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2.
Nature ; 537(7618): 50-6, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27582220

RESUMO

Alzheimer's disease (AD) is characterized by deposition of amyloid-ß (Aß) plaques and neurofibrillary tangles in the brain, accompanied by synaptic dysfunction and neurodegeneration. Antibody-based immunotherapy against Aß to trigger its clearance or mitigate its neurotoxicity has so far been unsuccessful. Here we report the generation of aducanumab, a human monoclonal antibody that selectively targets aggregated Aß. In a transgenic mouse model of AD, aducanumab is shown to enter the brain, bind parenchymal Aß, and reduce soluble and insoluble Aß in a dose-dependent manner. In patients with prodromal or mild AD, one year of monthly intravenous infusions of aducanumab reduces brain Aß in a dose- and time-dependent manner. This is accompanied by a slowing of clinical decline measured by Clinical Dementia Rating-Sum of Boxes and Mini Mental State Examination scores. The main safety and tolerability findings are amyloid-related imaging abnormalities. These results justify further development of aducanumab for the treatment of AD. Should the slowing of clinical decline be confirmed in ongoing phase 3 clinical trials, it would provide compelling support for the amyloid hypothesis.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/psicologia , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Anticorpos Monoclonais Humanizados/uso terapêutico , Placa Amiloide/tratamento farmacológico , Placa Amiloide/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Amiloide/efeitos dos fármacos , Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/farmacocinética , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Ensaios Clínicos Fase III como Assunto , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Modelos Biológicos , Placa Amiloide/patologia , Agregação Patológica de Proteínas/tratamento farmacológico , Solubilidade
3.
J Pharmacol Exp Ther ; 350(1): 110-23, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24756303

RESUMO

Multiple sclerosis (MS) is an autoimmune-inflammatory disease of the central nervous system (CNS) with prominent demyelination and axonal injury. While most MS therapies target the immunologic response, there is a large unmet need for treatments that can promote CNS repair. LINGO-1 (leucine-rich repeat and Ig-containing Nogo receptor interacting protein-1) is a membrane protein selectively expressed in the CNS that suppresses myelination, preventing the repair of damaged axons. We are investigating LINGO-1 antagonist antibodies that lead to remyelination as a new paradigm for treatment of individuals with MS. The anti-LINGO-1 Li81 antibody,BIIB033, is currently in clinical trials and is the first MS treatment targeting CNS repair. Here, to elucidate the mechanism of action of the antibody, we solved the crystal structure of the LINGO-1-Li81 Fab complex and used biochemical and functional studies to investigate structure-function relationships. Li81 binds to the convex surface of the leucine-rich repeat domain of LINGO-1 within repeats 4-8. Fab binding blocks contact points used in the oligomerization of LINGO-1 and produces a stable complex containing two copies each of LINGO-1 and Fab that results from a rearrangement of contacts stabilizing the quaternary structure of LINGO-1. The formation of the LINGO-1-Li81 Fab complex masks functional epitopes within the Ig domain of LINGO-1 that are important for its biologic activity in oligodendrocyte differentiation. These studies provide new insights into the structure and biology of LINGO-1 and how Li81 monoclonal antibody can block its function.


Assuntos
Anticorpos Monoclonais/metabolismo , Fragmentos Fab das Imunoglobulinas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Diferenciação Celular/imunologia , Feminino , Humanos , Estrutura Molecular , Esclerose Múltipla/tratamento farmacológico , Oligodendroglia/imunologia , Ligação Proteica , Estrutura Quaternária de Proteína , Ratos
4.
J Neurosci ; 31(44): 15861-9, 2011 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-22049429

