RESUMEN
Minor histocompatibility antigens are the main targets of donor-derived T-cells after allogeneic stem cell transplantation. Identification of these antigens and understanding their biology are a key requisite for more insight into how graft vs. leukemia effect and graft vs. host disease could be separated. We here identified four new HLA class II-restricted minor histocompatibility antigens using whole genome association scanning. For one of the new antigens, i.e., LB-PIP4K2A-1S, we measured strong T-cell recognition of the donor variant PIP4K2A-1N when pulsed as exogenous peptide, while the endogenously expressed variant in donor EBV-B cells was not recognized. We showed that lack of T-cell recognition was caused by intracellular cleavage by a protease named asparagine endopeptidase (AEP). Furthermore, microarray gene expression analysis showed that PIP4K2A and AEP are both ubiquitously expressed in a wide variety of healthy tissues, but that expression levels of AEP were lower in primary acute myeloid leukemia (AML). In line with that, we confirmed low activity of AEP in AML cells and demonstrated that HLA-DRB1*03:01 positive primary AML expressing LB-PIP4K2A-1S or its donor variant PIP4K2A-1N were both recognized by specific T-cells. In conclusion, LB-PIP4K2A-1S not only represents a novel minor histocompatibility antigen but also provides evidence that donor T-cells after allogeneic stem cell transplantation can target the autologous allelic variant as leukemia-associated antigen. Furthermore, it demonstrates that endopeptidases can play a role in cell type-specific intracellular processing and presentation of HLA class II-restricted antigens, which may be explored in future immunotherapy of AML.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Leucemia Mieloide Aguda , Antígenos de Histocompatibilidad Menor , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Variación Genética , Efecto Injerto vs Leucemia/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/metabolismo , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/inmunología , Antígenos de Histocompatibilidad Menor/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/inmunología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismoRESUMEN
Mice homozygous for several Tln2 gene targeted alleles are viable and fertile. Here we show that although the expression of talin2 protein is drastically reduced in muscle from these mice, other tissues continue to express talin2 albeit at reduced levels. We therefore generated a Tln2 allele lacking the entire coding sequence (Tln2(cd)). Tln2(cd/cd) mice were viable and fertile, and the genotypes of Tln2(cd/+) intercrosses were at the expected Mendelian ratio. Tln2(cd/cd) mice showed no major difference in body mass or the weight of the major organs compared to wild-type, although they displayed a mildly dystrophic phenotype. Moreover, Tln2(cd/cd) mouse embryo fibroblasts showed no obvious defects in cell adhesion, migration or proliferation. However, the number of Tln2(cd/cd) pups surviving to adulthood was variable suggesting that such mice have an underlying defect.
Asunto(s)
Desarrollo Embrionario/genética , Fertilidad , Talina/fisiología , Animales , Peso Corporal , Adhesión Celular , Movimiento Celular , Proliferación Celular , Femenino , Fibroblastos/fisiología , Eliminación de Gen , Masculino , Ratones , Ratones Noqueados , Distrofias Musculares/genética , Distrofias Musculares/patología , Talina/genéticaRESUMEN
Talin binds to and activates integrins and is thought to couple them to cytoskeletal actin. However, functional studies on talin have been restricted by the fact that most cells express two talin isoforms. Here we show that human umbilical vein endothelial cells (HUVEC) express only talin1, and that talin1 knockdown inhibited focal adhesion (FA) assembly preventing the cells from maintaining a spread morphology, a phenotype that was rescued by GFP-mouse talin1. Thus HUVEC offer an ideal model system in which to conduct talin structure/function studies. Talin contains an N-terminal FERM domain (comprised of F1, F2 and F3 domains) and a C-terminal flexible rod. The F3 FERM domain binds beta-integrin tails, and mutations in F3 that inhibited integrin binding (W359A) or activation (L325R) severely compromised the ability of GFP-talin1 to rescue the talin1 knockdown phenotype despite the presence of a second integrin-binding site in the talin rod. The talin rod contains several actin-binding sites (ABS), and mutations in the C-terminal ABS that reduced actin-binding impaired talin1 function, whereas those that increased binding resulted in more stable FAs. The results show that both the N-terminal integrin and C-terminal actin-binding functions of talin are essential to cell spreading and FA assembly. Finally, mutations that relieve talin auto-inhibition resulted in the rapid and excessive production of FA, highlighting the importance of talin regulation within the cell.
