Polyglutamine-Related Aggregates Can Serve as a Potent Antigen Source for Cross-Presentation by Dendritic Cells.

Protective MHC class I-dependent immune responses require an overlap between repertoires of proteins directly presented on target cells and cross-presented by professional APC, specifically dendritic cells. How stable proteins that rely on defective ribosomal proteins for direct presentation are captured for cell-to-cell transfer remains enigmatic. In this study, we address this issue using a combination of in vitro (C57BL/6-derived mouse cell lines) and in vivo (C57BL/6 mouse strains) approaches involving stable and unstable versions of OVA model Ags displaying defective ribosomal protein-dependent and -independent Ag presentation, respectively. Apoptosis, but not necrosis, of donor cells was found associated with robust global protein aggregate formation and captured stable proteins permissive for cross-presentation. Potency of aggregates to serve as Ag source was directly demonstrated using polyglutamine-equipped model substrates. Collectively, our data implicate global protein aggregation in apoptotic cells as a mechanism that ensures the overlap between MHC class I epitopes presented directly or cross-presented by APC and demonstrate the unusual ability of dendritic cells to process stable protein aggregates.

SQSTM1/p62-mediated autophagy compensates for loss of proteasome polyubiquitin recruiting capacity.

Protein homeostasis in eukaryotic cells is regulated by 2 highly conserved degradative pathways, the ubiquitin-proteasome system (UPS) and macroautophagy/autophagy. Recent studies revealed a coordinated and complementary crosstalk between these systems that becomes critical under proteostatic stress. Under physiological conditions, however, the molecular crosstalk between these 2 pathways is still far from clear. Here we describe a cellular model of proteasomal substrate accumulation due to the combined knockdown of PSMD4/S5a and ADRM1, the 2 proteasomal ubiquitin receptors. This model reveals a compensatory autophagic pathway, mediated by a SQSTM1/p62-dependent clearance of accumulated polyubiquitinated proteins. In addition to mediating the sequestration of ubiquitinated cargos into phagophores, the precursors to autophagosomes, SQSTM1 is also important for polyubiquitinated aggregate formation upon proteasomal inhibition. Finally, we demonstrate that the concomitant stabilization of steady-state levels of ATF4, a rapidly degraded transcription factor, mediates SQSTM1 upregulation. These findings provide new insight into the molecular mechanisms by which selective autophagy is regulated in response to proteasomal overflow.

Inherent asymmetry in the 26S proteasome is defined by the ubiquitin receptor RPN13.

The 26S double-capped proteasome is assembled in a hierarchic event that is orchestrated by dedicated set of chaperons. To date, all stoichiometric subunits are considered to be present in equal ratios, thus providing symmetry to the double-capped complex. Here, we show that although the vast majority (if not all) of the double-capped 26S proteasomes, both 19S complexes, contain the ubiquitin receptor Rpn10/S5a, only one of these 19S particles contains the additional ubiquitin receptor Rpn13, thereby defining asymmetry in the 26S proteasome. These results were validated in yeast and mammals, utilizing biochemical and unbiased AQUA-MS methodologies. Thus, the double-capped 26S proteasomes are asymmetric in their polyubiquitin binding capacity. Our data point to a potential new role for ubiquitin receptors as directionality factors that may participate in the prevention of simultaneous substrates translocation into the 20S from both 19S caps.

The direction of protein entry into the proteasome determines the variety of products and depends on the force needed to unfold its two termini.

Poorly structured domains in proteins enhance their susceptibility to proteasomal degradation. To learn whether the presence of such a domain near either end of a protein determines its direction of entry into the proteasome, directional translocation was enforced on several proteasome substrates. Using archaeal PAN-20S complexes, mammalian 26S proteasomes, and cultured cells, we identified proteins that are degraded exclusively from either the C or N terminus and some showing no directional preference. This property results from interactions of the substrate's termini with the regulatory ATPase and could be predicted based on the calculated relative stabilities of the N and C termini. Surprisingly, the direction of entry into the proteasome affected markedly the spectrum of peptides released and consequently influenced the efficiency of MHC class I presentation. Thus, easily unfolded termini are translocated first, and the direction of translocation influences the peptides generated and presented to the immune system.

