RESUMO
Lycium barbarum (LB) is a famous traditional Chinese medicinal plant as well as food supplement possessing various pharmacological functions such as anti-aging and antioxidant effects. The Parkinson's disease (PD)-related kinase Pink1 plays vital role in maintaining the neuron cell homeostasis, having been recognized as a potential target for the development of anti-PD drugs. In this work, the neuroprotective effects of methanol extract of LB fruit (LBFE) were investigated using a Drosophila PD model (PINK1B9) and a human neuroblastoma SH-SY5Y cell line. We found that when LBFE was supplied to the PINK1B9 flies at 6, 12, and 18 days of age, it raised the ATP and dopamine levels at all ages, extended life span, improved motor behavior, and rescued olfactory deficits of the PINK1B9 flies. In addition, histopathological examinations indicated that muscle atrophy in thoraces of the mutant flies was significantly repaired. Finally, LBFE was able to rescue the SH-SY5Y cells against MPP+-induced neurotoxicity. This work reports for the first time the anti-PD potential of L. barbarum fruit extract in PINK1 mutant fruit flies, presenting a new viewpoint for studing the mechanism of action of LBFE.
Assuntos
Proteínas de Drosophila , Lycium , Neuroblastoma , Fármacos Neuroprotetores , Doença de Parkinson , Animais , Humanos , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Doença de Parkinson/genética , Fármacos Neuroprotetores/farmacologia , Lycium/metabolismo , Modelos Genéticos , Extratos Vegetais/farmacologia , Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/farmacologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/farmacologiaRESUMO
Antibiotic resistance is a growing threat to human health exacerbated by a lack of new antibiotics. We now describe a series of substituted diamines that produce rapid bactericidal activity against both Gram-positive and Gram-negative bacteria, including methicillin-resistant Staphylococcus aureus and stationary-phase bacteria. These compounds reduce biofilm formation and promote biofilm dispersal in Pseudomonas aeruginosa. The most potent analogue, 3 (1,13-bis{[(2,2-diphenyl)-1-ethyl]thioureido}-4,10-diazatridecane), primarily acts by depolarization of the cytoplasmic membrane and permeabilization of the bacterial outer membrane. Transmission electron microscopy confirmed that 3 disrupts membrane integrity rapidly. Compound 3 is also synergistic with kanamycin, demonstrated by the checkerboard method and by time-kill kinetic experiments. In human cell toxicity assays, 3 showed limited adverse effects against the HEK293T human kidney embryonic cells and A549 human adenocarcinoma cells. In addition, 3 produced no adverse effects on Caenorhabditis elegans development, survival, and reproduction. Collectively, diamines related to 3 represent a new class of broad-spectrum antibacterials against drug-resistant pathogens.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Diaminas/química , Animais , Antibacterianos/efeitos adversos , Biofilmes/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/crescimento & desenvolvimento , Permeabilidade da Membrana Celular/efeitos dos fármacos , Técnicas de Química Sintética , Diaminas/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Interações Medicamentosas , Escherichia coli/efeitos dos fármacos , Células HEK293/efeitos dos fármacos , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacosRESUMO
Tamarixia triozae (Burks) (Hymenoptera: Eulophidae) is an important parasitoid of the potato or tomato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Trizoidae), a serious pest of potato (Solanum tuberosum L.), tomato (Solanum lycopersicum L.), and other solanaceous vegetables in many countries. To produce a marketable crop, insecticides are required when B. cockerelli populations reach economically damaging levels. We evaluated 11 commonly used insecticides for their effects on T. triozae. Glass-surface residues of spinetoram, imidacloprid-cyfluthrin, abamectin, and tolfenpyrad caused 100% mortality of T. triozae in 72 h, and the leaf residue of spinetoram was extremely toxic to T. triozae adults; even 15-d-old residues caused 100% mortality. Cyantraniliprole, fenpyroximate, pymetrozine, spirotetramat, spiromesifen, and chenopodium oil did not cause significant mortality in either glass surface or leaf-residue bioassays. Ingestion of spinetoram, abamectin, and imidacloprid+cyfluthrin (Leverage) by the adults resulted in 100% mortality in 12 h, and tolfenpyrad, 75.0% mortality in 12 h; whereas chenopodium oil and pymetrozine showed moderate effects on adult survival. Ingestion of abamectin, imidacloprid-cyfluthrin, and spinetoram killed all adults in the first day of treatment, whereas female adults in the treatment of pymetrozine lived 80.8 d, which was similar to those in the control. Ingestion of abamectin, imidacloprid-cyfluthrin, chenopodium oil, and spinetoram killed all male adults in the first day, whereas ingestion of other insecticides did not cause significant mortality, but reduced percent parasitism. Abamectin, imidacloprid-cyfluthrin, and spinetoram had the most deleterious effects on T. triozae, and have the least potential for use in integrated control programs using this parasitoid.
