RESUMO
PARP-1 is a key early responder to DNA damage in eukaryotic cells. An allosteric mechanism links initial sensing of DNA single-strand breaks by PARP-1's F1 and F2 domains via a process of further domain assembly to activation of the catalytic domain (CAT); synthesis and attachment of poly(ADP-ribose) (PAR) chains to protein sidechains then signals for assembly of DNA repair components. A key component in transmission of the allosteric signal is the HD subdomain of CAT, which alone bridges between the assembled DNA-binding domains and the active site in the ART subdomain of CAT. Here we present a study of isolated CAT domain from human PARP-1, using NMR-based dynamics experiments to analyse WT apo-protein as well as a set of inhibitor complexes (with veliparib, olaparib, talazoparib and EB-47) and point mutants (L713F, L765A and L765F), together with new crystal structures of the free CAT domain and inhibitor complexes. Variations in both dynamics and structures amongst these species point to a model for full-length PARP-1 activation where first DNA binding and then substrate interaction successively destabilise the folded structure of the HD subdomain to the point where its steric blockade of the active site is released and PAR synthesis can proceed.
Assuntos
Poli(ADP-Ribose) Polimerase-1/química , Regulação Alostérica , Amidas/química , Domínio Catalítico , Cristalografia por Raios X , Dano ao DNA , Ativação Enzimática , Modelos Moleculares , Mutação , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/química , Domínios ProteicosRESUMO
The activation loop (A-loop) plays a key role in regulating the catalytic activity of protein kinases. Phosphorylation in this region enhances the phosphoryl transfer rate of the kinase domain and increases its affinity for ATP. Furthermore, the A-loop possesses autoinhibitory functions in some kinases, where it collapses onto the protein surface and blocks substrate binding when unphosphorylated. Due to its flexible nature, the A-loop is usually disordered and untraceable in kinase domain crystal structures. The resulting lack of structural information is regrettable as it impedes the design of drug A-loop contacts, which have proven favourable in multiple cases. Here, we characterize the binding with A-loop engagement between type 1.5 kinase inhibitor 'example 172' (EX172) and Mer tyrosine kinase (MerTK). With the help of crystal structures and binding kinetics, we portray how the recruitment of the A-loop elicits a two-step binding mechanism which results in a drug-target complex characterized by high affinity and long residence time. In addition, the type 1.5 compound possesses excellent kinome selectivity and a remarkable preference for the phosphorylated over the dephosphorylated form of MerTK. We discuss these unique characteristics in the context of known type 1 and type 2 inhibitors and highlight opportunities for future kinase inhibitor design.
Assuntos
Trifosfato de Adenosina/química , Inibidores de Proteínas Quinases/química , c-Mer Tirosina Quinase/antagonistas & inibidores , c-Mer Tirosina Quinase/química , Humanos , Estrutura Secundária de ProteínaRESUMO
Protein arginine methyltransferases (PRMTs) are of great interest for the development of therapeutics due to their involvement in a number of malignancies, such as lung and colon cancer. PRMT5 catalyzes the formation of symmetrical dimethylarginine of a wide variety of substrates and is responsible for the majority of this mark within cells. To gain insight into the mechanism of PRMT5 inhibition, we co-expressed the human PRMT5:MEP50 complex (hPRMT5:MEP50) in insect cells for a detailed mechanistic study. In this report, we carry out steady state, product, and dead-end inhibitor studies that show hPRMT5:MEP50 uses a rapid equilibrium random order mechanism with EAP and EBQ dead-end complexes. We also provide evidence of ternary complex formation in solution using hydrogen/deuterium exchange mass spectrometry. Isotope exchange and intact protein mass spectrometry further rule out ping-pong as a potential enzyme mechanism, and finally, we show that PRMT5 exhibits a pre-steady state burst that corresponds to an initial slow turnover with all four active sites of the hetero-octamer being catalytically active.
