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ATP-binding cassette (ABC) transporters shuttle diverse substrates across biological membranes. They play a role in many physiological processes but are also the reason for antibiotic resistance of microbes and multi drug resistance in cancer, and their dysfunction can lead to serious diseases. Transport is achieved through an ATP-driven closure of the two nucleotide binding sites (NBSs) which induces a transition between an inward-facing (IF) and an outward-facing (OF) conformation of the connected transmembrane domains (TMDs). In contrast to this forward transition, the reverse transition (OF-to-IF) that involves Mg2+-dependent ATP hydrolysis and release is less understood. This is particularly relevant for heterodimeric ABC transporters with asymmetric NBSs. These transporters possess an ATPase active consensus NBS (c-NBS) and a degenerate NBS (d-NBS) with little or no ATPase activity.
Crucial details regarding function and mechanism of the transport cycle remain elusive.
Here, these open questions were addressed using pulse electron-electron double resonance (PELDOR or DEER) spectroscopy of the heterodimeric ABC exporter TmrAB.
To better understand the transport cycle, the underlying kinetics of the conformational transitions need to be elucidated. By introducing paramagnetic nitroxide (NO) spin probes at key positions of TmrAB and employing time-resolved PELDOR spectroscopy, the forward transition could be followed over time and the rate constants for the conformational transition at the TMDs and NBSs were characterized.
The temperature dependence of these rate constants was further analyzed to determine for the first time the activation energy of conformational changes in a large membrane protein. For TMD opening and c-NBS dimerization, values of 75 ± 27 kJ/mol and 56 ± 3 kJ/mol, respectively were found. These values agree with reported activation energies of peptide transport and peptide dissociation in other ABC transporters, suggesting that the forward transition may be the rate-limiting step for substrate translocation.
The functional relevance of asymmetric NBSs is so far not well understood. By combining Mg2+-to-Mn2+ substitution with Mn2+-NO and NO-NO PELDOR spectroscopy, the binding of ATP-Mn2+, the conformation of the NBSs, and the conformation of the TMDs could be simultaneously monitored for the first time. These results reveal an asymmetric post-hydrolytic state. Time-resolved investigation showed that ATP hydrolysis at the active c-NBS triggers the reverse transition, whereas opening of the impaired d-NBS regulates the return to the IF conformation.
The peptide loading complex (PLC) is a central machinery in adaptive immunity ensuring antigen presentation by major histocompatibility complex class I (MHC I) molecules to immune cells. If nucleated cells present foreign antigenic peptides from various origins (e.g., viral infected or cancer cells) on their cell surface they are targeted and eliminated by effector cells of the immune system to protect the organism against the hazard. The antigen presentation process starts with proteasomal degradation. Peptide loading and quality control of most, if not all, MHC I is performed by the PLC. Despite the main components, architecture, and general functions of this labile and multi-subunit assembly have been described, knowledge about the inner mechanics of MHC I loading and quality control in the PLC is limited. Detailed structural insights into the interactions and functions of key elements are lacking. In this PhD thesis, structural and functional aspects of the PLC in peptide loading and quality control of MHC I are unraveled, and the PLC was analyzed from an evolutionary perspective.
First, composition and architecture of native PLC isolated from different mammalian species was analyzed. Comparison of detergent-solubilized PLC from cow and sheep spleens with PLC isolated from human source showed a compositional conservation in mammals, with the central components TAP, ERp57, tapasin, calreticulin, and the MHC I heterodimer were conserved in these species. Negative-stain electron microscopy (EM) analyses revealed an identical overall architecture of PLCs from human, sheep, and cow with two major densities at opposing sides of the plane of the detergent micelle corresponding to endoplasmic reticulum (ER) luminal and cytosolic domains. Interestingly, the glucose-regulated protein 78 (GRP78) was associated only with the PLC from sheep and cow as revealed by mass spectrometry. This ER chaperone is involved in initial folding steps of MHC I but was not co-purified with human PLC, rendering it an interesting target for future functional and in-depth structural studies.
The human PLC was stabilized by reconstitution in membrane mimicking systems that replace the detergent, which is necessary to solubilize the complex. This stabilization allowed detailed structural analysis by single-particle cryogenic electron microscopy (cryo-EM). The structure of the MHC I editing module in the PLC, composed of tapasin, ERp57, calreticulin, MHC I, and β-2-microglobulin (β2m), was solved at an overall resolution of 3.7 Å. Within the structure, two important features were visualized: (i) the editing loop of tapasin, which is directly involved in peptide proofreading of MHC I; (ii) the A-branch of the Asn86 tethered N-linked glycan on MHC I. Both features are crucial elements in the quality control and peptide editing process on MHC I. The editing loop interacts with the peptide binding groove in MHC I. It disturbs the interaction between a cargo peptide C terminus and the F-pocket in the binding groove by displacing Tyr84 and the helices α1 and α2. The helix displacement widens the F-pocket which allows a faster peptide exchange on MHC I. The glycan is bound in its monoglucosylated form (Glc1Man9GlcNAc2) by the lectin domain of calreticulin. The A-branch of this glycan is stretched between MHC I Asn86 and the lectin domain, leading to the hypothesis that the glycan will be released from calreticulin once MHC I is loaded with a favored peptide (pMHC I).
