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1.
J Neurochem ; 137(5): 820-37, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27016395

RESUMO

Huntington's disease (HD) is an inherited and fatal polyglutamine neurodegenerative disorder caused by an expansion of the CAG triplet repeat coding region within the HD gene. Progressive dysfunction and loss of striatal GABAergic medium spiny neurons (MSNs) may account for some of the characteristic symptoms in HD patients. Interestingly, in HD, MSNs expressing neuropeptide Y (NPY) are spared and their numbers is even up-regulated in HD patients. Consistent with this, we report here on increased immuno-linked NPY (IL-NPY) levels in human cerebrospinal fluid (hCSF) from HD patients (Control n = 10; early HD n = 9; mid HD n = 11). As this antibody-based detection of NPY may provide false positive differences as a result of the antibody-based detections of only fragments of NPY, the initial finding was validated by investigating the proteolytic stability of NPY in hCSF using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and selective inhibitors. A comparison between resulting NPY-fragments and detailed epitope analysis verified significant differences in IL-NPY1-36/3-36 and NPY1-30 levels between HD patients and control subjects with no significant differences between early vs mid HD cases. Ex vivo degradomics analysis demonstrated that NPY is initially degraded to NPY1-30 by cathepsin D in both HD patients and control subjects. Yet, NPY1-30 is then further differentially hydrolyzed by thimet oligopeptidase (TOP) in HD patients and by neprilysin (NEP) in control subjects. Furthermore, altered hCSF TOP-inhibitor Dynorphin A1-13 (Dyn-A1-13 ) and TOP-substrate Dyn-A1-8 levels indicate an impaired Dyn-A-TOP network in HD patients. Thus, we conclude that elevated IL-NPY-levels in conjunction with TOP-/NEP-activity/protein as well as Dyn-A1-13 -peptide levels may serve as a potential biomarker in human CSF of HD. Huntington's disease (HD) patients' cerebrospinal fluid (CSF) exhibits higher neuropeptide Y (NPY) levels. Further degradomics studies show that CSF-NPY is initially degraded to NPY1-30 by Cathepsin D. The NPY1-30 fragment is then differentially degraded in HD vs control involving Neprilysin (NEP), Thimet Oligopeptidase (TOP), and TOP-Dynorphin-A network. Together, these findings may help in search for HD biomarkers.


Assuntos
Doença de Huntington/líquido cefalorraquidiano , Doença de Huntington/diagnóstico , Neuropeptídeo Y/líquido cefalorraquidiano , Fragmentos de Peptídeos/líquido cefalorraquidiano , Proteólise , Adulto , Idoso , Animais , Biomarcadores/líquido cefalorraquidiano , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Ratos
2.
Bioorg Chem ; 60: 98-101, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25981125

RESUMO

Phosphate ions and glutaminyl cyclase (QC) both catalyze the formation of pyroglutamate (pE, pGlu) from N-terminal glutamine residues of peptides and proteins. Here, we studied the mechanism of glutamine cyclization using kinetic secondary deuterium and solvent isotope effects. The data suggest that proton transfer(s) are rate determining for the spontaneous reaction, and that phosphate and QC are accelerating the reaction by promoting synchronized proton transfers in a concerted mechanism. Thus, non-enzymatic and enzymatic catalysis of pyroglutamate formation exploit a similar mode of transition-state stabilization.


Assuntos
Aminoaciltransferases/metabolismo , Drosophila melanogaster/enzimologia , Fosfatos/metabolismo , Ácido Pirrolidonocarboxílico/metabolismo , Animais , Ciclização , Drosophila melanogaster/metabolismo , Glutamina/metabolismo , Prótons
3.
Amino Acids ; 44(4): 1205-14, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23344882

RESUMO

The formation of isoaspartate (isoAsp) from asparaginyl or aspartyl residues is a spontaneous post-translational modification of peptides and proteins. Due to isopeptide bond formation, the structure and possibly function of peptides and proteins is altered. IsoAsp modifications within the peptide chain have been reported for many cytosolic proteins. Amyloid peptides (Aß) deposited in Alzheimer's disease may carry an N-terminal isoAsp-modification. Here, we describe a quantitative investigation of isoAsp-formation from N-terminal Asn and Asp using model peptides similar to the Aß N-terminus. The study is based on a newly developed separation of peptides using capillary electrophoresis (CE). 1H NMR was employed to validate the basic finding of N-terminal isoAsp-formation from Asp and Asn. Thereby, the isomerization of Asn at neutral pH (0.6 day(-1), peptide NGEF) is approximately six times faster than that within the peptide chain (AANGEF). The difference in velocity between Asn and Asp isomerization is approximately 50-fold. In contrast to N-terminal Asn, Asp isomerization is significantly accelerated at acidic pH. The kinetic solvent isotope (kD2O/kH2O) effect of 2.46 suggests a rate-limiting proton transfer in isoAsp-formation. The proton inventory is consistent with transfer of one proton in the transition state, supporting the previous notion of rate-limiting deprotonation of the peptide backbone amide during succinimide-intermediate formation. The study provides evidence for a spontaneous N-terminal isoAsp-formation within peptides and might explain the accumulation of N-terminal isoAsp in amyloid deposits.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Asparagina/química , Ácido Aspártico/química , Ácido Isoaspártico/química , Doença de Alzheimer/patologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Peptídeos beta-Amiloides/metabolismo , Asparagina/metabolismo , Ácido Aspártico/metabolismo , Eletroforese Capilar , Humanos , Concentração de Íons de Hidrogênio , Ácido Isoaspártico/metabolismo , Isomerismo , Cinética , Placa Amiloide , Processamento de Proteína Pós-Traducional
4.
J Neurochem ; 121(5): 774-84, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22375951