Asunto(s)
Células Endoteliales/metabolismo , Integrinas/metabolismo , Talina/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Células Endoteliales/citología , Células Endoteliales/fisiología , Adhesiones Focales/fisiología , Técnicas de Silenciamiento del Gen , Humanos , Integrinas/química , Integrinas/genética , Ratones , Fenotipo , Talina/química , Talina/genética , Transfección , Venas Umbilicales/citología , Regulación hacia ArribaRESUMEN
The nuclear envelope (NE) LINC complex, in mammals comprised of SUN domain and nesprin proteins, provides a direct connection between the nuclear lamina and the cytoskeleton, which contributes to nuclear positioning and cellular rigidity. SUN1 and SUN2 interact with lamin A, but lamin A is only required for NE localization of SUN2, and it remains unclear how SUN1 is anchored. Here, we identify emerin and short nesprin-2 isoforms as novel nucleoplasmic binding partners of SUN1/2. These have overlapping binding sites distinct from the lamin A binding site. However, we demonstrate that tight association of SUN1 with the nuclear lamina depends upon a short motif within residues 209-228, a region that does not interact significantly with known SUN1 binding partners. Moreover, SUN1 localizes correctly in cells lacking emerin. Importantly then, the major determinant of SUN1 NE localization has yet to be identified. We further find that a subset of lamin A mutations, associated with laminopathies Emery-Dreifuss muscular dystrophy (EDMD) and Hutchinson-Gilford progeria syndrome (HGPS), disrupt lamin A interaction with SUN1 and SUN2. Despite this, NE localization of SUN1 and SUN2 is not impaired in cell lines from either class of patients. Intriguingly, SUN1 expression at the NE is instead enhanced in a significant proportion of HGPS but not EDMD cells and strongly correlates with pre-lamin A accumulation due to preferential interaction of SUN1 with pre-lamin A. We propose that these different perturbations in lamin A-SUN protein interactions may underlie the opposing effects of EDMD and HGPS mutations on nuclear and cellular mechanics.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas de la Membrana/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/fisiología , Distrofia Muscular de Emery-Dreifuss/patología , Membrana Nuclear/metabolismo , Proteínas Nucleares/fisiología , Progeria/patología , Proteínas de Unión a Telómeros/fisiología , Animales , Núcleo Celular/metabolismo , Femenino , Fibroblastos/metabolismo , Humanos , Lamina Tipo A/química , Ratones , Distrofia Muscular de Emery-Dreifuss/metabolismo , Células 3T3 NIH , Progeria/metabolismo , Isoformas de Proteínas , Estructura Terciaria de ProteínaRESUMEN
The talin rod contains approximately 11 vinculin binding sites (VBSs), each defined by hydrophobic residues in a series of amphipathic helices that are normally buried within the helical bundles that make up the rod. Consistent with this, talin failed to compete for binding of the vinculin Vd1 domain to an immobilized talin polypeptide containing a constitutively active VBS. However, talin did bind to GST-Vd1 in pull-down assays, and isothermal titration calorimetry measurements indicate a K(d) of approximately 9 mum. Interestingly, Vd1 binding exposed a trypsin cleavage site in the talin rod between residues 898 and 899, indicating that there are one or more active VBSs in the N-terminal part of the talin rod. This region comprises a five helix bundle (residues 482-655) followed by a seven-helix bundle (656-889) and contains five VBSs (helices 4, 6, 9, 11, and 12). The single VBS within 482-655 is cryptic at room temperature. In contrast, talin 482-889 binds Vd1 with high affinity (K(d) approximately 0.14 mum), indicating that one or more of the four VBSs within 656-889 are active, and this likely represents the vinculin binding region in intact talin. In support of this, hemagglutinin-tagged talin 482-889 localized efficiently to focal adhesions, whereas 482-655 did not. Differential scanning calorimetry showed a strong negative correlation between Vd1 binding and helical bundle stability, and a 755-889 mutant with a more stable fold bound Vd1 much less well than wild type. We conclude that the stability of the helical bundles that make up the talin rod is an important factor determining the activity of the individual VBSs.
