All living organisms are made out of cells, which serve as the fundamental units of life. Protein molecules, comprised of amino acids, carry out diverse functions across various cellular... Show moreAll living organisms are made out of cells, which serve as the fundamental units of life. Protein molecules, comprised of amino acids, carry out diverse functions across various cellular compartments. Proteins are tightly controlled in their synthesis, folding, localization and degradation to ensure proper functioning. Dysregulation of protein control mechanisms within the cell can lead to cellular dysfunction, disease or eventually cell death. Post-translational modifications (PTMs) are the addition of a chemical group to an existing protein to regulate its function, localization, stability or interaction. In this thesis we will dive deeper into PTMs SUMO and ubiquitin, with the aim to understand the biochemistry of these proteins in different aspects of cellular function and human pathophysiology. An interesting aspect of the conjugation process lies in its reversibility, which is governed by specific proteases known as SENPs in the case of SUMO. However, only SENP6 and SENP7 have the ability to depolymerize SUMO2/3 chains by an insertion in their conserved catalytic domains. We delve into the promising horizon that lies ahead of a small yet profoundly impactful post-translational modifying protein. Despite its modest size, this protein wields a significant influence on fundamental cellular processes Show less
Nowadays, therapeutic antibodies are the major and fastest growing class of biotherapeuticals. Since their invention, they are continuously developed to improve structural and functional... Show moreNowadays, therapeutic antibodies are the major and fastest growing class of biotherapeuticals. Since their invention, they are continuously developed to improve structural and functional characteristics. The high complexity of recently generated antibody derivatives, with various modifications induced during the manufacturing process itself leads to many proteoform variants of the desired product. These proteoforms can potentially exhibit altered activity. Therefore, an adequate characterization of the proteoforms, the assessment of their impact and careful monitoring of critical species is indispensable in order to guarantee effective and safe biopharmaceuticals. As the landscape of next-generation Ab formats continuously evolves, it is likewise of great importance to further develop appropriate analytical methods for their thorough attribute analysis. Hence, the focus of the research performed in this thesis is the development of multi-level approaches for the in-depth, primarily MS-based characterization of biopharmaceuticals to overcome the present restrictions and challenges arising e.g. by the implementation of complex Ab formats. Show less
This thesis presents the first synthetic peptides ADP-ribosylated on serine, threonine, tyrosine, arginine and cysteine. Besides synthetic peptides, this thesis discusses the first synthetic route... Show moreThis thesis presents the first synthetic peptides ADP-ribosylated on serine, threonine, tyrosine, arginine and cysteine. Besides synthetic peptides, this thesis discusses the first synthetic route towards ADP-ribosylated nucleic acids. Furthermore, two photoaffinity probes for PARP1 have been developed and assessed in living cells and two activity based probes have been synthesized, designed for CD38. Show less
Apelt, K.; Lans, H.; Scharer, O.D.; Luijsterburg, M.S. 2021
Global genome nucleotide excision repair (GG-NER) eliminates a broad spectrum of DNA lesions from genomic DNA. Genomic DNA is tightly wrapped around histones creating a barrier for DNA repair... Show moreGlobal genome nucleotide excision repair (GG-NER) eliminates a broad spectrum of DNA lesions from genomic DNA. Genomic DNA is tightly wrapped around histones creating a barrier for DNA repair proteins to access DNA lesions buried in nucleosomal DNA. The DNA-damage sensors XPC and DDB2 recognize DNA lesions in nucleosomal DNA and initiate repair. The emerging view is that a tight interplay between XPC and DDB2 is regulated by post-translational modifications on the damage sensors themselves as well as on chromatin containing DNA lesions. The choreography between XPC and DDB2, their interconnection with post-translational modifications such as ubiquitylation, SUMOylation, methylation, poly(ADP-ribos)ylation, acetylation, and the functional links with chromatin remodelling activities regulate not only the initial recognition of DNA lesions in nucleosomes, but also the downstream recruitment and necessary displacement of GG-NER factors as repair progresses. In this review, we highlight how nucleotide excision repair leaves a mark on chromatin to enable DNA damage detection in nucleosomes. Show less
