Light control of the peptide-loading complex synchronizes antigen translocation and MHC I trafficking

Abstract

Antigen presentation via major histocompatibility complex class I (MHC I) molecules is essential to mount an adaptive immune response against pathogens and cancerous cells. To this end, the transporter associated with antigen processing (TAP) delivers snippets of the cellular proteome, resulting from proteasomal degradation, into the ER lumen. After peptide loading and editing by the peptide-loading complex (PLC), stable peptide-MHC I complexes are released for cell surface presentation. Since the process of MHC I trafficking is poorly defined, we established an approach to control antigen presentation by introduction of a photo-caged amino acid in the catalytic ATP-binding site of TAP. By optical control, we initiate TAP-dependent antigen translocation, thus providing new insights into TAP function within the PLC and MHC I trafficking in living cells. Moreover, this versatile approach has the potential to be applied in the study of other cellular pathways controlled by P-loop ATP/GTPases.

Efficient Amber Suppression via Ribosomal Skipping for In Situ Synthesis of Photoconditional Nanobodies

“…Recent advances in ribosome profiling and other technologies demonstrated reduced fidelity in early phases of translation. − We rationalized that the early amber codon at position Tyr37 is prone to such inaccuracy. In a recent study, we observed slightly increased amber suppression efficiency by using a bicistronic translation approach . In order to circumvent the Nb mCherry background expression, we developed a new approach inspired by the translation strategy of the foot-and-mouth disease virus (Figure a, construct V).…”

“…To analyze PA Nb mCherry binding, we co-expressed the cognate target protein (EGFP), which can be fused to many reference proteins via a multiple cloning site. As an initial reference, we used the ER-resident transporter associated with antigen processing (TAP1). , Second, we connected both gene constructs via the foot-and-mouth disease virus ribosomal skipping site (F2A), which allows bicistronic translation of two proteins (target and nanobody) in a single ribosome passage (Figure a). , Polycistronic mRNAs are a valuable strategy for the expression of certain genes in mammalian cells that is gaining increasing recognition . Parallel protein synthesis was monitored using flow cytometry to detect the two fluorescent reporters (Figure S4).…”

“…TAP1 or H2B was cloned into the multiple cloning site via endonuclease restriction. The TAP1 gene was amplified by PCR originating from a previously generated plasmid encoding TAP1 myc‑SBP | 2A |TAP2 mVenus‑His10 . A primer pair binding an upstream NheI restriction site and inserting a downstream BamHI restriction site was applied.…”

“…Genetic code expansion connects the versatility of chemical synthesis to protein expression in living systems. − Protein modifications can be inserted site-specifically via natural translation machineries . By reprogramming the genetic code, non-canonical amino acids bearing a modification of choice are used in ribosomal polypeptide synthesis. − Non-canonical amino acids have many applications in protein engineering, as they can be equipped with isotopes for structural studies, − photoreactive groups and post-translational modifications for functional studies, − reactive groups for bio-orthogonal coupling, ,,− photocross-linkers, ,,− infrared-active probes to follow conformational dynamics, ,− fluorescent dyes for imaging, − biotin analogues for high-affinity interactions with streptavidin, and stable phosphotyrosine analogues for analysis of signal transduction. , Site-specific incorporation is achieved by suppressing a stop codon with an additional aminoacyl-tRNA-synthetase (aaRS)/tRNA pair. ,,, In bacterial or mammalian cells, the rarest amber codon (TAG) is most often used to minimize suppression throughout the proteome. ,,,, However, the underlying processes of the genetic code expansion are complex and hardly understood. , For efficient protein synthesis, an i...…”

Spatiotemporal Activation of Protein O-GlcNAcylation in Living Cells

“…This photoactivation strategy has been widely adopted in probing protein functions across different protein families. 4 Chemical modification of serine and/or threonine residues with O-GlcNAc commonly occurs on intracellular proteins. 5 Accumulating evidence has demonstrated that O-GlcNAc plays a pivotal role in controlling protein functions, ranging from subcellular localization, to protein stability, to protein−protein interactions.…”