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1.
J Biol Chem ; 286(6): 4555-65, 2011 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-21118805

RESUMO

The Josephin domain is a conserved cysteine protease domain found in four human deubiquitinating enzymes: ataxin-3, the ataxin-3-like protein (ATXN3L), Josephin-1, and Josephin-2. Josephin domains from these four proteins were purified and assayed for their ability to cleave ubiquitin substrates. Reaction rates differed markedly both among the different proteins and for different substrates with a given protein. The ATXN3L Josephin domain is a significantly more efficient enzyme than the ataxin-3 domain despite their sharing 85% sequence identity. To understand the structural basis of this difference, the 2.6 Å x-ray crystal structure of the ATXN3L Josephin domain in complex with ubiquitin was determined. Although ataxin-3 and ATXN3L adopt similar folds, they bind ubiquitin in different, overlapping sites. Mutations were made in ataxin-3 at selected positions, introducing the corresponding ATXN3L residue. Only three such mutations are sufficient to increase the catalytic activity of the ataxin-3 domain to levels comparable with that of ATXN3L, suggesting that ataxin-3 has been subject to evolutionary restraints that keep its deubiquitinating activity in check.


Assuntos
Endopeptidases/química , Evolução Molecular , Complexos Multiproteicos/química , Proteínas do Tecido Nervoso/química , Proteínas Nucleares/química , Dobramento de Proteína , Proteínas Repressoras/química , Ubiquitina/química , Ataxina-3 , Cristalografia por Raios X , Endopeptidases/genética , Endopeptidases/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Relação Estrutura-Atividade , Ubiquitina/genética , Ubiquitina/metabolismo
2.
Protein Expr Purif ; 82(1): 6-10, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22100524

RESUMO

Conotoxin PrIIIE is a 22-amino acid peptide containing three disulfide bonds isolated from the venom of Conus parius Reeve. It is a non-competitive antagonist of the mammalian muscle nicotinic acetylcholine receptor (nAChR). We fused the PrIIIE to small ubiquitin-like modifier (SUMO) and expressed the fusion protein in an Escherichia coli strain with an oxidizing cytoplasm. We purified the fusion protein by immobilized metal affinity chromatography and further purified PrIIIE from cleaved SUMO using cation exchange chromatography. The yield of peptide was 1.5mg/L of culture. The recombinant peptide is functional, as demonstrated by two-electrode voltage clamp experiments. This system may prove valuable for future structure-function studies.


Assuntos
Conotoxinas/genética , Conotoxinas/isolamento & purificação , Caramujo Conus/genética , Escherichia coli/genética , Sequência de Aminoácidos , Animais , Cromatografia de Afinidade , Conotoxinas/química , Conotoxinas/metabolismo , Caramujo Conus/química , Caramujo Conus/metabolismo , Expressão Gênica , Camundongos , Dados de Sequência Molecular , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Xenopus
3.
Proc Natl Acad Sci U S A ; 106(8): 2635-40, 2009 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-19202070

RESUMO

Lymphangioleiomyomatosis (LAM) is an often fatal disease primarily affecting young women in which tuberin (TSC2)-null cells metastasize to the lungs. The mechanisms underlying the striking female predominance of LAM are unknown. We report here that 17-beta-estradiol (E(2)) causes a 3- to 5-fold increase in pulmonary metastases in male and female mice, respectively, and a striking increase in circulating tumor cells in mice bearing tuberin-null xenograft tumors. E(2)-induced metastasis is associated with activation of p42/44 MAPK and is completely inhibited by treatment with the MEK1/2 inhibitor, CI-1040. In vitro, E(2) inhibits anoikis of tuberin-null cells. Finally, using a bioluminescence approach, we found that E(2) enhances the survival and lung colonization of intravenously injected tuberin-null cells by 3-fold, which is blocked by treatment with CI-1040. Taken together these results reveal a new model for LAM pathogenesis in which activation of MEK-dependent pathways by E(2) leads to pulmonary metastasis via enhanced survival of detached tuberin-null cells.


