Progress in biological imaging is intrinsically linked to advances in labeling methods. The explosion in the development of high-resolution and super-resolution imaging calls for new approaches to label targets with small probes. These should allow to faithfully report the localization of the target within the imaging resolution – typically nowadays a few nanometers - and allow access to any epitope of the target, in the native cellular and tissue environment. We report here the development of a complete labeling and imaging pipeline using genetic code expansion and non-canonical amino acids in neurons that allows to fluorescently label masked epitopes in target transmembrane proteins in live neurons, both in dissociated culture and organotypic brain slices. This allows us to image the differential localization of two AMPA receptor (AMPAR) auxiliary subunits of the transmembrane AMPAR regulatory protein family in complex with their partner with a variety of methods including widefield, confocal, and dSTORM super-resolution microscopy.

The discovery of UBA6 in 2007 has challenged the ubiquitin field, as for a long time, the ubiquitin-activating enzyme 1 (UBA1) was thought to be the sole E1 responsible for the activation of ubiquitin. UBA6 shares 40% sequence identity with UBA1 in humans. However, UBA6 is present only in mammals, zebrafish, and sea urchins. UBA6 and UBA1 use a different spectrum of E2 enzymes and direct ubiquitin to different subsets of E3 enzymes and consequently substrates. Since UBA1 initiates 99% of the ubiquitylation events, UBA6 is presumably responsible for a subset of substrates. Moreover, UBA6 is an unusual E1 enzyme as it activates both ubiquitin and FAT10. FAT10 is a ubiquitin-like modifier and consists of two ubiquitin-like domains arranged in tandem. FAT10 is present in vertebrates only, and its expression is synergistically induced by the pro-inflammatory cytokines interferon γ (INFγ) and tumor necrosis factor α (TNFα). Furthermore, FAT10 is the only ubiquitin-like modifier besides ubiquitin, which can directly target proteins for proteasomal degradation. The involvement of UBA6-mediated ubiquitylation and FATylation in a broad spectrum of cellular pathways and diseases, along with the ongoing preclinical development of E1 inhibitors such as MNL4924 (Pevonedistat) and MLN7243 (TAK-243), has spurred increased research efforts to develop additional E1 inhibitors. UBA6, with its more limited role in catalyzing ubiquitin activation and being the sole E1 for FAT10, is considered to be a possible drug target. However, to reach this goal, the dual specificity of UBA6 needs to be understood first. Inhibition of UBA6-mediated FATylation while not affecting ubiquitylation or vice versa will not only provide a possibility to further investigate the roles that UBA6 plays in downstream pathways, but also open a window for targeting either the ubiquitylation or FATylation of UBA6 with specific inhibitors. In this thesis, the crystal structure of UBA6 in complex with FAT10 will be described. In the UBA6-FAT10 complex, the C-terminal domain of FAT10 interacts with UBA6 in a similar manner as ubiquitin with UBA1, while its N-terminal domain binds to the 3-helix bundle inserted into the inactive adenylation domain of UBA6. The structure was corroborated by functional studies, which, surprisingly, identified UBA6 residues specifically abrogating the activation of either ubiquitin or FAT10. These results provide the foundation to study the individual roles that UBA6 is playing in the activation of either ubiquitin or FAT10 in downstream cellular pathways. In addition, a high-throughput screening (HTS) assay for the identification of compounds inhibiting UBA6 was established, which provides the starting point to identify small molecules exclusively inhibiting the UBA6-activated ubiquitylation or FATylation. Lastly, FATylation shares a common UBA6-USE1 cascade with UBA6-activated ubiquitylation, and several studies demonstrated that the cooperation between UBA6 and USE1 is functionally crucial in different pathways. Given that USE1 is currently one of the main dedicated E2 enzymes for UBA6, the underlying mechanism regarding the transfer is unclear yet. To visualize the transthioesterification between UBA6 and USE1, a cross-linking strategy was applied to form the binary UBA6-USE1 complex. Besides, to improve the yield and homogeneity of the complex samples, a non-catalytical cysteine variant of USE1 (3M_USE1) was generated, thus representing promising starting point samples that can be used for cryo-EM screening and x-ray crystallography. Ultimately, visualizing the catalytic transfer of ubiquitin/FAT10 from UBA6 to USE1 would provide insights into the molecular basis of UBA6-USE1 pathway mediated ubiquitylation and FATylation.