Assuntos
Inseticidas/toxicidade , Vespas/efeitos dos fármacos , Agricultura , Animais , Agentes de Controle Biológico , Feminino , Hemípteros/parasitologia , Solanum lycopersicum , Masculino , Resíduos de Praguicidas/toxicidade , Medição de Risco , Solanum tuberosumRESUMO
OBJECTIVE: To compare the chemosensitivity of pirarubicin (THP) and epirubicin (EPI) in primary breast cancer (PBC) cells so as to examine their differential chemosensitivity to THP and EPI by CD-DST (collagen gel droplet embedded culture-drug sensitivity test) system; To detect the differences in the short-term clinical efficacy and side effects between TAC (docetaxel + pirarubicin + cyclophosphamide) and TEC (docetaxel + epirubicin + cyclophosphamide) as the neoadjuvant chemotherapy regimens and the long-term clinical efficacy of CAF (cyclophosphamide + pirarubicin + fluorouracil) and CEF (cyclophosphamide + epirubicin + fluorouracil) as the chemotherapy regimens in breast cancer; To evaluate the feasibility of THP as an adjuvant chemotherapeutic regimen in the treatment of breast cancer. METHODS: From January 2008 to January 2009, a total of 129 fresh breast cancer samples were collected. The differential chemosensitivity of cultured PBC cells to THP and EPI was measured by CD-DST test. And 139 cases of PBC patients inIIb-IIIc phase were randomly divided into two groups: TAC and TEC groups. After 4-6 cycles of neoadjuvant chemotherapy, the primary lesion, axillary lymph nodes and side effects were assessed; The clinical data and survival status of 1241 cases of PBC patients treated at our hospital from 2003 to 2006 were collected and divided into CAF and CEF groups according to their chemotherapeutic regimens. Long-term prognosis was compared between two groups. RESULTS: There was no significant difference of chemosensitivity between THP and EPI in PBC cells (P = 0.743); The overall response rate (RR) of neoadjuvant chemotherapy was 87.8%; the clinical objective responses, pathologic complete remission (pCR), clinical complete remission (cCR), clinical partial remission (cPR) and stable disease (SD) of groups TAC and TEC were 88.7%, 11.3%, 28.2%, 60.6%, 11.3% vs 86.8%, 10.3%, 26.5%, 60.3%, 13.2% respectively. There was no significant difference between two groups (P > 0.05). No significant differences existed between two groups in such side effects as leukopenia, thrombocytopenia, constipation, cardiotoxity and hepatorenal dysfunction (P > 0.05). The gastrointestinal reactions of nausea and vomiting was less frequent in the TAC group than that in the TEC group (46.5% vs 66.2%, P = 0.019); There was no significant difference in 5-year disease-free survival rate (79% vs 78%) and overall survival rate between two groups (85% vs 82%, P > 0.05). CONCLUSION: There were no significant differences in chemosensitivity, clinical efficacy of neoadjuvant chemotherapy, side effects or long-term efficacy between THP and EPI. Both pirarubicin and epirubicin may be used as conventional chemotherapy in breast cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/análogos & derivados , Epirubicina/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Ciclofosfamida/uso terapêutico , Doxorrubicina/uso terapêutico , Feminino , Fluoruracila/uso terapêutico , Humanos , Pessoa de Meia-Idade , Terapia Neoadjuvante , Estadiamento de Neoplasias , Estudos Retrospectivos , Tamoxifeno/uso terapêutico , Adulto JovemRESUMO
