Contribution of proteasome-catalyzed peptide cis-splicing to viral targeting by CD8⁺ T cells in HIV-1 infection

Peptides generated by proteasome-catalyzed splicing of noncontiguous amino acid sequences have been shown to constitute a source of nontemplated human leukocyte antigen class I (HLA-I) epitopes, but their role in pathogen-specific immunity remains unknown. CD8⁺ T cells are key mediators of HIV type 1 (HIV-1) control, and identification of novel epitopes to enhance targeting of infected cells is a priority for prophylactic and therapeutic strategies. To explore the contribution of proteasome-catalyzed peptide splicing (PCPS) to HIV-1 epitope generation, we developed a broadly applicable mass spectrometry-based discovery workflow that we employed to identify spliced HLA-I–bound peptides on HIV-infected cells. We demonstrate that HIV-1–derived spliced peptides comprise a relatively minor component of the HLA-I–bound viral immunopeptidome. Although spliced HIV-1 peptides may elicit CD8⁺ T cell responses relatively infrequently during infection, CD8⁺ T cells primed by partially overlapping contiguous epitopes in HIV-infected individuals were able to cross-recognize spliced viral peptides, suggesting a potential role for PCPS in restricting HIV-1 escape pathways. Vaccine-mediated priming of responses to spliced HIV-1 epitopes could thus provide a novel means of exploiting epitope targets typically underutilized during natural infection

Molecular Characterization of Mammalian De Novo DNA Methyltransferase Chromatin Recruitment

Precise deposition of CpG methylation is critical for mammalian development and tissue homeostasis and is often dysregulated in human diseases. The genomic localization of de novo DNA methyltransferases 3A (DNMT3A) and 3B (DNMT3B) is facilitated in part by chromatin “reader” domains to establish DNA methylation patterns genome-wide. Recent work has highlighted a role for the PWWP reader domain in directing recruitment of DNMT3B to actively transcribed gene bodies. However, our understanding of de novo DNA methyltransferase chromatin recruitment remains incomplete. Here I demonstrate using a variety of biochemical and cellular techniques that DNMT3A genomic localization is regulated by competing chromatin recruitment pathways. Under steady-state conditions, DNMT3A is targeted to non-coding intergenic regions through PWWP reader domain recognition of H3K36me2, an abundant histone post-translational modification (PTM) in cells. Following depletion of cellular H3K36me2 levels, DNMT3A is re-targeted to actively transcribed gene bodies through PWWP domain interaction with H3K36me3, thereby resembling the genomic localization of DNMT3B. Lastly, loss of PWWP domain reader functionality reveals the presence of a previously uncharacterized chromatin reader domain within DNMT3A that serves as an alternative targeting mechanism to facilitate de novo methylation of Polycomb-regulated regions, including a subset of CpG islands. Perturbations to these recruitment mechanisms may underlie the pathology of developmental overgrowth and undergrowth syndromes associated with distinct germline mutations in DNMT3A and may also contribute to the altered DNA methylation landscapes observed in diverse cancers

Flucloxacillin Protein Binding Leads to the Presentation of Novel MHC Peptides

Flucloxacillin is a β-lactam antibiotic associated with a high incidence of idiosyncratic drug-induced liver reactions. Although expression of HLA-B*57:01 increases susceptibility, little is known of the pathological mechanisms involved in the induction of the clinical phenotype. Off-target protein modification is suspected to drive the reaction, either through non-immune mediated pathways, through the modification of peptides that are presented to T cells by the risk allele, or both. In this thesis, the characterization of proteins haptenated by flucloxacillin was performed using proteomic techniques. As protein haptenation, followed by antigen processing and presentation of the drug-derived antigenic determinants may drive the adverse event, the immunopeptidome of HLA-B*57:01 in the presence and absence of flucloxacillin was determined. In order to detect flucloxacillin modified proteins, an antibody specific for flucloxacillin was generated. Characterization of the antibody to determine cross reactivity and selectivity was determined. Anti-flucloxacillin antibody was used to identify flucloxacillin modified proteins in a number of immortalised cell lines and primary human hepatocytes. Western blot, immunocytochemistry and mass spectrometry were used for the detection, localisation and characterization, respectively, of drug modified proteins. C1R-B*57:01, B-lymphoblast cells transfected with HLA-B*57:01, were incubated with flucloxacillin for 48h. HLA peptide complexes were subsequently eluted and processed for mass spectrometric analysis. Finally, bioinformatic pipelines were generated to assist in the characterization of flucloxacillin-modified MHC peptides to allow a more high-throughput approach to immunopeptidomic data analysis. The generation of a high titre flucloxacillin specific antibody was successful, with no cross reactivity with proteins. Isoxazole ring containing β-lactams did cross react, indicating this was the site of recognition. Intracellular protein modification was identified in all the cell lines examined, including primary human hepatocytes. Using the liver cell line HepaRG, localization within the bile canaliculi was observed. The function of the major hepatocellular efflux transporters MRP2 and P-gp was increased in the presence of flucloxacillin in a time-dependant manner. Modification of master regulators of MAP Kinase signalling molecules was detected. Flucloxacillin also modified HLA-B*57:01 protein directly, which could lead to neo-antigens being presented. In depth analysis of the global repertoire of peptides was interrogated. Flucloxacillin was found to alter the C-terminal amino acid on the majority of peptides, where an increase in phenylalanine and a decrease in tryptophan was observed. Peptides unique to flucloxacillin treatment were theoretically weaker binders to HLA-B*57:01, indicating flucloxacillin may assist in their stabilization. Of the peptides eluted from flucloxacillin treated C1R-B*57:01 cells, 7 were fully annotated to show flucloxacillin-lysine covalent binding, with other partially annotated peptides indicating modifications. In this thesis a wide range of off-target protein modification has been determined, including proteins involved in regulatory signalling pathways. The localization of flucloxacillin was identified to occur in the site of clinical disease during flucloxacillin-induced liver injury. It was also demonstrated that drug-modified peptides are presented by HLA-B*57:01 and that global repertoires are altered by flucloxacillin. Further investigation into the immunogenicity of haptenated proteins in the onset of iDILI is required to determine the role of these peptides in drug hypersensitivity

