RESUMO
ELISA assays are commonly used for drug screening by racing laboratories but are known to suffer from limited specificity. Inaccurate ELISA screening results are typically produced by non-specific antibody interactions or by the retention of chromogenic material in the sample well due to sample degradation. While confirmation of drug positives can be achieved by mass spectrometry, the follow-up of inaccurate ELISA screening results represents an unnecessary cost in staff time and reagents. This is particularly true in the case of rhEPO screening using sandwich ELISA assays, where the confirmation method requires up to 3 days to perform. While most racing laboratories purchase commercial ELISA kits, these products can be customised to provide increased specificity for enhanced screening of positive samples. The specificity of commercial sandwich ELISA kits can be improved by a variety of mechanisms including the addition of competing analyte specific antibodies, substitution of capture antibodies or by performing ELISA analysis with and without capture antibodies. Non-specific signals in difficult matrices such as canine urine can also be reduced by the addition of BSA solutions to the ELISA plate prior to the addition of samples.
RESUMO
Previous liquid chromatography/mass spectrometry (LC/MS) methods for the detection of insulin and other similar peptide hormones in equine plasma relied on the use of antibody affinity extraction. As a result, these methods were not suitable for routine high-throughput analysis. A solid-phase extraction (SPE) method incorporating size exclusion as well as reversed-phase interactions allows the selective extraction of peptide hormones such as adrenocorticotropic hormone (ACTH), insulin and their synthetic analogues from equine plasma with approximately 80% extraction efficiencies. This extraction was combined with on-column derivatisation with acetic anhydride, followed by tryptic digestion and analysis by micro-LC/MSMS for high-sensitivity peptide hormone detection. The analysis of tryptic peptides provides greater sensitivity and more robust chromatography compared with the analysis of intact insulin and ACTH. For quantitative analysis, isotopically labelled internal standards of target peptides can be prepared in the laboratory through the use of deuterated acetic anhydride. The utility of the method was assessed for the analysis of ACTH and insulin in samples from horses suffering from pituitary pars intermedia dysfunction (PPID).
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Hyaluronic acid (HA) has been commonly used to treat osteoarthritis and joint injuries in horses and dogs since the 1970s. HA is a polysaccharide made up of alternating N-acetyl-d-glucosamine and d-glucuronic acid residues, with polymeric molecules achieving molecular weights as high as 20 MDa. High molecular weight HA forms a viscous hydrogel when dissolved in water, making HA solutions distinct from most other pharmaceutical preparations. Clear viscous solutions are often encountered during stable and kennel inspections, but in the absence of an analytical method, it is not possible to identify if these substances contain HA or other unknown compounds. This paper presents a simple method for the identification of HA in seized materials based on chemical hydrolysis followed by Hypercarb chromatography and MS/MS analysis.
RESUMO
Gonadotropin-releasing hormone (GnRH) and its synthetic analogues are considered banned substances by the racing industry. GnRH is used as a pharmaceutical to regulate the female oestrous cycle, but the hormone is also thought to increase the production of testosterone in male animals. Using liquid chromatography in conjunction with high-resolution mass spectrometry (LC-HRMS) and data-independent acquisition (DIA), a method is presented for the detection of intact and truncated peptides of GnRH and its analogues down to the low picogram level in equine urine. The study of the catabolism of GnRH and analogues in plasma, combined with the analysis of urine from administration studies, reveals a common pattern of peptide catabolites that can be used to guide the design of MS-based screens for this class of drugs. This culminated in the successful detection of the peptide in two out-of-competition canine urine samples.
Assuntos
Hormônio Liberador de Gonadotropina , Testosterona , Animais , Masculino , Feminino , Cavalos , Cães , Espectrometria de Massas , Cromatografia Líquida/métodos , Detecção do Abuso de Substâncias/veterinária , Detecção do Abuso de Substâncias/métodosRESUMO
The proteotypic human EPO peptides YLLEAK (T4), SLTTLLR (T11), TITADTFR (T14), and VYSNFLR (T17) are often used to confirm the presence of recombinant human EPO (rhEPO) in equine samples. Each of these peptides contains one or more isomeric leucine or isoleucine amino acids, raising the possibility that a simple leucine/isoleucine substitution could lead to a false identification when compared with a rhEPO reference standard. To examine this possibility variants of these four peptides were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These studies indicate that confirmation of rhEPO in equine samples by immuno-affinity capture and LC-MS/MS analysis is true and accurate. It was also found that chromatography played a greater role in determining LC-MS/MS specificity than tandem mass spectrometry and that, in the case of more hydrophilic peptides, the accuracy of peptide identification could be enhanced by the inclusion of 13 C and 15 N labelled peptide internal standards.
