RESUMO
The Arp2/3 complex is a seven-protein assembly that is critical for actin nucleation and branching in cells. Here we report the reconstitution of active human Arp2/3 complex after expression of all seven subunits in insect cells. Expression of partial complexes revealed that a heterodimer of the p34 and p20 subunits constitutes a critical structural core of the complex, whereas the remaining subunits are peripherally located. Arp3 is crucial for nucleation, consistent with it being a structural component of the nucleation site. p41, p21, and p16 contribute differently to nucleation and stimulation by ActA and WASP, whereas p34/p20 bind actin filaments and likely function in actin branching. This study reveals that the nucleating and organizing functions of Arp2/3 complex subunits are separable, indicating that these activities may be differentially regulated in cells.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas do Citoesqueleto , Citoesqueleto de Actina/ultraestrutura , Proteína 2 Relacionada a Actina , Proteína 3 Relacionada a Actina , Actinas/química , Actinas/isolamento & purificação , Humanos , Substâncias Macromoleculares , Modelos Biológicos , Polímeros/química , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Using the yeast two-hybrid system, we have identified a novel 62 kDa coiled-coil protein that specifically interacts with the GTP-bound form of Rab5, a small GTPase that regulates membrane traffic in the early endocytic pathway. This protein shares 42% sequence identity with Rabaptin-5, a previously identified effector of Rab5, and we therefore named it Rabaptin-5beta. Like Rabaptin-5, Rabaptin-5beta displays heptad repeats characteristic of coiled-coil proteins and is recruited on the endosomal membrane by Rab5 in a GTP-dependent manner. However, Rabaptin-5beta has features that distinguish it from Rabaptin-5. The relative expression levels of the two proteins varies in different cell types. Rabaptin-5beta does not heterodimerize with Rabaptin-5, and forms a distinct complex with Rabex-5, the GDP/GTP exchange factor for Rab5. Immunodepletion of the Rabaptin-5beta complex from cytosol only partially inhibits early endosome fusion in vitro, whereas the additional depletion of the Rabaptin-5 complex has a stronger inhibitory effect. Fusion activity can mostly be recovered by addition of the Rabaptin-5 complex alone, but maximal fusion efficiency requires the presence of both Rabaptin-5 and Rabaptin-5beta complexes. Our results suggest that Rab5 binds to at least two distinct effectors which cooperate for optimal endocytic membrane docking and fusion.
Assuntos
Proteínas de Transporte/metabolismo , Endossomos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Fusão de Membrana/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Transporte Vesicular , Sequência de Aminoácidos , Animais , Proteínas de Transporte/análise , Proteínas de Transporte/genética , Sistema Livre de Células , Clonagem Molecular/métodos , Citosol/química , Dimerização , Endossomos/química , Células HeLa , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Leveduras/genética , Proteínas rab5 de Ligação ao GTPRESUMO
Antigen-selected T cell receptor (TCR) repertoires vary in complexity from very limited to extremely diverse. We have previously characterized two different CD8 T cell responses, which are restricted by the same mouse major histocompatibility complex (MHC) class I molecule, H-2 Kd. The TCR repertoire in the response against a determinant from Plasmodium berghei circumsporozoite protein (PbCS; region 252-260) is very diverse, whereas TCRs expressed by clones specific for a determinant in region 170-179 of HLA-CW3 (human) MHC class I molecule show relatively limited structural diversity. We had already demonstrated that cytolytic T lymphocyte (CTL) clones specific for the PbCS peptide display diverse patterns of antigen recognition when tested with a series of single Ala-substituted PbCS peptides or mutant. H-2 Kd molecules. We now show that CW3-specific CTL clones display much less diverse patterns of recognition. Our earlier functional studies with synthetic peptide variants suggested that the optimal peptides recognized were 9 (or 8) residues long for PbCS and 10 residues long for CW3. We now present more direct evidence that the natural CW3 ligand is indeed a 10-mer. Our functional data together with molecular modeling suggest that the limited TCR repertoire selected during the CW3 response is not due to a paucity of available epitopes displayed at the surface of the CW3 peptide/Kd complex. We discuss other factors, such as the expression of similar self MHC peptide sequences, that might be involved in trimming this TCR repertoire.
Assuntos
Epitopos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Apresentação de Antígeno , Citotoxicidade Imunológica , Epitopos/genética , Antígenos H-2/imunologia , Antígenos HLA/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Camundongos , Modelos Imunológicos , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes/imunologia , Subpopulações de Linfócitos T/imunologiaRESUMO
Vesicular transport is a dynamic process that requires coordinated interactions between membrane and cytoskeleton. The mechanisms and molecules integrating these interactions are unclear. A Rho protein, RhoD, might provide a molecular link between membrane traffic and the cytoskeleton. Activated RhoD causes rearrangements of the actin cytoskeleton and cell surface, and governs early endosome motility and distribution.