OBJECTIVES: Previously we demonstrated the neuroprotective effect of montelukast, a cysteinyl leukotriene receptor-1 (CysLT(1) ) antagonist, on acute brain injury after focal cerebral ischaemia in mice. In this study, we have determined its effect on chronic brain injury after focal cerebral ischaemia in mice and rats. METHODS: After transient focal cerebral ischaemia was induced by middle cerebral artery occlusion, montelukast was intraperitoneally injected in mice or orally administered to rats for five days. Behavioural dysfunction, brain infarct volume, brain atrophy and neuron loss were determined to evaluate brain lesions. KEY FINDINGS: Montelukast (0.1 mg/kg) attenuated behavioural dysfunction, brain infarct volume, brain atrophy and neuron loss in mice, which was similar to pranlukast, another CysLT(1) receptor antagonist. Oral montelukast (0.5 mg/kg) was effective in rats and was more effective than edaravone, a free radical scavenger. CONCLUSION: Montelukast protected mice and rats against chronic brain injury after focal cerebral ischaemia, supporting the therapeutic potential of CysLT(1) receptor antagonists.
Assuntos
Acetatos/uso terapêutico , Antipirina/análogos & derivados , Lesão Encefálica Crônica/patologia , Lesão Encefálica Crônica/prevenção & controle , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/patologia , Degeneração Neural/tratamento farmacológico , Quinolinas/uso terapêutico , Acetatos/administração & dosagem , Acetatos/farmacologia , Administração Oral , Animais , Antipirina/uso terapêutico , Atrofia/prevenção & controle , Comportamento Animal/efeitos dos fármacos , Lesão Encefálica Crônica/complicações , Cromonas/uso terapêutico , Ciclopropanos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Edaravone , Sequestradores de Radicais Livres/uso terapêutico , Injeções Intraperitoneais , Antagonistas de Leucotrienos/farmacologia , Antagonistas de Leucotrienos/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos ICR , Quinolinas/administração & dosagem , Quinolinas/farmacologia , Ratos , Ratos Sprague-Dawley , SulfetosRESUMO
BACKGROUND: No published study has addressed the effect of genistein postconditioning on gastric ischemia-reperfusion (GI-R) injury in rats. AIM: To examine whether capsaicin receptor-mediated genistein postconditioning protects against gastric ischemia-reperfusion injury via the PI3K/Akt signal pathway. METHODS AND RESULTS: Chloraldurat-anesthetized rats underwent occlusion of the celiac artery for 30 min, followed by 60 min of reperfusion. Based on this animal model of gastric ischemia-reperfusion injury, genistein at doses of 100, 500 or 1,000 µg/kg was administered via peripheral vein 5 min before reperfusion. The dose of 500 µg/kg was optimal for postconditioning, at which the severity of I-R-induced gastric injury significantly decreased. Immunohistochemistry also showed that gastric mucosal cell apoptosis decreased. Capsazepine (CPZ), a specific antagonist for the capsaicin receptor, was administered (1,000 µg/kg, i.v.) just before ischemia. Capsaicin (50 mg/kg, s.c.) once a day for 4 days reversed the protective effects of genistein. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting showed increased calcitonin gene-related peptide (CGRP) expression in genistein group but not in capsazepine or capsaicin group. CGRP inhibitor CGRP8-37 also prevented the effects of genistein in decreasing gastric mucosal injury index. In addition, PI3K inhibitor LY294002 (1.5 mg/kg) reversed the protective effect of genistein. Compared with genistein group, Western blots also demonstrated decreased Akt phosphorylation in LY294002 group. CONCLUSION: Our data suggest that capsaicin receptors mediated the protective effects of genistein postconditioning. CGRP secreted by activated capsaicin-sensitive neurons played an important role in the protective effects of genistein. PI3K/Akt pathway was also involved in the protective effects of genistein.
