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Yoshizumi Ishino - One of the best experts on this subject based on the ideXlab platform.
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the archaeal dna primase biochemical characterization of the P41 p46 complex frompyrococcus furiosus
Journal of Biological Chemistry, 2001Co-Authors: Kayoko Komori, Arnaud A. Bocquier, Sonoko Ishino, Daisuke Kohda, Isaac K. O. Cann, Yoshizumi IshinoAbstract:Abstract We characterized the primase complex of the hyperthermophilic archaeon, Pyrococcus furiosus. The two proteins, PfuP41 and Pfup46, have similar sequences to the p48 and p58 subunits, respectively, of the eukaryotic DNA polymerase α-primase complex. Unlike previously reported primases, the PfuP41 preferentially utilizes deoxyribonucleotides for its de novo synthesis, and moreover, it synthesizes up to several kilobases in length in a template-dependent manner (Bocquier, A., Liu, L., Cann, I., Komori, K., Kohda, D., and Ishino, Y. (2001) Curr. Biol.11, 452–456). The P41-p46 complex showed higher DNA binding activity than the catalytic P41 subunit alone. In addition, the amount of DNA synthesized by the P41-p46 complex was much more abundant and shorter in length than that by PfuP41 alone. The activity for RNA primer synthesis, which was not detected with PfuP41, was observed from the reaction using the P41-p46 complex in vitro. The in vitro replication of M13 single-stranded DNA by the P. furiosus proteins was stimulated by ATP. Observation of the labeled primers by using [γ-32P]ATP in the substrates suggests ATP as the preferable initiating nucleotide for the P41-p46 complex. These results show that the primer synthesis activity of PfuP41 is regulated by Pfup46, and the P41-p46 complex may function as the primase in the DNA replication machinery of P. furiosus, in a similar fashion to the eukaryotic polymerase α-primase complex.
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the archaeal dna primase biochemical characterization of the P41 p46 complex from pyrococcus furiosus
Journal of Biological Chemistry, 2001Co-Authors: Lidong Liu, Arnaud A. Bocquier, Kayoko Komori, Sonoko Ishino, Daisuke Kohda, Isaac K. O. Cann, Yoshizumi IshinoAbstract:Abstract We characterized the primase complex of the hyperthermophilic archaeon, Pyrococcus furiosus. The two proteins, PfuP41 and Pfup46, have similar sequences to the p48 and p58 subunits, respectively, of the eukaryotic DNA polymerase α-primase complex. Unlike previously reported primases, the PfuP41 preferentially utilizes deoxyribonucleotides for its de novo synthesis, and moreover, it synthesizes up to several kilobases in length in a template-dependent manner (Bocquier, A., Liu, L., Cann, I., Komori, K., Kohda, D., and Ishino, Y. (2001) Curr. Biol.11, 452–456). The P41-p46 complex showed higher DNA binding activity than the catalytic P41 subunit alone. In addition, the amount of DNA synthesized by the P41-p46 complex was much more abundant and shorter in length than that by PfuP41 alone. The activity for RNA primer synthesis, which was not detected with PfuP41, was observed from the reaction using the P41-p46 complex in vitro. The in vitro replication of M13 single-stranded DNA by the P. furiosus proteins was stimulated by ATP. Observation of the labeled primers by using [γ-32P]ATP in the substrates suggests ATP as the preferable initiating nucleotide for the P41-p46 complex. These results show that the primer synthesis activity of PfuP41 is regulated by Pfup46, and the P41-p46 complex may function as the primase in the DNA replication machinery of P. furiosus, in a similar fashion to the eukaryotic polymerase α-primase complex.
James H Miller - One of the best experts on this subject based on the ideXlab platform.
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the proteolytic environment involved in mhc class ii restricted antigen presentation can be modulated by the P41 form of invariant chain
Journal of Immunology, 1996Co-Authors: Beatrice Fineschi, Kazuyasu Sakaguchi, E Appella, James H MillerAbstract:During biosynthesis, MHC class II associates with invariant chain which exists in two forms, p31 and P41. Both forms of invariant chain prevent peptide binding to class II, facilitate transport, and enhance class II localization to Ag-processing compartments. In spite of these shared functions, presentation of some Ags can be selectively enhanced by expression of P41. Here we show that P41 can function as a protease inhibitor: 1) the functional and biochemical consequences of P41 expression can be mimicked by inhibiting cysteine proteases in vivo; 2) the amount of intracellular active cysteine proteases is dramatically decreased in P41-positive cells; and 3) a polypeptide corresponding to the P41-unique region is a potent inhibitor of cathepsin L in vitro. These data suggest that P41 can enhance Ag presentation by reducing the proteolytic activity of the Ag-processing compartment, thus protecting a subset of antigenic epitopes from excessive degradation.
