Malini Raghavan
University Of Michigan At Ann Arbor
Project start date: 1999-01-15
Project end date: 2013-12-31
Sponsored Links Excellgen http://Excellgen.com
INTERACTIONS & MECHANISMS OF FUNCTION OF THE TAP COMPLEX
Malini Raghavan, Assistant Professor
Microbiology And Immunologyuniversity Of Michigan At Ann Arbor
3003 South State Street, Room 1040
ann Arbor, Mi 481091274
Grant 5R01AI044115-03 from National Institute Of Allergy And Infectious Diseases IRG: IMB
Abstract: The transporters associated with antigen processing (TAP) are crucial components of the class I major histocompatibility complex (MHC) antigen presentation pathway. TAP is a complex of two subunits, TAP1 and TAP2, that functions to transport peptides from the cytosol to the endoplasmic reticulum (ER) for class I MHC antigen presentation. TAP1 and TAP2 both comprise a transmembrane domain and an ATP binding domain (NBD). The studies proposed here will result in a better understanding of TAP function and insights into molecular mechanisms by which the immune system combats viruses and cancers. In the first of the proposed studies, the interactions of ATP with human TAP subunits will be analyzed and structural changes that are induced as a consequence of ATP binding will be probed using proteolysis, circular dichroism and surface plasmon resonance-based assays. In particular, experiments are proposed to determine whether nucleotide binding alters the stability of a complex that we observe between the NBD of TAP1 and TAP2. In the second of the proposed studies, the question of whether ATP binding and hydrolysis by both TAP1 and TAP2 subunits is required for peptide translocation by TAP is addressed, by the generation and characterization of mutants that are predicted to alter the ATPase activities of one of the TAP subunits. The basis for these studies is the observation of natural mutations in human and rodent TAP2 sequences at sites that are highly conserved and that have been shown to be crucial for ATPase activity and transport function of other members of the ATP binding cassette (ABC) family of transmembrane transporters (of which TAP is a member). In the last of proposed studies, the interactions of the TAP complex with the human class I MHC molecule HLA -B27 and the newly discovered TAP-associated protein, tapasin will be examined. Studies in other laboratories have indicated that newly synthesized class I MHC molecules form complexes with TAP in the ER, and that this interaction is bridged or stabilized by tapasin. By expressing different combinations of the proteins TAP1 TAP2, tapasin, and the class I MHC heavy and light chains in insect cells, the molecular nature of the TAP/tapasin/class I MHC complexes will be elucidated, and insights will be obtained into whether other unknown components are required for these interactions, as well as for class I MHC antigen presentation. Finally, analysis of TAP interactions with HLA-B27 that are proposed will provide insight into the molecular mechanisms that underlie the association of B27 with autoimmune arthritic diseases
Keywords: MHC class I antigen, antigen presentation, membrane transport protein, protein structure /function adenosinetriphosphatase, enzyme activity circular dichroism, laboratory mouse, surface plasmon resonance
Project start date: 1999-01-15
Project end date: 2003-12-31
5R01AI044115-03 (2001): $172391
5R01AI044115-02 (2000): $167370
5R01AI044115-05 (2003): $182893
5R01AI044115-08 (2006): $295135
5R01AI044115-07 (2005): $303772
5R01AI044115-09 (2007): $286048
INTERACTIONS AND MECHANISMS OF FUNCTION OF THE TAP COMPLEX
Malini Raghavan, Associate Professor
University Of Michigan At Ann Arbor, 1040 Wolverine Tower, Ann Arbor, Mi 48109-1274
Grant 5R01AI044115-11 from National Institute Of Allergy And Infectious Diseases
Abstract: Assembly of major histocompatibility complex (MHC) class I molecules occurs within the endoplasmic reticulum (ER) of cells. Newly synthesized MHC class I molecules are recruited into interactions with the transporter associated with antigen processing (TAP), tapasin, ERp57, protein disulfide isomerase, calnexin and calreticulin. This complex of accessory proteins can be considered a molecular machine whose job it is to (i) pump the peptide products of protein degradation into the region of MHC class I assembly (ii) recruit unassembled MHC class I (iii) facilitate MHC class I-peptide assembly and (iv) ensure regulated release of optimally loaded MHC class I. Much remains to be understood about the workings of this intricate molecular machine, which has been the focus of our research for the past eleven years. Based on our previous work with the TAP transporter, we are able to propose a detailed model for how ATP binding and hydrolysis couple to peptide binding and transport. In the proposed studies we will examine effects of TAP substrates on nucleotide binding and exchange by TAP, and on interactions between the nucleotide binding domains (NBD). A model for the peptide-binding site of TAP will also be examined. These investigations will allow for better