RESUMO
Presenilin and signal peptide peptidase are multispanning intramembrane-cleaving proteases with a conserved catalytic GxGD motif. Presenilin comprises the catalytic subunit of γ-secretase, a protease responsible for the generation of amyloid-ß peptides causative of Alzheimer disease. Signal peptide peptidase proteins are implicated in the regulation of the immune system. Both protease family proteins have been recognized as druggable targets for several human diseases, but their detailed structure still remains unknown. Recently, the x-ray structures of some archaeal GxGD proteases have been determined. We review the recent progress in biochemical and biophysical probing of the structure of these atypical proteases.
Assuntos
Proteínas de Membrana/química , Presenilinas/química , Serina Endopeptidases/química , Doença de Alzheimer/imunologia , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/imunologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/imunologia , Peptídeos beta-Amiloides/metabolismo , Animais , Archaea/química , Archaea/imunologia , Archaea/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/imunologia , Proteínas Arqueais/metabolismo , Domínio Catalítico , Humanos , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Presenilinas/imunologia , Presenilinas/metabolismo , Estrutura Terciária de Proteína , Serina Endopeptidases/imunologia , Serina Endopeptidases/metabolismo , Relação Estrutura-AtividadeRESUMO
Bacillus Calmette-Guérin (BCG), the antituberculosis vaccine, localizes within immature phagosomes of macrophages and dendritic cells (APCs), and avoids lysosomal degradation. BCG-derived antigenic peptides are thus inefficiently processed by APCs, and we investigated alternate mechanisms of Ag processing. Proteomics identified that BCG phagosomes are enriched for nicastrin, APH, and presenilin components of γ-secretase, a multimeric protease. Using an in vitro Ag presentation assay and BCG-infected APCs, we found γ-secretase components to cleave BCG-derived Ag85B to produce a peptide epitope, which, in turn, primed IL-2 release from Ag85B-specific T cell hybridoma. siRNA knockdown or chemical inhibition of γ-secretase components using L685458 decreased the ability of BCG or Mycobacterium tuberculosis-infected APCs to present Ag85B. In addition, L685485 inhibition of γ-secretase led to a decreased ability of BCG-dendritic cells to immunize mice and induce Ag85B-specific CD4 T cells in vivo. Because BCG and M. tuberculosis sequester within APCs preventing immune recognition, γ-secretase components appear to fortuitously process the immunodominant Ag85B, facilitating immune recognition.
Assuntos
Secretases da Proteína Precursora do Amiloide/imunologia , Apresentação de Antígeno/imunologia , Antígenos de Bactérias/imunologia , Vacina BCG/imunologia , Epitopos de Linfócito T/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Células Apresentadoras de Antígenos/microbiologia , Antígenos de Bactérias/metabolismo , Endopeptidases/imunologia , Endopeptidases/metabolismo , Imunofluorescência , Antígenos de Histocompatibilidade Classe II/imunologia , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium bovis/imunologia , Mycobacterium bovis/metabolismo , Fagossomos/imunologia , Fagossomos/metabolismo , Presenilinas/imunologia , Presenilinas/metabolismo , Proteômica , Linfócitos T/metabolismoRESUMO
The study of membrane protein function and structure requires their successful detection, expression, solubilization, and/or reconstitution, which poses a challenging task and relies on the availability of suitable tools. Several research groups have successfully applied Nanobodies in the purification, as well as the functional and structural characterization of membrane proteins. Nanobodies are small, single-chain antibody fragments originating from camelids presenting on average a longer CDR3 which enables them to bind in cavities and clefts (such as active and allosteric sites). Notably, Nanobodies generally bind conformational epitopes making them very interesting tools to stabilize, dissect, and characterize specific protein conformations. In the clinic, several Nanobodies are under evaluation either as potential drug candidates or as diagnostic tools. In recent years, we have successfully generated high-affinity, conformation-sensitive anti-γ-secretase Nanobodies. γ-Secretase is a multimeric membrane protease involved in processing of the amyloid precursor protein with high clinical relevance as mutations in its catalytic subunit (Presenilin) cause early-onset Alzheimer's disease. Advancing our knowledge on the mechanisms governing γ-secretase intramembrane proteolysis through various strategies may lead to novel therapeutic avenues for Alzheimer's disease. In this chapter, we present the strategies we have developed and applied for the screening and characterization of anti-γ-secretase Nanobodies. These protocols could be of help in the generation of Nanobodies targeting other membrane proteins.
