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1.
Biol Chem ; 397(1): 45-55, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26351917

RESUMO

Secretory peptides and proteins are frequently modified by pyroglutamic acid (pE, pGlu) at their N-terminus. This modification is catalyzed by the glutaminyl cyclases QC and isoQC. Here, we decipher the roles of the isoenzymes by characterization of IsoQC-/- mice. These mice show a significant reduction of glutaminyl cyclase activity in brain and peripheral tissue, suggesting ubiquitous expression of the isoQC enzyme. An assay of substrate conversion in vivo reveals impaired generation of the pGlu-modified C-C chemokine ligand 2 (CCL2, MCP-1) in isoQC-/- mice. The pGlu-formation was also impaired in primary neurons, which express significant levels of QC. Interestingly, however, the formation of the neuropeptide hormone thyrotropin-releasing hormone (TRH), assessed by immunohistochemistry and hormonal analysis of hypothalamic-pituitary-thyroid axis, was not affected in isoQC-/-, which contrasts to QC-/-. Thus, the results reveal differential functions of isoQC and QC in the formation of the pGlu-peptides CCL2 and TRH. Substrates requiring extensive prohormone processing in secretory granules, such as TRH, are primarily converted by QC. In contrast, protein substrates such as CCL2 appear to be primarily converted by isoQC. The results provide a new example, how subtle differences in subcellular localization of enzymes and substrate precursor maturation might influence pGlu-product formation.


Assuntos
Aminoaciltransferases/metabolismo , Administração Oral , Aminoaciltransferases/deficiência , Animais , Células Cultivadas , Glucose/administração & dosagem , Teste de Tolerância a Glucose , Inflamação/induzido quimicamente , Inflamação/metabolismo , Isoenzimas/metabolismo , Lipopolissacarídeos/administração & dosagem , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Ácido Pirrolidonocarboxílico/metabolismo , Especificidade por Substrato
2.
BMC Neurosci ; 14: 108, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-24083638

RESUMO

BACKGROUND: Posttranslational modifications of beta amyloid (Aß) have been shown to affect its biophysical and neurophysiological properties. One of these modifications is N-terminal pyroglutamate (pE) formation. Enzymatic glutaminyl cyclase (QC) activity catalyzes cyclization of truncated Aß(3-x), generating pE3-Aß. Compared to unmodified Aß, pE3-Aß is more hydrophobic and neurotoxic. In addition, it accelerates aggregation of other Aß species. To directly investigate pE3-Aß formation and toxicity in vivo, transgenic (tg) ETNA (E at the truncated N-terminus of Aß) mice expressing truncated human Aß(3-42) were generated and comprehensively characterized. To further investigate the role of QC in pE3-Aß formation in vivo, ETNA mice were intercrossed with tg mice overexpressing human QC (hQC) to generate double tg ETNA-hQC mice. RESULTS: Expression of truncated Aß(3-42) was detected mainly in the lateral striatum of ETNA mice, leading to progressive accumulation of pE3-Aß. This ultimately resulted in astrocytosis, loss of DARPP-32 immunoreactivity, and neuronal loss at the sites of pE3-Aß formation. Neuropathology in ETNA mice was associated with behavioral alterations. In particular, hyperactivity and impaired acoustic sensorimotor gating were detected. Double tg ETNA-hQC mice showed similar Aß levels and expression sites, while pE3-Aß were significantly increased, entailing increased astrocytosis and neuronal loss. CONCLUSIONS: ETNA and ETNA-hQC mice represent novel mouse models for QC-mediated toxicity of truncated and pE-modified Aß. Due to their significant striatal neurodegeneration these mice can also be used for analysis of striatal regulation of basal locomotor activity and sensorimotor gating, and possibly for DARPP-32-dependent neurophysiology and neuropathology. The spatio-temporal correlation of pE3-Aß and neuropathology strongly argues for an important role of this Aß species in neurodegenerative processes in these models.


Assuntos
Aminoaciltransferases/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Corpo Estriado/enzimologia , Corpo Estriado/patologia , Degeneração Neural/enzimologia , Peptídeos beta-Amiloides/química , Animais , Comportamento Animal , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Degeneração Neural/patologia , Processamento de Proteína Pós-Traducional
3.
J Neurosci ; 31(36): 12790-801, 2011 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-21900558

RESUMO

Posttranslational amyloid-ß (Aß) modification is considered to play an important role in Alzheimer's disease (AD) etiology. An N-terminally modified Aß species, pyroglutamate-amyloid-ß (pE3-Aß), has been described as a major constituent of Aß deposits specific to human AD but absent in normal aging. Formed via cyclization of truncated Aß species by glutaminyl cyclase (QC; QPCT) and/or its isoenzyme (isoQC; QPCTL), pE3-Aß aggregates rapidly and is known to seed additional Aß aggregation. To directly investigate pE3-Aß toxicity in vivo, we generated and characterized transgenic TBA2.1 and TBA2.2 mice, which express truncated mutant human Aß. Along with a rapidly developing behavioral phenotype, these mice showed progressively accumulating Aß and pE3-Aß deposits in brain regions of neuronal loss, impaired long-term potentiation, microglial activation, and astrocytosis. Illustrating a threshold for pE3-Aß neurotoxicity, this phenotype was not found in heterozygous animals but in homozygous TBA2.1 or double-heterozygous TBA2.1/2.2 animals only. A significant amount of pE3-Aß formation was shown to be QC-dependent, because crossbreeding of TBA2.1 with QC knock-out, but not isoQC knock-out, mice significantly reduced pE3-Aß levels. Hence, lowering the rate of QC-dependent posttranslational pE3-Aß formation can, in turn, lower the amount of neurotoxic Aß species in AD.