understanding of how TAP can be manipulated to enhance or suppress immune responses, and will also allow for better predictions of immunodominant cytotoxic T lymphocyte (CTL) epitopes. Based on analyses of the assembly characteristics of various MHC class I allotypes in tapasin-deficient cells, it is our hypothesis that tapasin is essential for peptide loading of MHC class I allotypes that have slow intrinsic peptide loading kinetics. Peptide binding properties of tapasin dependent and independent MHC class I allotypes will be compared under different conditions. Our data suggest that tapasin is responsible for recruiting calreticulin and ERp57 into the peptide loading complex. Furthermore different conformational states of tapasin-ERp57 complexes had different activities in enhancing peptide loading of MHC class I molecules. We seek to better understand the nature of the differences. We also seek to understand the role of careticulin in tapasin-assisted MHC class I assembly. Although all MHC class I molecules appear to follow the same assembly route within the ER, closely related HLA-B allotypes differ dramatically in their intrinsic rates of assembly and ER exit. In the studies proposed here, we seek to classify high frequency HLA-B alleles as rapid or slow trafficking, and to also examine the functional consequences of rapid or slow trafficking upon antigen presentation and disease progression. It is our hypothesis that the trafficking phenotypes can impact both the CTL response and the NK cell response, which will be further examined. Together, these studies will allow for a better understanding of the different steps of the MHC class I assembly route, and will contribute to the development of more effective strategies to enhance CTL responses in infection and cancer. An understanding of the substrate interaction site of TAP, and of potential resting state conformations of TAP (inactive conformations) will be important for future designs of TAP inhibitors that could be of use in settings of transplantation and autoimmunity, and additionally in the design of inhibitors of other ABC transporters to overcome drug/antibiotic resistance. A better understanding of the mechanism of tapasin function could lead to new strategies for enhancing assembly of specific immunogenic peptides with MHC Class I molecules in infection and cancer. Finally, an understanding of how trafficking differences between HLA-B allotypes impact their antigen presenting ability will be important for better elucidating the effects of different HLA antigens on disease susceptibility, resolution, and progression
Keywords: 55-kDa High-Affinity Calcium Binding Protein; ABC Transport Protein; ABC Transporter Protein; ABC Transporters; AIDS; ATGN; ATP Hydrolysis; ATP-Binding Cassette Transporters; Acquired Immune Deficiency; Acquired Immune Deficiency Syndrome; Acquired Immuno-Deficiency Syndrome; Acquired Immunodeficiency Syndrome; Affect; African American; Afro American; Afroamerican; Alleles; Allelomorphs; American; Antibiotic Resistance; Antigen Presentation; Antigens; Autoimmune Status; Autoimmunity; Binding; Binding (Molecular Function); Binding Sites; Black Populations; Black or African American; CAB-63; CBP3 (Liver); CLN; CTL; Calcium-Binding Protein-3; Calnexin; Calregulin; Cancers; Cell surface; Cell-Mediated Lympholytic Cells; Cells; Cellular Membrane; Chaperone; Characteristics; Combining Site; Complex; Cytolytic T-Cell; Cytosol; Cytotoxic T Cell; Cytotoxic T-Lymphocytes; Cytotoxic cell; Data; Dehydrogenases; Dependence; Development; Diathesis; Disease; Disease Progression; Disease susceptibility; Disorder; Dissociation; Disulfide Interchange Enzyme; Disulfide Isomerase; Drugs; ERp57; ERp59 PDI; ERp60; Endoplasmic Reticulum; Ensure; Epitopes, T-Lymphocyte; Ergastoplasm; Erp59; European; Family; Frequencies (time pattern); Frequency; Future; GSBP; Glycosylation Site-Binding Protein; Goals; HACBP; HL-A Antigens; HLA A*0201 antigen; HLA Antigens; HLA-A*0201; HLA-B; HLA-B Antigens; Histocompatibility Complex; Histocompatibility Complices; Human; Human Leukocyte Antigens; Human, General; Hydrolysis; IP90 Protein; Immune Surveillance; Immune response; Immunologic Deficiency Syndrome, Acquired; Immunologic Surveillance; Immunological Surveillance; Infection; Integral Membrane Protein; Integral Membrane Protein IP90; Intrinsic Membrane Protein; Investigation; Jobs; K lymphocyte; Kinetic; Kinetics; Lead; Lectin; Leukocyte Antigens; MHC Receptor; Major Histocompatibility Complex; Major Histocompatibility Complex Receptor; Major Histocompatibility Complices; Malignant Neoplasms; Malignant Tumor; Man (Taxonomy); Man, Modern; Medication; Membrane Potentials; Modeling; Molecular Chaperones; Molecular Configuration; Molecular Conformation; Molecular Interaction; Molecular Machines; Molecular Stereochemistry; NK Cells; Natural Killer Cells; Nature; Nucleotides; Occupations; Oxidoreductase; PDI; Pathway interactions; Pattern; Pb element; Peptide Transport; Peptides; Pharmaceutic Preparations; Pharmaceutical Preparations; Phenotype; Phosphoprotein