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1.
FASEB J ; 33(4): 5076-5081, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30608876

RESUMO

The amyloid precursor-like protein 2 (APLP2) molecule is a type I transmembrane protein that is crucial for survival, cell-cell adhesion, neuronal development, myelination, cancer metastasis, modulation of metal, and glucose and insulin homeostasis. Moreover, the importance of the amyloid precursor protein (APP) family in biology and disease is very well known because of its central role in Alzheimer disease. In this study, we determined the crystal structure of the independently folded E2 domain of APLP2 and compared that with its paralogues APP and APLP2, demonstrating high overall structural similarities. The crystal structure of APLP2 E2 was solved as an antiparallel dimer, and analysis of the protein interfaces revealed a distinct mode of dimerization that differs from the previously reported dimerization of either APP or APLP1. Analysis of the APLP2 E2 metal binding sites suggested it binds zinc and copper in a similar manner to APP and APLP1. The structure of this key protein might suggest a relationship between the distinct mode of dimerization and its biologic functions.-Roisman, L. C., Han, S., Chuei, M. J., Connor, A. R., Cappai, R. The crystal structure of amyloid precursor-like protein 2 E2 domain completes the amyloid precursor protein family.


Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Precursor de Proteína beta-Amiloide/química , Sítios de Ligação , Dicroísmo Circular , Cristalografia por Raios X , Homeostase , Humanos , Relação Estrutura-Atividade
2.
Brain ; 134(Pt 4): 1089-100, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21436112

RESUMO

While considerable effort has focused on developing positron emission tomography ß-amyloid imaging radiotracers for the early diagnosis of Alzheimer's disease, no radiotracer is available for the non-invasive quantification of tau. In this study, we detail the characterization of (18)F-THK523 as a novel tau imaging radiotracer. In vitro binding studies demonstrated that (18)F-THK523 binds with higher affinity to a greater number of binding sites on recombinant tau (K18Δ280K) compared with ß-amyloid(1-42) fibrils. Autoradiographic and histofluorescence analysis of human hippocampal serial sections with Alzheimer's disease exhibited positive THK523 binding that co-localized with immunoreactive tau pathology, but failed to highlight ß-amyloid plaques. Micro-positron emission tomography analysis demonstrated significantly higher retention of (18)F-THK523 (48%; P < 0.007) in tau transgenic mice brains compared with their wild-type littermates or APP/PS1 mice. The preclinical examination of THK523 has demonstrated its high affinity and selectivity for tau pathology both in vitro and in vivo, indicating that (18)F-THK523 fulfils ligand criteria for human imaging trials.


Assuntos
Doença de Alzheimer/diagnóstico por imagem , Compostos de Anilina/farmacologia , Encéfalo/diagnóstico por imagem , Fluordesoxiglucose F18/farmacologia , Quinolinas/farmacologia , Compostos Radiofarmacêuticos/farmacologia , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Análise de Variância , Animais , Autorradiografia , Sítios de Ligação , Encéfalo/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Cintilografia
3.
Metallomics ; 11(1): 128-140, 2019 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-30465671

RESUMO

Abnormal protein structure and function have been implicated as the toxic species in many diseases including neurodegenerative diseases, such as Parkinson's. One key pathological hallmark in Parkinson's disease is the formation of Lewy bodies, of which alpha-synuclein is the major component. These Lewy bodies are formed by the aggregation and oligomerization of alpha-synuclein. The oligomeric form of the protein is suspected to be the main contributor to the neurotoxicity seen in the disease. The formation of toxic oligomers has been shown to occur through reactions with lipids, dopamine, hydrogen peroxide as well as metals. The interplay between metals and alpha-synuclein has also been proposed to cause oxidative stress, which promotes the formation of protein aggregates. Most studies investigating the relationship of Cu, Fe and Zn with alpha-synuclein have relied on the use of recombinant protein and there is little evidence that the interaction between metals and alpha-synuclein are physiologically relevant. To address this gap in our knowledge we have characterized the metal content and metal binding capacity of alpha-synuclein purified from human erythrocytes and brain tissue. In addition, we examined the ability of dityrosine cross-linked alpha-synuclein oligomers to bind Cu, Fe and Zn. Using size exclusion chromatography-inductively coupled plasma-mass spectrometry we demonstrated that native human alpha-synuclein, recombinant familial mutants and oligomers do not bind to significant amounts of metal even when they are added to the protein in excess.