For investigation of the glycan status of MHC I, intact protein liquid chromatography coupled mass spectrometry (LC-MS) was performed under denaturating conditions. An allosteric coupling between peptide loading and removal of the terminal glucose by α-Glucosidase II (GluII) was discovered. In addition, the PLC remained fully intact after peptide loading, which demonstrated GluII action on the PLC once MHC I is loaded.
With establishing GluII as transient interaction partner, this work deepens the knowledge of the molecular sociology of the PLC and how the PLC is involved in the endoplasmic reticulum quality control (ERQC). Further investigation of the ER aminopeptidases ERAP1 and ERAP2 showed that these enzymes neither alone nor together stably interact with the PLC. In contrast, both work independent from the PLC on free peptides in the ER.
LC-MS analysis of the PLC components revealed a very unusual glycosylation pattern of tapasin. Tapasin was observed with N-linked glycans ranging from the full glycan (Man9GlcNAc2) to heavily trimmed glycans, where only a single GlcNAc remained attached to Asn233. In the PLC, tapasin is probably shielded from degradation by ERQC and can remain functional and intact without a full N-linked glycan.
Mechanistic and structural insights into the quality control of the MHC I antigen processing pathway
(2022)
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...
Zika virus (ZIKV) is a member of the Flaviviridae family that received public attention and scientific interest after the outbreak in French Polynesia (2013-2014) and the epidemic in the Americas (2015-2016). Even though only 20% of infected people exhibit clinical manifestations and they are predominantly flu-like symptoms, these events unveiled neurological complications associated with ZIKV infection, such as the Guillain-Barré syndrome in adults and microcephaly in newborns. Lacking a preventive vaccine and a specific antiviral therapy against ZIKV allied to the fact that this pathogen is a re-emerging virus, uncovering and comprehending novel virus-host interactions is crucial to the identification of new antiviral targets and the development of innovative antiviral approaches. Previous research work uncovered that the Chinese hamster ovary (CHO) cells do not support ZIKV infection.459 As this cell line does not express endogenous epidermal growth factor receptor (EGFR), this study aimed to investigate whether EGFR and EGFR-dependent signaling are relevant for the ZIKV life cycle in vitro.
In the first part of the study, viral infection was investigated in CHO cells and compared to A549 cells, a highly ZIKV permissive cell line. After performing binding and entry assays, ZIKV entry, but not the attachment, was significantly decreased in CHO cells in comparison to A549 cells. Additionally, in A549-EGFR KO cells, ZIKV entry was diminished relatively to the off-target control. These results show the clear impact that the absence of EGFR has on viral entry, implicating EGFR during this process. Even though EGFR overexpression in CHO cells could not render these cells permissive to ZIKV infection, as demonstrated by the lack of viral infection after electroporation with in vitro transcribed capped ZIKV-Renilla luciferase RNA, it was possible to rescue ZIKV entry. These findings suggest that there are additional elements, which are not expressed in CHO cells, required for viral replication.
Furthermore, the impact of ZIKV infection on EGFR mRNA and protein levels as well as on the EGFR subcellular localization and distribution was evaluated. The relative number of EGFR specific transcripts continuously increased with ZIKV infection, whereas the EGFR protein level diminished at later times of infection. Moreover, changes in the subcellular localization of EGFR and its colocalization with the early endosomal marker EEA1 in ZIKV-infected cells revealed that ZIKV triggers EGFR internalization. The relevance of EGFR in the ZIKV entry process was further corroborated by the observation of EGFR internalization at 30 min post-infection (mpi) and to less extent at 60 mpi, which concurs with the expected time of ZIKV entry into the host cells.