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proteolysis of major histocompatibility complex class ii associated invariant chain is regulated by the alternatively spliced gene product P41
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Beatrice Fineschi, Lynne S Arneson, Marisa F Naujokas, James H MillerAbstract:Abstract Invariant chain (Ii) is an intracellular type II transmembrane glycoprotein that is associated with major histocompatibility complex class II molecules during biosynthesis. Ii exists in two alternatively spliced forms, p31 and P41. Both p31 and P41 facilitate folding of class II molecules, promote egress from the endoplasmic reticulum, prevent premature peptide binding, and enhance localization to proteolytic endosomal compartments that are thought to be the sites for Ii degradation, antigen processing, and class II-peptide association. In spite of the dramatic and apparently equivalent effects that p31 and P41 have on class II biosynthesis, the ability of invariant chain to enhance antigen presentation to T cells is mostly restricted to P41. Here we show that degradation of Ii leads to the generation of a 12-kDa amino-terminal fragment that in P41-positive, but not in p31-positive, cells remains associated with class II molecules for an extended time. Interestingly, we find that coexpression of the two isoforms results in a change in the pattern of p31 degradation such that endosomal processing of p31 also leads to extended association of a similar 12-kDa fragment with class II molecules. These data raise the possibility that P41 may have the ability to impart its pattern of proteolytic processing on p31 molecules expressed in the same cells. This would enable a small number of P41 molecules to modify the post-translational transport and/or processing of an entire cohort of class II-Ii complexes in a manner that could account for the unique ability of P41 to enhance antigen presentation.
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antigen presentation enhanced by the alternatively spliced invariant chain gene product P41
Nature, 1992Co-Authors: Mary Peterson, James H MillerAbstract:During biosynthesis, class II molecules of the major histocompatibility complex are associated with a nonpolymorphic protein called invariant chain, Ii, which facilitates folding of class II molecules and their exit from the endoplasmic reticulum, interferes with their association with peptide and directs their post-Golgi transport (refs 7-9). If Ii blocks class II loading with endogenous antigens in the endoplasmic reticulum and/or directs class II molecules to the exogenous antigen-loading compartment, then the co-expression of Ii should enhance the ability of class II molecules to present exogenous antigens to T cells. But data supporting a role for Ii in class II-restricted antigen presentation are controversial. Here we show that Ii can facilitate exogenous antigen presentation for a subset of antigens. Although all known functions of Ii have been ascribed to the principal form of Ii, p31, we find that in most cases antigen presentation is facilitated only by the alternatively spliced, minor form of Ii, P41.
Elizabeth K Bikoff - One of the best experts on this subject based on the ideXlab platform.
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requirement for endocytic antigen processing and influence of invariant chain and h 2m deficiencies in cns autoimmunity
Journal of Clinical Investigation, 2001Co-Authors: Alison Jane SLAVIN, Jeanne M Soos, Juan C Patarroyo, Elizabeth K Bikoff, Olaf Stuve, Adriano Fontana, Howard L Weiner, Scott S ZamvilAbstract:The role of processing in antigen (Ag) presentation and T cell activation in experimental allergic encephalomyelitis (EAE) was evaluated in wild-type mice, mice that selectively express either Ii p31 or P41, and mice completely deficient in Ii or H-2M. We demonstrate that processing of myelin oligodendrocyte glycoprotein (MOG) is required for presentation of the dominant encephalitogenic MOG epitope, p35-55. Ii p31- and P41-expressing mice developed EAE with similar incidence to wild-type mice, although P41 mice had a more severe course. Ag-presenting cells (APCs) from Ii- or H-2M–deficient mice could present p35-55, but not MOG, demonstrating that these APCs could not process native MOG. Ii- and H-2M–deficient mice were not susceptible to EAE by immunization with p35-55 or MOG or by adoptive transfer of encephalitogenic T cells. However, CD4+ T cells from p35-55–immunized H-2M–deficient mice proliferated, secreted IFN-γ, and transferred EAE to wild-type, but not H-2M–deficient, mice. Thus, EAE resistance in H-2M–deficient mice is not due to an inability of APCs to present p35-55, or an intrinsic defect in the encephalitogenic T cell repertoire, but reflects a defect in APC function. Our results indicate that processing is required for initial Ag presentation and CNS T cell activation and suggest that autopathogenic peptides of CNS autoantigen may not be readily available for presentation without processing.