Assuntos
Cobre/metabolismo , Ferro/metabolismo , Zinco/metabolismo , alfa-Sinucleína/metabolismo , Sítios de Ligação , Encéfalo/metabolismo , Química Encefálica , Cobre/análise , Eritrócitos/química , Eritrócitos/metabolismo , Humanos , Ferro/análise , Espectrometria de Massas/métodos , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Zinco/análise , alfa-Sinucleína/química
4.
Matrix Biol ; 59: 23-38, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27471094

RESUMO

Members of the CCN family of matricellular proteins are cytokines linking cells to the extracellular matrix. We report that CCN3 (Nov) and CCN5 (WISP2) are novel substrates of MMP14 (membrane-type 1-matrix metalloproteinase, MT1-MMP) that we identified using MMP14 "inactive catalytic domain capture" (ICDC) as a yeast two-hybrid protease substrate trapping platform in parallel with degradomics mass spectrometry screens for MMP14 substrates. CCN3 and CCN5, previously unknown substrates of MMPs, were biochemically validated as substrates of MMP14 and other MMPs in vitro-CCN5 was processed in the variable region by MMP14 and MMP2, as well as by MMP1, 3, 7, 8, 9 and 15. CCN1, 2 and 3 are proangiogenic factors yet we found novel opposing activity of CCN5 that was potently antiangiogenic in an aortic ring vessel outgrowth model. MMP14, a known regulator of angiogenesis, cleaved CCN5 and abrogated the angiostatic activity. CCN3 was also processed in the variable region by MMP14 and MMP2, and by MMP1, 8 and 9. In addition to the previously reported cleavages of CCN1 and CCN2 by several MMPs we found that MMPs 8, 9, and 1 process CCN1, and MMP8 and MMP9 also process CCN2. Thus, our study reveals additional and pervasive family-wide processing of CCN matricellular proteins/cytokines by MMPs. Furthermore, CCN5 cleavage by proangiogenic MMPs results in removal of an angiogenic brake held by CCN5. This highlights the importance of thorough dissection of MMP substrates that is needed to reveal higher-level control mechanisms beyond type IV collagen and other extracellular matrix protein remodelling in angiogenesis. SUMMARY: We find CCN family member cleavage by MMPs is more pervasive than previously reported and includes CCN3 (Nov) and CCN5 (WISP2). CCN5 is a novel antiangiogenic factor, whose function is abrogated by proangiogenic MMP cleavage. By processing CCN proteins, MMPs regulate cell responses angiogenesis in connective tissues.


Assuntos
Proteínas de Sinalização Intercelular CCN/química , Vetores Genéticos/metabolismo , Metaloproteinase 14 da Matriz/química , Proteína Sobre-Expressa em Nefroblastoma/química , Proteínas Repressoras/química , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Sinalização Intercelular CCN/genética , Proteínas de Sinalização Intercelular CCN/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Células HeLa , Humanos , Células MCF-7 , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Modelos Moleculares , Proteína Sobre-Expressa em Nefroblastoma/genética , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Técnicas do Sistema de Duplo-Híbrido
5.
PLoS One ; 10(2): e0116497, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25679387