Asunto(s)
Talina/química , Vinculina/química , Animales , Sitios de Unión , Calorimetría , Rastreo Diferencial de Calorimetría , Pollos , Cromatografía en Gel , Dicroismo Circular , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Adhesiones Focales , Glutatión Transferasa/metabolismo , Humanos , Cinética , Ratones , Células 3T3 NIH , Péptidos/química , Reacción en Cadena de la Polimerasa , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Temperatura , Tripsina/químicaRESUMEN
Although the endpoint of the class II antigen-processing pathway is well characterized, the processing events that lead to the production of class II major histocompatibility complex (MHC)/peptide complexes are not. It is generally assumed that protease action on native antigen substrates leads to unfolding and capture of either long or short peptides. Whether specific protease activities are needed for presentation of particular T-cell epitopes is largely unknown. Here, we review our recent studies that aim to identify the processing enzymes that initiate processing of different antigens. We suggest a general strategy that can potentially identify preferred relationships between substrates and processing enzymes in vitro and suggest ways in which these relationships can be tested in vivo. We draw heavily on the example of asparaginyl endopeptidase, which is involved in both productive and destructive processing of different antigen substrates. Overall, while there is undoubtedly redundancy in class II MHC antigen processing, the contributions of individual enzymes can be clearly dissected.
Asunto(s)
Presentación de Antígeno/inmunología , Cisteína Endopeptidasas/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Animales , Células Presentadoras de Antígenos/enzimología , Células Presentadoras de Antígenos/inmunología , Cisteína Endopeptidasas/metabolismo , HumanosRESUMEN
The adaptive immune response depends on the creation of suitable peptides from foreign antigens for display on MHC molecules to T lymphocytes. Similarly, MHC-restricted display of peptides derived from self proteins results in the elimination of many potentially autoreactive T cells. Different proteolytic systems are used to generate the peptides that are displayed as T cell epitopes on class I compared with class II MHC molecules. In the case of class II MHC molecules, the proteases that reside within the endosome/lysosome system of antigen-presenting cells are responsible; surprisingly, however, there are relatively few data on which enzymes are involved. Recently we have asked whether proteolysis is required simply in a generic sense, or whether the action of particular enzymes is needed to generate specific class II MHC-associated T cell epitopes. Using the recently identified mammalian asparagine endopeptidase as an example, we review recent evidence that individual enzymes can make clear and non-redundant contributions to MHC-restricted peptide display.
Asunto(s)
Endopeptidasas/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Secuencia de Aminoácidos , Animales , Compartimento Celular , Datos de Secuencia MolecularRESUMEN
Mammalian asparaginyl endopeptidase (AEP) or legumain is a recently discovered lysosomal cysteine protease that specifically cleaves after asparagine residues. How this unusually specific lysosomal protease is itself activated is not fully understood. Using purified recombinant pro-enzyme, we show that activation is autocatalytic, requires sequential removal of C- and N-terminal pro-peptides at different pH thresholds, and is bimolecular. Removal of the N-terminal propeptide requires cleavage after aspartic acid rather than asparagine. Cellular processing, either of exogenously added AEP precursor or of pulse-labeled endogenous precursor, introduces at least one further cleavage to yield the final mature lysosomal enzyme. We also provide evidence that in living cells, there is clear compartmental heterogeneity in terms of AEP activation status. Moreover, we show that human monocyte-derived dendritic cells harbor inactive proforms of AEP that become activated upon maturation of dendritic cells with lipopolysaccharide.