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The P41 isoform of invariant chain is a chaperone for cathepsin L
The EMBO Journal, 2001Co-Authors: Ana-maria Lennon-duménil, Ann H. Erickson, Rebecca A. Roberts, Karine Valentijn, Elizabeth K Bikoff, Peter J Peters, Herman S. Overkleeft, Christoph Driessen, Hidde L Ploegh, Paula Wolf BryantAbstract:The P41 splice variant of major histocompatibility complex (MHC) class II-associated invariant chain (Ii) contains a 65 aa segment that binds to the active site of cathepsin L (CatL), a lysosomal cysteine protease involved in MHC class II-restricted antigen presentation. This segment is absent from the predominant form of Ii, p31. Here we document the in vivo significance of the P41–CatL interaction. By biochemical means and electron microscopy, we demonstrate that the levels of active CatL are strongly reduced in bone marrow-derived antigen-presenting cells that lack P41. This defect mainly concerns the mature two-chain forms of CatL, which depend on P41 to be expressed at wild-type levels. Indeed, pulse–chase analysis suggests that these mature forms of CatL are degraded by endocytic proteases when P41 is absent. We conclude that P41 is required for activity of CatL by stabilizing the mature forms of the enzyme. This suggests that P41 is not merely an inhibitor of CatL enzymatic activity, but serves as a chaperone to help maintain a pool of mature enzyme in late-endocytic compartments of antigen-presenting cells.
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in vivo functions mediated by the P41 isoform of the mhc class ii associated invariant chain
Journal of Immunology, 1997Co-Authors: Norma T Takaesu, J A Lower, Deborah Yelon, Elizabeth J Robertson, Elizabeth K BikoffAbstract:We used a "hit and run" gene targeting strategy to generate mice expressing only the P41 isoform of the conserved invariant (Ii) chain associated with MHC class II molecules. In contrast to mutants expressing only p31 Ii chain, a small proportion of A(alpha)b A(beta)b molecules produced by these animals have reduced mobilities in SDS-PAGE and appear incompletely processed. Nonetheless, class II surface expression, peptide occupancy, CD4+ T cell maturation, and proliferative responses toward intact protein Ags are efficiently reconstituted. Moreover, spleen cells exclusively expressing P41 or p31 alone display equivalent dose-response curves in Ag presentation assays. Similar conclusions were reached analyzing mutants expressing two independent MHC haplotypes. Overall, these results demonstrate that Ii chain functional activities as a class II-specific chaperone are largely shared by p31 and P41 isoforms in the intact animal. Mutant mouse strains producing only p31 or P41 under control of endogenous regulatory elements responsible for constitutive and inducible Ii chain expression should prove useful for dissecting the contributions of these isoforms to diverse CD4+ T cell responses in vivo, such as those responsible for Ab production, inflammatory responses, autoimmune diseases, and protection against infectious agents.
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major histocompatibility class ii peptide occupancy antigen presentation and cd4 t cell function in mice lacking the P41 isoform of invariant chain
Immunity, 1995Co-Authors: Norma T Takaesu, J A Lower, Elizabeth J Robertson, Elizabeth K BikoffAbstract:Abstract We used a "hit and run" gene targeting strategy to generate mice expressing only the p31 isoform of the conserved invariant (li) chain associated with major histocompatibility complex (MHC) class II molecules. Spleen cells from these mice appear indistinguishable from wild type with respect to class II subunit assembly, transport, peptide acquisition, surface expression, and the ability to present intact protein antigens. Moreover, these mutant mice have normal numbers of thymic and peripheral CD4 + T cells, and intact CD4 + T-dependent proliferative responses towards a soluble antigen. In short, MHC class II expression and function are surprisingly unaffected in mice lacking P41 invariant chain, implying that the p31 and P41 isoforms may be functionally redundant in the intact animal.
Vito Turk - One of the best experts on this subject based on the ideXlab platform.