RESUMO

α-synuclein (α-syn) is a major component of the intracellular inclusions called Lewy bodies, which are a key pathological feature in the brains of Parkinson's disease patients. The neurotransmitter dopamine (DA) inhibits the fibrillisation of α-syn into amyloid, and promotes α-syn aggregation into SDS-stable soluble oligomers. While this inhibition of amyloid formation requires the oxidation of both DA and the methionines in α-syn, the molecular basis for these processes is still unclear. This study sought to define the protein sequences required for the generation of oligomers. We tested N- (α-syn residues 43-140) and C-terminally (1-95) truncated α-syn, and found that similar to full-length protein both truncated species formed soluble DA:α-syn oligomers, albeit 1-95 had a different profile. Using nuclear magnetic resonance (NMR), and the N-terminally truncated α-syn 43-140 protein, we analysed the structural characteristics of the DA:α-syn 43-140 dimer and α-syn 43-140 monomer and found the dimerisation interface encompassed residues 43 to 60. Narrowing the interface to this small region will help define the mechanism by which DA mediates the formation of SDS-stable soluble DA:α-syn oligomers.


Assuntos
Dopamina/metabolismo , Fragmentos de Peptídeos/química , Multimerização Proteica , alfa-Sinucleína/química , Humanos , Fragmentos de Peptídeos/metabolismo , Estrutura Secundária de Proteína , Proteólise , Solubilidade , Tripsina/metabolismo , alfa-Sinucleína/metabolismo
6.
Nucl Med Biol ; 39(7): 1042-8, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22503458

RESUMO

PURPOSE: Amyloid-ß (Aß) plaques are a major pathological hallmark of Alzheimer's disease (AD). The noninvasive detection of Aß plaques may increase the accuracy of clinical diagnosis as well as monitor therapeutic interventions. While [(11)C]-PiB is the most widely used Aß positron emission tomography (PET) radiotracer, due to the short half-life of (11)C (20 min), its application is limited to centers with an on-site cyclotron and (11)C radiochemistry expertise. Therefore, novel [(18)F] (half-life 110 min)-labeled Aß PET tracers have been developed. We have demonstrated that [(18)F]-florbetaben-PET can differentiate individuals diagnosed with AD from healthy elderly, Parkinson's disease and frontotemporal lobe dementia (FTLD-tau) patients. While [(18)F]-florbetaben-PET retention matched the reported postmortem distribution of Aß plaques, the nature of [(18)F]-florbetaben binding to other pathological lesions comprising misfolded proteins needs further assessment. The objective of this study was to determine whether Florbetaben selectively binds to Aß plaques in postmortem tissue specimens containing mixed pathological hallmarks (i.e., tau and α-synuclein aggregates). METHOD: Human AD, FTLD-tau and dementia with Lewy bodies (DLB) brain sections were analyzed by [(18)F]-florbetaben autoradiography and [(3)H]-florbetaben high-resolution emulsion autoradiography and [(19)F]-florbetaben fluorescence microscopy. RESULTS: Both autoradiographical analyses demonstrated that Florbetaben exclusively bound Aß plaques in AD brain sections at low nanomolar concentrations. Furthermore, at concentrations thousand-folds higher than those during a PET scan, [(19)F]-florbetaben did not bind to α-synuclein or tau aggregates in DLB and FTLD-tau brain sections, respectively. Detection of [(19)F]-florbetaben staining by fluorescence microscopy in several AD brain regions demonstrated that Florbetaben identified Aß plaques in all brain regions examined. CONCLUSION: This study provides further evidence that [(18)F]-florbetaben-PET is a highly selective radiotracer to assess Aß plaque deposition in the brain.


Assuntos
Compostos de Anilina/metabolismo , Imagem Molecular , Placa Amiloide/diagnóstico , Placa Amiloide/metabolismo , Estilbenos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Placa Amiloide/patologia , Traçadores Radioativos , Especificidade por Substrato , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Proteínas tau/química , Proteínas tau/metabolismo
7.
Brain Imaging Behav ; 3(3): 246-61, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22005989