Assuntos
Eritropoetina , Espectrometria de Massas em Tandem , Animais , Cromatografia Líquida/métodos , Eritropoetina/análise , Cavalos , Humanos , Isoleucina , Leucina , Proteínas Recombinantes/análise , Espectrometria de Massas em Tandem/métodosRESUMO
Venoms were first identified as potential doping agents by the racing industry in 2007 when three vials of cobra venom were seized during an inspection of a stable at Keeneland Racecourse in the USA. Venoms are a complex mixture of proteins, peptides, and other substances with a wide range of biological effects, including inhibiting the transmission of nervous and muscular impulses. As an example of this, cobratoxin, an α-neurotoxin found in cobra venom, is claimed to be an effective treatment for pain. Recent analysis of seized samples identified venom from two different species of snake. Proteomic analysis identified the first sample as cobra venom, while the second sample, in a vial labeled "Conotoxin", was identified as venom from a many banded krait. Cobratoxin, conotoxins, and bungarotoxins (a component of krait venom) are all α-neurotoxins, suggesting a common application for all three venom proteins as potential pain blocking medications. Using a peptide based on the nicotinic acetylcholine receptor, a one-step affinity purification method was developed for the detection of α-neurotoxins in plasma.
Assuntos
Dopagem Esportivo/prevenção & controle , Neurotoxinas/análise , Detecção do Abuso de Substâncias/métodos , Animais , Bungarotoxinas/análise , Bungarotoxinas/sangue , Proteínas Neurotóxicas de Elapídeos/análise , Proteínas Neurotóxicas de Elapídeos/sangue , Conotoxinas/análise , Conotoxinas/sangue , Cavalos , Neurotoxinas/sangue , Proteômica/métodos , Receptores Nicotínicos/metabolismo , Detecção do Abuso de Substâncias/veterináriaRESUMO
CJC-1295 is a peptide-based drug that stimulates the production of growth hormone (GH) from the pituitary gland. It incorporates a functional maleimido group at the C-terminus that allows it to covalently bind plasma proteins such as serum albumin. These CJC-1295-protein conjugates have a much greater half-life compared to the unconjugated peptide and are capable of stimulating GH production for more than six days in humans after a single administration. Conjugated CJC-1295 is difficult to detect in blood by mass spectrometry due to its low abundance, high molecular weight, and conjugation to a range of different protein substrates. Previously we described a screening procedure for the detection of CJC-1295 in equine plasma using an immuno-PCR assay. Here we demonstrate the confirmation of CJC-1295 in equine plasma by LC-MS/MS after immuno-affinity capture and tryptic digestion. Using this method, CJC-1295 was identified down to concentrations as low as 180 pg/mL in 1 mL of equine plasma.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/análogos & derivados , Hormônios/sangue , Cavalos/sangue , Fragmentos de Peptídeos/sangue , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Cromatografia Líquida/métodos , Hormônio Liberador de Hormônio do Crescimento/análise , Hormônio Liberador de Hormônio do Crescimento/sangue , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Hormônios/análise , Hormônios/metabolismo , Cavalos/metabolismo , Limite de Detecção , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Albumina Sérica/metabolismo , Detecção do Abuso de Substâncias/métodosRESUMO
Adenosine monophosphate-activated protein kinase (AMPK) regulates multiple signaling pathways involved in glucose and lipid metabolism in response to changes in hormonal and nutrient status. Cell culture studies have shown that AMPK phosphorylation and inhibition of the rate-limiting enzyme in the mevalonate pathway 3-hydroxy-3-methylglutaryl (HMG) coenzyme A (CoA) reductase (HMGCR) at serine-871 (Ser871; human HMGCR Ser872) suppresses cholesterol synthesis. In order to evaluate the role of AMPK-HMGCR signaling in vivo, we generated mice with a Ser871-alanine (Ala) knock-in mutation (HMGCR KI). Cholesterol synthesis was significantly suppressed in wild-type (WT) but not in HMGCR KI hepatocytes in response to AMPK activators. Liver cholesterol synthesis and cholesterol levels were significantly up-regulated in HMGCR KI mice. When fed a high-carbohydrate diet, HMGCR KI mice had enhanced triglyceride synthesis and liver steatosis, resulting in impaired glucose homeostasis. Conclusion: AMPK-HMGCR signaling alone is sufficient to regulate both cholesterol and triglyceride synthesis under conditions of a high-carbohydrate diet. Our findings highlight the tight coupling between the mevalonate and fatty acid synthesis pathways as well as revealing a role of AMPK in suppressing the deleterious effects of a high-carbohydrate diet.