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Inhibitory P41 isoform of invariant chain and its potential target enzymes cathepsins L and H in distinct population of macrophages in human lymph nodes
Immunology, 2004Co-Authors: Vito Turk, Tadeja Bevec, Ana Schweiger, Rastko Golouh, Tina Zavašnik-bergantAbstract:Activation of the CD4+ T-cell mediated immune response relies on the proteolytic capacity of enzymes involved in modulating major histocompatibility complex (MHC) II-associated antigen presentation in antigen-presenting cells (APC). The MHC II-associated chaperone molecule P41 isoform of invariant chain (inhibitory P41 Ii) has been suggested to regulate stability and activity of cathepsin L in these APC. In the present study the human lymph node distribution of non-inhibitory p31 Ii and inhibitory P41 Ii have been compared by differential labelling, using two specific monoclonal antibodies. The distribution of P41 Ii, but not p31 Ii, matched the distribution of cathepsins L and H in subcapsular and cortical sinuses and germinal centres. Co-localization of P41 Ii with cathepsin H was confirmed in strongly CD68 + sinus-lining macrophages, acting as APC. Furthermore, P41 Ii was determined together with cathepsins L and H in tingible body macrophages, highly phagocytic, but not antigen-presenting cells inside germinal centres. With respect to the physiological function that these two populations of macrophages have in human lymph nodes, our results support a regulatory function of P41 Ii towards cathepsins L and H in human macrophages, associated with the processes of phagocytosis rather than antigen presentation.
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immunochemical localisation of cathepsin s cathepsin l and mhc class ii associated P41 isoform of invariant chain in human lymph node tissue
Biological Chemistry, 2001Co-Authors: V Zavanikbergant, Rastko Golouh, Vito Turk, Andreja Sekirnik, Janko KosAbstract:Antigen presentation by MHC class II molecules requires cysteine proteases (CP) for two convergent proteolytic processes: stepwise degradation of the invariant chain (Ii) and generation of immunogenic peptides. Their activity is controlled by intracellular CP inhibitors, including presumably the P41 isoform of invariant chain (P41 Ii), which is in vitro a potent inhibitor of cathepsin L but not of cathepsin S. In order to evaluate the inhibitory potential of P41 Ii in antigen-presenting cells (APC), these three proteins were stained in lymph node tissue using specific monoclonal and polyclonal antibodies. The most abundant labelling was observed in subcapsular (cortical) and trabecular sinuses of the lymph node. In this area the most frequent APC were macrophages, as confirmed by the CD68 cell marker. Using confocal fluorescence microscopy, co-localisation of P41 Ii with cathepsin S, but not with cathepsin L was found in these cells. Our results are consistent with the hypothesis that cathepsin S participates in degradation of the invariant chain, but they do not support the association between cathepsin L and P41 Ii in APC.
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the P41 fragment story
Iubmb Life, 1999Co-Authors: Dusan Turk, Gregor Guncar, Vito TurkAbstract:The discovery of a fragment from the P41 form of invariant chain tightly bound to cathepsin L provided the first direct link between MHC class II molecules and the regulation of activity of lysosomal cysteine proteases. We recently determined the crystal structure of this P41 invariant chain fragment in complex with cathepsin L [EMBO J. 18, 793-803 (1999)]. This structure explains the specificity of the observed interactions and actually provides a tool, which can be utilized by means of molecular biology, to explore and understand the specificity of thyroglobulin type I domains and thus allow the design of specific inhibitors of papain-like cysteine proteases. The structure further supports the hypothesis that the thyroglobulin type I and II domains present in various proteins, sometimes in multiple repeats, are regulatory elements of the processing of these proteins by proteolytic cleavage.
