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
Drug candidates with a covalent binding mode have gained interest since the approval of multiple covalent anticancer drugs, but were long avoided due to concerns regarding promiscuous reactivity... Show moreDrug candidates with a covalent binding mode have gained interest since the approval of multiple covalent anticancer drugs, but were long avoided due to concerns regarding promiscuous reactivity with off-target proteins. In this dissertation, the scope and versatility of the newly discovered in situ thiol–alkyne reaction is evaluated: the nonactivated alkynes exhibit an unprecedented target reactivity with excellent thiol selectivity, thus potentially outperforming currently used cysteine-reactive warheads. Chapter 1 starts with the history of (ir)reversible covalent inhibition, the reactivity of (non)activated alkynes, and the serendipitous discovery of the thiol–alkyne reaction. Established technologies for direct detection of covalent protein–drug adducts are reviewed in Chapter 2, and in Chapter 3 a detailed guide for the evaluation of (ir)reversible covalent inhibitors to obtain relevant kinetic parameters is provided, accompanied by kinetic simulations and step-wise protocols for enzymatic activity assays. In Chapter 4, the nitrile warhead in reversible CatK inhibitor odanacatib (ODN) is replaced with alkyne warheads to investigate whether it an irreversible covalent adduct is formed with cysteine protease cathepsin K (CatK) despite having a small recognition element. In Chapter 5, we evaluate if nonactivated alkynes can target noncatalytic cysteine residues by replacing the irreversible covalent acrylamide warhead in EGFR/HER2 inhibitor neratinib by an alkyne warhead. In Chapter 6, the impact of substituents on the alkyne warhead is explored using a panel of ubiquitin-based ABPs bearing substituents on the propargylamide warhead. Finally, the most important findings are summarized in Chapter 7, and placed in the context of covalent drug discovery. Show less
Cells are home to a variety of organelles, each with a specialized role that supports cellular homeostasis. As the largest cellular organelle, the endoplasmic reticulum (ER) plays a coordinating... Show moreCells are home to a variety of organelles, each with a specialized role that supports cellular homeostasis. As the largest cellular organelle, the endoplasmic reticulum (ER) plays a coordinating role in intracellular architecture by communicating with other organelles through membrane contact sites. This thesis describes the function of an ER contact site-localized E3 ubiquitin ligase, RNF26, that controls the positioning of other organelles in space and time. Through a combination of microcopy and biochemistry, we have identified key regulators of RNF26 that together control essential cell biological processes such as growth hormone trafficking and proteostasis in response to cellular stress, and describe the role of the ER and ubiquitination on other processes such as calcium trafficking and antigen presentation. Altogether, this work unravels the molecular machinery central to intracellular architecture and its consequences for organelle biology. Show less
Cells constitute the tissues of our body and are responsible for producing various changes in response to different situations. For instance, the repair of damaged DNA. DNA resides within the cell... Show moreCells constitute the tissues of our body and are responsible for producing various changes in response to different situations. For instance, the repair of damaged DNA. DNA resides within the cell nucleus and can be transcribed and translated into proteins, which play vital roles in numerous cellular processes. The cell relies on modifying existing proteins to carry out essential functions. These modifications can involve the conjugation of small molecules such as Ubiquitin (Ub) or Small Ubiquitin-like Modifiers (SUMOs), leading to protein degradation, conformational changes or intracellular relocation of critical proteins. The conjugation of these small molecules involves a well-orchestrated sequence of enzymatic activities performed by dedicated enzymes: E1 (activating), E2 (conjugating) and E3 (ligase). Among these, the E3 ligase enzymes hold significant importance as they confer substrate specificity.In this thesis, we have developed an advanced Mass-Spectrometry technology called TULIP2 (Targets for Ubiquitin Ligases Identified by Proteomics 2), which facilitates the identification of Ubiquitination targets for specific E3 ligases of interest. Using this technology, we have investigated the BRCA1-BARD1 E3 ligase and explore the in vivo role of the E2 UBE2D3. Furthermore, we have adapted the TULIP2 technology to create the SUMO Activated Target Traps (SATTs), enabling the identification of an E3-specific SUMO proteome. Show less