pp90; Population; Professional Postions; Property; Property, LOINC Axis 2; Protein Cleavage; Protein Degradation, Metabolic; Protein Degradation, Regulatory; Protein Disulfide Isomerase; Protein Turnover; Proteins; Proteolysis; Pump; RACK1 (peptide); Reactive Site; Receptors, Antigen, T-Cell; Recruitment Activity; Reductases; Refractory; Research; Resistance to antibiotics; Resistance, Antibiotic; Resistant to antibiotics; Resolution; Resting Potentials; Role; Route; S-S rearrangase; Site; Substrate Interaction; Sulfhydryl-Disulfide Interchange Enzyme; Surveillances, Immunologic; Surveillances, Immunological; T-Cell Epitopes; T-Cell Receptor; T-Cells; T-Lymphocyte; T-Lymphocyte Epitopes; T-Lymphocyte and NK-Cell; T-Lymphocyte and Natural Killer Cell; T-Lymphocytes, Cytotoxic; TAP-A protein; Testing; Thiol-Disulfide Transhydrogenase; Thymus-Dependent Lymphocytes; Transmembrane Potentials; Transmembrane Protein; Transplantation; Trypanothione-Glutathione Thioltransferase; Variant; Variation; Viral; Work; antibiotic resistant; antigenic peptide transporter; base; black American; cC1qR Protein; calreticulin; conformation; conformational state; design; designing; disease/disorder; disease/disorder proneness/risk; drug/agent; dsbA Gene Product; dsbA Protein; dsbC Gene Product; dsbD Gene Product; gene product; heavy metal Pb; heavy metal lead; host response; immunogen; immunogenic; immunoresponse; inhibitor; inhibitor/antagonist; insight; liability to disease; malignancy; mutant; neoplasm/cancer; pathway; peptide I; protein degradation; protein function; public health relevance; recruit; response; reticulum cell; self recognition (immune); social role; tapasin; thymus derived lymphocyte; trafficking; transplant; transporter associated with antigen processing; tumor; xprA Gene Product
Relevance: An understanding of the substrate interaction site of TAP, and of potential resting state conformations of TAP (inactive conformations) will be important for future designs of TAP inhibitors that could be of use in settings of transplantation and autoimmunity, and additionally in the design of inhibitors of other ABC transporters to overcome drug/antibiotic resistance. A better understanding of the mechanism of tapasin function could lead to new strategies for enhancing assembly of specific immunogenic peptides with MHC Class I molecules in infection and cancer. Finally, an understanding of how trafficking differences between HLA-B allotypes impact their antigen presenting ability will be important for better elucidating the effects of different HLA antigens on disease susceptibility, resolution, and progression
Project start date: 1999-01-15
Project end date: 2013-12-31
Budget start date: 1-JAN-2010
Budget end date: 31-DEC-2010
PFA/PA: PA-07-070
5R01AI044115-11 (2010): $347547
Grants awarded to Malini Raghavan
CALRETICULIN´S FUNCTIONS IN THE ADAPTIVE IMMUNE RESPONSE
Malini Raghavan, Associate Professor
University Of Michigan At Ann Arbor, 1040 Wolverine Tower, Ann Arbor, Mi 48109-1274
Grant 3R01AI066131-03S1 from National Institute Of Allergy And Infectious Diseases
Abstract: This award is issued in response to Notice OD-09-060, Recovery Act Administrative Supplements Providing Summer Research Experiences for Students and Science Educators. Calreticulin is an endoplasmic reticulum (ER) chaperone that promotes folding and assembly of glycoproteins, including major histocompatibility complex (MHC) class I molecules. Calreticulin also has the capacity to direct exogenous antigens onto the MHC class I antigen presentation pathway, a phenomenon called cross-presentation. As a lectin, Calreticulin interacts with monoglucosylated core glycans on glycoproteins. Under certain conditions, Calreticulin is able to bind polypeptide components of substrates. Calcium depletion and heat-treatment expose calreticulin´s polypeptide binding site and enhance Calreticulin binding to polypeptide substrates in vitro and in vivo cells. These treatments also induce Calreticulin dimerization and oligomerization. The formation of Calreticulin dimers is additionally induced by other types of ER stress, including virus infection and tunicamycin treatment. It is our hypothesis that these conformational transitions and polypeptide-binding properties are important for calreticulin´s protein folding and cross-priming functions in cells. The first specific aim explores the role of polypeptide binding by Calreticulin during MHC class I folding and assembly in cells. We propose partial proteolysis and mass spectrometry-based approaches to identify Calreticulin sub-domains that are mobilized by calcium depletion. Conserved hydrophobic residues of Calreticulin, that are predicted to be surface-exposed, will be mutated to alanines. Mutants that display defects in interactions with polypeptide components of MHC class I heavy chains in vitro, as well as other mutants with defects in binding oligosaccharide substrates, will be expressed in calreticulin-deficient cells, and assessed for the ability to facilitate MHC class I folding and assembly. Together, these studies will allow