In the remaining part of the study, the influence of ZIKV infection in EGFR-dependent signaling as well as the contribution of EGFR and EGFR signaling for viral infection were studied. Activation of EGFR and the MAPK/ERK signaling cascade was detected as early as 5 mpi and ceased within 30 mpi in ZIKV-infected cells. Taking into account that EGFR internalization was observed at 30 mpi in infected cells, the activation of EGFR and ERK and subsequent dephosphorylation within this period go along with this previous observation. Vice-versa, inhibition of the activation of EGFR and the MAPK/ERK pathway declines ZIKV infection. On the one hand, inhibition of EGFR activation by Erlotinib affected ZIKV entry, as a consequence of impaired EGFR internalization. On the other hand, Raf and MEK inhibitors reduced ZIKV infection without disturbing viral replication or viral entry. These data suggest that the activation of the MAPK/ERK signaling cascade is necessary for a step of the viral life cycle before the onset of genome replication and morphogenesis and after viral entry. The importance of EGFR signaling was additionally investigated by the determination of EGFR half-life in ZIKV-infected cells upon EGF stimulation. While the EGFR half-life was similar in uninfected and Uganda-infected cells, a delay in EGFR degradation was observed in French Polynesia-infected cells. This observation might indicate an extended usurpation of the EGFR signaling since EGFR seems to still be active in the endosomes. Moreover, disruption of lipid rafts by MβCD, a cholesterol-depleting agent, hampered ZIKV entry. In uninfected cells, MβCD treatment led to the activation of EGFR, but at the same time prevented EGFR internalization, indicating that EGFR activation exclusively is not sufficient for an efficient ZIKV entry and further supporting the importance of EGFR internalization during the ZIKV entry process.
Taken together, this study uncovers EGFR as a relevant host factor in the early stages of ZIKV infection, providing novel insights into the ZIKV entry process. Since numerous monoclonal antibodies and substances that target EGFR are licensed, repurposing these compounds might be a helpful tool for the establishment of an antiviral therapy in case of ZIKV re-emergence.
The health status of every nucleated cell in the human body is monitored through peptides presented by major histocompatibility complex class I (MHC I) to T-cell receptors of CD8+ T-cells. Thereby, the adaptive immune system ensures the recognition and elimination of infected or cancerous cells. MHC I molecules comprise the polymorphic heavy chain (hc) and the light chain β2-microglobulin (β2m). More than 13,000 allomorphs of the MHC I hc have been identified. All MHC I hcs associate with β2m but differ in their binding preferences for peptides, ensuring the presentation of a large peptide pool. After maturation of MHC I hc/β2m heterodimers in the endoplasmic reticulum (ER), most of the peptide-deficient MHC I molecules are recruited to the peptide-loading complex (PLC). There, they go through peptide loading and editing before they are released as stable peptide-MHC I (pMHC I) complexes and traffic to the cell surface for antigen presentation.
During the stringent quality control of MHC I peptide loading and editing within the PLC, the chaperone tapasin in conjunction with the oxidoreductase ERp57 stabilizes peptide-receptive MHC I molecules and alters the peptide cargo for high immunogenicity by catalyzing peptide-exchange. The tapasin-homologue TAP-binding protein related (TAPBPR) is involved in downstream quality control, editing the peptide repertoire of MHC I molecules that slipped through peptide proofreading by tapasin. Both chaperones were shown to adopt similar binding-modes for MHC I, suggesting related mechanisms of peptide editing. Nevertheless, the MHC I specific chaperones operate in different subcellular locations with differing assistance. While TAPBPR mediates peptide-exchange solely in the peptide-poor environment of the cis-Golgi and ER-Golgi intermediate compartment (ERGIC), tapasin functions mainly within the PLC together with ERp57 and the lectin-like chaperone calreticulin. Calreticulin with its lectin-, arm- and C-terminal domain contacts the MHC I heterodimer, ERp57 and the C-terminal domain of tapasin, respectively. Notably, the interaction site between calreticulin and tapasin has not yet been elucidated experimentally at molecular detail. The depletion of tapasin leads to a compromised immune response and a change in the pool of peptide cargo. The numerous MHC I allomorphs vary in their plasticity and their dependence on tapasin for the loading of optimal peptides. Moreover, the conformational plasticity of MHC I correlates with their dependence on tapasin. However, the molecular basis on how tapasin edits the various MHC I allomorphs and the structural features that are essential for peptide exchange catalysis at atomic resolution remained elusive.
In the first part of this thesis, the trimeric complex of tapasin–ERp57/calreticulin was analyzed. To this end, laser induced liquid bead ionization mass spectrometry (LILBID-MS) was performed as part of a collaboration and revealed the trimeric assembly for tapasin–ERp57 and calreticulin. Furthermore, additional to a wildtype construct of calreticulin, a second construct, lacking the acidic helix of calreticulin that was found to come to close contact with tapasin, was utilized for isothermal titration calorimetry (ITC). A micromolar affinity of wildtype calreticulin to tapasin–ERp57 was determined. Previous biochemical and NMR studies utilizing the P-domain of calreticulin and solely ERp57 provided a micromolar affinity for the complex of calreticulin and ERp57. In this study, no interaction of calreticulin lacking the acidic helix with tapasin–ERp57 could be measured by ITC. However, these results undergo with findings that calreticulin lacking the acidic helix impairs the function of the PLC. Most likely, the negatively charged acidic helix is located in a groove of tapasin, carrying a more positive charge. Taken together, the functional data demonstrates the importance of the acidic helix of calreticulin for assembly of the trimeric subunit of calreticulin/tapasin–ERp57.