RESUMO

With the advent of new therapeutic strategies aimed at reducing ß-amyloid (Aß) burden in the brain to potentially prevent or delay functional and irreversible cognitive loss, there is increased interest in developing agents that allow assessment of Aß burden in vivo. Molecular neuroimaging techniques such as positron emission tomography (PET), in conjunction with related biomarkers in plasma and cerebrospinal fluid, are proving valuable in the early and differential diagnosis of Alzheimer's disease (AD). (11)C-PiB PET has proven useful in the discrimination of dementias, showing significantly higher PiB retention in grey matter of AD patients when compared with healthy controls or patients with frontotemporal dementia. (11)C-PiB PET also appears to be more accurate than FDG for the diagnosis of AD. Despite apparently underestimating the Aß burden in the brain, (11)C-PiB PET is an optimal method to differentiate healthy controls from AD, matching histopathological reports in aging and dementia and reflecting the true regional density of Aß plaques in cortical areas. High striatal Aß deposition seems to be typical for carriers of familial forms of AD, whilst ApoE ε4 carriers, independent of diagnosis or disease severity, present with higher Aß burden than non- ε4 carriers. Characterization of the binding properties of PiB has shown that despite binding to other misfolded proteins in vitro, PiB is extremely selective for Aß at the concentrations achieved during a PET scan. Aß burden as assessed by PET does not correlate with measures of cognition or cognitive decline in AD. Approximately 30% of apparently healthy older people, and 50-60% of people with mild cognitive impairment, present with cortical (11)C-PiB retention. In these groups, Aß burden does correlate with episodic memory and rate of memory decline. These observations suggest that Aß deposition is not part of normal ageing, supporting the hypothesis that Αß deposition occurs well before the onset of symptoms and is likely to represent preclinical AD. Further longitudinal observations, coupled with different disease-specific tracers and biomarkers are required not only to confirm this hypothesis, but also to better elucidate the role of Αß deposition in the course of Alzheimer's disease.

8.
PLoS One ; 2(3): e312, 2007 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-17375198

RESUMO

We identify matrix metalloproteinase (MMP)-8, the polymorphonuclear (PMN) leukocyte collagenase, as a critical mediator initiating lipopolysaccharide (LPS)-responsiveness in vivo. PMN infiltration towards LPS is abrogated in Mmp8-null mice. MMP-8 cleaves LPS-induced CXC chemokine (LIX) at Ser(4)-Val(5) and Lys(79)-Arg(80). LIX bioactivity is increased upon N-terminal cleavage, enhancing intracellular calcium mobilization and chemotaxis upon binding its cognate receptor, CXCR2. As there is no difference in PMN chemotaxis in Mmp8-null mice compared with wild-type mice towards synthetic analogues of MMP-8-cleaved LIX, MMP-8 is not essential for extravasation or cell migration in collagenous matrices in vivo. However, with biochemical redundancy between MMPs 1, 2, 9, and 13, which also cleave LIX at position 4 approximately 5, it was surprising to observe such a markedly reduced PMN infiltration towards LPS and LIX in Mmp8-/- mice. This lack of physiological redundancy in vivo identifies MMP-8 as a key mediator in the regulation of innate immunity. Comparable results were found with CXCL8/IL-8 and CXCL5/ENA-78, the human orthologues of LIX. MMP-8 cleaves CXCL8 at Arg(5)-Ser(6) and at Val(7)-Leu(8) in CXCL5 to activate respective chemokines. Hence, rather than collagen, these PMN chemoattractants are important MMP-8 substrates in vivo; PMN-derived MMP-8 cleaves and activates LIX to execute an in cis PMN-controlled feed-forward mechanism to orchestrate the initial inflammatory response and promote LPS responsiveness in tissue.


Assuntos
Quimiotaxia/fisiologia , Lipopolissacarídeos/farmacologia , Metaloproteinase 8 da Matriz/metabolismo , Neutrófilos/fisiologia , Animais , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Quimiocinas CXC/efeitos dos fármacos , Quimiocinas CXC/genética , Quimiocinas CXC/fisiologia , Quimiotaxia/efeitos dos fármacos , Humanos , Metaloproteinase 8 da Matriz/deficiência , Metaloproteinase 8 da Matriz/genética , Camundongos , Camundongos Knockout , Neutropenia/fisiopatologia , Neutrófilos/efeitos dos fármacos , Cicatrização
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