us to refine our working model for the calreticulin-substrate interaction cycle, in which alternating interactions with oligosaccharide and polypeptide components of substrates are proposed. We will attempt to crystallize truncated versions of Calreticulin that have enhanced ability to bind polypeptide substrates, and also crystallize Calreticulin complexes with chicken IgY fragments. The second specific aim will explore mechanisms of calreticulin-mediated cross-presentation. Intracellular trafficking of Calreticulin and calreticulin-associated peptides during cross-presentation will be assessed, to investigate the hypothesis of an endosome-trans Golgi network-ER trafficking route. The requirement for Calreticulin for cross-presentation of antigens associated with apoptotic cells will also be assessed. Finally, the effects of ER stress on Calreticulin trafficking, cell surface expression, and interactions with receptors on antigen presenting cells will be assessed. Understanding the molecular mechanisms of calreticulin´s functions, and elucidation of conditions that enhance calreticulin´s T cell priming activities, will facilitate more effective design of vaccines
Keywords: 55-kDa High-Affinity Calcium Binding Protein; APC; APC receptor; ATGN; Alanine; Alanine, L-Isomer; Antigen Presentation; Antigen Presentation Pathway; Antigen Processing and Presentation; Antigen-Presenting Cells; Antigens; Apoptotic; Binding; Binding (Molecular Function); Binding Sites; Biochemical; Blood Coagulation Factor IV; CAB-63; CBP3 (Liver); CLN; Ca++ element; Calcium; Calcium-Binding Protein-3; Calnexin; Calregulin; Cell surface; Cells; Cellular Stress; Chaperone; Chickens; Class I Antigens; Class I Major Histocompatibility Antigens; Coagulation Factor IV; Combining Site; Complex; Complex Class 1; Cross Presentation; Cross-Priming; Defect; Dimerization; Disulfides; ERp60; Endoplasmic Reticulum; Endosomes; Ergastoplasm; Factor IV; Gallus domesticus; Gallus gallus; Gallus gallus domesticus; Glycans; Glycoproteins; HACBP; Heating; Histocompatibility Antigens Class I; Histocompatibility Complex; Histocompatibility Complices; IP90 Protein; IgY; Immune response; Immunity; Immunofluorescence; Immunofluorescence Immunologic; Immunologic Accessory Cells; Immunologic, Immunofluorescence; In Vitro; Integral Membrane Protein IP90; L-Alanine; Lectin; Link; Location; MHC Class I Molecule; MHC Class I Protein; MHC class I antigen; Major Histocompatibility Complex; Major Histocompatibility Complex Class 1; Major Histocompatibility Complices; Mass Spectrum; Mass Spectrum Analysis; Mediating; Modeling; Molecular; Molecular Chaperones; Molecular Configuration; Molecular Conformation; Molecular Interaction; Molecular Stereochemistry; Monocytes / Macrophages / APC; Mutate; Oligosaccharides; Pathway interactions; Peptides; Phagocytes; Phagocytic Cell; Phosphoprotein pp90; Photometry/Spectrum Analysis, Mass; Physiologic; Physiological; Polysaccharides; Property; Property, LOINC Axis 2; Protein Cleavage; Protein Dimerization; Proteins; Proteolysis; Quality Control; Reactive Site; Receptor Protein; Receptosomes; Role; Route; Site; Spectrometry, Mass; Spectroscopy, Mass; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Structure; Substrate Interaction; Surface; T-Cells; T-Lymphocyte; TGN; Thymus-Dependent Lymphocytes; Tunicamycin; Vaccine Design; Viral Diseases; Virus Diseases; Work; accessory cell; activated protein C receptor; amebocyte; base; cC1qR Protein; calreticulin; conformation; conformational conversion; conformational state; conformational transition; dimer; endoplasmic reticulum stress; endothelial cell protein C receptor; experiment; experimental research; experimental study; gene product; host response; immunogen; immunoglobulin Y; immunoresponse; in vitro activity; in vivo; insight; mutant; pathway; polypeptide; protein folding; receptor; research study; social role; thymus derived lymphocyte; trafficking; trans-Golgi Network; uptake; viral infection; virus infection
Project start date: 2009-07-14
Project end date: 2010-09-30
Budget start date: 14-JUL-2009
Budget end date: 30-SEP-2010
3R01AI066131-03S1 (2009): $22866
INTERACTIONS AND MECHANISMS OF FUNCTION OF THE TAP COMPLEX
Malini Raghavan, Associate Professor
University Of Michigan At Ann Arbor, 1040 Wolverine Tower, Ann Arbor, Mi 48109-1274
Grant 3R01AI044115-10A2S1 from National Institute Of Allergy And Infectious Diseases