Assuntos
Endossomos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas/metabolismo , Proteínas rho de Ligação ao GTP , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Linhagem Celular , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Humanos , Camundongos , Microscopia de Vídeo , Dados de Sequência Molecular , Mutação , Receptores da Transferrina/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
The homeodomain of the Antennapedia molecule (AntpHD) spontaneously crosses cellular membranes and can be used to deliver up to 50 additional amino acids to the cytoplasm. We exploited this approach to deliver antigenic peptides to the MHC class I processing and presentation pathway. AntpHD-based fusion peptides expressing the 170-179 HLA-Cw3 CTL epitope (pCw3) were produced in bacteria. Incubation of these fusion peptides with H-2d target cells resulted in efficient delivery to the cytosol as indicated by protease resistance and confocal microscopy. Moreover, this introduction of an exogenous Ag resulted in sensitization of the cell to lysis by a CTL clone specific for the 170-179 HLA-Cw3-derived peptide. Sensitivity of the Ag processing to brefeldin A but not to chloroquine is consistent with the delivery of AntpHD fusion peptides to the conventional class I-associated processing pathway. Immunization of DBA/2 (H-2d) mice with AntpHD pCw3 fusion peptide in the presence of SDS primed H-2Kd-restricted HLA-Cw3-specific CTL. Similar results were obtained with AntpHD fusion peptides expressing the 147-156 influenza nucleoprotein peptide. The strategy outlined in this paper provides a new approach for introducing molecules into the MHC class I Ag-presenting pathway. This approach has clear relevance to the design of synthetic peptide-based vaccines.
Assuntos
Apresentação de Antígeno , Citotoxicidade Imunológica , Drosophila/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Proteínas de Homeodomínio/imunologia , Proteínas Nucleares , Linfócitos T Citotóxicos/imunologia , Fatores de Transcrição , Sequência de Aminoácidos , Animais , Proteína do Homeodomínio de Antennapedia , Apresentação de Antígeno/efeitos dos fármacos , Brefeldina A , Células Clonais/imunologia , Ciclopentanos/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Citotoxicidade Imunológica/genética , Proteínas de Drosophila , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/farmacologia , Ativação Linfocitária/genética , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Dodecilsulfato de Sódio/farmacologia , Linfócitos T Citotóxicos/efeitos dos fármacosRESUMO
Transport of an immunogenic self-peptide from the second domain of the mouse major histocompatibility complex (MHC) H-2Kd class I molecule is blocked at the TAP1-TAP2 peptide pump level due to its amino acid sequence and is not presented to cytolytic T lymphocytes (CTL). We demonstrate that first, TAP1-TAP2 pumps can restrict antigen presentation by selecting against internal peptide motifs which are not involved in peptide binding to MHC class I molecules. Second, some molecules targeted to the endoplasmic reticulum are processed for MHC class I presentation in the cytosol. Third, some abundantly expressed immunogenic self-peptides are cytosolically sequestered. The advantage for the host, in terms of the peripheral T cell repertoire is that the spared CTL can be used to recognize foreign antigens. It is, however, anticipated that this advantage will be exploited by pathogens to evade immune surveillance by similar strategies.
Assuntos
Células Apresentadoras de Antígenos/metabolismo , Antígenos HLA-C/química , Peptídeos/metabolismo , Linfócitos T Citotóxicos/imunologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Transporte Biológico Ativo , Primers do DNA/química , Epitopos , Rearranjo Gênico da Cadeia alfa dos Receptores de Antígenos dos Linfócitos T , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Antígenos HLA-C/metabolismo , Camundongos , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Peptídeos/química , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Relação Estrutura-AtividadeRESUMO
The major histocompatibility complex (MHC) class I molecules expressed at the cell surface are associated with a large number of different peptides so that the density of a given MHC-peptide complex is relatively low. Here we describe the properties of MHC class I molecules genetically attached to a single antigenic peptide. Cells expressing these fusion proteins are recognized by T cells specific for the particular MHC-peptide complex. Coculture of naive splenocytes with cells expressing these MHC-peptide fusion proteins and the B7.1 antigen allows the induction of primary cytotoxic T lymphocytes (CTL) in vitro. Injection of these cells into naive mice enhances the frequency of specific CTL precursors and protects against a subsequent challenge with a tumor or a virus bearing the antigenic peptide. Soluble MHC-peptide fusions were also produced in which all three components, that is, the heavy chain, beta 2-microglobulin and the peptide, have fused into a single-chain protein. The availability of MHC class I molecules bound to a single peptide provides valuable tools for the manipulation of CTL responses and the analysis of the selection processes in the thymus.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Superfície/metabolismo , Sequência de Bases , Células CHO , Linhagem Celular , Células Clonais , Cricetinae , Antígenos de Histocompatibilidade Classe I/química , Hibridomas , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/imunologia , Baço/citologia , Baço/imunologia , Linfócitos T Citotóxicos/imunologia , Microglobulina beta-2/metabolismoAssuntos
Alquil e Aril Transferases , Endocitose/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas rab de Ligação ao GTP , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Transporte Biológico Ativo , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Clatrina/metabolismo , Citosol/metabolismo , Cães , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/isolamento & purificação , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Fígado/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas rab5 de Ligação ao GTPRESUMO
DBA/2 (H-2d) HLA-B7 x human beta 2-microglobulin transgenic and DBA/2 nontransgenic mice stimulated by DBA/2 HLA-A3 x human beta 2-microglobulin transgenic mouse spleen cells developed potent H-2Kd restricted cytolytic responses with recognition of a peptide from the second domain of the HLA-A3 H chain. These H-2Kd-restricted responses obliterated, as a rule, cytolytic responses with direct recognition of the HLA-A3 molecules, even in HLA-B7 transgenic mice. These immunodominant H-2Kd-restricted responses could be abrogated in DBA/2 HLA-A24 mice because of cross-tolerance, the HLA-A3 derived-H-2Kd presented peptide being shared by several (including A24) HLA class I H chain allelic variants. Under such experimental circumstances, strong CTL responses with exclusive direct recognition of HLA-A3 molecules constantly developed. Further analysis of these responses in six DBA/2 HLA-A24 responder mice indicated that a large fraction of the mouse V beta and V alpha genes could be used to mount such CTL responses. Thus, by combining classical HLA class I transgenesis and selective destruction of H-2K and H-2D genes, it should be possible to derive useful strains of mice for the study of HLA class I-restricted CTL responses.