The research described in this thesis aims at the development of ubiquitin-based research tools to study the enzymes of the ubiquitination pathway, the ligase enzymes and the deubiquitinating... Show moreThe research described in this thesis aims at the development of ubiquitin-based research tools to study the enzymes of the ubiquitination pathway, the ligase enzymes and the deubiquitinating enzymes. These enzymes are responsible for the conjugation and the removal of the post-translational modifier ubiquitin. This small protein is involved in almost all cellular processes, and when conjugated onto a substrate protein it can signal for degradation and influence the localization, interaction, stability and activity of the protein. Therefore, the dysregulation of these processes can have detrimental effects of cell organization and survival which in turn has implications in numerous processes related to diseases. Hence, it is important to fully understand the ubiquitination pathway and how to interact with it. The ubiquitin-based research tools described in this thesis aim to shine light on parts of this pathway. Ranging from the selectivity and specificity of DUBs for specific Ub linkages in competition and the catalytic efficiency of these proteolytic cleavage processes to the selectivity and activity of ligases and the activity of DUBs in cells. All ubiquitin research tools are based on synthetic ubiquitin modified with unnatural amino acids, neutron-encoded amino acids, point mutations and/or fluorescent labels, in order to study the characteristics of the enzymes in vitro. Show less
This work involves the development of novel technologies to interrogate the complex interactions between eight differently linked diubiquitin chains and the ubiquitin binding domains (UBDs) or full... Show moreThis work involves the development of novel technologies to interrogate the complex interactions between eight differently linked diubiquitin chains and the ubiquitin binding domains (UBDs) or full-length proteins that are able to recognize them in a linkage-specific manner. Chapter 2 describes the development of a high-throughput (HT) fluorescence polarization assay used to discover alpha-helical ubiquitin binding domains. This leads to validation of known interacting binding domains and the discovery of a novel set of K6-linkage specific UBDs. In Chapter 3, all eight diubiquitin proteins are prepared using click chemistry that renders the diubiquitin proteins immune to proteolysis by deubiquitinases (DUBs). This allowed for pulldown efforts in different cell lysates and identification of novel linkage-specific interacting proteins using tandem mass spectrometry. The procedure of this effort is outlined in Chapter 4. The study in Chapter 5 details the interaction of one of the major hits found in Chapter 3: the linkage-specific interaction of K27 diubiquitin and UCHL3. The development of a panel of specific assay reagents combined with X-ray crystallography and kinetic modelling lead to a proposed model in which the consequences of the interaction between K27Ub2 and UCHL3 are explained. In Chapter 6 future directions are offered. Show less
Berlin, I.; Sapmaz, A.; Stevenin, V.; Neefjes, J. 2023
The endolysosomal system comprises a dynamic constellation of vesicles working together to sense and interpret environmental cues and facilitate homeostasis. Integrating extracellular information... Show moreThe endolysosomal system comprises a dynamic constellation of vesicles working together to sense and interpret environmental cues and facilitate homeostasis. Integrating extracellular information with the internal affairs of the cell requires endosomes and lysosomes to be proficient in decision-making: fusion or fission; recycling or degradation; fast transport or contacts with other organelles. To effectively discriminate between these options, the endolysosomal system employs complex regulatory strategies that crucially rely on reversible post-translational modifications (PTMs) with ubiquitin (Ub) and ubiquitin-like (Ubl) proteins. The cycle of conjugation, recognition and removal of different Ub-and Ubl-modified states informs cellular protein stability and behavior at spatial and temporal resolution and is thus well suited to finetune macromolecular complex assembly and function on endolysosomal membranes. Here, we discuss how ubiquitylation (also known as ubiquitination) and its biochemical relatives orchestrate endocytic traffic and designate cargo fate, influence membrane identity transitions and support formation of membrane contact sites (MCSs). Finally, we explore the opportunistic hijacking of Ub and Ubl modification cascades by intracellular bacteria that remodel host trafficking pathways to invade and prosper inside cells. Show less
In this thesis we explore the Ubiquitin code using purification methods coupled with mass spectrometry. We overview the available methods and current knowledge of of Ubiquitin and SUMO... Show moreIn this thesis we explore the Ubiquitin code using purification methods coupled with mass spectrometry. We overview the available methods and current knowledge of of Ubiquitin and SUMO modifications with examples of substrates where the precise site of modification is important, and substrates where the modification site seems interchangeable.We show that the deubiquitinating enzymes regulate a separate subset of the ubiquitinome than the proteasome. The PARylating enzyme PARP1 show increased activity after ubiquitination, which is regulated by deubiquitinating enzymes.Furthermore, we developed an in-vivo tool to study SUMO dependent interaction using proximity labeling which can be used for microscopy or identification by mass spectrometry.Finally, we investigate binders of Ubiquitin and SUMO chains with specific linkages and elucidate a preference for some chain linkages based on biological pathway of the binder. We also explore possible binding domains and predict the structure of some chain binding complexes using protein docking. Show less