Abstract: This award is issued in response to Notice OD-09-060, Recovery Act Administrative Supplements Providing Summer Research Experiences for Students and Science Educators. Assembly of major histocompatibility complex (MHC) class I molecules occurs within the endoplasmic reticulum (ER) of cells. Newly synthesized MHC class I molecules are recruited into interactions with the transporter associated with antigen processing (TAP), tapasin, ERp57, protein disulfide isomerase, calnexin and calreticulin. This complex of accessory proteins can be considered a molecular machine whose job it is to (i) pump the peptide products of protein degradation into the region of MHC class I assembly (ii) recruit unassembled MHC class I (iii) facilitate MHC class I-peptide assembly and (iv) ensure regulated release of optimally loaded MHC class I. Much remains to be understood about the workings of this intricate molecular machine, which has been the focus of our research for the past eleven years. Based on our previous work with the TAP transporter, we are able to propose a detailed model for how ATP binding and hydrolysis couple to peptide binding and transport. In the proposed studies we will examine effects of TAP substrates on nucleotide binding and exchange by TAP, and on interactions between the nucleotide binding domains (NBD). A model for the peptide-binding site of TAP will also be examined. These investigations will allow for better understanding of how TAP can be manipulated to enhance or suppress immune responses, and will also allow for better predictions of immunodominant cytotoxic T lymphocyte (CTL) epitopes. Based on analyses of the assembly characteristics of various MHC class I allotypes in tapasin-deficient cells, it is our hypothesis that tapasin is essential for peptide loading of MHC class I allotypes that have slow intrinsic peptide loading kinetics. Peptide binding properties of tapasin dependent and independent MHC class I allotypes will be compared under different conditions. Our data suggest that tapasin is responsible for recruiting calreticulin and ERp57 into the peptide loading complex. Furthermore different conformational states of tapasin-ERp57 complexes had different activities in enhancing peptide loading of MHC class I molecules. We seek to better understand the nature of the differences. We also seek to understand the role of careticulin in tapasin-assisted MHC class I assembly. Although all MHC class I molecules appear to follow the same assembly route within the ER, closely related HLA-B allotypes differ dramatically in their intrinsic rates of assembly and ER exit. In the studies proposed here, we seek to classify high frequency HLA-B alleles as rapid or slow trafficking, and to also examine the functional consequences of rapid or slow trafficking upon antigen presentation and disease progression. It is our hypothesis that the trafficking phenotypes can impact both the CTL response and the NK cell response, which will be further examined. Together, these studies will allow for a better understanding of the different steps of the MHC class I assembly route, and will contribute to the development of more effective strategies to enhance CTL responses in infection and cancer. An understanding of the substrate interaction site of TAP, and of potential resting state conformations of TAP (inactive conformations) will be important for future designs of TAP inhibitors that could be of use in settings of transplantation and autoimmunity, and additionally in the design of inhibitors of other ABC transporters to overcome drug/antibiotic resistance. A better understanding of the mechanism of tapasin function could lead to new strategies for enhancing assembly of specific immunogenic peptides with MHC Class I molecules in infection and cancer. Finally, an understanding of how trafficking differences between HLA-B allotypes impact their antigen presenting ability will be important for better elucidating the effects of different HLA antigens on disease susceptibility, resolution, and progression
Keywords: 55-kDa High-Affinity Calcium Binding Protein; ABC Transport Protein; ABC Transporter Protein; ABC Transporters; AIDS; ATGN; ATP Hydrolysis; ATP-Binding Cassette Transporters; Acquired Immune Deficiency; Acquired Immune Deficiency Syndrome; Acquired Immuno-Deficiency Syndrome; Acquired Immunodeficiency Syndrome; Affect; African American; Afro American; Afroamerican; Alleles; Allelomorphs; American; Antibiotic Resistance; Antigen Presentation; Antigens; Autoimmune Status; Autoimmunity; Binding; Binding (Molecular Function); Binding Sites; Black Populations; Black or African American; CAB-63; CBP3 (Liver); CLN; CTL; Calcium-Binding Protein-3; Calnexin; Calregulin; Cancers; Cell surface; Cell-Mediated Lympholytic Cells; Cells; Cellular Membrane; Chaperone; Characteristics; Combining Site; Complex; Cytolytic T-Cell; Cytosol; Cytotoxic T Cell; Cytotoxic T-Lymphocytes; Cytotoxic cell; Data; Dehydrogenases; Dependence; Development; Diathesis; Disease; Disease Progression; Disease susceptibility; Disorder; Dissociation; Disulfide Interchange Enzyme; Disulfide Isomerase; Drugs; ERp57; ERp59 PDI; ERp60; Endoplasmic Reticulum; Ensure; Epitopes, T-Lymphocyte; Ergastoplasm; Erp59; European; Family; Frequencies (time pattern); Frequency; Future; GSBP; Glycosylation Site-Binding Protein; Goals; HACBP; HL-A Antigens; HLA A*0201 antigen; HLA Antigens; HLA-A*0201; HLA-B; HLA-B Antigens; Histocompatibility Complex; Histocompatibility Complices; Human; Human Leukocyte Antigens; Human, General; Hydrolysis; IP90 Protein; Immune Surveillance; Immune response; Immunologic Deficiency Syndrome, Acquired; Immunologic Surveillance; Immunological Surveillance; Infection; Integral Membrane