Assuntos
Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Medição da Troca de Deutério , Inibidores Enzimáticos/farmacologia , Humanos , Técnicas In Vitro , Cinética , Espectrometria de Massas , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteína-Arginina N-Metiltransferases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
The rat lungworm Angiostrongylus cantonensis is a nematode parasite that can cause potentially fatal eosinophilic meningitis in humans. The life cycle of A. cantonensis involves multiple hosts, with the most common terminal hosts being rodents and intermediate hosts comprising gastropods. One such gastropod is the invasive island apple snail Pomacea maculata, which is native to South America but is currently established in several states in the USA, including South Carolina. It has been identified as an intermediate host for A. cantonensis in several locations in Louisiana. The ability of the island apple snail to serve as an intermediate host for A. cantonensis poses significant potential threats to human health, yet no studies to date have determined the prevalence of this parasite in island apple snails in South Carolina. The objective of this study was to investigate the frequency of occurrence of A. cantonensis in South Carolina island apple snails by using a real-time PCR assay. One-hundred individuals from each of three distinct stormwater retention ponds were tested, and no positive detections were found. Determining the prevalence of A. cantonensis in island apple snails is critical in accurately informing the public as to the risks involved in handling and/or consuming island apple snails.
Assuntos
Angiostrongylus cantonensis/fisiologia , Distribuição Animal , Caramujos/parasitologia , Animais , Espécies Introduzidas , South CarolinaRESUMO
The heterotrimeric AMP-activated protein kinase (AMPK) has a key role in regulating cellular energy metabolism; in response to a fall in intracellular ATP levels it activates energy-producing pathways and inhibits energy-consuming processes. AMPK has been implicated in a number of diseases related to energy metabolism including type 2 diabetes, obesity and, most recently, cancer. AMPK is converted from an inactive form to a catalytically competent form by phosphorylation of the activation loop within the kinase domain: AMP binding to the γ-regulatory domain promotes phosphorylation by the upstream kinase, protects the enzyme against dephosphorylation, as well as causing allosteric activation. Here we show that ADP binding to just one of the two exchangeable AXP (AMP/ADP/ATP) binding sites on the regulatory domain protects the enzyme from dephosphorylation, although it does not lead to allosteric activation. Our studies show that active mammalian AMPK displays significantly tighter binding to ADP than to Mg-ATP, explaining how the enzyme is regulated under physiological conditions where the concentration of Mg-ATP is higher than that of ADP and much higher than that of AMP. We have determined the crystal structure of an active AMPK complex. The structure shows how the activation loop of the kinase domain is stabilized by the regulatory domain and how the kinase linker region interacts with the regulatory nucleotide-binding site that mediates protection against dephosphorylation. From our biochemical and structural data we develop a model for how the energy status of a cell regulates AMPK activity.
Assuntos
Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/metabolismo , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Animais , Sítios de Ligação , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Cinética , Magnésio/metabolismo , Mamíferos , Modelos Moleculares , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Ligação Proteica , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , TermodinâmicaRESUMO
Dysregulation of histone methyl transferase nuclear receptor-binding SET domain 2 (NSD2) has been implicated in several hematological and solid malignancies. NSD2 is a large multidomain protein that carries histone writing and histone reading functions. To date, identifying inhibitors of the enzymatic activity of NSD2 has proven challenging in terms of potency and SET domain selectivity. Inhibition of the NSD2-PWWP1 domain using small molecules has been considered as an alternative approach to reduce NSD2-unregulated activity. In this article, we present novel computational chemistry approaches, encompassing free energy perturbation coupled to machine learning (FEP/ML) models as well as virtual screening (VS) activities, to identify high-affinity NSD2 PWWP1 binders. Through these activities, we have identified the most potent NSD2-PWWP1 binder reported so far in the literature: compound 34 (pIC50 = 8.2). The compounds identified herein represent useful tools for studying the role of PWWP1 domains for inhibition of human NSD2.
