Dual Topology of the Melanocortin-2 Receptor Accessory Protein Is Stable.

Melanocortin 2 receptor accessory protein (MRAP) facilitates trafficking of melanocortin 2 (MC2) receptors and is essential for ACTH binding and signaling. MRAP is a single transmembrane domain protein that forms antiparallel homodimers. These studies ask when MRAP first acquires this dual topology, whether MRAP architecture is static or stable, and whether the accessory protein undergoes rapid turnover. To answer these questions, we developed an approach that capitalizes on the specificity of bacterial biotin ligase, which adds biotin to lysine in a short acceptor peptide sequence; the distinct mobility of MRAP protomers of opposite orientations based on their N-linked glycosylation; and the ease of identifying biotin-labeled proteins. We inserted biotin ligase acceptor peptides at the N- or C-terminal ends of MRAP and expressed the modified proteins in mammalian cells together with either cytoplasmic or endoplasmic reticulum-targeted biotin ligase. MRAP assumed dual topology early in biosynthesis in both CHO and OS3 adrenal cells. Once established, MRAP orientation was stable. Despite its conformational stability, MRAP displayed a half-life of under 2 h in CHO cells. The amount of MRAP was increased by the proteasome inhibitor MG132 and MRAP underwent ubiquitylation on lysine and other amino acids. Nonetheless, when protein synthesis was blocked with cycloheximide, MRAP was rapidly degraded even when MG132 was included and all lysines were replaced by arginines, implicating non-proteasomal degradation pathways. The results show that although MRAP does not change orientations during trafficking, its synthesis and degradation are dynamically regulated.

Adrenocorticotropic Hormone (ACTH) Responses Require Actions of the Melanocortin-2 Receptor Accessory Protein on the Extracellular Surface of the Plasma Membrane.

The melanocortin-2 (MC2) receptor is a G protein-coupled receptor that mediates responses to ACTH. The MC2 receptor acts in concert with the MC2 receptor accessory protein (MRAP) that is absolutely required for ACTH binding and signaling. MRAP has a single transmembrane domain and forms a highly unusual antiparallel homodimer that is stably associated with MC2 receptors at the plasma membrane. Despite the physiological importance of the interaction between the MC2 receptor and MRAP, there is little understanding of how the accessory protein works. The dual topology of MRAP has made it impossible to determine whether highly conserved and necessary regions of MRAP are required on the intracellular or extracellular face of the plasma membrane. The strategy used here was to fix the orientation of two antiparallel MRAP molecules and then introduce inactivating mutations on one side of the membrane or the other. This was achieved by engineering proteins containing tandem copies of MRAP fused to the amino terminus of the MC2 receptor. The data firmly establish that only the extracellular amino terminus (Nout) copy of MRAP, oriented with critical segments on the extracellular side of the membrane, is essential. The transmembrane domain of MRAP is also required in only the Nout orientation. Finally, activity of MRAP-MRAP-MC2-receptor fusion proteins with inactivating mutations in either MRAP or the receptor was rescued by co-expression of free wild-type MRAP or free wild-type receptor. These results show that the basic MRAP-MRAP-receptor signaling unit forms higher order complexes and that these multimers signal.

Evaluating the Role of HLA-DM in MHC Class II-Peptide Association Reactions.

Ag presentation by MHC class II (MHC II) molecules to CD4(+) T cells plays a key role in the regulation of the adaptive immune response. Loading of antigenic peptides onto MHC II is catalyzed by HLA-DM (DM), a nonclassical MHC II molecule. The mechanism of DM-facilitated peptide loading is an outstanding problem in the field of Ag presentation. In this study, we systemically explored possible kinetic mechanisms for DM-catalyzed peptide association by measuring real-time peptide association kinetics using fluorescence polarization assays and comparing the experimental data with numerically modeled peptide association reactions. We found that DM does not facilitate peptide association by stabilizing peptide-free MHC II against aggregation. Moreover, DM does not promote transition of an inactive peptide-averse conformation of MHC II to an active peptide-receptive conformation. Instead, DM forms an intermediate with MHC II that binds peptide with faster kinetics than MHC II in the absence of DM. In the absence of peptides, interaction of MHC II with DM leads to inactivation and formation of a peptide-averse form. This study provides novel insights into how DM efficiently catalyzes peptide loading during Ag presentation.

A broadly applicable approach to T cell epitope identification: application to improving tumor associated epitopes and identifying epitopes in complex pathogens.

Epitopes are a hallmark of the antigen specific immune response. The identification and characterization of epitopes is essential for modern immunologic studies, from investigating cellular responses against tumors to understanding host/pathogen interactions especially in the case of bacteria with intracellular residence. Here, we have utilized a novel approach to identify T cell epitopes exploiting the exquisite ability of particulate antigens, in the form of beads, to deliver exogenous antigen to both MHC class I and class II pathways for presentation to T cell hybridomas. In the current study, we coupled this functional assay with two distinct protein expression libraries to develop a methodology for the characterization of T cell epitopes. One set of expression libraries containing single amino acid substitutions in a defined epitope sequence was interrogated to identify epitopes with enhanced T cell stimulation for a MHC class I epitope. The second expression library is comprised of the majority of open reading frames from the intracellular pathogen and potential biowarfare agent, Francisella tularensis. By automating aspects of this technology, we have been able to functionally screen and identify novel T cell epitopes within F. tularensis. We have also expanded upon these studies to generate a novel expression vector that enables immunization of recombinant protein into mice, which has been utilized to facilitate T cell epitope discovery for proteins that are critically linked to Francisella pathogenicity. This methodology should be applicable to a variety of systems and other pathogens.

Identification of T-cell epitopes in Francisella tularensis using an ordered protein array of serological targets.

Francisella tularensis is a Gram-negative intracellular bacterium that is the causative agent of tularaemia. Concerns regarding its use as a bioterrorism agent have led to a renewed interest in the biology of infection, host response and pathogenesis. A robust T-cell response is critical to confer protection against F. tularensis. However, characterization of the cellular immune response has been hindered by the paucity of tools to examine the anti-Francisella immune response at the molecular level. We set out to combine recent advances of genomics with solid-phase antigen delivery coupled with a T-cell functional assay to identify T-cell epitopes. A subset of clones, encoding serological targets, was selected from an F. tularensis SchuS4 ordered genomic library and subcloned into a bacterial expression vector to test the feasibility of this approach. Proteins were expressed and purified individually employing the BioRobot 3000 in a semi-automated purification method. The purified proteins were coupled to beads, delivered to antigen-presenting cells for processing, and screened with Francisella-specific T-cell hybridomas of unknown specificity. We identified cellular reactivity against the pathogenicity protein IglB, and the chaperone proteins GroEL and DnaK. Further analyses using genetic deletions and synthetic peptides were performed to identify the minimal peptide epitopes. Priming with the peptide epitopes before infection with F. tularensis LVS increased the frequency of antigen-specific CD4 T cells as assessed by intracellular interferon-γ staining. These results illustrate the feasibility of screening an arrayed protein library that should be applicable to a variety of pathogens.