Protein; Integral Membrane Protein IP90; Intrinsic Membrane Protein; Investigation; Jobs; K lymphocyte; Kinetic; Kinetics; Lead; Lectin; Leukocyte Antigens; MHC Receptor; Major Histocompatibility Complex; Major Histocompatibility Complex Receptor; Major Histocompatibility Complices; Malignant Neoplasms; Malignant Tumor; Man (Taxonomy); Man, Modern; Medication; Membrane Potentials; Modeling; Molecular Chaperones; Molecular Configuration; Molecular Conformation; Molecular Interaction; Molecular Machines; Molecular Stereochemistry; NK Cells; Natural Killer Cells; Nature; Nucleotides; Occupations; Oxidoreductase; PDI; Pathway interactions; Pattern; Pb element; Peptide Transport; Peptides; Pharmaceutic Preparations; Pharmaceutical Preparations; Phenotype; Phosphoprotein pp90; Population; Professional Postions; Property; Property, LOINC Axis 2; Protein Cleavage; Protein Degradation, Metabolic; Protein Degradation, Regulatory; Protein Disulfide Isomerase; Protein Turnover; Proteins; Proteolysis; Pump; RACK1 (peptide); Reactive Site; Receptors, Antigen, T-Cell; Recruitment Activity; Reductases; Refractory; Research; Resistance to antibiotics; Resistance, Antibiotic; Resistant to antibiotics; Resolution; Resting Potentials; Role; Route; S-S rearrangase; Site; Substrate Interaction; Sulfhydryl-Disulfide Interchange Enzyme; Surveillances, Immunologic; Surveillances, Immunological; T-Cell Epitopes; T-Cell Receptor; T-Cells; T-Lymphocyte; T-Lymphocyte Epitopes; T-Lymphocyte and NK-Cell; T-Lymphocyte and Natural Killer Cell; T-Lymphocytes, Cytotoxic; TAP-A protein; Testing; Thiol-Disulfide Transhydrogenase; Thymus-Dependent Lymphocytes; Transmembrane Potentials; Transmembrane Protein; Transplantation; Trypanothione-Glutathione Thioltransferase; Variant; Variation; Viral; Work; antibiotic resistant; antigenic peptide transporter; base; black American; cC1qR Protein; calreticulin; conformation; conformational state; design; designing; disease/disorder; disease/disorder proneness/risk; drug/agent; dsbA Gene Product; dsbA Protein; dsbC Gene Product; dsbD Gene Product; gene product; heavy metal Pb; heavy metal lead; host response; immunogen; immunogenic; immunoresponse; inhibitor; inhibitor/antagonist; insight; liability to disease; malignancy; mutant; neoplasm/cancer; pathway; peptide I; protein degradation; protein function; public health relevance; recruit; response; reticulum cell; self recognition (immune); social role; tapasin; thymus derived lymphocyte; trafficking; transplant; transporter associated with antigen processing; tumor; xprA Gene Product
Relevance: An understanding of the substrate interaction site of TAP, and of potential resting state conformations of TAP (inactive conformations) will be important for future designs of TAP inhibitors that could be of use in settings of transplantation and autoimmunity, and additionally in the design of inhibitors of other ABC transporters to overcome drug/antibiotic resistance. A better understanding of the mechanism of tapasin function could lead to new strategies for enhancing assembly of specific immunogenic peptides with MHC Class I molecules in infection and cancer. Finally, an understanding of how trafficking differences between HLA-B allotypes impact their antigen presenting ability will be important for better elucidating the effects of different HLA antigens on disease susceptibility, resolution, and progression
Project start date: 2009-07-14
Project end date: 2010-09-30
Budget start date: 14-JUL-2009
Budget end date: 30-SEP-2010
PFA/PA: PA-07-070
3R01AI044115-10A2S1 (2009): $22866
INTERACTIONS & MECHANISMS OF FUNCTION OF THE TAP COMPLEX
Malini Raghavan, Assistant Professor
Microbiology And Immunologyuniversity Of Michigan At Ann Arbor
3003 South State Street, Room 1040
ann Arbor, Mi 481091274
Grant 1R01AI044115-01 from National Institute Of Allergy And Infectious Diseases IRG: IMB
Abstract: The transporters associated with antigen processing (TAP) are crucial components of the class I major histocompatibility complex (MHC) antigen presentation pathway. TAP is a complex of two subunits, TAP1 and TAP2, that functions to transport peptides from the cytosol to the endoplasmic reticulum (ER) for class I MHC antigen presentation. TAP1 and TAP2 both comprise a transmembrane domain and an ATP binding domain (NBD). The studies proposed here will result in a better understanding of TAP function and insights into molecular mechanisms by which the immune system combats viruses and cancers. In the first of the proposed studies, the interactions of ATP with human TAP subunits will be analyzed and structural changes that are induced as a consequence of ATP binding will be probed using proteolysis, circular dichroism and surface plasmon resonance-based assays. In particular, experiments are proposed to determine whether nucleotide binding alters the stability of a complex that we observe between the NBD of TAP1 and TAP2. In the second of the proposed studies, the question of whether ATP binding and hydrolysis by both TAP1 and TAP2 subunits is required for peptide translocation by TAP is