In the main part of this study an MHC I–tapasin–ERp57 complex was structurally analyzed. Therefore, a photo-triggered approach was chosen to assemble the transient complex of MHC I–tapasin–ERp57. Various allomorphs were screened for complex formation with the tapasin–ERp57 heterodimer after photocleavage by size exclusion chromatography (SEC), resulting in mouse MHC I H2-Db as the suited allomorph. Microseed matrix screening was performed. Crystals diffracting X-rays to a resolution of 2.7 Å were obtained showing one tetrameric tapasin–ERp57–MHC I complex per asymmetric unit.
The MHC I-chaperone structure shows molecular rearrangements upon MHC I engagement and unveils structural features of tapasin, involved in peptide-exchange catalysis...
Mechanism of the MHC I chaperone TAPBPR and its role in promoting UGGT1-mediated quality control
(2022)
Information about the health status of most nucleated cells is provided through peptides presented on major histocompatibility complex I (pMHC I) on the cell surface. T cell receptors of CD8+ T cells constantly monitor these complexes and allow the immune system to detect and eliminate infected or cancerous cells. Antigenic peptides displayed on MHC I are typically derived from the cellular proteome and are translocated into the lumen of the endoplasmic reticulum (ER) by the ATP-binding cassette (ABC) transporter associated with antigen processing (TAP), which is part of the peptide-loading complex (PLC). In a process called peptide editing, the MHC I-dedicated chaperone tapasin (Tsn) selects peptides for their ability to form stable complexes with MHC I. While initial peptide loading is catalyzed in the confines of the PLC, the second quality control is mediated by TAPBPR, operating in the peptide-depleted cis-Golgi network. TAPBPR was shown to have a more fine-tuning effect on the presented peptide repertoire rather than initial peptide selection. The fundamental mechanism of peptide editing was illuminated by two crystal structures of TAPBPR in complex with peptide-receptive MHC I. Notably, one of these structures reported a structural element that inserted into the peptidebinding pocket. The so-called scoop loop was assumed to be involved in mediating peptide exchange but the underlying mechanism remained undefined. Additionally, latest results suggested that TAPBPR mediates the interaction of the glucosyltransferase UGGT1 with peptide-receptive MHC. To expand the current knowledge of quality control processes in the antigen presentation pathway, the contribution of the scoop loop in peptide editing and the role of TAPBPR in UGGT1-mediated quality control needs to be elucidated. In the first part of this study, TAPBPR proteins with various loop lengths were designed to scrutinize the contribution of the scoop loop in chaperoning peptidereceptive MHC I. In a light-driven approach, the ability of TAPBPR variants to form stable complexes with peptide-free MHC I was tested. These results demonstrated that in a peptide-depleted environment, the scoop loop is of critical importance for TAPBPR to chaperone intrinsically unstable, peptidereceptive MHC I clients. Moreover, fluorescence polarization-based assays allowed the pursuit of peptide exchange in different, native-like environments. Peptide displacement activities of TAPBPR variants illustrated that catalyzed peptide editing is primarily induced by structural elements outside the scoop loop. In a peptide-depleted environment, the scoop loop occupies the position of the peptide C-terminus and acts as an internal peptide surrogate. By combining complex formation and fluorescence polarization experiments, the scoop loop of TAPBPR was shown to be critically important in stabilizing empty MHC I and functions as an internal peptide selector. In the second part of this study, a novel in-vitro glucosylation assay was established to examine the role of TAPBPR in UGGT1-catalyzed re-glucosylation of TAPBPR-bound MHC I clients. Therefore, a peptide-free MHC I-TAPBPR complex with defined glycan species was designed which served as physiological substrate for UGGT1. By subjecting the recombinantly expressed HLA-A*68:02- TAPBPR complex and UGGT1 proteins to the new in-vitro system, UGGT1 was shown to catalyze the transfer of a glucose residue to the N-linked glycan of TAPBPR-bound Man9GlcNAc2-HLA-A*68:02. Moreover, a high-affinity, photocleavable peptide was applied to dissociate the MHC I-chaperone complex. However, in the absence of TAPBPR, no glucosyltransferase activity was observed. Generation of peptide-free MHC I through UV illumination also showed no activity, and only the addition of TAPBPR could restore UGGT1-mediated reglucosylation of the empty MHC I. Independent of the peptide status of HLAA*68:02, the combination of protein glycoengineering and LC-MS analysis implicated that UGGT1 exclusively acts on TAPBPR-chaperoned HLA-A*68:02. The newly established system provided insights into the function of TAPBPR during UGGT1-catalyzed re-glucosylation activity and quality control of MHC I. Taken together, the scoop loop allows TAPBPR to function as MHC I chaperone through stabilizing peptide-receptive MHC I. In a peptide-depleted environment, the loop structure serves as an internal peptide surrogate and can only be dislodged by a high-affinity peptide. Based on these findings, TAPBPR fulfills a dual function in the second level of quality control. On the one hand, TAPBPR functions as peptide editor, shaping the repertoire of presented peptides. On the other hand, TAPBPR mediates peptide-receptive MHC I clients to the folding sensor UGGT1. Here, TAPBPR is essential to promote UGGT1-catalyzed reglucosylation of the N-linked glycan, giving MHC I a second chance to be loaded with an optimal peptide cargo in the peptide loading complex.