Assuntos
Precursor de Proteína beta-Amiloide/biossíntese , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Transtornos Heredodegenerativos do Sistema Nervoso/patologia , Hipocampo/patologia , Ácido Pirrolidonocarboxílico/metabolismo , Envelhecimento/patologia , Envelhecimento/psicologia , Doença de Alzheimer/patologia , Animais , Comportamento Animal , Encéfalo/patologia , Ensaio de Imunoadsorção Enzimática , Gliose/patologia , Transtornos Heredodegenerativos do Sistema Nervoso/psicologia , Humanos , Imuno-Histoquímica , Cinética , Potenciação de Longa Duração/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Microscopia Eletrônica , Plasticidade Neuronal/genética , Plasticidade Neuronal/fisiologia , Fenótipo , Equilíbrio Postural/fisiologia , Processamento de Proteína Pós-Traducional , Reflexo de Sobressalto/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
4.
J Biol Chem ; 286(16): 14199-208, 2011 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-21330373

RESUMO

Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate (pGlu) residues at the N terminus of peptides and proteins. Hypothalamic pGlu hormones, such as thyrotropin-releasing hormone and gonadotropin-releasing hormone are essential for regulation of metabolism and fertility in the hypothalamic pituitary thyroid and gonadal axes, respectively. Here, we analyzed the consequences of constitutive genetic QC ablation on endocrine functions and on the behavior of adult mice. Adult homozygous QC knock-out mice are fertile and behave indistinguishably from wild type mice in tests of motor function, cognition, general activity, and ingestion behavior. The QC knock-out results in a dramatic drop of enzyme activity in the brain, especially in hypothalamus and in plasma. Other peripheral organs like liver and spleen still contain QC activity, which is most likely caused by its homolog isoQC. The serum gonadotropin-releasing hormone, TSH, and testosterone concentrations were not changed by QC depletion. The serum thyroxine was decreased by 24% in homozygous QC knock-out animals, suggesting a mild hypothyroidism. QC knock-out mice were indistinguishable from wild type with regard to blood glucose and glucose tolerance, thus differing from reports of thyrotropin-releasing hormone knock-out mice significantly. The results suggest a significant formation of the hypothalamic pGlu hormones by alternative mechanisms, like spontaneous cyclization or conversion by isoQC. The different effects of QC depletion on the hypothalamic pituitary thyroid and gonadal axes might indicate slightly different modes of substrate conversion of both enzymes. The absence of significant abnormalities in QC knock-out mice suggests the presence of a therapeutic window for suppression of QC activity in current drug development.


Assuntos
Aminoaciltransferases/genética , Hipogonadismo/genética , Hipotireoidismo/genética , Aminoaciltransferases/metabolismo , Animais , Desenho de Fármacos , Células-Tronco Embrionárias/citologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Ácido Pirrolidonocarboxílico/química , Testosterona/metabolismo , Tireotropina/metabolismo
5.
Acta Neuropathol ; 112(3): 267-76, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16791600

RESUMO

Disturbance of intracellular trafficking plays a major role in several neurodegenerative disorders including Alzheimer or Parkinson's disease. The Chediak-Higashi syndrome (CHS), a life-threatening autosomal recessive disease with frequent mutations in the LYST gene, and its animal model, the beige mouse, are both characterized by lysosomal defects with accumulation of giant lysosomes. Clinically they manifest as hypopigmentation, abnormal bleeding and increased susceptibility to infection with various degrees of involvement of the nervous system. In the course of a recessive N-ethyl-N-nitrosurea (ENU) mutagenesis screen, we identified the first murine missense mutation in the lysosomal trafficking regulator gene (Lyst(Ing3618)) located at a highly conserved position in the WD40 protein domain. Nearly all described human Lyst alleles lead to protein truncation and fatal childhood CHS. Only four different missense mutations have been reported in patients with adolescent or adult forms of CHS involving the nervous system. Interestingly, the Lyst(Ing3618) model presents with a predominant neurodegenerative phenotype with progressive degeneration and loss of Purkinje cells and lacks severe impairment of the immune system. Therefore, the Lyst(Ing3618 )allele could represent a new model for adult CHS with neurological impairment. It could also provide an important tool to elucidate the role of neuronal lysosomal trafficking in the pathophysiology of neurodegeneration.


Assuntos
Proteínas dos Microfilamentos/genética , Mutação de Sentido Incorreto/fisiologia , Degeneração Neural/genética , Proteínas/genética , Células de Purkinje/patologia , Sequência de Aminoácidos , Animais , Calbindinas , Mapeamento Cromossômico , Clonagem Molecular , DNA/genética , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Dados de Sequência Molecular , Degeneração Neural/patologia , Reação do Ácido Periódico de Schiff , Fenótipo , Proteína G de Ligação ao Cálcio S100/metabolismo , Proteínas de Transporte Vesicular
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