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© 2015 Macmillan Publishers Limited. All rights reserved.A current challenge in life sciences is to image cell membrane receptors while characterizing their specific interactions with various ligands. Addressing this issue has been hampered by the lack of

Abstract Background In contrast to mammals, amphibians, such as adult urodeles (for example, newts) and anuran larvae (for example, Xenopus) can regenerate their spinal cord after injury. However, the cellular and molecular mechanisms involved in this process are still poorly understood. Results Here, we report that tail amputation results in a global increase of Sox2 levels and proliferation of Sox2+ cells. Overexpression of a dominant negative form of Sox2 diminished proliferation of spinal cord resident cells affecting tail regeneration after amputation, suggesting that spinal cord regeneration is crucial for the whole process. After spinal cord transection, Sox2+ cells are found in the ablation gap forming aggregates. Furthermore, Sox2 levels correlated with regenerative capabilities during metamorphosis, observing a decrease in Sox2 levels at non-regenerative stages. Conclusions Sox2+ cells contribute to the regeneration of spinal cord after tail amputation and transection. Sox2 levels decreases during metamorphosis concomitantly with the lost of regenerative capabilities. Our results lead to a working hypothesis in which spinal cord damage activates proliferation and/or migration of Sox2+ cells, thus allowing regeneration of the spinal cord after tail amputation or reconstitution of the ependymal epithelium after spinal cord transection.

To eliminate infected and cancerous cells, antigen processing and presentation play a pivotal role through the recognition of antigenic peptides displayed on Major Histocompatibility Complex class I (MHC I) molecules. Here, we developed a photostimulated antigen release system that enables the temporal inception of antigen flux. Simple and effective photocaging of the human immunodeficiency virus (HIV)-Nef73-derived epitope, a representative high-affinity MHC I ligand, was provided by steric hindrance to block the recognition by the transporter associated with antigen processing (TAP) in the peptide loading complex (PLC). In response to light, a heteronomous release of antigens and subsequent translocation in various scenarios is demonstrated, including a TAP-related ATP-binding cassette (ABC) transporter reconstituted in liposomes and the native PLC in the endoplasmic reticulum (ER) membrane of human cells. The photochemically induced 'burst' of antigens opens new opportunities for a mechanistic analysis of the antigen translocation machinery and will help to provide insights into antigen processing pathways via an on-demand, subcellular pulse-chase release of antigens., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

ABC transporters are found in all organisms and almost every cellular compartment. They mediate the transport of various solutes across membranes, energized by ATP binding and hydrolysis. Dysfunctions can result in severe diseases, such as cystic fibrosis or antibiotic resistance. In type IV ABC transporters, each of the two nucleotide-binding domains is connected to a transmembrane domain by two coupling helices, which are part of cytosolic loops. Although there are many structural snapshots of different conformations, the interdomain communication is still enigmatic. Therefore, we analyzed the function of three conserved charged residues in the intracytosolic loop 1 of the human homodimeric, lysosomal peptide transporter TAPL (transporter associated with antigen processing-like). Substitution of D278 in coupling helix 1 by alanine interrupted peptide transport by impeding ATP hydrolysis. Alanine substitution of R288 and D292, both localized next to the coupling helix 1 extending to transmembrane helix 3, reduced peptide transport but increased basal ATPase activity. Surprisingly, the ATPase activity of the R288A variant dropped in a peptide-dependent manner, whereas ATPase activity of wildtype and D292A was unaffected. Interestingly, R288A and D292A mutants did not differentiate between ATP and GTP in respect of hydrolysis. However, in contrast to wildtye TAPL, only ATP energized peptide transport. In sum, D278 seems to be involved in bidirectional interdomain communication mediated by network of polar interactions, whereas the two residues in the cytosolic extension of transmembrane helix 3 are involved in regulation of ATP hydrolysis, most likely by stabilization of the outward-facing conformation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

T cell receptor (TCR) clustering and formation of an immune synapse are crucial for TCR signaling. However, limited information is available about these dynamic assemblies and their connection to transmembrane signaling. In this work, TCR clustering is controlled via plug-and-play nanotools based on an engineered irreversible conjugation pair and a peptide-loaded major histocompatibility complex (pMHC) molecule to compare receptor assembly in a ligand (pMHC)-induced or ligand-independent manner. A streptavidin-binding peptide displayed in both tools enabled their anchoring in streptavidin-pre-structured matrices. Strikingly, pMHC-induced clustering in the confined regions exhibit higher density and dynamics than the ligand-free approach, indicating that the size and architecture of the pMHC ligand influences TCR assembly. This approach enables the control of membrane receptor clustering with high specificity and provides the possibility to explore different modalities of receptor activation., (© 2024 The Author(s). Advanced Materials published by Wiley‐VCH GmbH.)