Protein mimetic amyloid inhibitor potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function

Missense mutations in p53 are severely deleterious and occur in over 50% of all human cancers. The majority of these mutations are located in the inherently unstable DNA-binding domain (DBD), many of which destabilize the domain further and expose its aggregation-prone hydrophobic core, prompting self-assembly of mutant p53 into inactive cytosolic amyloid-like aggregates. Screening an oligopyridylamide library, previously shown to inhibit amyloid formation associated with Alzheimer\u2019s disease and type II diabetes, identified a tripyridylamide, ADH-6, that abrogates self-assembly of the aggregation-nucleating subdomain of mutant p53 DBD. Moreover, ADH-6 targets and dissociates mutant p53 aggregates in human cancer cells, which restores p53\u2019s transcriptional activity, leading to cell cycle arrest and apoptosis. Notably, ADH-6 treatment effectively shrinks xenografts harboring mutant p53, while exhibiting no toxicity to healthy tissue, thereby substantially prolonging survival. This study demonstrates the successful application of a bona fide small-molecule amyloid inhibitor as a potent\ua0anticancer agent

Combinatorial optimization for affinity proteomics

Biochemical test development can significantly benefit from combinatorial optimization. Multiplex assays do require complex planning decisions during implementation and subsequent validation. Due to the increasing complexity of setups and the limited resources, the need to work efficiently is a key element for the success of biochemical research and test development. The first approached problem was to systemically pool samples in order to create a multi-positive control sample. We could show that pooled samples exhibit a predictable serological profile and by using this prediction a pooled sample with the desired property. For serological assay validation it must be shown that the low, medium, and high levels can be reliably measured. It is shown how to optimally choose a few samples to achieve this requirements. Finally the latter methods were merged to validate multiplexed assays using a set of pooled samples. A novel algorithm combining fast enumeration and a set cover formulation has been introduced. The major part of the thesis deals with optimization and data analysis for Triple X Proteomics - immunoaffinity assays using antibodies binding short linear, terminal epitopes of peptides. It has been shown that the problem of choosing a minimal set of epitopes for TXP setups, which combine mass spectrometry with immunoaffinity enrichment, is equivalent to the well-known set cover problem. TXP Sandwich immunoassays capture and detect peptides by combining the C-terminal and N-terminal binders. A greedy heuristic and a meta-heuristic using local search is presented, which proves to be more efficient than pure ILP formulations. All models were implemented in the novel Java framework SCPSolver, which is applicable to many problems that can be formulated as integer programs. While the main design goal of the software was usability, it also provides a basic modelling language, easy deployment and platform independence. One question arising when analyzing TXP data was: How likely is it to observe multiple peptides sharing the same terminus? The algorithms TXP-TEA and MATERICS were able to identify binding characteristics of TXP antibodies from data obtained in immunoaffinity MS experiments, reducing the cost of such analyses. A multinomial statistical model explains the distributions of short sequences observed in protein databases. This allows deducing the average optimal length of the targeted epitope. Further a closed-from scoring function for epitope enrichment in sequence lists is derived

Development and application of quantitative proteomics strategies to analyze molecular mechanisms of neurodegeneration

Neurodegenerative disorders such as Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, Amyotrophic Lateral Sclerosis or Prion diseases are chronic, incurable and often fatal. A cardinal feature of all neurodegenerative disorders is the accumulation of misfolded and aggregated proteins. Depending on the disease, these aggregated proteins are cell type specific and have distinct cellular localizations, compositions and structures. Despite intensive research, the contribution of protein misfolding and aggregation to the cell type specific toxicity is not completely understood. In recent years, quantitative proteomics has matured into an exceptionally powerful technology providing accurate quantitative information on almost all cellular proteins as well as protein interactions, modifications, and subcellular localizations, which cannot be addressed by other omics technologies. The aim of this thesis is to investigate key features of neurodegeneration such as misfolded proteins and toxic protein aggregates with cutting edge proteomics, presenting a technological “proof of concept” and novel insights into the (patho)physiology of neurodegeneration