Asunto(s)
Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Animales , Asparagina/química , Ácido Aspártico/química , Células CHO , Línea Celular , Células Cultivadas , Clonación Molecular , Cricetinae , Células Dendríticas/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Lipopolisacáridos/química , Lisosomas/metabolismo , Microscopía Fluorescente , Modelos Biológicos , Datos de Secuencia Molecular , Monocitos/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Péptidos/química , Estructura Terciaria de Proteína , Conejos , Ovinos , Factores de Tiempo , TransfecciónRESUMEN
The invariant chain (Ii) chaperone for MHC class II molecules is crucial for their effective function. Equally important is its removal. Cathepsins S or L are known to be required for the final stages of Ii removal in different APCs, but the enzymes which initiate Ii processing have not been identified. Here we show that this step can be performed in B lymphocytes by asparagine endopeptidase (AEP), which targets different asparagine residues in the lumenal domain of human and mouse invariant chain. Inhibition of AEP activity slows invariant chain processing and hinders the expression of an antigenic peptide engineered to replace the groove binding region of Ii (CLIP). However, the initiation of Ii removal can also be performed by other proteases, reflecting the importance of this step.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B/metabolismo , Cisteína Endopeptidasas/fisiología , Antígenos de Histocompatibilidad Clase II/metabolismo , Animales , Presentación de Antígeno , Células Presentadoras de Antígenos/metabolismo , Linfocitos B/metabolismo , Línea Celular , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , RatonesRESUMEN
IL-12 is a 75 kDa heterodimeric cytokine composed of two disulfide-linked subunits, p35 and p40, which plays an important role in the regulation of the immune response. We tested the hypothesis that thiol antioxidants might interfere with dimerization of the two IL-12 subunits. We thus studied the effect of reduced glutathione (GSH) and N-acetyl-cysteine (NAC) on IL-12 p75 production by human THP-1 cell stimulated with IFN-gamma and Staphylococcus aureus Cowan strain I (SAC), using ELISAs specific for IL-12 p75 or the p40 subunit. NAC and GSH, but not cystine, at concentrations of 5-10 mM inhibited production of IL-12 p75 but not of the p40 subunit. NAC did not inhibit p40 or p35 mRNA expression in dendritic cells or THP-1 cells, or NF-kappa B activation in THP-1 cells. The effect of NAC was specific for IL-12 p75, as NAC did not affect induction of MHC class II expression by IFN-gamma-stimulated THP-1 cells. IL-12 dimer formation appears to be reduced by NAC also in vivo, because pretreatment with NAC (1 g/kg, orally), before LPS injection in mice, inhibited peak IL-12 p75 serum levels without affecting those of p40. We conclude that thiol levels regulate IL-12 p75 production and that assembly of the heterodimer is a step that might represent a target for pharmacological intervention.
Asunto(s)
Antioxidantes/farmacología , Interleucina-12/química , Compuestos de Sulfhidrilo/farmacología , Acetilcisteína/farmacología , Línea Celular , Dimerización , Disulfuros , Glutatión/farmacología , Humanos , Interferón gamma/farmacología , Interleucina-12/biosíntesis , Interleucina-12/genética , Subunidades de Proteína , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Transcripción Genética/efectos de los fármacosRESUMEN
Little is known about the processing of putative human autoantigens and why tolerance is established to some T cell epitopes but not others. Here we show that a principal human HLA-DR2-restricted epitope--amino acids 85-99 of myelin basic protein, MBP(85-99)--contains a processing site for the cysteine protease asparagine endopeptidase (AEP). Presentation of this epitope by human antigen-presenting cells is inversely proportional to the amount of cellular AEP activity: inhibition of AEP in living cells greatly enhances presentation of the MBP(85-99) epitope, whereas overexpression of AEP diminishes presentation. These results indicate that central tolerance to this encephalitogenic MBP epitope may not be established because destructive processing limits its display in the thymus. Consistent with this hypothesis, AEP is expressed abundantly in thymic antigen-presenting cells.