RESUMO
CJC-1295 is a 30 amino acid peptide-based drug that stimulates the release of growth hormone (GH) from the pituitary gland. It is unique among performance-enhancing peptides due to the presence of a reactive maleimidopropionic acid group that covalently links the peptide to free thiols on the surface of plasma proteins. Once conjugated, CJC-1295 remains active in the bloodstream for significantly longer than non-conjugated peptide-based drugs that are rapidly excreted. Conjugation of CJC-1295 to plasma proteins prevents its detection by top-down mass-spectrometry-based peptide screening protocols as it effectively becomes a macromolecular protein with an undefined molecular weight. Using a pair of monoclonal antibodies raised against the CJC-1295 peptide, we present an immuno-polymerase chain reaction (I-PCR) assay that is capable of detecting the CJC-1295-protein conjugate at concentrations down to 0.8 pg/mL. Detection of endogenous equine GHRH necessitated a screening threshold for CJC-1295 in equine plasma of 50 pg/mL. The effectiveness of the assay for controlling the illicit use of CJC-1295 was confirmed in equine blood samples after administration in thoroughbred race horses.
Assuntos
Hormônio Liberador de Hormônio do Crescimento/análogos & derivados , Cavalos/sangue , Fragmentos de Peptídeos/sangue , Animais , Anticorpos Monoclonais/química , Hormônio Liberador de Hormônio do Crescimento/administração & dosagem , Hormônio Liberador de Hormônio do Crescimento/sangue , Imunoensaio/métodos , Limite de Detecção , Fragmentos de Peptídeos/administração & dosagem , Reação em Cadeia da Polimerase/métodos , Detecção do Abuso de Substâncias/métodos , Ressonância de Plasmônio de Superfície/métodosRESUMO
The recombinant human erythropoietins epoetin alfa (Eprex®), darbepoetin (Aranesp®) and methoxy polyethylene glycol-epoetin beta (Mircera®) were administered to greyhounds for 7, 10 and 14 days respectively. Blood and urine samples were collected and analysed for erythropoietin by ELISA, LC-MS/MS and western blotting. Limits of confirmation in plasma for western blotting and LC-MS/MS methods ranged from a low of 2.5mIU/mL, and closely matched the sensitivity of ELISA screening.
Assuntos
Dopagem Esportivo/métodos , Eritropoetina/análise , Sequência de Aminoácidos , Animais , Western Blotting , Cromatografia Líquida de Alta Pressão , Reações Cruzadas , Darbepoetina alfa , Cães , Ensaio de Imunoadsorção Enzimática , Epoetina alfa , Eritropoetina/farmacocinética , Humanos , Limite de Detecção , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas Recombinantes/análise , Proteínas Recombinantes/farmacocinética , Padrões de ReferênciaRESUMO
A synthetic Interleukin-1 receptor antagonist peptide with the sequence Acetyl-Phe-Glu-Trp-Thr-Pro-Gly-Tyr-Trp-Gln-Pro-Tyr-Ala-Leu-Pro-Leu-OH has been identified in a vial seized during a stable inspection. The use of peptide-based Interleukin-1 receptor antagonists as anti-inflammatory agents has not been previously reported, making this peptide the first in a new class of sports doping peptides. The peptide has been characterized by high-resolution mass spectrometry and a detection method developed based on solid-phase extraction and liquid chromatography - triple quadrupole mass spectrometry. Using in vitro and in vivo models to study the properties of the peptide after administration, the peptide was shown to be highly unstable in plasma and was not detected in urine after administration in a rat. The poor stability of the peptide makes detection challenging but also suggests that it has limited effectiveness as an anti-inflammatory drug. Copyright © 2015 John Wiley & Sons, Ltd.