RESUMO

Growing evidence supports the hypothesis that soluble, diffusible forms of the amyloid ß-peptide (Aß) are pathogenically important in Alzheimer's disease (AD) and thus have both diagnostic and therapeutic salience. To learn more about the dynamics of soluble Aß economy in vivo, we used microdialysis to sample the brain interstitial fluid (ISF), which contains the most soluble Aß species in brain at steady state, in >40 wake, behaving APP transgenic mice before and during the process of Aß plaque formation (age 3-28 months). Diffusible forms of Aß, especially Aß(42), declined significantly in ISF as mice underwent progressive parenchymal deposition of Aß. Moreover, radiolabeled Aß administered at physiological concentrations into ISF revealed a striking difference in the fate of soluble Aß in plaque-rich (vs plaque-free) mice: it clears more rapidly from the ISF and becomes more associated with the TBS-extractable pool, suggesting that cerebral amyloid deposits can rapidly sequester soluble Aß from the ISF. Likewise, acute γ-secretase inhibition in plaque-free mice showed a marked decline of Aß(38), Aß(40), and Aß(42), whereas in plaque-rich mice, Aß(42) declined significantly less. These results suggest that most of the Aß(42) that populates the ISF in plaque-rich mice is derived not from new Aß biosynthesis but rather from the large reservoir of less soluble Aß(42) in brain parenchyma. Together, these and other findings herein illuminate the in vivo dynamics of soluble Aß during the development of AD-type neuropathology and after γ-secretase inhibition and help explain the apparent paradox that CSF Aß(42) levels fall as humans develop AD.


Assuntos
Envelhecimento/patologia , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/patologia , Líquido Extracelular/metabolismo , Placa Amiloide/fisiopatologia , Vigília , Fatores Etários , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Análise de Variância , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática/métodos , Glicerol/metabolismo , Isótopos de Iodo/farmacocinética , Lactase/metabolismo , Camundongos , Camundongos Transgênicos , Microdiálise/métodos , Dinâmica não Linear , Ácido Pirúvico/metabolismo
5.
Biochemistry ; 50(42): 9023-35, 2011 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-21919498

RESUMO

The amyloid ß-peptide (Aß), strongly implicated in the pathogenesis of Alzheimer's disease (AD), is produced from the amyloid ß-protein precursor (APP) through consecutive proteolysis by ß- and γ-secretases. The latter protease contains presenilin as the catalytic component of a membrane-embedded aspartyl protease complex. Missense mutations in presenilin are associated with early-onset familial AD, and these mutations generally both decrease Aß production and increase the ratio of the aggregation-prone 42-residue form (Aß42) to the 40-residue form (Aß40). The connection between these two effects is not understood. Besides Aß40 and Aß42, γ-secretase produces a range of Aß peptides, the result of initial cutting at the ε site to form Aß48 or Aß49 and subsequent trimming every three or four residues. Thus, γ-secretase displays both overall proteolytic activity (ε cutting) and processivity (trimming) toward its substrate APP. Here we tested whether a decrease in total activity correlates with decreased processivity using wild-type and AD-mutant presenilin-containing protease complexes. Changes in pH, temperature, and salt concentration that reduced the overall activity of the wild-type enzyme did not consistently result in increased proportions of longer Aß peptides. Low salt concentrations and acidic pH were notable exceptions that subtly alter the proportion of individual Aß peptides, suggesting that the charged state of certain residues may influence processivity. Five different AD mutant complexes, representing a broad range of effects on overall activity, Aß42:Aß40 ratios, and ages of disease onset, were also tested, revealing again that changes in total activity and processivity can be dissociated. Factors that control initial proteolysis of APP at the ε site apparently differ significantly from factors affecting subsequent trimming and the distribution of Aß peptides.


Assuntos
Doença de Alzheimer/enzimologia , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/genética , Mutagênese Sítio-Dirigida , Presenilina-1/química , Presenilina-1/genética , Processamento de Proteína Pós-Traducional/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/biossíntese , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Células CHO , Domínio Catalítico/genética , Linhagem Celular , Cricetinae , Ativação Enzimática/genética , Humanos , Fragmentos de Peptídeos/biossíntese , Presenilina-1/metabolismo , Presenilina-2/química , Presenilina-2/genética , Presenilina-2/metabolismo , Proteólise , Especificidade por Substrato/genética
6.
Cytoskeleton (Hoboken) ; 67(2): 120-33, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20169536

