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The adaptive immune system is able to detect and destroy cells that are malignantly transformed or infected by intracellular pathogens. Specific immune responses against these cells are elicited by antigenic peptides that are presented on major histocompatibility complex class I (MHC I) molecules and recognized by cytotoxic T lymphocytes at the cell surface. Since these MHC I-presented peptides are generated in the cytosol by proteasomal protein degradation, they can be metaphorically described as a window providing immune cells with insights into the state of the cellular proteome. A crucial element of MHC I antigen presentation is the peptide-loading complex (PLC), a multisubunit machinery, which contains as key constituents the transporter associated with antigen processing (TAP) and the MHC I-specific chaperone tapasin (Tsn). While TAP recognizes and shuttles the cytosolic antigenic peptides into the endoplasmic reticulum (ER), Tsn samples peptides in the ER for their ability to form stable complexes with MHC I, a process called peptide proofreading or peptide editing. Through its selection of peptides that improve MHC I stability, Tsn contributes to the hierarchy of immunodominant peptide epitopes. Despite the fact that it concerns a key event in adaptive immunity, insights into the catalytic mechanism of peptide proofreading carried out by Tsn have only lately been gained via biochemical, biophysical, and structural studies. Furthermore, a Tsn homolog called TAP-binding protein-related (TAPBPR) has only recently been demonstrated to function as a second MHC I-specific chaperone and peptide proofreader. Although TAPBPR is PLC-independent and has a distinct allomorph specificity, it is likely to share a common catalytic mechanism with Tsn. This review focuses on the current knowledge of the multivalent protein–protein interactions and the concomitant dynamic molecular processes underlying peptide-proofreading catalysis. We do not only derive a model that highlights the common mechanistic principles shared by the MHC I editors Tsn and TAPBPR, and the MHC II editor HLA-DM, but also illustrate the distinct quality control strategies employed by these chaperones to sample epitopes. Unraveling the mechanistic underpinnings of catalyzed peptide proofreading will be crucial for a thorough understanding of many aspects of immune recognition, from infection control and tumor immunity to autoimmune diseases and transplant rejection.
The transporter associated with antigen processing (TAP) selectively translocates antigenic peptides into the endoplasmic reticulum. Loading onto major histocompatibility complex class I molecules and proofreading of these bound epitopes are orchestrated within the macromolecular peptide-loading complex, which assembles on TAP. This heterodimeric ABC-binding cassette (ABC) transport complex is therefore a major component in the adaptive immune response against virally or malignantly transformed cells. Its pivotal role predestines TAP as a target for infectious diseases and malignant disorders. The development of therapies or drugs therefore requires a detailed comprehension of structure and function of this ABC transporter, but our knowledge about various aspects is still insufficient. This review highlights recent achievements on the structure and dynamics of antigenic peptides in complex with TAP. Understanding the binding mode of antigenic peptides in the TAP complex will crucially impact rational design of inhibitors, drug development, or vaccination strategies.
Super-resolution fluorescence microscopy revolutionizes cell biology research and provides novel insights on how proteins are organized at the nanoscale and in the cellular context. In order to extract a maximum of information, specialized tools for image analysis are necessary. Here, we introduce the LocAlization Microscopy Analyzer (LAMA), a comprehensive software tool that extracts quantitative information from single-molecule super-resolution imaging data. LAMA allows characterizing cellular structures by their size, shape, intensity, distribution, as well as the degree of colocalization with other structures. LAMA is freely available, platform-independent and designed to provide direct access to individual analysis of super-resolution data.
The full-length translation-regulating add adenine riboswitch (Asw) from Vibrio vulnificus has a more complex conformational space than its isolated aptamer domain. In addition to the predicted apo (apoA) and holo conformation that feature the conserved three-way junctional purine riboswitch aptamer, it adopts a second apo (apoB) conformation with a fundamentally different secondary structure. Here, we characterized the ligand-dependent conformational dynamics of the full-length add Asw by NMR and by single-molecule FRET (smFRET) spectroscopy. Both methods revealed an adenine-induced secondary structure switch from the apoB-form to the apoA-form that involves no tertiary structural interactions between aptamer and expression platform. This strongly suggests that the add Asw triggers translation by capturing the apoA-form secondary structure in the holo state. Intriguingly, NMR indicated a homogenous, docked aptamer kissing loop fold for apoA and holo, while smFRET showed persistent aptamer kissing loop docking dynamics between comparably stable, undocked and docked substates of the apoA and the holo conformation. Unraveling the folding of large junctional riboswitches thus requires the integration of complementary solution structural techniques such as NMR and smFRET.
