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For its immune system to function properly, the human body relies on a multitude of signaling processes executed by many different types cells. The most common one of these cell types is the neutrophil. Neutrophil cells patrol the blood and once encountering pathogens, they react to kill it making use of their armory contained in different types of granules. The neutrophil serine proteases are an important part of this armory and they can, for instance, be released in the phagosome containing a phagocytosed pathogen to be killed. Proteases, like the neutrophil serine proteases, are enzymes that hydrolyze peptide bonds and as such cut their substrate peptides. They can have a variety of functions but need to be tightly controlled as aberrant activity of a protease in question may lead to disease. For example, aberrant control of the neutrophil serine proteases can play a part in diseases such as chronic obstructive pulmonary disease, cancer and Alzheimer dementia. This regulation, however, means that protease presence does not necessarily equal protease activity. Therefore, activity-based probes have been developed as a special tool enabling distinguishing of active from inactive protease. To achieve this activity-direction, these small molecule probes contain an electrophilic group that reacts with the active site residue of an active protease. The probes eventually stay connected to the protease and usually also contain a tag, either a dye or a group allowing for the attachment of a dye, to make the protease-probe complex visible. Furthermore, quenched probes exist which turn fluorescent only after having reacted with a protease. In this work we design, develop and apply new activity-based probes directed against the neutrophil serine proteases. In chapter 2, we develop amino phosphinates as irreversible inhibitors for serine proteases. These are similar to amino phosphonates which find wide application as serine protease inhibitors. We establish a facile synthetic path towards the phosphinates and incorporate different carbon substituents at the phosphorus and evaluate their influence on activity, thereby assessing their reactivity and fit into the protease active site. We show that phosphinates incorporating a phenyl substituent are better inhibitors than analogous phosphonates and perform docking experiments giving evidence on a preferred configuration at the phosphorus. We then go on to use these novel phosphinate inhibitors as reactive group for serine protease activity-based probes in chapter 3. The phosphinates are fused to a linker and a tag and then used on neutrophil serine proteases neutrophil elastase, proteinase 3 and cathepsin G as well as the digestive protease chymotrypsin. We are able to separate different diastereomers of some of the probes and show that these possess distinct labeling capacities providing indirect proof for the docking results of parent inhibitors. Finally, we use some of these probes for imaging of neutrophil serine proteases in primary human neutrophils. Chapter 4 is directly related to the one before and follows a similar path. Here we apply the same phosphinate warhead but use it to develop quenched activity-based probes. These contain a quencher furnishing a dark molecule. The quencher gets expelled from the molecule upon reaction with protease restoring fluorescence. We make quenched probes for neutrophil elastase and cathepsin G, show that they can be used for purified protease labeling and labeling of protease in neutrophil lysates. Furthermore, we show unquenching of one of the probes by neutrophil elastase. This chapter establishes the phosphinate scaffold as a new and only the second group that allows synthesis of quenched activity-based probes for serine proteases. Lastly, in chapter 5 we develop quenched activity-based probes for the three most abundant neutrophil serine proteases, this time using the already established mixed phosphonate group as the reactive group. We make a set of probes, demonstrate labeling of purified protease and in neutrophil lysates as well as in the supernatant of neutrophils stimulated for degranulation. We then go on to use the probe designed to target neutrophil elastase in a live-cell imaging experiment of neutrophils undergoing NETosis. As we can see background fluorescence in this experiment, we go on to improve the probe, fitting a different quencher and dye and end with establishing its labeling capacity. Finally, we show using this new probe in a similar live-cell experiment of neutrophils undergoing NETosis. However, this experiment needs to be improved and is thus placed in chapter 6, the general discussion, as an outlook to future experiments we plan to perform.
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In this project the student will theoretically analyze how cell division is spatially and temporally coordinated. Using computational modeling, the student will first uncover how multiple functional motifs (such as bistable switches) can optimally work together to create different desired cell responses in time. Second, the student will develop models to show how the cell cycle is dynamically coordinated in space. Special attention will be paid to studying how the spatial regulation of cell division is influenced by changes in its surroundings, such as differences in size and shape of the cells, as well as in its internal structure (i.e. cytoskeleton, nucleus, and centrosomes). The modeling efforts are expected to interact synergistically with experiments that are carried out by others in the lab.