RESUMO

Cellular processes propelled by actin polymerization require rapid disassembly of filaments, and then efficient recycling of ADF/cofilin-bound ADP-actin monomers back to an assembly-competent ATP-bound state. How monomer recharging is regulated in vivo is still not well understood, but recent work suggests the involvement of the ubiquitous actin-monomer binding protein Srv2/CAP. To better understand Srv2/CAP mechanism, we explored the contribution of its WH2 domain, the function of which has remained highly elusive. We found that the WH2 domain binds to actin monomers and, unlike most other WH2 domains, exhibits similar binding affinity for ATP-actin and ADP-actin (K(d) approximately 1.5 microM). Mutations in the WH2 domain that impair actin binding disrupt the ability of purified full-length Srv2/CAP to catalyze nucleotide exchange on ADF/cofilin-bound actin monomers and accelerate actin turnover in vitro. The same mutations impair Srv2/CAP function in vivo in regulating actin organization, cell growth, and cell morphogenesis. Thus, normal cell growth and organization depend on the ability of Srv2/CAP to recharge actin monomers, and the WH2 domain plays a central role in this process. Our data also reveal that while most isolated WH2 domains inhibit nucleotide exchange on actin, WH2 domains in the context of intact proteins can help promote nucleotide exchange.


Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Análise Mutacional de DNA , Microscopia de Fluorescência , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Coelhos , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos
7.
J Biol Chem ; 284(16): 10923-34, 2009 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-19201756

RESUMO

Srv2/cyclase-associated protein is expressed in virtually all plant, animal, and fungal organisms and has a conserved role in promoting actin depolymerizing factor/cofilin-mediated actin turnover. This is achieved by the abilities of Srv2 to recycle cofilin from ADP-actin monomers and to promote nucleotide exchange (ATP for ADP) on actin monomers. Despite this important and universal role in facilitating actin turnover, the mechanism underlying Srv2 function has remained elusive. Previous studies have demonstrated a critical functional role for the G-actin-binding C-terminal half of Srv2. Here we describe an equally important role in vivo for the N-terminal half of Srv2 in driving actin turnover. We pinpoint this activity to a conserved patch of surface residues on the N-terminal dimeric helical folded domain of Srv2, and we show that this functional site interacts with cofilin-actin complexes. Furthermore, we show that this site is essential for Srv2 acceleration of cofilin-mediated actin turnover in vitro. A cognate Srv2-binding site is identified on a conserved surface of cofilin, suggesting that this function likely extends to other organisms. In addition, our analyses reveal that higher order oligomerization of Srv2 depends on its N-terminal predicted coiled coil domain and that oligomerization optimizes Srv2 function in vitro and in vivo. Based on these data, we present a revised model for the mechanism by which Srv2 promotes actin turnover, in which coordinated activities of its N- and C-terminal halves catalyze sequential steps in recycling cofilin and actin monomers.


Assuntos
Fatores de Despolimerização de Actina/química , Fatores de Despolimerização de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Difosfato de Adenosina/análogos & derivados , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Despolimerização de Actina/genética , Actinas/genética , Proteínas Adaptadoras de Transdução de Sinal , Difosfato de Adenosina/química , Difosfato de Adenosina/genética , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas do Citoesqueleto/genética , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência
8.
J Cell Sci ; 120(Pt 7): 1225-34, 2007 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-17376963

RESUMO

Profilin and cyclase-associated protein (CAP, known in yeast as Srv2) are ubiquitous and abundant actin monomer-binding proteins. Profilin catalyses the nucleotide exchange on actin monomers and promotes their addition to filament barbed ends. Srv2/CAP recycles newly depolymerized actin monomers from ADF/cofilin for subsequent rounds of polymerization. Srv2/CAP also harbors two proline-rich motifs and has been suggested to interact with profilin. However, the mechanism and biological role of the possible profilin-Srv2/CAP interaction has not been investigated. Here, we show that Saccharomyces cerevisiae Srv2 and profilin interact directly (K(D) approximately 1.3 microM) and demonstrate that a specific proline-rich motif in Srv2 mediates this interaction in vitro and in vivo. ADP-actin monomers and profilin do not interfere with each other's binding to Srv2, suggesting that these three proteins can form a ternary complex. Genetic and cell biological analyses on an Srv2 allele (srv2-201) defective in binding profilin reveals that a direct interaction with profilin is not essential for Srv2 cellular function. However, srv2-201 causes a moderate increase in cell size and partially suppresses the cell growth and actin organization defects of an actin binding mutant profilin (pfy1-4). Together these data suggest that Srv2 is an important physiological interaction partner of profilin.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Profilinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Actinas/análise , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Alelos , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Tamanho Celular , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/isolamento & purificação , Imunoprecipitação , Cinética , Mutação , Profilinas/genética , Prolina/química , Ligação Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Espectrometria de Fluorescência
9.
Mol Biol Cell ; 16(7): 3128-39, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15872087