Assuntos
Antígenos H-2/genética , Antígenos HLA-A/imunologia , Antígeno HLA-A3/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Sequência de Bases , Antígenos H-2/imunologia , Antígeno HLA-A24 , Antígeno HLA-B7/genética , Humanos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Dados de Sequência Molecular , RNA/análise , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Microglobulina beta-2/genéticaRESUMO
Peptides derived from HLA-Cw3 and HLA-A24 within region 170-179 differ by a single substitution, at position 173, and are both presented by the class I major histocompatibility complex molecule H-2Kd for recognition by murine cytolytic T lymphocytes (CTLs). As a first approach to understand the way T cell receptors (TCRs) intact with the HLA peptides, we have analyzed the TCR selection by, and recognition of, the two HLA antigenic sites. First, we have compared the TCR repertoires selected by HLA-Cw3 and HLA-A24, not only by sequencing the TCRs carried by CTL clones isolated and grown in vitro, but also by analyzing the TCRs expressed in vivo by peritoneal exudate lymphocytes from immune animals. Second, we have compared the TCR crossrecognition of HLA-A24 by CTLs selected by HLA-Cw3 with that of HLA-Cw3 by CTLs selected by HLA-A24. The combined analysis of TCR selection by and recognition of these two related HLA antigenic sites provides evidence that the TCR beta junctional regions interact with the amino-terminal part of the HLA peptides.
Assuntos
Antígenos HLA-A/análise , Antígenos HLA-C/análise , Antígenos de Histocompatibilidade Classe I/análise , Complexo Principal de Histocompatibilidade/imunologia , Receptores de Antígenos de Linfócitos T/análise , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , DNA/análise , DNA/genética , Antígenos HLA-A/genética , Antígenos HLA-A/imunologia , Antígeno HLA-A24 , Antígenos HLA-C/genética , Antígenos HLA-C/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Complexo Principal de Histocompatibilidade/genética , Camundongos , Dados de Sequência Molecular , Peritônio/citologia , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/química , Linfócitos T/imunologia , Linfócitos T/ultraestruturaRESUMO
We previously showed that H-2Kd-restricted cytotoxic T lymphocyte (CTL) clones specific for a single nonapeptide derived from the Plasmodium berghei circumsporozoite (PbCS) protein displayed T cell receptors (TCRs) of highly diverse primary structure. We have now analyzed the TCR repertoire of CTLs that recognize a peptide derived from the human class I major histocompatibility complex (MHC) molecule HLA-Cw3 in association with the same murine class I MHC molecule H-2Kd. We first sequenced the TCR alpha and beta genes of the CTL clone Cw3/1.1 and, based on this genomic analysis, the TCR alpha and beta cDNA junctional regions of 23 independent H-2Kd-restricted CTL clones specific for HLA-Cw3. The results show that the TCR chains display very limited heterogeneity, both in terms of V alpha, J alpha, V beta, and J beta segments, and in terms of length and sequence of the CDR3 alpha and beta loops. The TCR repertoire used in vivo was then analyzed by harvesting CTL populations from the peritoneal cavity of immune mice. The peritoneal exudate lymphocytes (PELs) displayed HLA-Cw3-specific cytolytic activity in the absence of any stimulation in vitro. Remarkably, most of these freshly isolated PELs expressed TCRs that shared the same structural features as those from HLA-Cw3-reactive CTL clones. Thus, our results show that a peptide from HLA-Cw3 presented by H-2Kd selects CTLs that bear TCRs of very limited diversity in vivo. When taken together with the high diversity of the TCRs specific for the PbCS peptide, these findings suggest that natural tolerance to self peptides presented by class I MHC molecules may substantially reduce the size of the TCR repertoire of CTLs specific for antigenic peptides homologous to self.