Ubiquitin and SUMO modify thousands of substrates to regulate most cellular processes. System-wide identifi-cation of ubiquitin and SUMO substrates provides global understanding of their cellular... Show moreUbiquitin and SUMO modify thousands of substrates to regulate most cellular processes. System-wide identifi-cation of ubiquitin and SUMO substrates provides global understanding of their cellular functions. In this review, we discuss the biological importance of site-specific modifications by ubiquitin and SUMO regulating the DNA damage response, protein quality control and cell cycle progression. Furthermore we discuss the machinery responsible for these modifications and methods to purify and identify ubiquitin and SUMO modified sites by mass spectrometry. We provide a framework to aid in the selection of appropriate purification, digestion and acquisition strategies suited to answer different biological questions. We highlight opportunities in the field for employing innovative technologies, as well as discuss challenges and long-standing questions in the field that are difficult to address with the currently available tools, emphasizing the need for further innovation. Show less
Proteins play an essential role in almost all the processes of a living organism, and post-translational modifications (PTM) can regulate their structure, location, function, and fate. Ubiquitin... Show moreProteins play an essential role in almost all the processes of a living organism, and post-translational modifications (PTM) can regulate their structure, location, function, and fate. Ubiquitin and ISG15 are two of the most versatile and common PTM modifiers in mammalian cells. Aberrant modification of Ubiquitin and ISG15 in cells results in various diseases, such as cancers and microbial infections. This dissertation introduces the development of small-molecule inhibitors against ISG15 deconjugating enzyme USP18 and ubiquitin deconjugating enzyme OTUB2 separately, and the identification of USP16 as a dual deconjugating enzyme that cleaves Ubiquitin and ISG15 from substrates. These research achievements are supposed to deepen the understanding of biology, regulation, and biochemical mechanisms of Ubiquitin and ISG15 deconjugating enzymes, thus paving the way for deconjugating enzymes-targeted therapeutics development in the future. Show less
DNA damage-induced SUMOylation serves as a signal for two antagonizing proteins that both stimulate repair of DNA double-strand breaks (DSBs). Here, we demonstrate that the SUMO-dependent... Show moreDNA damage-induced SUMOylation serves as a signal for two antagonizing proteins that both stimulate repair of DNA double-strand breaks (DSBs). Here, we demonstrate that the SUMO-dependent recruitment of the deubiquitylating enzyme ataxin-3 to DSBs, unlike recruitment of the ubiquitin ligase RNF4, additionally depends on poly [ADP-ribose] polymerase 1 (PARP1)-mediated poly(ADP-ribosyl)ation (PARylation). The co-dependence of ataxin-3 recruitment on PARylation and SUMOylation temporally confines ataxin-3 to DSBs immediately after occurrence of DNA damage. We propose that this mechanism ensures that ataxin-3 prevents the premature removal of DNA repair proteins only during the early phase of the DSB response and does not interfere with the subsequent timely displacement of DNA repair proteins by RNF4. Thus, our data show that PARylation differentially regulates SUMO-dependent recruitment of ataxin-3 and RNF4 to DSBs, explaining how both proteins can play a stimulatory role at DSBs despite their opposing activities. Show less
Ubiquitin (Ub) is a small post-translational modifier protein involved in a myriad of biochemical processes including DNA damage repair, proteasomal proteolysis, and cell cycle control. Ubiquitin... Show moreUbiquitin (Ub) is a small post-translational modifier protein involved in a myriad of biochemical processes including DNA damage repair, proteasomal proteolysis, and cell cycle control. Ubiquitin signalling pathways have not been completely deciphered due to the complex nature of the enzymes involved in ubiquitin conjugation and deconjugation. Hence, probes and assay reagents are important to get a better understanding of this pathway. Recently, improvements have been made in synthesis procedures of Ub derivatives. In this perspective, we explain various research reagents available and how chemical synthesis has made an important contribution to Ub research. Show less