RESUMO

Abp1 is a multidomain protein that regulates the Arp2/3 complex and links proteins involved in endocytosis to the actin cytoskeleton. All of the proposed cellular functions of Abp1 involve actin filament binding, yet the actin binding site(s) on Abp1 have not been identified, nor has the importance of actin binding for Abp1 localization and function in vivo been tested. Here, we report the crystal structure of the Saccharomyces cerevisiae Abp1 actin-binding actin depolymerizing factor homology (ADFH) domain and dissect its activities by mutagenesis. Abp1-ADFH domain and ADF/cofilin structures are similar, and they use conserved surfaces to bind actin; however, there are also key differences that help explain their differential effects on actin dynamics. Using point mutations, we demonstrate that actin binding is required for localization of Abp1 in vivo, the lethality caused by Abp1 overexpression, and the ability of Abp1 to activate Arp2/3 complex. Furthermore, we genetically uncouple ABP1 functions that overlap with SAC6, SLA1, and SLA2, showing they require distinct combinations of activities and interactions. Together, our data provide the first structural and functional view of the Abp1-actin interaction and show that Abp1 has distinct cellular roles as an adapter, linking different sets of ligands for each function.


Assuntos
Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiologia , Actinas/química , Actinas/metabolismo , Alelos , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Endocitose , Proteínas de Fluorescência Verde/metabolismo , Immunoblotting , Cinética , Ligantes , Proteínas dos Microfilamentos/genética , Microscopia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Mutação , Fenótipo , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Fatores de Tempo
10.
Mol Biol Cell ; 15(11): 5158-71, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15356265

RESUMO

Cyclase-associated protein (CAP), also called Srv2 in Saccharomyces cerevisiae, is a conserved actin monomer-binding protein that promotes cofilin-dependent actin turnover in vitro and in vivo. However, little is known about the mechanism underlying this function. Here, we show that S. cerevisiae CAP binds with strong preference to ADP-G-actin (Kd 0.02 microM) compared with ATP-G-actin (Kd 1.9 microM) and competes directly with cofilin for binding ADP-G-actin. Further, CAP blocks actin monomer addition specifically to barbed ends of filaments, in contrast to profilin, which blocks monomer addition to pointed ends of filaments. The actin-binding domain of CAP is more extensive than previously suggested and includes a recently solved beta-sheet structure in the C-terminus of CAP and adjacent sequences. Using site-directed mutagenesis, we define evolutionarily conserved residues that mediate binding to ADP-G-actin and demonstrate that these activities are required for CAP function in vivo in directing actin organization and polarized cell growth. Together, our data suggest that in vivo CAP competes with cofilin for binding ADP-actin monomers, allows rapid nucleotide exchange to occur on actin, and then because of its 100-fold weaker binding affinity for ATP-actin compared with ADP-actin, allows other cellular factors such as profilin to take the handoff of ATP-actin and facilitate barbed end assembly.


Assuntos
Actinas/química , Difosfato de Adenosina/química , Proteínas de Ciclo Celular/fisiologia , Proteínas do Citoesqueleto/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Fatores de Despolimerização de Actina , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Alelos , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Dicroísmo Circular , Proteínas do Citoesqueleto/metabolismo , Dimerização , Relação Dose-Resposta a Droga , Glutationa Transferase/metabolismo , Cinética , Proteínas dos Microfilamentos/química , Dados de Sequência Molecular , Mutação , Fenótipo , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Temperatura , Fatores de Tempo
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