Assuntos
Desenho de Fármacos , Histona-Lisina N-Metiltransferase , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/química , Ligantes , Humanos , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Relação Estrutura-Atividade , Aprendizado de Máquina , Modelos Moleculares , Domínios ProteicosRESUMO
PRMT5, a type 2 arginine methyltransferase, has a critical role in regulating cell growth and survival in cancer. With the aim of developing MTA-cooperative PRMT5 inhibitors suitable for MTAP-deficient cancers, herein we report our efforts to develop novel "MTA-cooperative" compounds identified through a high-throughput biochemical screening approach. Optimization of hits was achieved through structure-based design with a focus on improvement of oral drug-like properties. Bioisosteric replacement of the original thiazole guanidine headgroup, spirocyclization of the isoindolinone amide scaffold to both configurationally and conformationally lock the bioactive form, and fine-tuning of the potency, MTA cooperativity, and DMPK properties through specific substitutions of the azaindole headgroup were conducted. We have identified an orally available in vivo lead compound, 28 ("AZ-PRMT5i-1"), which shows sub-10 nM PRMT5 cell potency, >50-fold MTA cooperativity, suitable DMPK properties for oral dosing, and significant PRMT5-driven in vivo efficacy in several MTAP-deficient preclinical cancer models.
RESUMO
MAT2a is a methionine adenosyltransferase that synthesizes the essential metabolite S-adenosylmethionine (SAM) from methionine and ATP. Tumors bearing the co-deletion of p16 and MTAP genes have been shown to be sensitive to MAT2a inhibition, making it an attractive target for treatment of MTAP-deleted cancers. A fragment-based lead generation campaign identified weak but efficient hits binding in a known allosteric site. By use of structure-guided design and systematic SAR exploration, the hits were elaborated through a merging and growing strategy into an arylquinazolinone series of potent MAT2a inhibitors. The selected in vivo tool compound 28 reduced SAM-dependent methylation events in cells and inhibited proliferation of MTAP-null cells in vitro. In vivo studies showed that 28 was able to induce antitumor response in an MTAP knockout HCT116 xenograft model.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/química , Metionina Adenosiltransferase/antagonistas & inibidores , Sítio Alostérico , Animais , Proliferação de Células , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Técnicas de Inativação de Genes , Células HCT116 , Meia-Vida , Humanos , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Camundongos , Simulação de Dinâmica Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Quinazolinas/química , Quinazolinas/metabolismo , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Ratos , S-Adenosilmetionina/metabolismo , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
Mer is a member of the TAM (Tyro3, Axl, Mer) kinase family that has been associated with cancer progression, metastasis, and drug resistance. Their essential function in immune homeostasis has prompted an interest in their role as modulators of antitumor immune response in the tumor microenvironment. Here we illustrate the outcomes of an extensive lead-generation campaign for identification of Mer inhibitors, focusing on the results from concurrent, orthogonal high-throughput screening approaches. Data mining, HT (high-throughput), and DECL (DNA-encoded chemical library) screens offered means to evaluate large numbers of compounds. We discuss campaign strategy and screening outcomes, and exemplify series resulting from prioritization of hits that were identified. Concurrent execution of HT and DECL screening successfully yielded a large number of potent, selective, and novel starting points, covering a range of selectivity profiles across the TAM family members and modes of kinase binding, and offered excellent start points for lead development.
Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , c-Mer Tirosina Quinase/antagonistas & inibidores , Animais , Cristalografia por Raios X , Mineração de Dados , Descoberta de Drogas , Humanos , Modelos Moleculares , c-Mer Tirosina Quinase/química , c-Mer Tirosina Quinase/metabolismoRESUMO
Poly-ADP-ribose-polymerase (PARP) inhibitors have achieved regulatory approval in oncology for homologous recombination repair deficient tumors including BRCA mutation. However, some have failed in combination with first-line chemotherapies, usually due to overlapping hematological toxicities. Currently approved PARP inhibitors lack selectivity for PARP1 over PARP2 and some other 16 PARP family members, and we hypothesized that this could contribute to toxicity. Recent literature has demonstrated that PARP1 inhibition and PARP1-DNA trapping are key for driving efficacy in a BRCA mutant background. Herein, we describe the structure- and property-based design of 25 (AZD5305), a potent and selective PARP1 inhibitor and PARP1-DNA trapper with excellent in vivo efficacy in a BRCA mutant HBCx-17 PDX model. Compound 25 is highly selective for PARP1 over other PARP family members, with good secondary pharmacology and physicochemical properties and excellent pharmacokinetics in preclinical species, with reduced effects on human bone marrow progenitor cells in vitro.