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crystal structure of mhc class ii associated P41 ii fragment bound to cathepsin l reveals the structural basis for differentiation between cathepsins l and s
The EMBO Journal, 1999Co-Authors: Gregor Guncar, Vito Turk, Galina Pungercic, Ivica Klemencic, Dusan TurkAbstract:The lysosomal cysteine proteases cathepsins S and L play crucial roles in the degradation of the invariant chain during maturation of MHC class II molecules and antigen processing. The P41 form of the invariant chain includes a fragment which specifically inhibits cathepsin L but not S. The crystal structure of the P41 fragment, a homologue of the thyroglobulin type-1 domains, has been determined at 2.0 A resolution in complex with cathepsin L. The structure of the P41 fragment demonstrates a novel fold, consisting of two subdomains, each stabilized by disulfide bridges. The first subdomain is an alpha-helix-beta-strand arrangement, whereas the second subdomain has a predominantly beta-strand arrangement. The wedge shape and three-loop arrangement of the P41 fragment bound to the active site cleft of cathepsin L are reminiscent of the inhibitory edge of cystatins, thus demonstrating the first example of convergent evolution observed in cysteine protease inhibitors. However, the different fold of the P41 fragment results in additional contacts with the top of the R-domain of the enzymes, which defines the specificity-determining S2 and S1' substrate-binding sites. This enables inhibitors based on the thyroglobulin type-1 domain fold, in contrast to the rather non-selective cystatins, to exhibit specificity for their target enzymes.
Kayoko Komori - One of the best experts on this subject based on the ideXlab platform.
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the archaeal dna primase biochemical characterization of the P41 p46 complex frompyrococcus furiosus
Journal of Biological Chemistry, 2001Co-Authors: Kayoko Komori, Arnaud A. Bocquier, Sonoko Ishino, Daisuke Kohda, Isaac K. O. Cann, Yoshizumi IshinoAbstract:Abstract We characterized the primase complex of the hyperthermophilic archaeon, Pyrococcus furiosus. The two proteins, PfuP41 and Pfup46, have similar sequences to the p48 and p58 subunits, respectively, of the eukaryotic DNA polymerase α-primase complex. Unlike previously reported primases, the PfuP41 preferentially utilizes deoxyribonucleotides for its de novo synthesis, and moreover, it synthesizes up to several kilobases in length in a template-dependent manner (Bocquier, A., Liu, L., Cann, I., Komori, K., Kohda, D., and Ishino, Y. (2001) Curr. Biol.11, 452–456). The P41-p46 complex showed higher DNA binding activity than the catalytic P41 subunit alone. In addition, the amount of DNA synthesized by the P41-p46 complex was much more abundant and shorter in length than that by PfuP41 alone. The activity for RNA primer synthesis, which was not detected with PfuP41, was observed from the reaction using the P41-p46 complex in vitro. The in vitro replication of M13 single-stranded DNA by the P. furiosus proteins was stimulated by ATP. Observation of the labeled primers by using [γ-32P]ATP in the substrates suggests ATP as the preferable initiating nucleotide for the P41-p46 complex. These results show that the primer synthesis activity of PfuP41 is regulated by Pfup46, and the P41-p46 complex may function as the primase in the DNA replication machinery of P. furiosus, in a similar fashion to the eukaryotic polymerase α-primase complex.
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the archaeal dna primase biochemical characterization of the P41 p46 complex from pyrococcus furiosus
Journal of Biological Chemistry, 2001Co-Authors: Lidong Liu, Arnaud A. Bocquier, Kayoko Komori, Sonoko Ishino, Daisuke Kohda, Isaac K. O. Cann, Yoshizumi IshinoAbstract:Abstract We characterized the primase complex of the hyperthermophilic archaeon, Pyrococcus furiosus. The two proteins, PfuP41 and Pfup46, have similar sequences to the p48 and p58 subunits, respectively, of the eukaryotic DNA polymerase α-primase complex. Unlike previously reported primases, the PfuP41 preferentially utilizes deoxyribonucleotides for its de novo synthesis, and moreover, it synthesizes up to several kilobases in length in a template-dependent manner (Bocquier, A., Liu, L., Cann, I., Komori, K., Kohda, D., and Ishino, Y. (2001) Curr. Biol.11, 452–456). The P41-p46 complex showed higher DNA binding activity than the catalytic P41 subunit alone. In addition, the amount of DNA synthesized by the P41-p46 complex was much more abundant and shorter in length than that by PfuP41 alone. The activity for RNA primer synthesis, which was not detected with PfuP41, was observed from the reaction using the P41-p46 complex in vitro. The in vitro replication of M13 single-stranded DNA by the P. furiosus proteins was stimulated by ATP. Observation of the labeled primers by using [γ-32P]ATP in the substrates suggests ATP as the preferable initiating nucleotide for the P41-p46 complex. These results show that the primer synthesis activity of PfuP41 is regulated by Pfup46, and the P41-p46 complex may function as the primase in the DNA replication machinery of P. furiosus, in a similar fashion to the eukaryotic polymerase α-primase complex.