RESUMO

Pyroglutamate (pGlu)-modified amyloid peptides have been identified in sporadic and familial forms of Alzheimer's disease (AD) and the inherited disorders familial British and Danish Dementia (FBD and FDD). In this study, we characterized the aggregation of amyloid-ß protein Aß37, Aß38, Aß40, Aß42 and ADan species in vitro, which were modified by N-terminal pGlu (pGlu-Aß3-x, pGlu-ADan) or possess the intact N-terminus (Aß1-x, ADan). The pGlu-modification confers rapid formation of oligomers and short fibrillar aggregates. In accordance with these observations, the pGlu-modified Aß38, Αß40 and Αß42 species inhibit hippocampal long term potentiation of synaptic response, but pGlu-Aß3-42 showing the highest effect. Among the unmodified Aß peptides, only Aß1-42 exhibites such propensity, which was similar to pGlu-Aß3-38 and pGlu-Aß3-40. Likewise, the amyloidogenic peptide pGlu-ADan impaired synaptic potentiation more pronounced than N-terminal unmodified ADan. The results were validated using conditioned media from cultivated HEK293 cells, which express APP variants favoring the formation of Aß1-x, Aß3-x or N-truncated pGlu-Aß3-x species. Hence, we show that the ability of different amyloid peptides to impair synaptic function apparently correlates to their potential to form oligomers as a common mechanism. The pGlu-modification is apparently mediating a higher surface hydrophobicity, as shown by 1-anilinonaphtalene-8-sulfonate fluorescence, which enforces potential to interfere with neuronal physiology.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Hipocampo/metabolismo , Potenciação de Longa Duração/fisiologia , Ácido Pirrolidonocarboxílico/metabolismo , Peptídeos beta-Amiloides/química , Animais , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Hipocampo/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ácido Pirrolidonocarboxílico/química
5.
J Pept Sci ; 18(11): 691-5, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23001756

RESUMO

The aggregation of the Aß plays a fundamental role in the pathology of AD. Recently, N-terminally modified Aß species, pE-Aß, have been described as major constituents of Aß deposits in the brains of AD patients. pE-Aß has an increased aggregation propensity and shows increased toxicity compared with Aß1-40 and Aß1-42. In the present work, high-resolution NMR spectroscopy was performed to study pE-Aß3-40 in aqueous TFE-containing solution. Two-dimensional TOCSY and NOESY experiments were performed. On the basis of NOE and chemical shift data, pE-Aß3-40 was shown to contain two helical regions formed by residues 14-22 and 30-36. This is similar as previously described for Aß1-40. However, the secondary chemical shift data indicate decreased helical propensity in pE-Aß3-40 when compared with Aß1-40 under exactly the same conditions. This is in agreement with the observation that pE-Aß3-40 shows a drastically increased tendency to form ß-sheet-rich structures under more physiologic conditions. Structural studies of pE-Aß are crucial for better understanding the structural basis of amyloid fibril formation in the brain during development of AD, especially because an increasing number of reports indicate a decisive role of pE-Aß for the pathogenesis of AD.


Assuntos
Peptídeos beta-Amiloides/química , Espectroscopia de Ressonância Magnética , Ácido Pirrolidonocarboxílico/química , Doença de Alzheimer/patologia , Humanos , Isoformas de Proteínas/química
6.
Biochemistry ; 50(28): 6280-8, 2011 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-21671571

RESUMO

Formation of N-terminal pyroglutamate (pGlu or pE) from glutaminyl or glutamyl precursors is catalyzed by glutaminyl cyclases (QC). As the formation of pGlu-amyloid has been linked with Alzheimer's disease, inhibitors of QCs are currently the subject of intense development. Here, we report three crystal structures of N-glycosylated mammalian QC from humans (hQC) and mice (mQC). Whereas the overall structures of the enzymes are similar to those reported previously, two surface loops in the neighborhood of the active center exhibit conformational variability. Furthermore, two conserved cysteine residues form a disulfide bond at the base of the active center that was not present in previous reports of hQC structure. Site-directed mutagenesis suggests a structure-stabilizing role of the disulfide bond. At the entrance to the active center, the conserved tryptophan residue, W(207), which displayed multiple orientations in previous structure, shows a single conformation in both glycosylated human and murine QCs. Although mutagenesis of W(207) into leucine or glutamine altered substrate conversion significantly, the binding constants of inhibitors such as the highly potent PQ50 (PBD150) were minimally affected. The crystal structure of PQ50 bound to the active center of murine QC reveals principal binding determinants provided by the catalytic zinc ion and a hydrophobic funnel. This study presents a first comparison of two mammalian QCs containing typical, conserved post-translational modifications.


