Nanoscale organization of the MHC I peptide-loading complex in human dendritic cells

Koller, Nicole; Höllthaler, Philipp; Barends, Martina; Döring, Marius; Spahn, Christoph; Durán, Verónica; Costa, Bibiana; Becker, J. Ole; Heilemann, Mike; Kalinke, Ulrich; Tampé, Robert

doi:10.1007/s00018-022-04472-2

Cited by 4 publications

(3 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…presented below, this approach may allow to study PLC assembly, peptide loading, and decay in a realistic membrane environment with free diffusion. In the future, this approach will provide opportunities to analyze clustering of PLCs [103,104] , which can be structurally resolved by cryo-ET. The active NBDs could further allow trapping in different conformations, which may further unravel the mechanistic details of the peptide loading complex.…”

Section: Plc Reconstitution In Membrane Mimeticsmentioning

confidence: 99%

“…This is possible since antibodies can be labeled with other molecules, such as fluorophores or affinity tags like biotin. [201] Ultimately, even when labeled with small molecule fluorophores, antibodies remain comparatively large molecules that, on the one hand, exhibit low spatial localization accuracy of the target molecule in high-resolution microscopy [104] and, on the other hand, do not penetrate well into tissue. Also, due to steric constraints, some epitopes may not be accessible.…”

Section: Nanobodies To Modulate and Track Cellular Processesmentioning

confidence: 99%

“…Eventually, this light-guided approach may allow highresolution structural analyses and visualization of PLCs clustering in proteoliposomes by cryo-ET measurements, which would otherwise potentially be suppressed by ICP47. [103,104]…”

Section: Semisynthetic Viral Inhibitor For Light Control Of the Mhc I...mentioning

confidence: 99%

See 2 more Smart Citations

Mechanistic and structural insights into the quality control of the MHC I antigen processing pathway

Winter¹

View full text Add to dashboard Cite

The human body is permanently exposed to its environment and thus to viruses and other pathogens, which require a flexible response and defense. Alongside to the innate immune system, the adaptive immune system provides highly specialized protection against these threats. The major histocompatibility complex class I (MHC I) antigen presentation system is a cornerstone of the adaptive immune system and a major constituent of cellular immunity. Pathogens such as viruses that invade a cell will leave traces in the form of proteins and peptides which are degraded and loaded onto MHC I molecules. MHC I peptide loading is performed by peptide loading complex (PLC) in the membrane of the endoplasmic reticulum as part of a multifaceted and comprehensive quality control machinery. Monitored by multiple layers of quality assurance, the MHC I molecules consequently display the immune status of the cell on its surface. In this context, the captured fragment of the virus serves as a call for help issued by the cell, alerting the adaptive immune system to the infection to mount an appropriate immune response. The three-dimensional structure as well as the mechanistic details of parts of this complex machinery were characterized in the context of this dissertation. Among other tools, light-modulable nanotools were developed in this thesis, which permit external regulation of cellular processes in temporal and spatial resolution. Furthermore, methods and model systems for the biochemical characterization of cellular signaling cascades, proteins, as well as entire cell organelles were developed, which are likely to influence the field of cellular immunity and protein biochemistry in the future. This cumulative work comprises a total of six publications whose scientific key advances will be briefly outlined in this abstract. In the introduction, the scientific background as well as the current state of research and methodological background knowledge are conveyed. The results section condenses the main aspects of the publications and links them to each other. Further details can be retrieved from the attached original publications. In “Semisynthetic viral inhibitor for light control of the MHC I peptide loading complex, Winter, Domnick et al., Angew Chem Int Ed 2022” a photocleavable viral inhibitor of the peptide loading complex was produced by semi-synthesis. This nanotool was shown to be suitable for both purifying the PLC from human Raji cells as well as reactivating it in a light-controlled manner. Thus, this tool establishes the isolation of a fully intact and functional peptide loading complex for biochemical characterization. In addition, a novel flow cytometric analysis pipeline for microsomes was developed, allowing cellular vesicles to be characterized with single organelle resolution, similar to cells. In “Molecular basis of MHC I quality control in the peptide loading complex, Domnick, Winter et al., Nat Commun 2022” the peptide loading complex was reconstituted into large nanodiscs, and a cryo-EM structural model of the editing module at 3.7 Å resolution was generated. By combining the structural model with in vitro glycan editing assays, an allosteric coupling between peptide-MHC I assembly and glycan processing was revealed, extending the known model of MHC I loading and dissociation from the PLC. These mechanisms provide a prototypical example for endoplasmic reticulum quality control. In a related context, in “Structure of an MHC I–tapasin–ERp57 editing complex defines chaperone promiscuity, Müller, Winter et al., Nat Commun 2022” a recombinantly assembled editing module comprised of MHC I-tapasin-ERp57 was crystallized for X-ray structural biology. The resulting crystal structure at a resolution of 2.7 Å permitted the precise identification of characteristic features of the editing module and particularly of the peptide proofreading mechanism of tapasin. This study provided pivotal insights into the tapasin-mediated peptide editing of different MHC I allomorphs as well as similarities to TAPBPR-based MHC I peptide proofreading. In “TAPBPR is necessary and sufficient for UGGT1-mediated quality control of MHC I, Sagert, Winter et al. (in preparation)” novel insights concerning the peptide proofreader TAPBPR and its close interplay with the folding sensor and glucosyltransferase UGGT1 were obtained. It was shown that TAPBPR is an integral part of the second level of endoplasmic quality control and is indispensable for effective MHC I coordination by UGGT1. In “Light-guided intrabodies for on-demand in situ target recognition in human cells, Joest, Winter et al., Chem Sci 2021” intracellular nanobodies were equipped with a photocaged target recognition domain by genetic code expansion via amber suppression. These intrabodies, acting as high-affinity binding partners endowed with a fluorophore, could be used in a light-triggered approach to instantaneously visualize their target molecule...