Assuntos
Genisteína/uso terapêutico , Precondicionamento Isquêmico/métodos , Fitoestrógenos/uso terapêutico , Traumatismo por Reperfusão/prevenção & controle , Canais de Cátion TRPV/fisiologia , Animais , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Genisteína/administração & dosagem , Marcação In Situ das Extremidades Cortadas , Morfolinas/farmacologia , Fitoestrógenos/administração & dosagem , Ratos , Ratos Sprague-Dawley , Regulação para Cima/fisiologiaRESUMO
PlsY is a recently discovered acyltransferase that executes an essential step in membrane phospholipid biosynthesis in Gram- positive bacteria. By using a bioisosteric replacement approach to generate substrate-based inhibitors of PlsY as potential novel antibacterial agents, a series of stabilized acyl phosphate mimetics, including acyl phosphonates, acyl alpha,alpha-difluoromethyl phosphonates, acyl phosphoramides, reverse amide phosphonates, acyl sulfamates, and acyl sulfamides were designed and synthesized. Several acyl phosphonates, phosphoramides, and sulfamates were identified as inhibitors of PlsY from Streptococcus pneumoniae and Bacillus anthracis. As anticipated, these inhibitors were competitive inhibitors with respect to the acyl phosphate substrate. Antimicrobial testing showed the inhibitors to have generally weak activity against Gram-positive bacteria with the exception of some acyl phosphonates, reverse amide phosphonates, and acyl sulfamates, which had potent activity against multiple strains of B. anthracis.
Assuntos
Aciltransferases/antagonistas & inibidores , Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , Bactérias Gram-Positivas/efeitos dos fármacos , Fosfatos/química , Aciltransferases/metabolismo , Antibacterianos/síntese química , Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Avaliação Pré-Clínica de Medicamentos , Bactérias Gram-Positivas/enzimologia , Bactérias Gram-Positivas/metabolismo , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Fosfatos/síntese química , Fosfatos/farmacologia , Streptococcus pneumoniae/efeitos dos fármacosRESUMO
The double bond in anaerobic unsaturated fatty acid (UFA) biosynthesis is introduced by the FabA dehydratase/isomerase of the bacterial type II fatty acid biosynthetic pathway. A DeltafabA mutant of Pseudomonas aeruginosa grew aerobically, but required a UFA supplement for anaerobic growth. Wild-type cells produced 18:1Delta11 as the principal UFA, whereas the DeltafabA strain produced only 16:1Delta9. The double bond in the 16:1Delta9 was introduced after phospholipid formation and was localized in the sn-2 position. Two predicted membrane proteins, DesA and DesB, possessed the conserved histidine clusters characteristic of fatty acid desaturases. The DeltafabADeltadesA double mutant required exogenous fatty acids for growth but the DeltafabAdesB double mutant did not. Exogenous stearate was converted to 18:1Delta9 and supported the growth of DeltafabADeltadesA double mutant. A DeltafabADeltadesAdesB triple mutant was unable to desaturate exogenous stearate and was an UFA auxotroph. We detected a 2.5-fold increase in desA expression in DeltafabA mutants, whereas desB expression was derepressed by the deletion of the gene encoding a transcriptional repressor DesT. These data add two aerobic desaturases to the enzymes used for fatty acid metabolism in proteobacteria: DesA, a 2-position phospholipid Delta9-desaturase that supplements the anaerobic FabA pathway, and DesB, an inducible acyl-CoA Delta9-desaturase whose expression is repressed by DesT.