Protein biosynthesis is a fundamental process across all domains of life. Polypeptides are produced by translating the genetic information of the messenger RNA (mRNA) into amino acids. This elaborate procedure is divided into the four distinct phases: initiation, elongation, termination, and ribosome recycling. The phases are controlled and regulated by a multitude of translation factors. During initiation, the ribosome assembles on the mRNA. Initiation factors (IFs) bind to the small ribosomal subunit (SSU) and assist the recruitment of mRNA and initiator transfer RNA (tRNA), which delivers the first amino acid methionine. After positioning the SSU at the start codon of the mRNA, additional IFs support the joining of the large ribosomal subunit (LSU). Next, elongation factors (EFs) deliver amino-acylated tRNAs (aa-tRNAs) to the translating ribosome and assist kinetic proofreading and ribosome subunit translocation after the catalytic transfer of the polypeptide onto the aa-tRNA. When a stop codon is reached, translation is terminated by release factors (RFs) that hydrolyze the peptidyl-tRNA to release the nascent protein chain. Afterwards, the ribosome is recycled in Eukaryotes and Archaea by the conserved and essential factor ABCE1, which splits the ribosome into the LSU and SSU. ABCE1 remains bound to the SSU forming the post-splitting complex (post-SC). mRNA translation closes into a cycle by recruitment of IFs to the post-SC and the start of a new round of initiation. The post-SC presents the platform for translation initiation. However, the role of ABCE1 in initiation remains elusive. Therefore, the main goal of my thesis was to unravel the molecular mechanism of ABCE1 on the post-SC and during initiation complex (IC) assembly.
Using a reconstituted system, the high-resolution structure of the archaeal post-SC was solved by cryogenic electron microscopy (cryo-EM) following the native splitting route. It was the first complete model of an archaeal SSU at atomic resolution and revealed a previously undescribed ribosomal protein, which we termed eS21. The hinge 2 region of ABCE1 was identified to be the major interaction interface that anchors to the SSU. Functional characterization of single residue mutations in hinge 2 unraveled essential interactions with the ribosomal RNA backbone of the SSU. Sensing of SSU-binding was found to be allosterically transmitted to the nucleotide-binding sites (NBSs) for integration into the ATPase cycle of ABCE1.
Reconstitution of the archaeal translation apparatus allowed for dissection of IC assembly in the presence of ABCE1. Three different ICs were resolved by cryo-EM. The results were in accordance with recent structural findings of eukaryotic translation initiation and highlighted that the involvement of ABCE1 is conserved.
In a semi-native approach, recombinant ABCE1 was pulled-down from crenarchaeal cell lysates. Mass spectrometric analysis of co-immunoprecipitated ribosomal complexes identified the association of numerous translation factors to the post-SC in a cellular context. The establishment of the genetic toolbox of the acidothermophilic Sulfolobus acidocaldarius allowed the homologous expression of ABCE1. Pull-down of native ABCE1 revealed similar ribosomal complexes as the semi-native and reconstituted approaches. Together, my results gave first physiological relevance of ABCE1 involvement in mRNA translation initiation in Archaea. Native archaeal ABCE1-ICs were vitrified for structural analysis by cryo-EM. Thereby, future structural analysis will allow to analyze the interactions of ABCE1 on native ICs and identify its role in IC assembly.
To address the molecular process of IC assembly, the binding affinity of aIF1 to the SSU was determined by fluorescence polarization. Similar studies will allow for a detailed functional analysis on IF recruitment to the SSU in presence of ABCE1.
mRNA surveillance and ribosome-associated quality control (RQC) mechanisms evolved to ensure cell viability. The pathways overcome ribosome stalling and defective translation components. Stalled ribosomes are terminated by special RFs, which do not hydrolyze the peptidyl-tRNA, but allow dissociation of the ribosome by ABCE1. Faulty messages are degraded via mRNA decay pathways and the LSU is rescued by RQC factors. Recently, the bacterial RQC factor MutS2 was identified to specifically target collided di- and polysomes but its molecular mechanism remains unknown. In this thesis, initial functional analyses showed tri-phosphate specific nucleotide binding of MutS2. While the dissociation of collided disomes by MutS2 could not be observed, the results pave the way for future in vitro studies of bacterial RQC factors acting on specific ribosome populations.
