Malini Raghavan - US grants

9707382 Raghavan This work focuses on one of the major players of the cellular antigen processing machinery, the transporters associated with antigen processing (TAP). The TAP proteins are members of the ATP binding cassette (ABC) family of transmembrane transporters. The long term goal of this research is to gain insight into the mechanisms involved in substrate transport by ABC transporters. The TAP proteins will be investigated as the prototype transporter. Recombinant TAP proteins will be purified from insect cell membranes and reconstituted into artificial membranes to measure transport activities under strictly controlled conditions. The coupling between peptide and nucleotide binding by TAP will also be studied. Finally, soluble TAP fragments will be obtained for structural studies. A family of proteins called ABC transporters use the energy from ATP to transport various substances across cell membranes. This project uses biochemical and biophysical studies to understand the mechanism by which these transporters function.

[unreadable] DESCRIPTION (provided by applicant): The transporter associated with antigen processing complex comprises three subunits, TAP1, TAP2, and tapasin. Of these, the TAP1 and TAP2 subunits belong to the ATP binding cassette (ABC) family of transmembrane transporters, and function to translocate peptides across the endoplasmic reticulum (ER) membrane. Tapasin interacts with both TAP subunits, and is required for enhancing the structural stability of the TAP1/TAP2 complex. The ER-luminal domain of tapasin also interacts with major histocompatibility complex (MHC) class I molecules, facilitating their assembly. In the proposed studies, we first focus on the molecular events in the cytosol, and will investigate the functions of TAP1 and TAP2 nucleotide binding domains (NBD) during peptide translocation by TAP complexes. We examine evidence for the existence of conformations of the TAP NBD that resemble NBD interactions in the DNA repair enzyme Rad50. In such an interaction, each TAP nucleotide binding site would be comprised of residues from the Walker A motif of one NBD and the signature motif of the second NBD. Using viral inhibitors of TAP, TAP substrates, and appropriately designed TAP1 and TAP2 mutants expressed in insect cells, we will attempt to reconstruct the sequence of molecular events that occur during a TAP catalytic cycle. In the second part of the proposed studies, the focus is upon the ER-luminal face of TAP complexes, in particular on the dynamics of peptide/MHC class I and tapasin/MHC class I interactions. We have been able to demonstrate direct binding between various peptide-deficient MHC class I molecules and tapasin. In the proposed studies, we investigate a competitive displacement model for tapasin function, in which peptides and tapasin compete for class I heavy chain binding. We will examine which domains of tapasin and MHC class I are important for complex formation. We also examine the hypotheses that the tapasin-dependence vs. independence of MHC class I molecules results from intrinsic properties of the class I molecule. We seek to understand the functional consequences of enhanced TAP/tapasin binding in the ER by particular class I molecules. Taken together, these studies will provide insights into how peptide transport into the ER, and peptide assembly with MHC class I molecules, are orchestrated within the cell [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): 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. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH 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.

DESCRIPTION (provided by applicant): Major histocompatibility complex (MHC) class I polymorphisms influence outcomes in a number of infectious diseases, cancers and inflammatory diseases. In human immunodeficiency virus (HIV) infections, among all genetic factors known to influence progression to acquired immunodeficiency syndrome (AIDS), the strongest associations link to human MHC class I genes. MHC class I molecules bind to peptide antigens and present these antigens to CD8 T cells. MHC class I molecules are also ligands for inhibitory receptors of NK cells Three sets of genes encode human MHC class I molecules which are the human leukocyte antigens (HLA) A, B and C. These genes are highly polymorphic, with the HLA-B genes being the most variable. It is generally assumed that HLA-disease associations link to the peptide binding characteristics of individual HLA class I molecules. However, it remains largely unknown whether and how differences in assembly characteristics or stabilities of HLA class I molecules influence immunological outcomes. A set of objectives of this application is to examine such influences. A central hypothesis is that the observed assembly and stability differences between HLA-B allotypes influence their cell surface expression, and their abilities to mediate CD8 T cell and NK cell responses. In Aim 1, we show that dependence on the assembly factor tapasin is quite variable among HLA-B molecules. Tapasin-dependent assembly is highly prevalent within the HLA-Bw4 serotype, whereas many HLA-B molecules of HLA-Bw6 serotype are tapasin-independent for their assembly. HLA-Bw4 molecules but not HLA-Bw6 molecules are ligands for inhibitory receptors of natural killer (NK) cells, which are responsive to cell surface expression densities of MHC class I molecules. To further examine whether the HLA-Bw4/HLA- Bw6 segregation in tapasin dependence reflects altered cell surface expression of different allotypes, quantitative flow cytometry will be used to compare cell surface expression densities of HLA-B molecules in primary human CD4 T cells under basal conditions, and following infections with HIV-1. In Aim 2, we show varying conformational stabilities of soluble peptide-deficient HLA-B molecules during their refolding, and variable levels of expression of HLA-B molecules on cells deficient in the transporter associated with antigen processing (TAP). Based on these findings, we will examine variable recognition of HLA-B molecules by endoplasmic reticulum (ER) quality control factors, and differing requirements for other assembly factors. The molecular basis for stability differences of the empty HLA-B proteins will be elucidated. In Aim 3, we will examine whether the observed HLA-B assembly and stability differences influence the breadth and stability of peptide selection by HLA-B molecules. These analyses will be undertaken by comparing different HLA-B- restricted CD8 T cell responses against peptide antigens spanning the HIV proteome. Taken together, these studies are significant towards defining variations in immune functions of different HLA-B molecules, which are relevant towards vaccine design and infectious disease outcomes.