Assuntos
Anti-Inflamatórios/sangue , Anti-Inflamatórios/urina , Proteína Antagonista do Receptor de Interleucina 1/sangue , Proteína Antagonista do Receptor de Interleucina 1/urina , Peptídeos/sangue , Peptídeos/urina , Receptores de Interleucina-1/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/análise , Cromatografia Líquida , Dopagem Esportivo , Estabilidade de Medicamentos , Cavalos , Proteína Antagonista do Receptor de Interleucina 1/administração & dosagem , Proteína Antagonista do Receptor de Interleucina 1/análise , Peptídeos/administração & dosagem , Peptídeos/análise , Ratos , Detecção do Abuso de Substâncias , Espectrometria de Massas em TandemRESUMO
Isolating, purifying, and identifying proteins in complex biological matrices are often difficult, time consuming, and unreliable. Herein we describe a rapid screening technique for proteins in biological matrices that combines selective protein isolation with direct surface enhanced Raman spectroscopy (SERS) detection. Magnetic core gold nanoparticles were synthesized, characterized, and subsequently functionalized with recombinant human erythropoietin (rHuEPO)-specific antibody. The functionalized nanoparticles were used to capture rHuEPO from horse blood plasma within 15 min. The selective binding between the protein and the functionalized nanoparticles was monitored by SERS. The purified protein was then released from the nanoparticles' surface and directly spectroscopically identified on a commercial nanopillar SERS substrate. ELISA independently confirmed the SERS identification and quantified the released rHuEPO. Finally, the direct SERS detection of the extracted protein was successfully demonstrated for in-field screening by a handheld Raman spectrometer within 1 min sample measurement time. FROM THE CLINICAL EDITOR: The rapid detection of recombinant human erythropoietin (rHuEPO) is important in competitive sports to screen for doping offences. In this article, the authors reported their technique of direct surface enhanced Raman spectroscopy (SERS) detection using magnetic core gold nanoparticles functionalized with recombinant human erythropoietin-specific antibody. The findings should open a new way for future detection of other proteins.
Assuntos
Eritropoetina/sangue , Eritropoetina/isolamento & purificação , Ouro/química , Nanopartículas de Magnetita/química , Imãs/química , Análise Espectral Raman/métodos , Animais , Anticorpos Imobilizados/química , Cavalos , Humanos , Detecção do Abuso de Substâncias/métodosRESUMO
The co-transporter activity of Na(+)-K(+)-2Cl(-) 1 (NKCC1) is dependent on phosphorylation. In this study we show the energy-sensing kinase AMPK inhibits NKCC1 activity. Three separate AMPK activators (AICAR, Phenformin and A-769662) inhibited NKCC1 flux in a variety of nucleated cells. Treatment with A-769662 resulted in a reduction of NKCC1(T212/T217) phosphorylation, and this was reversed by treatment with the non-selective AMPK inhibitor Compound C. AMPK dependence was confirmed by treatment of AMPK null mouse embryonic fibroblasts, where A-769662 had no effect on NKCC1 mediated transport. AMPK was found to directly phosphorylate a recombinant human-NKCC1 N-terminal fragment (1-293) with the phosphorylated site identified as S77. Mutation of Serine 77 to Alanine partially prevented the inhibitory effect of A-769662 on NKCC1 activity. In conclusion, AMPK can act to reduce NKCC1-mediated transport. While the exact mechanism is still unclear there is evidence for both a direct effect on phosphorylation of S77 and reduced phosphorylation of T212/217.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Fenformin/farmacologia , Pironas/farmacologia , Ribonucleotídeos/farmacologia , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Tiofenos/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Alanina/metabolismo , Aminoimidazol Carboxamida/farmacologia , Animais , Compostos de Bifenilo , Linhagem Celular , Cães , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Camundongos , Fosforilação , Mutação Puntual , Transporte Proteico/efeitos dos fármacos , Serina/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/genéticaRESUMO
The growth hormone releasing peptides (GHRPs) hexarelin, ipamorelin, alexamorelin, GHRP-1, GHRP-2, GHRP-4, GHRP-5, and GHRP-6 are all synthetic met-enkephalin analogues that include unnatural D-amino acids. They were designed specifically for their ability to stimulate growth hormone release and may serve as performance enhancing drugs. To regulate the use of these peptides within the horse racing industry and by human athletes, a method is presented for the extraction, derivatization, and detection of GHRPs from equine and human urine. This method takes advantage of a highly specific solid-phase extraction combined with a novel derivatization method to improve the chromatography of basic peptides. The method was validated with respect to linearity, repeatability, intermediate precision, specificity, limits of detection, limits of confirmation, ion suppression, and stability. As proof of principle, all eight GHRPs or their metabolites could be detected in urine collected from rats after intravenous administration.