In the future, mRNA translation research must focus on complex quality control processes to comprehensively understand this fundamental cellular process in a holistic context.
T-cell development is a highly dynamic and stepwise process comprimising T lineage commitment, T-cell receptor (TCR) gene rearrangements and subsequent selection. From a quantitative point of view, only a few hundred progenitor cells migrate from the bone marrow into the thymus. Developing thymocytes (termed double negative (DN), CD4-CD8-) can be further divided into DN1-4 cells based on the expression of CD25 and CD44. These developmental events are interspersed by proliferative bursts which ultimately lead to the generation of millions of double positive (DP, CD4+CD8+) thymocytes that then undergo selection. As a consequence, a proportion of naïve T-cells evolves to ensure adaptive, but not autoreactive immunity.
Previous studies of our lab focused on the quantification of thymus colonization and identified thymus entry to be dependent on expression of the chemokine receptors CCR7 and CCR9 (Krueger et al., 2010; Ziętara et al., 2015). CCR7/9 double knockout (DKO) mice are almost completely devoid of the most immature thymocyte populations (DN1 and DN2), but show near normal DN3 cellularity. Interestingly, a similar defect during early development but a virtually complete recovery of later stages and total thymocyte numbers was also observed in thymi of miR-17~92 deficient mice. Here, a failure of prethymic IL-7 signaling dampens early T-cell development (Regelin et al., 2015). For this reason, we hypothesized a tight regulation of thymocyte population size through alterations in the underlying cell cycle kinetics.
In this thesis, we employed in vivo single- and dual-nucleoside pulse labeling combined with determination of DNA replication over time in different WT thymocyte subsets at steady-state. Based on this, we assessed alterations in cell cycle kinetics of CCR7/9 and miR-17~92 defcicient mice and identified compensatory mechanisms of thymocytes on the level of cell cycle phase distribution and cell cycle speed. In addition, single-cell RNA sequencing helped to obtain information on cell cycle dynamics of early thymocyte subsets, exemplarily shown for WT and CCR7/9 DKO mice. Lastly, we performed cell cycle analyses in a model of endogenous thymic repair upon sublethal total body irradiation which provided insight into intrathymic cell cycle regulation as an adjustable system to re-establish normal thymus cellularity.
In the second part of the thesis, we addressed the role of miR-21 in the thymus. In various studies, we and others identified miRNAs as key posttranscriptional regulators of the immune system and especially for T-cell development (Regelin et al. 2015; Mildner et al. 2017; Li et al. 2007; Ebert et al. 2009; Ziętara et al. 2013; Schaffert et al. 2015). The dynamic expression of miR-21 during T-cell development (Neilson et al. 2007; Kirigin et al. 2012; Kuchen et al. 2010) prompted us to hypothesize that miR-21 has a regulatory function in the thymus. A miR 21-knockout mouse model allowed us to study the role of this miRNA for the development of T-cells in the thymus and the maintenance of T-cells in the periphery. In addition, we performed competitive bone marrow chimera experiments in the context of miR-21 deficiency and overexpression. Further insights were provided by exploring the function of miR-21 in negative selection in vivo as well as in T-cell differentiation in coculture experiments in vitro. To unravel implications of miR-21 to regulate cellular stress responses, we assessed the contribution of miR-21 in a model of endogenous regeneration of the thymus after sublethal irradiation. We could not provide evidence for a prominent role for miR-21 during T-cell development. Together, our experiments revealed that miR-21 is largely dispensable for physiologic T-cell development despite high and dynamic expression in the thymus (Kunze Schumacher et al., 2018). The apparent discrepancy between dynamic expression but lack of a regulatory function in the thymus led us to conclude that miR-21 is rather fine tuning T-cell responses than controlling a developmental event.
The specific and precise arrangement of proteins and biomolecules in 3D is an important prerequisite for the study of cell migration, cellular signal transduction and the production of artificial tissue. In a variety of research approaches, proteins have been immobilized on rigid surfaces such as glass or gold to observe protein-protein or protein-cell interactions. While these commonly used analytical platforms offer advantages such as rapid washing steps and easy use, due to their rigidity and two-dimensionality, they cannot replicate the extracellular matrix (ECM) the native environment of cells. This severe deviation from the natural environment results in significant changes in cell structure and cellular processes such as the polarization of the cell, its morphology, and signal transduction. In order to maintain the functionality of the immobilized proteins, it is also enormously important that the proteins are oriented and anchored in the material under mild conditions.