Assuntos
Ácidos Graxos Insaturados/biossíntese , Proteínas de Membrana/metabolismo , Pseudomonas aeruginosa/enzimologia , Estearoil-CoA Dessaturase/metabolismo , Aerobiose/genética , Sequência de Aminoácidos , Membrana Celular/enzimologia , Ácidos Graxos Insaturados/genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Hidroliases/genética , Proteínas de Membrana/genética , Dados de Sequência Molecular , Família Multigênica , Pseudomonas aeruginosa/genética , Estearatos/metabolismo , Estearoil-CoA Dessaturase/genética , Especificidade por SubstratoRESUMO
Pantothenate kinase catalyzes a key regulatory step in coenzyme A biosynthesis, and there are four mammalian genes that encode isoforms of this enzyme. Pantothenate kinase isoform PanK3 is highly related to the previously characterized PanK1beta isoform (79% identical, 91% similar), and these two almost identical proteins are expressed most highly in the same tissues. PanK1beta and PanK3 had very similar molecular sizes, oligomeric form, cytoplasmic cellular location, and kinetic constants for ATP and pantothenate. However, these two PanK isoforms possessed distinct regulatory properties. PanK3 was significantly more sensitive to feedback regulation by acetyl-CoA (IC50 = 1 microm) than PanK1beta (IC50 = 10 microm), and PanK3 was stringently regulated by long-chain acyl-CoA (IC50 = 2 microm), whereas PanK1beta was not. Domain swapping experiments localized the difference in the two proteins to a 48-amino-acid domain, where they are the most divergent. Consistent with these more stringent regulatory properties, metabolic labeling experiments showed that coenzyme A (CoA) levels in cells overexpressing PanK3 were lower than in cells overexpressing an equivalent amount of PanK1beta. Thus, the distinct regulatory properties exhibited by the family of the pantothenate kinases allowed the rate of CoA biosynthesis to be controlled by regulatory signals from CoA thioesters involved in different branches of intermediary metabolism.
Assuntos
Coenzima A/química , Ésteres/química , Retroalimentação Fisiológica , Regulação Enzimológica da Expressão Gênica , Fosfotransferases (Aceptor do Grupo Álcool)/química , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Animais , Bioquímica/métodos , Western Blotting , Catálise , Linhagem Celular , Cromatografia , Cromatografia em Gel , Citoplasma/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Cinética , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Peptídeos/química , Isoformas de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Transcrição Gênica , TransfecçãoRESUMO
The key regulatory step in CoA biosynthesis in bacteria and mammals is pantothenate kinase (CoaA), which governs the intracellular concentration of CoA through feedback regulation by CoA and its thioesters. CoaA from Staphylococcus aureus (SaCoaA) has a distinct primary sequence that is more similar to the mammalian pantothenate kinases than the prototypical bacterial CoaA of Escherichia coli. In contrast to all known pantothenate kinases, SaCoaA activity is not feedback-regulated by CoA or CoA thioesters. Metabolic labeling of S. aureus confirms that CoA levels are not controlled by CoaA or at steps downstream from CoaA. The pantothenic acid antimetabolite N-heptylpantothenamide (N7-Pan) possesses potent antimicrobial activity against S. aureus and has multiple cellular targets. N7-Pan is a substrate for SaCoaA and is converted to the inactive butyldethia-CoA analog by the downstream pathway enzymes. The analog is also incorporated into acyl carrier protein and D-alanyl carrier protein, the prosthetic groups of which are derived from CoA. The inactivation of acyl carrier protein and the cessation of fatty acid synthesis are the most critical causes of growth inhibition by N7-Pan because the toxicity of the drug is ameliorated by supplementing the growth medium with fatty acids. The absence of feedback regulation at the pantothenate kinase step allows the accumulation of high concentrations of intracellular CoA, consistent with the physiology of S. aureus, which lacks glutathione and relies on the CoA/CoA disulfide reductase redox system for protection from oxidative damage.