Circadian clocks coordinate time-of-day-specific metabolic and physiological processes to maximize organismal performance and fitness. In addition to light and temperature, which are regarded as strong zeitgebers for circadian clock entrainment, metabolic input has now emerged as an important signal for clock entrainment and modulation. Circadian clock proteins have been identified to be substrates of O-GlcNAcylation, a nutrient sensitive post-translational modification (PTM), and the interplay between clock protein O-GlcNAcylation and other PTMs is now recognized as an important mechanism by which metabolic input regulates circadian physiology. To better understand the role of O-GlcNAcylation in modulating clock protein function within the molecular oscillator, we used mass spectrometry proteomics to identify O-GlcNAcylation sites of PERIOD (PER), a repressor of the circadian transcriptome and a critical biochemical timer of the Drosophila clock. In vivo functional characterization of PER O-GlcNAcylation sites indicates that O-GlcNAcylation at PER(S942) reduces interactions between PER and CLOCK (CLK), the key transcriptional activator of clock-controlled genes. Since we observe a correlation between clock-controlled daytime feeding activity and higher level of PER O-GlcNAcylation, we propose that PER(S942) O-GlcNAcylation during the day functions to prevent premature initiation of circadian repression phase. This is consistent with the period-shortening behavioral phenotype of per(S942A) flies. Taken together, our results support that clock-controlled feeding activity provides metabolic signals to reinforce light entrainment to regulate circadian physiology at the post-translational level. The interplay between O-GlcNAcylation and other PTMs to regulate circadian physiology is expected to be complex and extensive, and reach far beyond the molecular oscillator.

Histones control gene expression by regulating chromatin structure and function. The posttranslational modifications (PTMs) on the side chains of histones form the epigenetic landscape, which is tightly controlled by epigenetic modulator enzymes and further recognized by so-called reader domains. Histone microarrays have been widely applied to investigate histone–reader interactions, but not the transient interactions of Zn2+-dependent histone deacetylase (HDAC) eraser enzymes. Here, we synthesize hydroxamic acid-modified histone peptides and use them in femtomolar microarrays for the direct capture and detection of the four class I HDAC isozymes. Follow-up functional assays in solution provide insights into their suitability to discover HDAC substrates and inhibitors with nanomolar potency and activity in cellular assays. We conclude that similar hydroxamic acid-modified histone peptide microarrays and libraries could find broad application to identify class I HDAC isozyme-specific substrates and facilitate the development of isozyme-selective HDAC inhibitors and probes.

Background The human leucocyte antigen (HLA) complex controls adaptive immunity by presenting defined fractions of the intracellular and extracellular protein content to immune cells. Understanding the benign HLA ligand repertoire is a prerequisite to define safe T-cell-based immunotherapies against cancer. Due to the poor availability of benign tissues, if available, normal tissue adjacent to the tumor has been used as a benign surrogate when defining tumor-associated antigens. However, this comparison has proven to be insufficient and even resulted in lethal outcomes. In order to match the tumor immunopeptidome with an equivalent counterpart, we created the HLA Ligand Atlas, the first extensive collection of paired HLA-I and HLA-II immunopeptidomes from 227 benign human tissue samples. This dataset facilitates a balanced comparison between tumor and benign tissues on HLA ligand level. Methods Human tissue samples were obtained from 16 subjects at autopsy, five thymus samples and two ovary samples originating from living donors. HLA ligands were isolated via immunoaffinity purification and analyzed in over 1200 liquid chromatography mass spectrometry runs. Experimentally and computationally reproducible protocols were employed for data acquisition and processing. Results The initial release covers 51 HLA-I and 86 HLA-II allotypes presenting 90,428 HLA-I- and 142,625 HLA-II ligands. The HLA allotypes are representative for the world population. We observe that immunopeptidomes differ considerably between tissues and individuals on source protein and HLA-ligand level. Moreover, we discover 1407 HLA-I ligands from non-canonical genomic regions. Such peptides were previously described in tumors, peripheral blood mononuclear cells (PBMCs), healthy lung tissues and cell lines. In a case study in glioblastoma, we show that potential on-target off-tumor adverse events in immunotherapy can be avoided by comparing tumor immunopeptidomes to the provided multi-tissue reference. Conclusion Given that T-cell-based immunotherapies, such as CAR-T cells, affinity-enhanced T cell transfer, cancer vaccines and immune checkpoint inhibition, have significant side effects, the HLA Ligand Atlas is the first step toward defining tumor-associated targets with an improved safety profile. The resource provides insights into basic and applied immune-associated questions in the context of cancer immunotherapy, infection, transplantation, allergy and autoimmunity. It is publicly available and can be browsed in an easy-to-use web interface at https://hla-ligand-atlas.org .