An immobilization strategy that makes this possible is bioaffinity. For this, the specific interaction of a biomolecule with an interaction partner anchored on a surface is used to immobilize the biomolecule. Such an interaction is for example the nitrilotriacetic acid (NTA)/His-tag binding. NTA is a chelator molecule that, when bound to divalent metal ions such as Ni(II), forms an octahedral complex with oligohistidines. The oligo histidine-tag can be competed out of the complex by free histidine or imidazole due to structural similarity. This is exploited in immobilized metal affinity chromatography (IMAC). The binding of a monoNTA/His-tag complex (KD=10 µM) is not stable enough to be used for immobilizations. Therefore, multivalent variants of the chelator were developed, like trisNTA which has a high affinity for His6 tagged proteins (KD= 10 nM). The PA-trisNTA developed in a preliminary work was the first light-activatable system based on the trisNTA chelator head.
The aim of this work was to synthesize a new two-photon (2P) activatable trisNTA (TPA trisNTA) interaction molecule, to analyze its photophysical characteristics and to apply it for two- and three dimensional (2D/3D) biomolecule patterning. The final goal was to use TPA trisNTA for cellular applications in order to manipulate membrane protein organization. Therefore, TPA trisNTA was designed to maintain a stable autoinhibition enabling the immobilization of proteins under physiological conditions with high precision in the x/y, as well as z dimension only upon light activation. 2P activation brings some outstanding advantages: i) the use of near-infrared (NIR) light is less harmful to cells compared to ultraviolet (UV) light, ii) the longer wavelength allows the radiation to penetrate deeper into tissues, iii) the precision of focal irradiation is more accurate because only a focal volume (about 1 fL) is excited and, unlike UV light, scattered light does not lead to activation.
Several backbones for TPA-trisNTA were considered as 2P cleavable groups due to their 2P absorption ability and small size: 3 nitrodibenzofuran (NDBF), 6 bromo 7 hydroxycoumarin (Bhc), and 7 diethylaminocoumarin (DEAC). Initially, suitable synthetic routes were developed for the respective carbaldehydes, since these represented an important intermediate for both the construction of amino acid (aa) derivatives as well as ß hydroxy acids. ß Hydroxy acids were important intermediates because their photocleavage differs from aa derivatives. To establish the conversion from carbaldehydes to hydroxy acids via Reformatsky reaction, commercially available carbaldehydes of the nitroveratral (NV) or nitropiperonal (NP) group were used in addition. The conversion of NDBF, NV, NP proved to be difficult, whereas the ß-hydroxy acid was successfully synthesized from Bhc as well as from DEAC.
Starting from DEAC ß hydroxy acid, a Fmoc protected amino acid derivative was synthesized. To ensure high cleavage efficiency, the DEAC ß hydroxy acid was linked to monoFmoc ethylenediamine through a carbamate linker. Subsequently, the photocleavable group was successfully incorporated into the linker of TPA-trisNTA by solid-phase peptide synthesis (SPPS).
The functional principle of TPA-trisNTA, similar to PA-trisNTA, is based on the autoinhibition of the multivalent chelator head trisNTA, which is linked to an intramolecular oligohistidine sequence by a peptide linker. In presence of Ni(II) ions, trisNTA forms a metal ion-mediated complex with histidine, causing TPA-trisNTA to self-inactivate. The cleavage site is the DEAC based photocleavable amino acid. In contrast to PA-trisNTA, the incorporation of two photocleavable amino acids was omitted. Instead, only one photocleavable DEAC was incorporated in front of the His tag. To avoid a second DEAC group within the His tag, a His5 tag was used instead of an His6 tag. It is known from preliminary work that a His5 tag is sufficient to maintain autoinhibition in the presence of His6-tagged proteins of interest (POIs), but can be displaced from the complex after light-driven cleavage of the peptide backbone. Placement of a cysteine in the peptide linker between the trisNTA and the DEAC group allowed for permanent surface anchoring after photocleavage of the linker.
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Endolysosomal effectors and their relevance for antiviral activity against the Hepatitis E virus
(2021)
Mit über 20 Millionen registrierter Fälle pro Jahr, repräsentiert das Hepatitis-E-Virus (HEV) eine Hauptursache einer viralen Hepatitis weltweit und stellt ein erhebliches Risiko insbesondere für Schwangere und Immunsupprimierte dar. Jedoch sind Behandlungsoptionen stark limitiert und mit teils schweren Nebenwirkungen verbunden. Neue Erkenntnisse des Wechselspiels zwischen Wirtszelle und HEV werden deshalb benötigt, um neue antivirale Wirkstoffe zu entwickeln. Der Fokus der Arbeit wurde hierbei auf Effektoren des endosomalen Systems gesetzt, welches von HEV zur Freisetzung von Virionen genutzt wird.