Assuntos
Cromatografia Líquida/métodos , Hormônio Liberador de Hormônio do Crescimento/urina , Detecção do Abuso de Substâncias/métodos , Espectrometria de Massas em Tandem/métodos , Administração Intravenosa , Animais , Dopagem Esportivo/prevenção & controle , Hormônio Liberador de Hormônio do Crescimento/análogos & derivados , Hormônio Liberador de Hormônio do Crescimento/análise , Ensaios de Triagem em Larga Escala/métodos , Cavalos , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Sensibilidade e Especificidade , Extração em Fase SólidaRESUMO
The Dermorphins are a family of peptides that act as potent agonists of the opioid µ receptor. Originally identified as a seven amino acid peptide on the skin of the South American Phyllomedusa frog, peptide chemists have since developed a large number of Dermorphin variants, many with superior opioid activity to the original peptide. Dermorphins are unique among the peptide opioid agonists as they appear to have a limited ability to cross the blood brain barrier, producing effects on both the central and peripheral nervous systems. It is this ability of Dermorphins to provide central anaesthesia after intravenous or subcutaneous administration that allows their use as analogues of the opioid class of drugs. Recently, illicit use of the Dermorphin peptide in the racing industry has shown the need for an analytical method to control the use of these peptides. We present a high-throughput liquid chromatography-tandem mass spectrometry screen for 17 Dermorphin peptides in equine urine and plasma with limits of detection down to 5 pg/mL. The peptide extraction technique is also suitable for use in human urine.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Cavalos/sangue , Cavalos/urina , Peptídeos Opioides/sangue , Peptídeos Opioides/urina , Animais , Cromatografia Líquida , Dopagem Esportivo/prevenção & controle , Humanos , Limite de Detecção , Sensibilidade e Especificidade , Detecção do Abuso de Substâncias/métodos , Espectrometria de Massas em TandemRESUMO
The two members of the Rho-associated coiled-coil kinase (ROCK1 and 2) family are established regulators of actin dynamics that are involved in the regulation of the cell cycle as well as cell motility and invasion. Here, we discovered a novel signaling pathway whereby ROCK regulates microtubule (MT) acetylation via phosphorylation of the tubulin polymerization promoting protein 1 (TPPP1/p25). We show that ROCK phosphorylation of TPPP1 inhibits the interaction between TPPP1 and histone deacetylase 6 (HDAC6), which in turn results in increased HDAC6 activity followed by a decrease in MT acetylation. As a consequence, we show that TPPP1 phosphorylation by ROCK increases cell migration and invasion via modulation of cellular acetyl MT levels. We establish here that the ROCK-TPPP1-HDAC6 signaling pathway is important for the regulation of cell migration and invasion.
Assuntos
Movimento Celular/fisiologia , Microtúbulos/metabolismo , Transdução de Sinais/fisiologia , Quinases Associadas a rho/metabolismo , Acetilação , Linhagem Celular Tumoral , Desacetilase 6 de Histona , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fosforilação/genética , Quinases Associadas a rho/genéticaRESUMO
Heat induces Hsp70.1 (HSPA1) and Hsc70 (HSPA8) to form complex detergent insoluble cytoplasmic and nuclear structures that are distinct from the cytoskeleton and internal cell membranes. These novel structures have not been observed by earlier immunofluorescence studies as they are obscured by the abundance of soluble Hsp70.1/Hsc70 present in cells. While resistant to detergents, these Hsp70 structures display complex intracellular dynamics and are efficiently disaggregated by ATP, indicating that this pool of Hsp70.1/Hsc70 retains native function and regulation. Hsp70.1 promotes the repair of proteotoxic damage and cell survival after stress. In heated fibroblasts expressing Hsp70.1, Hsp70.1 and Hsc70 complexes are efficiently disaggregated before the cells undergo-heat induced apoptosis. In the absence of Hsp70.1, fibroblasts have increased rates of heat-induced apoptosis and maintain stable insoluble Hsc70 structures. The differences in the intracellular distribution of Hsp70.1 and Hsc70, combined with the ability of Hsp70.1, but not Hsc70, to promote the disaggregation of insoluble Hsp70.1/Hsc70 complexes, indicate that these two closely related proteins perform distinctly different cellular functions in heated cells.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Temperatura Alta , Apoptose/fisiologia , Linhagem Celular , Grânulos Citoplasmáticos/metabolismo , HumanosRESUMO