This thesis spans the development of activity-based probes targeting the enzymes of the Ubiquitin and Ubiquitin-like cascade, their application and the exploration of the biological function of an... Show moreThis thesis spans the development of activity-based probes targeting the enzymes of the Ubiquitin and Ubiquitin-like cascade, their application and the exploration of the biological function of an understudied modification—UFM1. While the first chapter describes the attempt to introduce an unnatural amino acid into proteins to enable covalent substrate capture, the subsequent chapter reports on a unique cascading activity-based probe capable of being relayed through the enzyme cascade. The repertoire of activity-based probes was later expanded to include the Ubiquitin-like modifiers SUMO and UFM1. Additionally, the enigmatic role of UFM1 was deciphered by adapting a proteomics method previously used for SUMO, to uncover UFM1-modifed substrates. This approach enabled the dissection of a relevant pathway governed by UFMylation—ribosomal function during SRP-mediated protein translocation. Show less
Deubiquitinases (DUBs) are a family of enzymes that regulate the ubiquitin signaling cascade by removing ubiquitin from specific proteins in response to distinct signals. DUBs that belong to the... Show moreDeubiquitinases (DUBs) are a family of enzymes that regulate the ubiquitin signaling cascade by removing ubiquitin from specific proteins in response to distinct signals. DUBs that belong to the metalloprotease family (metalloDUBs) contain Zn2+ in their active sites and are an integral part of distinct cellular protein complexes. Little is known about these enzymes because of the lack of specific probes. Described here is a Ub‐based probe that contains a ubiquitin moiety modified at its C‐terminus with a Zn2+ chelating group based on 8‐mercaptoquinoline, and a modification at the N‐terminus with either a fluorescent tag or a pull‐down tag. The probe is validated using Rpn11, a metalloDUB found in the 26S proteasome complex. This probe binds to metalloDUBs and efficiently pulled down overexpressed metalloDUBs from a HeLa cell lysate. Such probes may be used to study the mechanism of metalloDUBs in detail and allow better understanding of their biochemical processes. Show less
The capability of cells to divide is essential for all organisms, while uncontrolled cell proliferation can have detrimental effects resulting in diseases like cancer. Cell division is... Show moreThe capability of cells to divide is essential for all organisms, while uncontrolled cell proliferation can have detrimental effects resulting in diseases like cancer. Cell division is therefore tightly controlled by regulatory mechanisms. Post-translational modifications (PTMs) are able to directly change the function of a protein and thereby provide a quick functional switch. This thesis focusses on the roles of small ubiquitin-like modifiers (SUMOs) and their crosstalk with other post-translational modifications during cell division, at the proteome-wide level as well as the single target protein level. Show less
The work described in this thesis provides novel insights into the structural and (multi)functional characteristics of arterivirus PLP2. This enzyme plays an essential role in the viral replication... Show moreThe work described in this thesis provides novel insights into the structural and (multi)functional characteristics of arterivirus PLP2. This enzyme plays an essential role in the viral replication cycle by cleaving the viral replicase polyproteins. In addition, there were indications that PLP2 is able to influence certain cellular processes by cleaving ubiquitin. We have now shown that PLP2 indeed functions as a deubiquitinating enzyme (DUB) and that this activity is important for the suppression of the innate immune response in the cell. To be able to separate both functions of PLP2 we have solved the crystal structure of this enzyme in complex with ubiquitin. Based on this structure, we were able to design mutations in PLP2 that selectively disrupt the interaction with ubiquitin, without interfering with cleavage of the viral polyproteins. Using these mutants, we have demonstrated for the first time the importance of a viral DUB in the evasion of innate immunity in the context of an infection. The acquired knowledge can now be applied to the design of improved arterivirus vaccines and studies of other viral DUBs, including those encoded by the zoonotic coronaviruses that cause SARS and MERS. Show less
Post translational modifications (PTMs) are orchestrated by highly active and reversible enzymatic systems to regulate the functional diversity of proteins. Because of their dynamic nature, PTMs... Show morePost translational modifications (PTMs) are orchestrated by highly active and reversible enzymatic systems to regulate the functional diversity of proteins. Because of their dynamic nature, PTMs are used by the cell as a controllable system to regulate a wide variety of processes. Studying modifications of proteins will give us more insight in how the cell uses PTMs to regulate cellular processes and how different PTMs act together to adjust the function of proteins. The research described in this thesis focuses one of these PTMs, the Small ubiquitin-Like Modifier (SUMO). SUMOs are proteins that are covalently attached to lysines in target proteins. These studies have uncovered hundreds of SUMO target proteins and acceptor sites and revealed a role for SUMOylation in protein degradation, cell cycle progression and DNA repair Show less