addressed, by the generation and characterization of mutants that are predicted to alter the ATPase activities of one of the TAP subunits. The basis for these studies is the observation of natural mutations in human and rodent TAP2 sequences at sites that are highly conserved and that have been shown to be crucial for ATPase activity and transport function of other members of the ATP binding cassette (ABC) family of transmembrane transporters (of which TAP is a member). In the last of proposed studies, the interactions of the TAP complex with the human class I MHC molecule HLA -B27 and the newly discovered TAP-associated protein, tapasin will be examined. Studies in other laboratories have indicated that newly synthesized class I MHC molecules form complexes with TAP in the ER, and that this interaction is bridged or stabilized by tapasin. By expressing different combinations of the proteins TAP1 TAP2, tapasin, and the class I MHC heavy and light chains in insect cells, the molecular nature of the TAP/tapasin/class I MHC complexes will be elucidated, and insights will be obtained into whether other unknown components are required for these interactions, as well as for class I MHC antigen presentation. Finally, analysis of TAP interactions with HLA-B27 that are proposed will provide insight into the molecular mechanisms that underlie the association of B27 with autoimmune arthritic diseases
Keywords: MHC class I antigen, antigen presentation, membrane transport protein, protein structure /function adenosinetriphosphatase, enzyme activity circular dichroism, laboratory mouse, surface plasmon resonance
Project start date: 1999-01-15
Project end date: 2003-12-31
1R01AI044115-01 (1999): $162492
2R01AI044115-06A1 (2004): $303837
Calreticulin´s Functions In The Adaptive Immune Response
Malini Raghavan, Assistant Professor
Microbiology And Immunologyuniversity Of Michigan At Ann Arbor, 3003 South State Street, Room 1040, Ann Arbor, Mi 481091274
Grant 1R01AI066131-01A2 from National Institute Of Allergy And Infectious Diseases IRG: CMIB
Abstract: DESCRIPTION () Calreticulin is an endoplasmic reticulum (ER) chaperone that promotes folding and assembly of glycoproteins, including major histocompatibility complex (MHC) class I molecules. Calreticulin also has the capacity to direct exogenous antigens onto the MHC class I antigen presentation pathway, a phenomenon called cross-presentation. As a lectin, Calreticulin interacts with monoglucosylated core glycans on glycoproteins. Under certain conditions, Calreticulin is able to bind polypeptide components of substrates. Calcium depletion and heat-treatment expose calreticulin´s polypeptide binding site and enhance Calreticulin binding to polypeptide substrates in vitro and in vivo cells. These treatments also induce Calreticulin dimerization and oligomerization. The formation of Calreticulin dimers is additionally induced by other types of ER stress, including virus infection and tunicamycin treatment. It is our hypothesis that these conformational transitions and polypeptide-binding properties are important for calreticulin´s protein folding and cross-priming functions in cells. The first specific aim explores the role of polypeptide binding by Calreticulin during MHC class I folding and assembly in cells. We propose partial proteolysis and mass spectrometry-based approaches to identify Calreticulin sub-domains that are mobilized by calcium depletion. Conserved hydrophobic residues of Calreticulin, that are predicted to be surface-exposed, will be mutated to alanines. Mutants that display defects in interactions with polypeptide components of MHC class I heavy chains in vitro, as well as other mutants with defects in binding oligosaccharide substrates, will be expressed in calreticulin-deficient cells, and assessed for the ability to facilitate MHC class I folding and assembly. Together, these studies will allow us to refine our working model for the calreticulin-substrate interaction cycle, in which alternating interactions with oligosaccharide and polypeptide components of substrates are proposed. We will attempt to crystallize truncated versions of Calreticulin that have enhanced ability to bind polypeptide substrates, and also crystallize Calreticulin complexes with chicken IgY fragments. The second specific aim will explore mechanisms of calreticulin-mediated cross-presentation. Intracellular trafficking of Calreticulin and calreticulin-associated peptides during cross-presentation will be assessed, to investigate the hypothesis of an endosome-trans Golgi network-ER trafficking route. The requirement for Calreticulin for cross-presentation of antigens associated with apoptotic cells will also be assessed. Finally, the effects of ER stress on Calreticulin trafficking, cell surface expression, and interactions with receptors on antigen presenting cells will be assessed. Understanding the molecular mechanisms of calreticulin´s functions, and elucidation of conditions that enhance calreticulin´s T cell priming activities, will facilitate more effective design of vaccines.