Assuntos
DNA , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases , Humanos , Cristalografia por Raios X , DNA/química , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/química , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Especificidade por SubstratoRESUMO
OBJECTIVE: To report a case of recurrent Stenotrophomonas maltophilia ventilator-associated pneumonia (VAP) that was successfully treated with doxycycline and aerosolized colistin. CASE SUMMARY: A 28-year-old male was admitted with a severe head injury and required mechanical ventilation. The patient developed S. maltophilia VAP on hospital day 17, which was cured after 7 days of treatment with high-dose intravenous trimethoprim/sulfamethoxazole (TMP/SMX). However, on day 34, the patient developed recurrent S. maltophilia VAP that did not respond clinically or demonstrate eradication on follow-up culture after 10 days of TMP/SMX. At that time, TMP/SMX was discontinued and treatment was initiated with intravenous doxycycline and aerosolized colistin. The VAP episode was cured after 14 days of treatment with doxycycline/aerosolized colistin. DISCUSSION: S. maltophilia is an emerging cause of VAP in some centers. This organism is associated with high mortality rates and has few treatment options because it is intrinsically resistant to most drug classes. Recent data suggest that doxycycline and aerosolized colistin each are effective in treatment of other multidrug-resistant organisms, such as Pseudomonas aeruginosa and Acinetobacter baumannii. However, this is the first report describing the use of this antibiotic regimen for S. maltophilia. High-dose TMP/SMX is considered to be the drug of choice primarily based on excellent in vitro activity. Few data exist on how to treat patients who fail therapy with TMP/SMX or cannot receive that drug because of resistance, allergy, or adverse events. Thus, it is important to report alternative methods for treating this infection. CONCLUSIONS: The positive clinical response to doxycycline and aerosolized colistin seen in the patient described here suggests that this combination may be an alternative treatment in patients who fail initial treatment or cannot receive standard therapies.
Assuntos
Antibacterianos/uso terapêutico , Colistina/uso terapêutico , Doxiciclina/uso terapêutico , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Pneumonia Bacteriana/tratamento farmacológico , Pneumonia Associada à Ventilação Mecânica/tratamento farmacológico , Stenotrophomonas maltophilia , Administração por Inalação , Adulto , Aerossóis , Antibacterianos/administração & dosagem , Anti-Infecciosos/uso terapêutico , Colistina/administração & dosagem , Doxiciclina/administração & dosagem , Farmacorresistência Bacteriana Múltipla , Quimioterapia Combinada , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Injeções Intravenosas , Masculino , Pneumonia Bacteriana/microbiologia , Pneumonia Associada à Ventilação Mecânica/microbiologia , Combinação Trimetoprima e Sulfametoxazol/uso terapêuticoRESUMO
Polycomb repressive complex 2 (PRC2) silences gene expression through trimethylation of K27 of histone H3 (H3K27me3) via its catalytic SET domain. A missense mutation in the substrate of PRC2, histone H3K27M, is associated with certain pediatric brain cancers and is linked to a global decrease of H3K27me3 in the affected cells thought to be mediated by inhibition of PRC2 activity. We present here the crystal structure of human PRC2 in complex with the inhibitory H3K27M peptide bound to the active site of the SET domain, with the methionine residue located in the pocket that normally accommodates the target lysine residue. The structure and binding studies suggest a mechanism for the oncogenic inhibition of H3K27M. The structure also reveals how binding of repressive marks, like H3K27me3, to the EED subunit of the complex leads to enhancement of the catalytic efficiency of the SET domain and thus the propagation of this repressive histone modification.