Our cells are continuously challenged by numerous external as well as internal hazards that, if not dealt with in an appropriate manner, may interfere with critical processes and underlie disease.... Show moreOur cells are continuously challenged by numerous external as well as internal hazards that, if not dealt with in an appropriate manner, may interfere with critical processes and underlie disease. Therefore, they heavily rely on protective mechanisms that recognize and counteract potential risks. For example, the immune system provides a first line of defense against pathogens, which upon host invasion could cause serious illness. In addition, the well-coordinated networks of the DNA damage response detect and remove damaged nucleotides that could drive mutagenesis and thereby provoke inherited disorders, cancer or ageing-related diseases. The correct activation, execution and completion of the implicated pathways, as wel as their crosstalk, is of key importance and necessitates the well-timed and -positioned presence of proteins with the desired functionality. To a great extent, this is established by post-translational modifications (PTMs) that fine-tune protein functionality. We used a combination of biological, biochemical and microscopic approaches to determine the contribution of several PTMs to the regulation of the mechanisms that safeguard our cells. Show less
Cell division ends when two daughter cells physically separate via abscission, the cleavage of the intercellular bridge. It is not clear how the anti-parallel microtubule bundles bridging daughter... Show moreCell division ends when two daughter cells physically separate via abscission, the cleavage of the intercellular bridge. It is not clear how the anti-parallel microtubule bundles bridging daughter cells are severed. Here, we present a novel abscission mechanism. We identified chromokinesin KIF4A, which is adjacent to the midbody during cytokinesis, as being required for efficient abscission. KIF4A is regulated by post-translational modifications. We evaluated modification of KIF4A by the ubiquitin-like protein SUMO. We mapped lysine 460 in KIF4A as the SUMO acceptor site and employed CRISPR-Cas9-mediated genome editing to block SUMO conjugation of endogenous KIF4A. Failure to SUMOylate this site in KIF4A delayed cytokinesis. SUMOylation of KIF4A enhanced the affinity for the microtubule destabilizer stathmin 1 (STMN1). We here present a new level of abscission regulation through the dynamic interactions between KIF4A and STMN1 as controlled by SUMO modification of KIF4A. Show less
Small ubiquitin-like modifiers (SUMOs) are small proteins that can be covalently attached to hundreds of target proteins. This post-translational modification can alter protein binding sites and... Show moreSmall ubiquitin-like modifiers (SUMOs) are small proteins that can be covalently attached to hundreds of target proteins. This post-translational modification can alter protein binding sites and therefore influence function, localization or stability of the target protein. This thesis describes novel roles of SUMOylation in three important cellular processes, proteostasis, centromere integrity and the DNA damage response. SUMOylation promotes proteostasis after heat-shock in cooperation with the HSF-1-regulated chaperone network. Furthermore, we show that the SUMO specific isopeptidase SENP6 is responsible for the deSUMOylation of multiple subunits of the constitutive centromere-associated network (CCAN), an important protein complex that provides the basis for the assembly of the kinetochore during mitosis. Failure of deSUMOylation results in an accumulation of SUMO chains on the CCAN proteins and consequently less efficient localization to the centromere. SUMOylation has multiple previously described roles in the DNA damage response. Here, we demonstrate that the protein Cockayne Syndrome B (CSB) is the highest SUMOylated protein in response to UV-induced DNA damage. SUMOylation promotes efficient localization of CSB to the damage sites and contributes to efficient repair. Show less
Immunoglobulin G (IgG) represents the most abundant antibody class in the human circulation. IgG consists of two heavy chains and two light chains. Parts of the heavy chains, together with the... Show moreImmunoglobulin G (IgG) represents the most abundant antibody class in the human circulation. IgG consists of two heavy chains and two light chains. Parts of the heavy chains, together with the light chains, form two fragment antigen binding (Fab) moieties, whilst the remainders of the two heavy chains form the fragment crystallizable (Fc) moiety. Human IgGs are glycosylated at the highly conserved N-glycosylation site asparagine 297 in the CH2 domain of each heavy polypeptide chain of the Fc part. Fully galactosylated N-glycans are positioned between the Fc polypeptide chains, resulting in an open Fc conformation which is required for high affinity binding to Fc_ receptors. Small changes in the Fc glycosylation can already have a profound influence on the interaction of the Fc portion with receptors modulating the anti and pro-inflammatory properties of IgG. Mass spectrometry provides great opportunities for deta iled structural characterization of protein glycosylation including protein identification, determination of site-specific glycosylation profiles, and structural characterization of glycans at the level of released glycans and glycopeptides. In this thesis novel approaches for fast, miniaturized and high-throughput analysis of IgG Fc N-glycosylation are presented, and the utility of these methods has been demonstrated for clinically relevant research questions. Show less