Eine virale Infektion führt in der Zelle zur Produktion von Interferonen (IFNs) und weiters zu einer IFN-Antwort. Ein essenzielles Effektormolekül, welches HEV nachweislich effizient repressiert, ist die GTPase guanylate binding protein 1 (GBP1). In dieser Studie wurde beleuchtet, dass Letztere durch eine HEV-Infektion induziert wird. Zusätzlich reduziert die ektopische Expression von GBP1 sowohl die intrazelluläre Menge des HEV Kapsidproteins als auch die Menge freigesetzter Virionen. Mechanistisch liegt diesem Sachverhalt die GBP1-induzierte Inkorporation von Virionen in Lysosomen zugrunde, was schlussendlich deren Abbau nach sich zieht. Erkenntnisse über die Rolle verschiedener GBP1 Proteindomänen innerhalb des Mechanismus wurden unter Verwendung ektopischer Expression von GBP1-Mutanten erlangt. Inkorporation der Mutation R48A führt zum Verlust der GTPase-Aktivität. Andererseits führt eine Inkorporation der Mutation S73A zum Verlust der Homodimerisierung, was die nachfolgende Farnesylierung und gekoppelte Membranassoziation reduziert. Hierbei behält GBP1-R48A Fähigkeiten zur Induktion lysosomalen Abbaus von HEV bei, GBP1-S73A jedoch nicht. Dies wiederum bedeutet, dass eine GBP1 Homodimerisierung notwendig für den antiviralen Mechanismus ist, was eine Adapterfunktion des Moleküls für lysosomale Inkorporation nahelegt. Die Relevanz von GBP1 während einer IFNγ-Antwort wurde deshalb mittels siRNA-basiertem Silencing untersucht. Ähnlich der ektopischen Expression von GBP1 induziert IFNγ die lysosomale Degradation von HEV. In Abwesenheit von GBP1 jedoch, ist dieser Effekt signifikant geringer ausgeprägt, was zu einem Effizienzverlust von IFNy in Bezug auf dessen antiviralen Effekt bedeutet. Dies führte schlussendlich zur Identifizierung von GBP1 als essenziellen Restriktionsfaktor gegen HEV, was seine Rolle in Abhängigkeit seiner Homodimerisierung via Induktion lysosomalen Abbaus erfüllt.
Nebst der Induktion von GBP1, konnte eine Akkumulation von Cholesterin in Lysosomen durch IFNy nachgewiesen werden. Da dieses Lipid einen essenziellen Faktor für endosomale Reifung, Transport und Funktionalität darstellt, wurden Cholesterinspiegel und verbundene transkriptionelle Fußabdrücke im Kontext einer HEV Infektion untersucht. Letztere führt zu einer Dysregulation Cholesterin-assoziierter Genexpression, was eine Reduktion intrazellulären Cholesterins nach sich zieht. Auch in HEV infizierten Patienten liegt eine Abnahme des Serumcholesterins vor. Unter Modulation intrazellulären Cholesterins, wurde deutlich, dass die Inhibition der Cholesterinsynthese durch Simvastatin eine verstärkte Freisetzung von Virionen nach sich zieht, was ebenso in HEV infizierten Patienten nachweisbar war. Im Gegensatz hierzu zieht eine Erhöhung intrazellulären Cholesterins via Supplementierung von Lipoproteinpartikeln niedriger Dichte (LDL) oder 25-Hydroxycholesterin eine signifikante Reduktion des viralen Kapsidproteins und freigesetzter Virionen nach sich. Dem liegt eine verstärkte Inkorporation von HEV in Lysosomen mit anschließender Degradation zugrunde. Ob dieser Mechanismus pharmakologisch nutzbar ist, wurde mittels eines Screenings Lipid modulatorischer Medikamente untersucht. Der p-Glykoprotein Inhibitor PSC833 und besonders der PPARα-Agonist Fenofibrat stellten sich als äußerst effiziente Inhibitoren des HEV heraus. Beide führen zu einer Erhöhung und Akkumulation zellulären Cholesterins in vesikulären Strukturen. Dies zieht eine dramatische Erhöhung lysosomaler Lokalisation von HEV nach sich und führt letzten Endes zu einer signifikanten Reduktion freigesetzter Virionen.
Zusammenfassend konnten in dieser Studie essenzielle Funktionen von GBP1 in Bezug auf dessen restriktiven Effekt gegen HEV identifiziert werden. Weiters wurde dieses als entscheidender Wirtsfaktor für die IFNγ-Antwort gegen das Virus identifiziert. Andererseits legt diese Studie nahe, dass HEV niedrige Cholesterinspiegel innerhalb infizierter Zellen für die Freisetzung von Virionen benötigt. Andererseits sind erhöhte intrazelluläre Cholesterinspiegel schädlich für die virale Freisetzung, da der lysosomale Abbau von Virionen induziert wird. Dies führte zur erfolgreichen Entdeckung eines neuartigen antiviralen Wirkstoffes, welcher diesen cholesterinabhängigen Effekt effizient induziert: Fenofibrat.