NBC3 transport activity, it did so independently of the NBC3/CAII interaction and did not involve phosphorylation of NBC3Ct.
Assuntos
Equilíbrio Ácido-Base/fisiologia , Adenosina/análogos & derivados , Anidrase Carbônica II/metabolismo , Membrana Celular/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Epiteliais/enzimologia , Simportadores de Sódio-Bicarbonato/metabolismo , Equilíbrio Ácido-Base/efeitos dos fármacos , Adenosina/farmacologia , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Anidrase Carbônica II/genética , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/genética , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Humanos , Mutação/genética , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Simportadores de Sódio-Bicarbonato/genética , TransfecçãoRESUMO
Human AE1 performs electroneutral exchange of Cl(-) for HCO(3)(-) across the erythrocyte membrane. We examined the topology of the AE1 C-terminal region using cysteine-scanning mutagenesis and sulfhydryl-specific chemistry. Eighty individual cysteine residues, introduced into an otherwise cysteine-less mutant between Phe(806) and Cys(885), were expressed by transient transfection of HEK293 cells. Topology of the region was determined by comparing cysteine labeling with the membrane-permeant cysteine-directed reagent biotin maleimide, with or without prior labeling with the membrane-impermeant reagents, bromotrimethylammoniumbimane bromide (qBBr) and lucifer yellow iodoacetamide (LYIA). Phe(806)-Leu(835), Ser(852)-Ala(855), and Ile(872)-Cys(885) were labeled by biotin maleimide, suggesting their location in an aqueous environment. In contrast, Phe(836)-Lys(851) and Ser(856)-Arg(871) were not labeled by biotin maleimide and therefore localize to the plane of the bilayer, as transmembrane segments (TM). Labeling by qBBr revealed that Pro(815)-Lys(829) and Ser(852)-Ala(855) are accessible to the extracellular medium. Pro(815)-Lys(829) mutants were also labeled with LYIA. Mutants Ile(872)-Cys(885) were inaccessible to the extracellular medium and thus localized to the intracellular surface of AE1. Functional assays revealed that one face of each of two AE1 TMs was sensitive to mutation. Based on these results, we propose a topology model for the C-terminal region of the membrane domain of human AE1.