Assuntos
Sobrevivência Celular/fisiologia , Estrogênios/fisiologia , Neoplasias Pulmonares/patologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Anoikis/fisiologia , Benzamidas/farmacologia , Proteínas de Transporte/antagonistas & inibidores , Feminino , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Metástase Neoplásica , Ovariectomia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Ratos , Serina-Treonina Quinases TOR , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genética
4.
Proteins ; 77(4): 753-9, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19731378

RESUMO

The covalent attachment of different types of poly-ubiquitin chains signal different outcomes for the proteins so targeted. For example, a protein modified with Lys-48-linked poly-ubiquitin chains is targeted for proteasomal degradation, whereas Lys-63-linked chains encode nondegradative signals. The structural features that enable these different types of chains to encode different signals have not yet been fully elucidated. We report here the X-ray crystal structures of Lys-63-linked tri- and di-ubiquitin at resolutions of 2.3 and 1.9 A, respectively. The tri- and di-ubiquitin species adopt essentially identical structures. In both instances, the ubiquitin chain assumes a highly extended conformation with a left-handed helical twist; the helical chain contains four ubiquitin monomers per turn and has a repeat length of approximately 110 A. Interestingly, Lys-48 ubiquitin chains also adopt a left-handed helical structure with a similar repeat length. However, the Lys-63 architecture is much more open than that of Lys-48 chains and exposes much more of the ubiquitin surface for potential recognition events. These new crystal structures are consistent with the results of solution studies of Lys-63 chain conformation, and reveal the structural basis for differential recognition of Lys-63 versus Lys-48 chains.


Assuntos
Ubiquitinas/química , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Humanos , Técnicas In Vitro , Lisina/química , Metais/metabolismo , Modelos Moleculares , Poliubiquitina/química , Poliubiquitina/metabolismo , Estrutura Quaternária de Proteína , Proteínas Recombinantes/metabolismo , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitinas/metabolismo
5.
J Biol Chem ; 284(1): 174-181, 2009 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-19004831

RESUMO

O-Linked N-acetylglucosamine (O-GlcNAc) is a cytosolic and nuclear carbohydrate post-translational modification most abundant in brain. We recently reported uniquely extensive O-GlcNAc modification of proteins that function in synaptic vesicle release and post-synaptic signal transduction. Here we examined potential roles for O-GlcNAc in mouse hippocampal synaptic transmission and plasticity. O-GlcNAc modifications and the enzyme catalyzing their addition (O-GlcNAc transferase) were enriched in hippocampal synaptosomes. Pharmacological elevation or reduction of O-GlcNAc levels had no effect on Schaffer collateral CA1 basal hippocampal synaptic transmission. However, in vivo elevation of O-GlcNAc levels enhanced long term potentiation (LTP), an electrophysiological correlate to some forms of learning/memory. Reciprocally, pharmacological reduction of O-GlcNAc levels blocked LTP. Additionally, elevated O-GlcNAc led to reduced paired-pulse facilitation, a form of short term plasticity attributed to presynaptic mechanisms. Synapsin I and II are presynaptic proteins that increase synaptic vesicle availability for release when phosphorylated, thus contributing to hippocampal synaptic plasticity. Synapsins are among the most extensively O-GlcNAc-modified proteins known. Elevating O-GlcNAc levels increased phosphorylation of Synapsin I/II at serine 9 (cAMP-dependent protein kinase substrate site), serine 62/67 (Erk 1/2 (MAPK 1/2) substrate site), and serine 603 (calmodulin kinase II site). Activation-specific phosphorylation events on Erk 1/2 and calmodulin kinase II, two proteins required for CA1 hippocampal LTP establishment, were increased in response to elevation of O-GlcNAc levels. Thus, O-GlcNAc is a novel regulatory signaling component of excitatory synapses, with specific roles in synaptic plasticity that involve interplay with phosphorylation.


Assuntos
Acetilglucosamina/metabolismo , Hipocampo/metabolismo , Potenciação de Longa Duração/fisiologia , Transdução de Sinais/fisiologia , Vesículas Sinápticas/metabolismo , Sinaptossomos/metabolismo , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Aprendizagem/fisiologia , Masculino , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Fosforilação/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Sinapsinas/metabolismo
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