Antigen presentation via major histocompatibility complex class I (MHC-I) molecules is essential for surveillance by the adaptive immune system. Central to this process is the peptide-loading complex (PLC), which translocates peptides from the cytosol to the endoplasmic reticulum and catalyzes peptide loading and proofreading of peptide-MHC-I (pMHC-I) complexes. Despite its importance, the impact of individual PLC components on the presented pMHC-I complexes is still insufficiently understood. Here, we used stoichiometrically defined antibody-nanobody complexes and engineered soluble T cell receptors (sTCRs) to quantify different MHC-I allomorphs and defined pMHC-I complexes, respectively. Thereby, we uncovered distinct effects of individual PLC components on the pMHC-I surface pool. Knockouts of components of the PLC editing modules, namely tapasin, ERp57, or calreticulin, changed the MHC-I surface composition to a reduced proportion of HLA-A*02:01 presentation compensated by a higher ratio of HLA-B*40:01 molecules. Intriguingly, these knockouts not only increased the presentation of suboptimally loaded HLA-A*02:01 complexes but also elevated the presentation of high-affinity peptides overexpressed in the cytosol. Our findings suggest that the components of the PLC editing module serve a dual role, acting not only as peptide proofreaders but also as limiters for abundant peptides. This dual function ensures the presentation of a broad spectrum of antigenic peptides., Competing Interests: Competing interests statement:B.W. and D.K.C. indicate a potential conflict of interest as former and present employee of Immunocore Ltd, respectively. The remaining authors declare no competing financial interests.

Eukaryotic tRNA guanine transglycosylase (TGT) is an RNA-modifying enzyme which catalyzes the base exchange of the genetically encoded guanine 34 of tRNAs Asp,Asn,His,Tyr for queuine, a hypermodified 7-deazaguanine derivative. Eukaryotic TGT is a heterodimer comprised of a catalytic and a non-catalytic subunit. While binding of the tRNA anticodon loop to the active site is structurally well understood, the contribution of the non-catalytic subunit to tRNA binding remained enigmatic, as no complex structure with a complete tRNA was available. Here, we report a cryo-EM structure of eukaryotic TGT in complex with a complete tRNA, revealing the crucial role of the non-catalytic subunit in tRNA binding. We decipher the functional significance of these additional tRNA-binding sites, analyze solution state conformation, flexibility, and disorder of apo TGT, and examine conformational transitions upon tRNA binding., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Engineered antibodies are essential tools for research and advanced pharmacy. In the development of therapeutics, antibodies are excellent candidates as they offer both target recognition and modulation. Thanks to the latest advances in biotechnology, light-activated antibody fragments can be constructed to control spontaneous antigen interaction with high spatiotemporal precision. To implement conditional antigen binding, several optogenetic and optochemical engineering concepts have recently been developed. Here, we highlight the various strategies and discuss the features of opto-conditional antibodies. Each concept offers intrinsic advantages beneficial to different applications. In summary, the novel design approaches constitute a complementary toolset to promote current and upcoming antibody technologies with ultimate precision., Competing Interests: Declaration of interests The authors declare no competing financial interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

ATP-binding cassette (ABC) transporters shuttle diverse substrates across biological membranes. Transport is often achieved through a transition between an inward-facing (IF) and an outward-facing (OF) conformation of the transmembrane domains (TMDs). Asymmetric nucleotide-binding sites (NBSs) are present among several ABC subfamilies and their functional role remains elusive. Here we addressed this question using concomitant NO-NO, Mn 2+ -NO, and Mn 2+ -Mn 2+ pulsed electron-electron double-resonance spectroscopy of TmrAB in a time-resolved manner. This type-IV ABC transporter undergoes a reversible transition in the presence of ATP with a significantly faster forward transition. The impaired degenerate NBS stably binds Mn 2+ -ATP, and Mn 2+ is preferentially released at the active consensus NBS. ATP hydrolysis at the consensus NBS considerably accelerates the reverse transition. Both NBSs fully open during each conformational cycle and the degenerate NBS may regulate the kinetics of this process., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Adaptive immune responses are triggered by antigenic peptides presented on major histocompatibility complex class I (MHC I) at the surface of pathogen-infected or cancerous cells. Formation of stable peptide-MHC I complexes is facilitated by tapasin and TAPBPR, two related MHC I-specific chaperones that catalyze selective loading of suitable peptides onto MHC I in a process called peptide editing or proofreading. On their journey to the cell surface, MHC I complexes must pass a quality control step performed by UGGT1, which senses the folding status of the transiting N-linked glycoproteins in the endoplasmic reticulum (ER). UGGT1 reglucosylates non-native glycoproteins and thereby allows them to revisit the ER folding machinery. Here, we describe a reconstituted in-vitro system of purified human proteins that enabled us to delineate the function of TAPBPR during the UGGT1-catalyzed quality control and reglucosylation of MHC I. By combining glycoengineering with liquid chromatography-mass spectrometry, we show that TAPBPR promotes reglucosylation of peptide-free MHC I by UGGT1. Thus, UGGT1 cooperates with TAPBPR in fulfilling a crucial function in the quality control mechanisms of antigen processing and presentation., Competing Interests: LS, CW, IR, MZ, CT, RT No competing interests declared, (© 2023, Sagert et al.)

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.