Assuntos
Histonas/química , Lisina/química , Complexo Repressor Polycomb 2/química , Domínios Proteicos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Carcinogênese/genética , Domínio Catalítico , Cristalografia por Raios X , Proteína Potenciadora do Homólogo 2 de Zeste/química , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Lisina/genética , Lisina/metabolismo , Metilação , Modelos Moleculares , Mutação , Oncogenes/genética , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Ligação ProteicaRESUMO
The Polycomb repressive complexes PRC1 and PRC2 play a central role in developmental gene regulation in multicellular organisms. PRC1 and PRC2 modify chromatin by catalysing histone H2A lysine 119 ubiquitylation (H2AK119u1), and H3 lysine 27 methylation (H3K27me3), respectively. Reciprocal crosstalk between these modifications is critical for the formation of stable Polycomb domains at target gene loci. While the molecular mechanism for recognition of H3K27me3 by PRC1 is well defined, the interaction of PRC2 with H2AK119u1 is poorly understood. Here we demonstrate a critical role for the PRC2 cofactor Jarid2 in mediating the interaction of PRC2 with H2AK119u1. We identify a ubiquitin interaction motif at the amino-terminus of Jarid2, and demonstrate that this domain facilitates PRC2 localization to H2AK119u1 both in vivo and in vitro. Our findings ascribe a critical function to Jarid2 and define a key mechanism that links PRC1 and PRC2 in the establishment of Polycomb domains.
Assuntos
Histonas/metabolismo , Lisina/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Ubiquitinação , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Metilação de DNA , Metilação , Camundongos , Nucleossomos/metabolismo , Complexo Repressor Polycomb 2/química , Ligação Proteica , Domínios Proteicos , Inativação do Cromossomo X/genéticaRESUMO
PURPOSE: A case of brodifacoum exposure leading to coagulopathy lasting for approximately one year despite treatment with large doses of phytonadione is reported. SUMMARY: A 36-year-old man was diagnosed with severe coagulopathy. He was treated and discharged on 40 mg of oral phytonadione daily. The cause of the coagulopathy remained unknown at discharge, but the hematologist theorized that exposure to a vitamin K antagonist was likely the source of the patient's condition. The patient was rehospitalized one week later with an International Normalized Ratio (INR) of 5.9 despite self-reported medication compliance. Oral phytonadione was increased to 80 mg daily. The patient was seen at an outpatient hematology clinic for several months and continued on tapering dosages of oral phytonadione. A coagulopathy panel from the original hospitalization confirmed the presence of brodifacoum, though the method of exposure remained unclear. He was lost to follow-up until approximately nine months later, when he reported taking 10 mg daily of oral phytonadione and had an INR of 1. Oral phytonadione was discontinued. Two months later, his INR was greater than 9, despite an undetectable level of brodifacoum. He was rehospitalized with oropharyngeal hematoma approximately 1 year after the initial coagulopathy diagnosis. The patient was discharged on 40 mg oral phytonadione daily with outpatient follow-up. CONCLUSION: A patient with brodifacoum exposure ingested brodifacoum had coagulopathy that lasted approximately one year despite long-term treatment with large dosages of oral phytonadione. The coagulopathy persisted even when brodifacoum was undetectable in the serum. Long-term treatment with high-dose phytonadione is expensive, which may influence medication compliance.