Project start date: 2007-03-15
Project end date: 2011-02-28
1R01AI066131-01A2 (2007): $298459
5R01AI066131-04 (2010): $349857
3R01AI066131-03S2 (2009): $389120
Interactions And Mechanisms Of Function Of The TAP Complex
Malini Raghavan, Associate Professor
Microbiology And Immunologyuniversity Of Michigan At Ann Arbor
Grant 2R01AI044115-10A2 from National Institute Of Allergy And Infectious Diseases IRG: CMIB
Abstract: Assembly of major histocompatibility complex (MHC) class I molecules occurs within the endoplasmic reticulum (ER) of cells. Newly synthesized MHC class I molecules are recruited into interactions with the transporter associated with antigen processing (TAP), tapasin, ERp57, protein disulfide isomerase, calnexin and calreticulin. This complex of accessory proteins can be considered a molecular machine whose job it is to (i) pump the peptide products of protein degradation into the region of MHC class I assembly (ii) recruit unassembled MHC class I (iii) facilitate MHC class I-peptide assembly and (iv) ensure regulated release of optimally loaded MHC class I. Much remains to be understood about the workings of this intricate molecular machine, which has been the focus of our research for the past eleven years. Based on our previous work with the TAP transporter, we are able to propose a detailed model for how ATP binding and hydrolysis couple to peptide binding and transport. In the proposed studies we will examine effects of TAP substrates on nucleotide binding and exchange by TAP, and on interactions between the nucleotide binding domains (NBD). A model for the peptide-binding site of TAP will also be examined. These investigations will allow for better understanding of how TAP can be manipulated to enhance or suppress immune responses, and will also allow for better predictions of immunodominant cytotoxic T lymphocyte (CTL) epitopes. Based on analyses of the assembly characteristics of various MHC class I allotypes in tapasin-deficient cells, it is our hypothesis that tapasin is essential for peptide loading of MHC class I allotypes that have slow intrinsic peptide loading kinetics. Peptide binding properties of tapasin dependent and independent MHC class I allotypes will be compared under different conditions. Our data suggest that tapasin is responsible for recruiting calreticulin and ERp57 into the peptide loading complex. Furthermore different conformational states of tapasin-ERp57 complexes had different activities in enhancing peptide loading of MHC class I molecules. We seek to better understand the nature of the differences. We also seek to understand the role of careticulin in tapasin-assisted MHC class I assembly. Although all MHC class I molecules appear to follow the same assembly route within the ER, closely related HLA-B allotypes differ dramatically in their intrinsic rates of assembly and ER exit. In the studies proposed here, we seek to classify high frequency HLA-B alleles as rapid or slow trafficking, and to also examine the functional consequences of rapid or slow trafficking upon antigen presentation and disease progression. It is our hypothesis that the trafficking phenotypes can impact both the CTL response and the NK cell response, which will be further examined. Together, these studies will allow for a better understanding of the different steps of the MHC class I assembly route, and will contribute to the development of more effective strategies to enhance CTL responses in infection and cancer. An understanding of the substrate interaction site of TAP, and of potential resting state conformations of TAP (inactive conformations) will be important for future designs of TAP inhibitors that could be of use in settings of transplantation and autoimmunity, and additionally in the design of inhibitors of other ABC transporters to overcome drug/antibiotic resistance. A better understanding of the mechanism of tapasin function could lead to new strategies for enhancing assembly of specific immunogenic peptides with MHC Class I molecules in infection and cancer. Finally, an understanding of how trafficking differences between HLA-B allotypes impact their antigen presenting ability will be important for better elucidating the effects of different HLA antigens on disease susceptibility, resolution, and progression
Project start date: 1999-01-15
Project end date: 2013-12-31