Assuntos
Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/química , Biologia Molecular/métodos , Presenilinas/química , Anticorpos de Domínio Único/química , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/imunologia , Secretases da Proteína Precursora do Amiloide/imunologia , Secretases da Proteína Precursora do Amiloide/uso terapêutico , Domínio Catalítico/genética , Epitopos/imunologia , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Mutação , Presenilinas/genética , Presenilinas/imunologia , Presenilinas/uso terapêutico , Conformação Proteica , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/uso terapêutico , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/uso terapêutico , Relação Estrutura-AtividadeRESUMO
Molecular genetic studies of familial Alzheimer's disease by 1995 had clearly implicated three proteins as critical to Alzheimer's disease (AD), the amyloid-beta protein precursor (AbetaPP) and the two homologous presenilins, PS-1 and PS-2. To account for the roles of these proteins in AD, we had proposed that as an early and critical step in the mechanisms that lead to AD, the PS on the surface of a brain cell engages in a specific receptor-ligand intercellular interaction with AbetaPP on the surface of a neighboring cell. This cell-cell interaction is required to trigger off a cascade of processes that lead to the production of amyloid-beta (Abeta) from AbetaPP, leading to AD. At about this time, however, many established AD researchers had obtained data that appeared to disagree with our proposed mechanism. Their immediate objections to our proposal were based on their conclusions that 1) The PS proteins were exclusively intracellular, and were not expressed at the cell surface, and 2) The topography of the PS proteins in intracellular membranes exhibits either 6 or 8-TM spanning domains, not 7. Here we discuss the evidence for the 6-TM, 7-TM, 8-TM and other models of PS topography and offer possibilities for the differences in interpretation of the various sets of data. We review the experimental demonstration of the cell-surface expression and the 7-TM structure of PS, the functional consequences of this structure, and the findings that PS-1 and PS-2 are members of the superfamily of 7-TM heterotrimeric G-protein coupled receptors (GPCRs).
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Precursor de Proteína beta-Amiloide/metabolismo , Presenilinas/fisiologia , Doença de Alzheimer/imunologia , Anticorpos Monoclonais/imunologia , Encéfalo/metabolismo , Imunofluorescência/métodos , Humanos , Presenilina-1/imunologia , Presenilina-1/metabolismo , Presenilina-2/imunologia , Presenilina-2/metabolismo , Presenilinas/imunologia , Presenilinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
CD46 is a ubiquitously expressed type I transmembrane protein, first identified as a regulator of complement activation, and later as an entry receptor for a variety of pathogens. The last decade has also revealed the role of CD46 in regulating the adaptive immune response, acting as an additional costimulatory molecule for human T cells and inducing their differentiation into Tr1 cells, a subset of regulatory T cells. Interestingly, CD46 regulatory pathways are defective in T cells from patients with multiple sclerosis, asthma and rheumatoid arthritis, illustrating its importance in regulating T cell homeostasis. Indeed, CD46 expression at the cell surface is tightly regulated in many different cell types, highlighting its importance in several biological processes. Notably, CD46 is the target of enzymatic processing, being cleaved by metalloproteinases and by the presenilin/gamma secretase complex. This processing is required for its functions, at least in T cells. This review will summarize the latest updates on the regulation of CD46 expression and on its effects on T cell activation.
Assuntos
Ativação do Complemento/imunologia , Proteína Cofatora de Membrana/imunologia , Linfócitos T Reguladores/imunologia , Imunidade Adaptativa/imunologia , Animais , Humanos , Ativação Linfocitária/imunologia , Proteína Cofatora de Membrana/metabolismo , Proteína Cofatora de Membrana/fisiologia , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Presenilinas/imunologia , Presenilinas/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
The development of a monoclonal antibody (MAb) specific to Drosophila presenilin (Psn) proteins in vivo was the major aim of this study, since the absence of specific antibodies recognizing Psn proteins hampered our progress in understanding Psn functions during development, differentiation, and pathogenesis. By dot blot and immunofluorescence screenings, we found that MAb Psn2G6 specifically recognized Psn proteins in wing imaginal discs and brains of wild-type control W1118 larvae. MAb Psn2G6 also transgenically expressed a long form of wild-type Psn (Psn + 14 WT) proteins in wing imaginal discs of two independent transgenic lines. Transgenic expression of Psn + 14 WT proteins in psn(B3) larvae completely rescued the expression patterns of Psn proteins and the development of wing imaginal discs. In addition, neural hyperplasia observed in wing imaginal discs of psn(B3) larvae was also suppressed.