Assuntos
4-Hidroxicumarinas/intoxicação , Anticoagulantes/intoxicação , Transtornos da Coagulação Sanguínea/induzido quimicamente , Rodenticidas/intoxicação , Adulto , Antifibrinolíticos/administração & dosagem , Antifibrinolíticos/uso terapêutico , Transtornos da Coagulação Sanguínea/tratamento farmacológico , Transtornos da Coagulação Sanguínea/fisiopatologia , Seguimentos , Humanos , Coeficiente Internacional Normatizado , Masculino , Índice de Gravidade de Doença , Fatores de Tempo , Vitamina K 1/administração & dosagem , Vitamina K 1/uso terapêuticoRESUMO
AMP-activated protein kinase (AMPK) plays a major role in regulating cellular energy balance by sensing and responding to increases in AMP/ADP concentration relative to ATP. Binding of AMP causes allosteric activation of the enzyme and binding of either AMP or ADP promotes and maintains the phosphorylation of threonine 172 within the activation loop of the kinase. AMPK has attracted widespread interest as a potential therapeutic target for metabolic diseases including type 2 diabetes and, more recently, cancer. A number of direct AMPK activators have been reported as having beneficial effects in treating metabolic diseases, but there has been no structural basis for activator binding to AMPK. Here we present the crystal structure of human AMPK in complex with a small molecule activator that binds at a site between the kinase domain and the carbohydrate-binding module, stabilising the interaction between these two components. The nature of the activator-binding pocket suggests the involvement of an additional, as yet unidentified, metabolite in the physiological regulation of AMPK. Importantly, the structure offers new opportunities for the design of small molecule activators of AMPK for treatment of metabolic disorders.
Assuntos
Proteínas Quinases Ativadas por AMP/química , Regulação Enzimológica da Expressão Gênica , Monofosfato de Adenosina/química , Trifosfato de Adenosina/química , Sítio Alostérico , Sítios de Ligação , Carboidratos/química , Dicroísmo Circular , Cristalografia por Raios X , Células HEK293 , Humanos , Interferometria , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Treonina/químicaRESUMO
The SNF1 protein kinase complex plays an essential role in regulating gene expression in response to the level of extracellular glucose in budding yeast. SNF1 shares structural and functional similarities with mammalian AMP-activated protein kinase. Both kinases are activated by phosphorylation on a threonine residue within the activation loop segment of the catalytic subunit. Here we show that ADP is the long-sought metabolite that activates SNF1 in response to glucose limitation by protecting the enzyme against dephosphorylation by Glc7, its physiologically relevant protein phosphatase. We also show that the regulatory subunit of SNF1 has two ADP binding sites. The tighter site binds AMP, ADP, and ATP competitively with NADH, whereas the weaker site does not bind NADH, but is responsible for mediating the protective effect of ADP on dephosphorylation. Mutagenesis experiments suggest that the general mechanism by which ADP protects against dephosphorylation is strongly conserved between SNF1 and AMPK.
Assuntos
Difosfato de Adenosina/metabolismo , Ativação Enzimática/genética , Glucose/metabolismo , Proteína Fosfatase 1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transdução de Sinais , Difosfato de Adenosina/química , Adenilato Quinase/genética , Adenilato Quinase/metabolismo , Sequência de Aminoácidos , Domínio Catalítico/genética , Sequência Conservada , Regulação Fúngica da Expressão Gênica/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteína Fosfatase 1/genética , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato , Treonina/metabolismoRESUMO
OBJECTIVE: To create a self-sufficient, innovative method for providing cardiopulmonary resuscitation (CPR) education within a college of pharmacy using a student-driven committee, and disseminating CPR education into the community through a service learning experience. DESIGN: A CPR committee comprised of doctor of pharmacy (PharmD) students at the University of Tennessee College of Pharmacy provided CPR certification to all pharmacy students. The committee developed a service learning project by providing CPR training courses in the community. Participants in the course were required to complete an evaluation form at the conclusion of each training course. ASSESSMENT: The CPR committee successfully certified more than 1,950 PharmD students and 240 community members from 1996 to 2009. Evaluations completed by participants were favorable, with 99% of all respondents (n = 351) rating the training course as either "excellent" or "good" in each of the categories evaluated. CONCLUSION: A PharmD student-directed committee successfully provided CPR training to other students and community members as a service learning experience.