Assuntos
Aminoaciltransferases/química , Aminoaciltransferases/metabolismo , Sequência de Aminoácidos , Aminoaciltransferases/genética , Animais , Bovinos , Sequência Conservada , Cristalografia por Raios X , Ativação Enzimática/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Glicosilação , Humanos , Camundongos , Dados de Sequência Molecular , Pichia/enzimologia , Pichia/genética , Conformação Proteica , Processamento de Proteína Pós-Traducional/genética , Ratos , Ovinos
7.
Biol Chem ; 389(8): 1043-53, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18979629

RESUMO

Posttranslational modifications influence the structure, stability and biological activity of proteins. Most of the reactions are enzyme-catalyzed, but some, such as asparagine (Asn) and glutamine (Gln) deamidation and the isoaspartate (isoAsp) formation within peptide chains, occur spontaneously. It has been previously shown that certain peptide sequences form isoAsp quite fast if the Asp stretches are exposed to the protein surface, thereby potentially changing susceptibility to proteolysis at these sites. This tempted us to investigate the activity of exo- and endopeptidases against Asp- or isoAsp-containing substrates. Members of the prolyl oligopeptidase family were unable to cleave substrates after proline if isoAsp was placed in the P2-position. Caspases, usually accepting Asp at P1-position of their substrates, did not cleave isoAsp-containing sequences. Similarly, the metal-dependent aminopeptidase amino peptidase N did not turnover N-terminal isoAsp-containing substrates, nor could the endopeptidase matrix metalloproteinase 3 (MMP 3) hydrolyze a serum amyloid A protein-like substrate if the sequence contained isoAsp instead of Asp. Also, the highly specific enterokinase, usually clipping after a stretch of four Asp residues and a lysine in the P1 position, could not turnover substrates if the P2 amino acid was replaced by isoAsp. In contrast, acylamino acid-releasing enzyme and dipeptidyl peptidases 1, 2 and 4 hydrolyzed substrates containing the isoAsp-Ala motif.


Assuntos
Ácido Isoaspártico/metabolismo , Peptídeo Hidrolases/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática , Humanos , Hidrólise , Ácido Isoaspártico/química , Cinética , Estrutura Molecular , Especificidade por Substrato
8.
Biol Chem ; 384(12): 1583-92, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14719800

RESUMO

Glutaminyl cyclases (QC) catalyze the intramolecular cyclization of N-terminal glutamine residues of peptides and proteins. For a comparison of the substrate specificity of human and papaya QC enzymes, a novel continuous assay was established by adapting an existing discontinuous method. Specificity constants (kcat/Km) of dipeptides and dipeptide surrogates were higher for plant QC, whereas the selectivity for oligopeptides was similar for both enzymes. However, only the specificity constants of mammalian QC were dependent on size and composition of the substrates. Specificity constants of both enzymes were equally pH-dependent in the acidic pH-region, revealing a pKa value identical to the pKa of the substrate, suggesting similarities in the substrate conversion mode. Accordingly, both QCs converted the L-beta homoglutaminyl residue in the peptide H-beta homoGln-Phe-Lys-Arg-Leu-Ala-NH2 and the glutaminyl residues of the branched peptide H-Gln-Lys(Gln)-Arg-Leu-Ala-NH2 as well as the partially cyclized peptide H-Gln-cyclo(N epsilon-Lys-Arg-Pro-Ala-Gly-Phe). In contrast, only QC from C. papaya was able to cyclize a methylated glutamine residue, while this compound did not even inhibit human QC-catalysis, suggesting distinct substrate recognition pattern. The conversion of the potential physiological substrates [Gln1]-gastrin, [Gln1]-neurotensin and [Gln1]-fertilization promoting peptide indicates that human QC may play a key role in posttranslational modification of most if not all pGlu-containing hormones.


Assuntos
Aminoaciltransferases/metabolismo , Carica/enzimologia , Hormônio Liberador de Tireotropina/análogos & derivados , Aminoácidos/química , Aminoaciltransferases/química , Animais , Catálise , Bovinos , Ciclização , Dipeptídeos/metabolismo , Estabilidade Enzimática/efeitos dos fármacos , Gastrinas/metabolismo , Glutamato Desidrogenase/metabolismo , Glutamina/química , Glutamina/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ácidos Cetoglutáricos/metabolismo , Cinética , NAD/metabolismo , Neurotensina/metabolismo , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Concentração Osmolar , Ácido Pirrolidonocarboxílico/análogos & derivados , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato , Hormônio Liberador de Tireotropina/metabolismo
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