Assuntos
Coenzima A/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Staphylococcus aureus/enzimologia , Sequência de Aminoácidos , Antimetabólitos/metabolismo , Ácidos Graxos/biossíntese , Retroalimentação Fisiológica , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Epigallocatechin gallate (EGCG) is the major component of green tea extracts and possesses antibacterial, antiviral, and antitumor activity. Our study focused on validating the inhibition of the bacterial type II fatty acid synthesis system as a mechanism for the antibacterial effects of EGCG and related plant polyphenols. EGCG and the related tea catechins potently inhibited both the FabG and FabI reductase steps in the fatty acid elongation cycle with IC(50) values between 5 and 15 microm. The presence of the galloyl moiety was essential for activity, and EGCG was a competitive inhibitor of FabI and a mixed type inhibitor of FabG demonstrating that EGCG interfered with cofactor binding in both enzymes. EGCG inhibited acetate incorporation into fatty acids in vivo, although it was much less potent than thiolactomycin, a validated fatty acid synthesis inhibitor, and overexpression of FabG, FabI, or both did not confer resistance. A panel of other plant polyphenols was screened for FabG/FabI inhibition and antibacterial activity. Most of these inhibited both reductase steps, possessed antibacterial activity, and inhibited cellular fatty acid synthesis. The ability of the plant secondary metabolites to interfere with the activity of multiple NAD(P)-dependent cellular processes must be taken into account when assessing the specificity of their effects.
Assuntos
Acetiltransferases/antagonistas & inibidores , Oxirredutases do Álcool/antagonistas & inibidores , Catequina/análogos & derivados , Catequina/farmacologia , Flavonoides/farmacologia , Complexos Multienzimáticos/antagonistas & inibidores , Oxirredutases/antagonistas & inibidores , Fenóis/farmacologia , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Catequina/isolamento & purificação , Enoil-(Proteína de Transporte de Acila) Redutase (NADH) , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Proteínas de Escherichia coli , Ácido Graxo Sintase Tipo II , Flavonoides/isolamento & purificação , Estrutura Molecular , Fenóis/isolamento & purificação , Polifenóis , Chá/químicaRESUMO
Disabled (Dab) 1 and 2 are mammalian homologues of Drosophila DAB. Dab1 is a key cytoplasmic mediator in Reelin signaling that controls cell positioning in the developing central nervous system, whereas Dab2 is an adapter protein that plays a role in endocytosis. DAB family proteins possess an amino-terminal DAB homology (DH) domain that is similar to the phosphotyrosine binding/phosphotyrosine interaction (PTB/PI) domain. We have solved the structures of the DH domains of Dab2 (Dab2-DH) and Dab1 (Dab1-DH) in three different ligand forms, ligand-free Dab2-DH, the binary complex of Dab2-DH with the Asn-Pro-X-Tyr (NPXY) peptide of amyloid precursor protein (APP), and the ternary complex of Dab1-DH with the APP peptide and inositol 1,4,5-trisphosphate (Ins-1,4,5-P3, the head group of phosphatidylinositol-4,5-diphosphate (PtdIns-4,5-P2)). The similarity of these structures suggests that the rigid Dab DH domain maintains two independent pockets for binding of the APP/lipoprotein receptors and phosphoinositides. Mutagenesis confirmed the structural determinants specific for the NPXY sequence and PtdIns-4,5-P2 binding. NMR spectroscopy confirmed that the DH domain binds to Ins-1,4,5-P3 independent of the NPXY peptides. These findings suggest that simultaneous interaction of the rigid DH domain with the NPXY sequence and PtdIns-4,5-P2 plays a role in the attachment of Dab proteins to the APP/lipoprotein receptors and phosphoinositide-rich membranes.