Amidated gastrin-releasing peptide (GRP) is the prototypical autocrine growth factor. Nonamidated peptides derived from the C terminus of pro-GRP are also biologically active in colorectal cancer (CRC) cell lines in vitro, via a receptor distinct from the GRP receptor. The aims of this study were to measure the amounts of pro-GRP-derived peptides in human CRC cell lines and tumors, characterize the immunoreactive peptide, and investigate its effect on proliferation in vitro and in vivo. Pro-GRP-derived peptides were quantitated by region-specific ELISA in extracts of five human CRC cell lines and 20 tumors. The immunoreactive material was purified by HPLC and its mass and sequence established by mass spectrometry. The concentration of GRPamide was determined by RIA. Proliferation of DLD-1 cells and murine gastrointestinal mucosa was measured by [(3)H]-thymidine incorporation and mitotic index, respectively. In CRC cell extracts, ELISA for pro-GRP-derived peptides detected 3-152 fmol/10(6) cells. The immunoreactive peptide was purified and identified as pro-GRP42-98. Resected stage III tumors contained significantly less pro-GRP immunoreactivity than stage II tumors, and no amidated GRP was detected in cell lines or tumors. Stable transfection of DLD-1 cells with pro-GRP short hairpin RNA, or treatment with a monoclonal anti-pro-GRP antibody, significantly reduced proliferation. Pro-GRP42-98, pro-GRP47-68, and pro-GRP80-97 significantly stimulated mitosis in colonic, but not small intestinal, mucosa of 10-wk-old mice. We conclude that nonamidated peptides derived from the C terminus of pro-GRP are expressed in significant quantities in CRC cell lines and tumors and stimulate the proliferation of CRC cells and of normal colonic mucosa. Such peptides are attractive targets for novel CRC therapies.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Peptídeo Liberador de Gastrina/química , Animais , Linhagem Celular Tumoral , Proliferação de Células , Cromatografia Líquida de Alta Pressão/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Mucosa Intestinal/metabolismo , Espectrometria de Massas/métodos , Camundongos , Mitose , Peptídeos/química , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Radioimunoensaio/métodosRESUMO
Ca(2+)/calmodulin-dependent protein kinase kinase ß (CaMKKß) is a serine/threonine-directed kinase that is activated following increases in intracellular Ca(2+). CaMKKß activates Ca(2+)/calmodulin-dependent protein kinase I, Ca(2+)/calmodulin-dependent protein kinase IV, and the AMP-dependent protein kinase in a number of physiological pathways, including learning and memory formation, neuronal differentiation, and regulation of energy balance. Here, we report the novel regulation of CaMKKß activity by multisite phosphorylation. We identify three phosphorylation sites in the N terminus of CaMKKß, which regulate its Ca(2+)/calmodulin-independent autonomous activity. We then identify the kinases responsible for these phosphorylations as cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3 (GSK3). In addition to regulation of autonomous activity, we find that phosphorylation of CaMKKß regulates its half-life. We find that cellular levels of CaMKKß correlate with CDK5 activity and are regulated developmentally in neurons. Finally, we demonstrate that appropriate phosphorylation of CaMKKß is critical for its role in neurite development. These results reveal a novel regulatory mechanism for CaMKKß-dependent signaling cascades.
Assuntos
Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuritos/enzimologia , Animais , Células COS , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Chlorocebus aethiops , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Meia-Vida , Humanos , Proteínas do Tecido Nervoso/genética , Fosforilação/fisiologia , Estrutura Terciária de Proteína , Ratos , Transdução de Sinais/fisiologiaRESUMO
The adenosine monophosphate (AMP)-activated protein kinase (AMPK) regulates whole-body and cellular energy balance in response to energy demand and supply. AMPK is an αßγ heterotrimer activated by decreasing concentrations of adenosine triphosphate (ATP) and increasing AMP concentrations. AMPK activation depends on phosphorylation of the α catalytic subunit on threonine-172 (Thr(172)) by kinases LKB1 or CaMKKß, and this is promoted by AMP binding to the γ subunit. AMP sustains activity by inhibiting dephosphorylation of α-Thr(172), whereas ATP promotes dephosphorylation. Adenosine diphosphate (ADP), like AMP, bound to γ sites 1 and 3 and stimulated α-Thr(172) phosphorylation. However, in contrast to AMP, ADP did not directly activate phosphorylated AMPK. In this way, both ADP/ATP and AMP/ATP ratios contribute to AMPK regulation.