Biology subjects

Trowitzsch, S.

Publications and source records attributed to Trowitzsch, S..

4 recordsLinked to original sources

bioRxiv2025

Reconstitution of glycan-driven MHC I recycling reveals calreticulin as mediator between TAPBPR and tapasin

Protein folding in the endoplasmic reticulum (ER) is essential for about one-third of the mammalian proteome. N-linked glycosylation and subsequent glycan remodeling barcodes glycoproteins during their maturation in the ER. Major histocompatibility complex class I (MHC I) molecules, key for adaptive immunity, rely on a dedicated quality control cycle that involves specialized chaperones and glycan-modifying enzymes for their maturation and loading of immunogenic peptides. However, the functional interplay of the MHC I editors tapasin as part of the peptide-loading complex (PLC), TAP-binding protein-related (TAPBPR), the UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1), and calreticulin in glycan-dependent transfer of MHC I clients has not been determined in a reconstituted system. With isolated components, we show that transfer of peptide-receptive MHC I from the downstream quality control factor TAPBPR back to tapasin depends on the recognition of the monoglucosylated glycan of MHC I by calreticulin. While calreticulins C-terminal acidic helix is dispensable for disengaging reglucosylated MHC I from TAPBPR, it is essential for docking MHC I onto tapasin. Our data provide a mechanistic basis for glycan-surveillance by calreticulin necessary for retrograde trafficking of misfolded or suboptimally loaded MHC I that escaped the first quality control at the PLC and were trapped by TAPBPR. Such finetuned dynamic network of glycan-dependent and MHC I-specific chaperones guarantees maturation of MHC I molecules and highlight the fundamental processes driving ER protein quality control. TeaserOur study dissects the mechanistic network of dedicated chaperones and glycan modifiers that quality-control MHC I. Significance StatementThe immune system relies on major histocompatibility complex (MHC) class I molecules to present protein fragments from within cells, enabling detection of infection or disease. This study uncovers how a dynamic, glycan-dependent chaperone network in the endoplasmic reticulum (ER) orchestrates the recycling of misfolded or suboptimally loaded MHC I. Using isolated components, the work shows how monoglucosylation of MHC I glycans by UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) allows calreticulin to mediate transfer from the post-ER chaperone TAPBPR back to the ER-resident chaperone tapasin as part of the peptide-loading complex. These findings illuminate the coordinated action of TAPBPR, UGGT1, calreticulin, and tapasin-ERp57 in MHC I quality control, offering new insights in immune surveillance and how its disruption may contribute to disease.

immunology↗

bioRxiv2025

US6 hijacks the peptide-loading complex by trapping transporter-chaperone dynamics

Adaptive immune responses are initiated by major histocompatibility class I (MHC I) presentation of antigenic peptides on the cell surface. This process relies on the peptide-loading complex (PLC), a dynamic transporter-multichaperone assembly in the endoplasmic reticulum (ER), to ensure high-fidelity selection, editing, and loading of peptides onto MHC I heterodimers1. The PLC is the primary target for viral immune evasion2, elicited in particular by human cytomegalovirus (HCMV)3, causing lifelong infections with severe risks for immunocompromised individuals. While the overall architecture of the PLC is known4, how its activity is jeopardized by viral immune evasins remains unclear. Here, we present the 2.59-2.88 [A] cryogenic electron microscopy structure of native human PLC associated with the HCMV immune evasin US6. US6 inhibits the heterodimeric transporter associated with antigen processing (TAP1/2) by latching its transmembrane helix laterally onto TAP2 and using its central disulfide-rich domain to mimic a translocating peptide. This effectively plugs the ER-lumenal exit and locks TAP in an outward-facing open conformation with closed nucleotide-binding domains and asymmetrically occluded ATP and ADP. The structure also highlights the role of the unique N-terminal transmembrane domains of TAP as dynamic scaffolds that recruit the MHC I-specific chaperone tapasin by clamping its transmembrane helix to the core transmembrane domain of each transporter subunit. Our findings uncover the molecular mechanism of US6-mediated viral immune evasion and reveal potential targets for therapeutic modulation of antigen presentation in cancer and infectious diseases.

immunology↗

bioRxiv2022

Structural basis of substrate recognition by human tRNA splicing endonuclease TSEN

The heterotetrameric human transfer RNA (tRNA) splicing endonuclease (TSEN) catalyzes the excision of intronic sequences from precursor tRNAs (pre-tRNAs)1. Mutations in TSEN and its associated RNA kinase CLP1 are linked to the neurodegenerative disease pontocerebellar hypoplasia (PCH)2-8. The three-dimensional (3D) assembly of TSEN/CLP1, the mechanism of substrate recognition, and the molecular details of PCH-associated mutations are not fully understood. Here, we present cryo-electron microscopy structures of human TSEN with intron-containing pre-tRNATyrO_SCPLOWGTAC_SCPLOW and pre-tRNAArgO_SCPLOWTCTC_SCPLOW. TSEN exhibits broad structural homology to archaeal endonucleases9 but has evolved additional regulatory elements that are involved in handling and positioning substrate RNA. Essential catalytic residues of subunit TSEN34 are organized for the 3 splice site which emerges from a bulge-helix configuration. The triple-nucleotide bulge at the intron/3-exon boundary is stabilized by an arginine tweezer motif of TSEN2 and an interaction with the proximal minor groove of the helix. TSEN34 and TSEN54 define the 3 splice site by holding the tRNA body in place. TSEN54 adapts a bipartite fold with a flexible central region required for CLP1 binding. PCH-associated mutations are located far from pre-tRNA binding interfaces explaining their negative impact on structural integrity of TSEN without abrogating its catalytic activity in vitro10. Our work defines the molecular framework of pre-tRNA recognition and cleavage by TSEN and provides a structural basis to better understand PCH in the future.

biochemistry↗

bioRxiv2020

Assembly defects of the human tRNA splicing endonuclease contribute to impaired pre-tRNA processing in pontocerebellar hypoplasia

Introns of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. Here, we show that intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers independently of CLP1. Splice site recognition involves the mature domain and the anticodon-intron base pair of pre-tRNAs. The 2.1-[A] resolution X-ray crystal structure of a TSEN15-34 heterodimer and differential scanning fluorimetry analyses show that PCH mutations cause thermal destabilization. While endonuclease activity in recombinant mutant TSEN is unaltered, we observe assembly defects and reduced pre-tRNA cleavage activity resulting in an imbalanced pre-tRNA pool in PCH patient-derived fibroblasts. Our work defines the molecular principles of intron excision in humans and provides evidence that modulation of TSEN stability may contribute to PCH phenotypes.

biochemistry↗