show abstract

Section: Plc Reconstitution In Membrane Mimeticsmentioning

confidence: 99%

Section: Nanobodies To Modulate and Track Cellular Processesmentioning

confidence: 99%

See 1 more Smart Citation

Mechanistic and structural insights into the quality control of the MHC I antigen processing pathway

Winter¹

View full text Add to dashboard Cite

show abstract

The N-terminal Proline Hinge Motif Controls the Structure of Bovine Herpesvirus 1-encoded Inhibitor of the Transporter Associated with Antigen Processing Required for its Immunomodulatory Function

Graul

Karska

Wąchalska

et al. 2023

Journal of Molecular Biology

View full text Add to dashboard Cite

Dynamic interactome of the MHC I peptide loading complex in human dendritic cells

Barends

Koller

Schölz

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Dendritic cells (DCs) orchestrate immune responses by presenting antigenic peptides on major histocompatibility complex (MHC) molecules to T cells. Antigen processing and presentation via MHC I rely on the peptide-loading complex (PLC), a supramolecular machinery assembled around the transporter associated with antigen processing (TAP), which is the peptide transporter in the endoplasmic reticulum (ER) membrane. We studied antigen presentation in human DCs by isolating monocytes from blood and differentiating them into immature and mature DCs. We uncovered that during DC differentiation and maturation, additional proteins are recruited to the PLC, including B-cell receptor-associated protein 31 (BAP31), vesicle-associated membrane protein-associated protein A (VAPA), and extended synaptotagmin-1 (ESYT1). We demonstrated that these ER cargo export and contact site–tethering proteins colocalize with TAP and are within 40 nm proximity of the PLC, suggesting that the antigen processing machinery is located near ER exit- and membrane contact sites. While CRISPR/Cas9-mediated deletion of TAP and tapasin significantly reduced MHC I surface expression, single-gene deletions of the identified PLC interaction partners revealed a redundant role of BAP31, VAPA, and ESYT1 in MHC I antigen processing in DCs. These data highlight the dynamics and plasticity of PLC composition in DCs that previously was not recognized by the analysis of cell lines.

show abstract

Nanoscale organization of the MHC I peptide-loading complex in human dendritic cells

Cited by 4 publications

References 52 publications

Mechanistic and structural insights into the quality control of the MHC I antigen processing pathway

Mechanistic and structural insights into the quality control of the MHC I antigen processing pathway

The N-terminal Proline Hinge Motif Controls the Structure of Bovine Herpesvirus 1-encoded Inhibitor of the Transporter Associated with Antigen Processing Required for its Immunomodulatory Function

Dynamic interactome of the MHC I peptide loading complex in human dendritic cells

Contact Info

Product

Resources

About