? DESCRIPTION (provided by applicant): Alterations of endoplasmic reticulum (ER) homeostasis can result from mutations of chaperones or of their substrate proteins. Calreticulin is a calcium-binding ER chaperone that is important for the folding and assembly of many N-linked glycoproteins. Calreticulin is also found on the surface of macrophages and apoptotic cells, where it facilitates cellular phagocytosis. Much remains to be understood about the molecular mechanisms of calreticulin-dependent protein folding, including factors that regulate substrate binding and release in the ER. Furthermore, the extracellular functions of calreticulin are poorly understood, including the mechanisms relevant to cell-surface interactions of calreticulin, and to calreticulin-dependent phagocytosis. There is also little knowledge about the loss and gain of function of calreticulin mutants with altered calcium- binding domains that are frequently found in myleoproliferative neoplasms (MPN). Some of these gaps in knowledge will be addressed in this application. The main hypotheses are that ATP is a key regulator of calreticulin-substrate interactions and that distinct modes of protein and lipid recognition are central to the cellular functions of calreticulin. Using computational and experimental approaches, a model for the ATP binding site of calreticulin is presented. ATP binding is shown to destabilize calreticulin binding to cellular monoglucosylated major histocompatibility complex (MHC) class I molecules. The effect of ATP binding deficient mutants on the maturation of other substrates will be examined, including ?1-antitrypsin (AAT), its misfolded variant ATZ, and the low-density lipoprotein-related protein (LRP-1). The molecular mechanisms by which calreticulin induces the clearance of insoluble ATZ will be studied, examining the model that polypeptide recognition by calreticulin is relevant to this activity. The influences of substrates and ER factos upon nucleotide exchange and upon the ATPase activity of calreticulin will be studied. Preliminary data indicate that the C-terminal acidic domain of calreticulin, which contains low affinity calcium-binding sites, also contains binding sites for phosphatidylserine (PS) and apoptotic cells. Somatic calreticulin mutants that are frequently present in MPN have altered non-acidic C-termini. These mutations are predicted to not only alter calcium and PS binding, and calreticulin-dependent cellular phagocytosis, but also affect the conformation and chaperone activity of calreticulin, which will be studied. Based on the knowledge gained from these studies, we expect to develop strategies to enhance the formation of active proteins in protein misfolding disorders such as AAT deficiency, and to understand the pathogenic effects of calreticulin mutations in cancer.

Major histocompatibility complex (MHC) class I molecules bind to peptide antigens and present these antigens to CD8 T cells. Human MHC class I variants are encoded by the HLA-A, HLA-B and HLA-C genes. These genes are highly polymorphic. The polymorphisms influence outcomes in a number of infectious diseases, cancers and inflammatory diseases. Among the three HLA class I loci, HLA-B molecules are shown have dominant influences upon outcomes in multiple diseases. A given HLA class I allotype can bind to a large number of cellular and pathogen-derived peptides, collectively called the peptidome. The diversity of HLA-B peptidomes could be one factor underlying their different effects upon disease outcomes. A number of factors are predicted to influence peptidome diversity among HLA class I allotypes, including the structure of the peptide-binding site, the intracellular assembly mechanism and the intrinsic stability of the peptide-deficient form. Several HLA-B molecules are assembled via unconventional intracellular pathways that are suggestive of the presence of novel unconventional epitopes within their peptidomes. We propose to use liquid chromatography mass spectrometry (LC-MS) methods to quantify, characterize and understand the full breadth of the peptidomes of selected HLA-B variants. Based on existing MS datasets, we will also develop globally normalized methods to quantify and compare peptidome diversities of different HLA-B variants. The findings of this study will be significant towards epitope and HLA selection during vaccine design against cancers and infectious diseases. The studies will also establish methods to identify novel disease-relevant epitopes, and allow a better understanding of the relationships between HLA class I genotypes and disease outcomes.