ATP-binding cassette (ABC) transporters, a superfamily of integral membrane proteins, catalyse the translocation of substrates across the cellular membrane by ATP hydrolysis. Here we demonstrate by nucleotide turnover and binding studies based on 31P solid-state NMR spectroscopy that the ABC exporter and lipid A flippase MsbA can couple ATP hydrolysis to an adenylate kinase activity, where ADP is converted into AMP and ATP. Single-point mutations reveal that both ATPase and adenylate kinase mechanisms are associated with the same conserved motifs of the nucleotide-binding domain. Based on these results, we propose a model for the coupled ATPase-adenylate kinase mechanism, involving the canonical and an additional nucleotide-binding site. We extend these findings to other prokaryotic ABC exporters, namely LmrA and TmrAB, suggesting that the coupled activities are a general feature of ABC exporters.
Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s−1). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals.
The asymmetric unit of the title co-crystalline adduct, 1,3,6,8-tetraazatricyclo[4.4.1.13,8]dodecane (TATD)–4-iodophenol (1/2), C8H16N4·2C6H5IO, comprises a half molecule of the aminal cage polyamine plus a 4-iodophenol molecule. A twofold rotation axis generates the other half of the adduct. The components are linked by two intermolecular O—H⋯N hydrogen bonds. The adducts are further linked into a three-dimensional framework structure by a combination of N⋯I halogen bonds and weak non-conventional C—H⋯O and C—H⋯I hydrogen bonds.
In the title compound, C26H24N2O2, the oxazine moiety is fused to a naphthalene ring system. The asymmetric unit consists of one half of the molecule, which lies about an inversion centre. The C atoms of the ethylene spacer group adopt an antiperiplanar arrangement. The oxazine ring adopts a half-chair conformation. In the crystal, supramolecular chains running along the b axis are formed via short C—H⋯π contacts. The crystal studied was a non-merohedral twin with a fractional contribution of 0.168 (2) of the minor twin component.
The turnover of endoplasmic reticulum (ER) ensures the correct biological activity of its distinct domains. In mammalian cells, the ER is degraded via a selective autophagy pathway (ER-phagy), mediated by two specific receptors: FAM134B, responsible for the turnover of ER sheets and SEC62 that regulates ER recovery following stress. Here, we identified reticulon 3 (RTN3) as a specific receptor for the degradation of ER tubules. Oligomerization of the long isoform of RTN3 is sufficient to trigger fragmentation of ER tubules. The long N-terminal region of RTN3 contains several newly identified LC3-interacting regions (LIR). Binding to LC3s/GABARAPs is essential for the fragmentation of ER tubules and their delivery to lysosomes. RTN3-mediated ER-phagy requires conventional autophagy components, but is independent of FAM134B. None of the other reticulon family members have the ability to induce fragmentation of ER tubules during starvation. Therefore, we assign a unique function to RTN3 during autophagy.
The neuronal transcriptome changes dynamically to adapt to stimuli from the extracellular and intracellular environment. In this study, we adapted for the first time a click chemistry technique to label the newly synthesized RNA in cultured hippocampal neurons and intact larval zebrafish brain. Ethynyl uridine (EU) was incorporated into neuronal RNA in a time- and concentration-dependent manner. Newly synthesized RNA granules observed throughout the dendrites were colocalized with mRNA and rRNA markers. In zebrafish larvae, the application of EU to the swim water resulted in uptake and labeling throughout the brain. Using a GABA receptor antagonist, PTZ (pentylenetetrazol), to elevate neuronal activity, we demonstrate that newly transcribed RNA signal increased in specific regions involved in neurogenesis.