Induction of antitumor immunity by CTL epitopes genetically linked to membrane-anchored beta2-microglobulin.

Level and persistence of antigenic peptides presented by APCs on MHC class I (MHC-I) molecules influence the magnitude and quality of the ensuing CTL response. We recently demonstrated the unique immunological properties conferred on APCs by expressing beta2-microglobulin (beta2m) as an integral membrane protein. In this study, we explored membrane-anchored beta2m as a platform for cancer vaccines using as a model MO5, an OVA-expressing mouse B16 melanoma. We expressed in mouse RMA-S cells two H-2Kb binding peptides from MO5, OVA257-264, and TRP-2181-188, each genetically fused with the N terminus of membranal beta2m via a short linker. Specific Ab staining and T cell hybridoma activation confirmed that OVA257-264 was properly situated in the MHC-I binding groove. In vivo, transfectants expressing both peptides elicited stronger CTLs and conferred better protection against MO5 than peptide-saturated RMA-S cells. Cells expressing OVA257-264/beta2m were significantly superior to OVA257-264-charged cells in their ability to inhibit the growth of pre-established MO5 tumors. Our results highlight the immunotherapeutic potential of membranal beta2m as a universal scaffold for optimizing Ag presentation by MHC-I molecules.

Membrane-anchored beta 2-microglobulin stabilizes a highly receptive state of MHC class I molecules.

The magnitude of response elicited by CTL-inducing vaccines correlates with the density of MHC class I (MHC-I)-peptide complexes formed on the APC membrane. The MHC-I L chain, beta2-microglobulin (beta2m), governs complex stability. We reasoned that genetically converting beta2m into an integral membrane protein should exert a marked stabilizing effect on the resulting MHC-I molecules and enhance vaccine efficacy. In the present study, we show that expression of membranal human beta2m (hbeta2m) in mouse RMA-S cells elevates MHC-I thermal stability. RMA-S transfectants bind an exogenous peptide at concentrations 10(4)- to 10(6)-fold lower than parental RMA-S, as detected by complex-specific Abs and by T cell activation. Moreover, saturation of the transfectants' MHC-I by exogenous peptide occurs within 1 min, as compared with approximately 1 h required for parental cells. At saturation, however, level of peptide bound by modified cells is only 3- to 5-fold higher. Expression of native hbeta2m only results in marginal effect on the binding profile. Soluble beta2m has no effect on the accelerated kinetics, but the kinetics of transfectants parallel that of parental cells in the presence of Abs to hbeta2m. Ab inhibition and coimmunoprecipitation analyses suggest that both prolonged persistence of peptide-receptive H chain/beta2m heterodimers and fast heterodimer formation via lateral diffusion may contribute to stabilization. In vivo, peptide-loaded transfectants are considerably superior to parental cells in suppressing tumor growth. Our findings support the role of an allosteric mechanism in determining ternary MHC-I complex stability and propose membranal beta2m as a novel scaffold for CTL induction.

Chimeric beta2 microglobulin/CD3zeta polypeptides expressed in T cells convert MHC class I peptide ligands into T cell activation receptors: a potential tool for specific targeting of pathogenic CD8(+) T cells.

CD8(+) T cells are key mediators of transplant rejection and graft-versus-host disease and contribute to the pathogenesis of autoimmune diseases. We tested whether TCR ligands can be converted into T cell activation receptors, redirecting genetically modified T cells at pathogenic CD8(+) T cells. For this purpose we exploited the ability of the non-polymorphic beta(2) microglobulin light chain to pair with all MHC class I heavy chains. In this report we describe the design and expression in a T cell hybridoma of two modalities of beta(2) microglobulin polypeptides, fused with the transmembrane and intracellular portion of CD3zeta chain. In the absence of a particular antigenic peptide, the chimeric product associates with different endogenous MHC class I heavy chains and triggers T cell activation upon heavy chain cross-linking. When an antigenic peptide is covalently attached to the N-terminus of the chimeric polypeptide, transfectants express high level of surface peptide-class I complexes and respond to antibodies and target T cells in a peptide-specific manner. Our results provide the basis for a universal genetic approach aimed at antigen-specific immunotargeting of pathogenic CD8(+) T cells.