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.

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.

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, β2-microglobulin (β2m), governs complex stability. We reasoned that genetically converting β2m 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 β2m (hβ2m) in mouse RMA-S cells elevates MHC-I thermal stability. RMA-S transfectants bind an exogenous peptide at concentrations 104- to 106-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 ∼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 hβ2m only results in marginal effect on the binding profile. Soluble β2m has no effect on the accelerated kinetics, but the kinetics of transfectants parallel that of parental cells in the presence of Abs to hβ2m. Ab inhibition and coimmunoprecipitation analyses suggest that both prolonged persistence of peptide-receptive H chain/β2m 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 β2m as a novel scaffold for CTL induction.

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.