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Activity-based probes (ABPs) are small molecular tools that allow the detection and monitoring of enzyme activity by covalently binding onto the active site of the enzyme. They consist of a warhead that binds the enzyme, a recognition element that alters the selectivity of the probe and a detection tag that allows the visualization, quantification or purification of probe-labeled enzymes. ABPs are applied to elucidate enzyme function, enzyme localization and the involvement of enzymes in health and disease. They can furthermore be used as screening tools for drug candidates. To monitor the activity a single enzyme, the selectivity of an ABP has to be steered towards this target. This often requires a difficult and time-consuming synthetic process, and as a result, selective ABPs have only been developed for relatively few enzymes expressed in human cells. One enzyme family of which only a few members have selective ABPs are the serine hydrolases. To speed up the process of ABP development for serine hydrolases, solid-phase synthesis procedures that allow a fast synthesis of six classes of serine-reactive probes were developed in Chapter III. The application of these procedures is shown by the combinatorial on-resin synthesis of a library of ABPs. These probes bear a recognition element made up of an aromatic moiety and a piperidine/piperazine ring, and a serine reactive warhead. Two probes with a triazole urea warhead were found to be reactive and selective for acyl-protein thioesterases 1 and 2 (APT-1/2) in a screening on serine hydrolases present in mouse brain.Chapter IV describes the use of the solid-phase synthesis procedures established in Chapter III to synthesize a library of triazole urea ABPs, based on the APT-1/2 reactive ABPs found in the previous chapter. In this library, changes were made in the recognition elements of the probes to drive their reactivity towards one of the APT-isoforms and creating isoform-selective ABPs. Although this yielded no selective probes, several probes with a higher reactivity for both APT-isoforms than the original probes were discovered. In Chapter V, the development of ABPs for the SARS-CoV-2 main protease (Mpro) is described. Mpro is a cysteine protease that is the key player in the proteolytic maturation of the non-structural proteins of the SARS-CoV-2 virus by cleavage of its polyproteins. It has gained a lot of attention as a potential target for antiviral drugs to combat the Covid-19 pandemic. In this chapter, azapeptidic ABPs for Mpro are synthesized that can be fully made on resin. The recognition element of these probes consists of the preferred P1, P2 and P3 substrates of Mpro, of which the P1 residue is an aza-amino acid. The ABPs are capped with a cysteine-reactive warhead on the P1 aza-residue. A probe with a chloroacetamide warhead was found to be very potent in labelling Mpro and to have a very low detection limit. This probe could furthermore inhibit cell death in SARS-CoV-2 infected cells and can potentially be used for labeling Mpro in live cells. The mammalian polyamine transport system was a mystery at the start of this research project, as no proteins had been identified as polyamine transporters. Chapter VI describes the synthesis of azide-functionalized polyamines, which were converted to fluorescent probes through "click" chemistry with an alkyne-BODIPY fluorophore. Through a SAR study with polyamine probes with different lengths, different positionings of the fluorophore and a different amount of positive charges, it was found that probes in which the fluorophore is attached to an outer amine of the polyamine get taken best into cells. These polyamine probes have since been used by other research groups in the identification of polyamine transporters. In summary, this thesis describes the synthesis and validation of probes that target APT-1/2, SARS-CoV-2 Mpro and the polyamine transport system. These probes can be synthesized efficiently via solid-phase synthesis or click chemistry. The developed solid-phase synthesis procedures are applicable for the synthesis of probes for other targets as well.
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The ubiquitously expressed Cx43 isoform is one of the major building blocks of gap junction channels or hemichannels in the heart, brain and bone. Gap junction channels connect the cytoplasm of two neighboring cells, thereby exchanging signaling molecules and metabolites, whereas hemichannels mediate paracrine signaling by releasing ATP and other signaling molecules into the extracellular environment. The opening of Cx43 hemichannels must be tightly controlled to avoid loss of metabolic, energetic and ionic gradients, resulting in cell demise.The regulation of Cx43-based gap junctions has been extensively studied, revealing that their activity is controlled by intramolecular interactions between the C-terminal tail (CT) of Cx43 and the second part of the intracellular loop (L2). A “ball-and-chain” model was proposed in which binding of the CT tail to the L2 region results in closing of the Cx43-based gap junctional channels. Until recently, the mechanisms controlling Cx43-hemichannel activity remained unresolved. However, our lab found that similar loop/tail interactions exist in Cx43 hemichannels as in gap junctions but with an opposite functional outcome, whereby loop/tail interactions are essential for Cx43-hemichannel opening. Loss of loop/tail interactions, either by genetically deleting the CT of Cx43 or by physiological conditions (like increases in [Ca2+]i of 1 µM and higher) that elicit actomyosin contractility, results in loss of Cx43-hemichannel activity. This can be overcome by the last 9 amino acids of the CT (CT9), which targets the L2 region, and more specifically the Gap19 region within this sequence, in Cx43.First, we identified the residues present in the CT9 region responsible for L2 interaction and for mediating Cx43-hemichannel activity. We found that the interaction of the CT9 region with the L2 region critically depended on the presence of Asp378 and Asp379 residues and of Pro375 and Pro377 residues. Also, altering these residues in CT9 into Ala residues rendered TAT-CT9 (a cell-permeable version of CT9) ineffective in restoring the hemichannel activity of Cx43M239-based hemichannels (CT-deleted variant) and Cx43-hemichannel activity in conditions of actomyosin contractility. These findings correlate with the highly positively charged Gap19 region within the L2 domain, the presumed target of CT9.Second, despite the fact that only the last 9 amino acids were sufficient to restore the activity of Cx43-hemichannels lacking the complete CT, deletion of this region in the CT of Cx43 only mildly impacted the interaction with Gap19/L2. We therefore hypothesized that another region within the CT contributes to such interaction. Here, we identified the SH3-binding domain as a second region within the CT, contributing to the loop/tail interaction. TAT-SH3 peptide was able to restore the activity of CT-truncated Cx43-hemichannels or of Cx43-hemichannels in conditions of increased actomyosin contractility. Furthermore, the ability of TAT-SH3 to restore CT-truncated Cx43-hemichannels depended on the presence of Gap19, since deletion of the latter rendered the channels resistant to functional modulation by TAT-SH3. Deletion of either the SH3-binding domain or the CT9 domain in Cx43 reduced hemichannel activity, while deletion of both domains completely abolished Cx43-hemichannel activity.Third, we know that the CT of Cx43 is not only important for channel regulation, but is also important for Cx43 trafficking. Here, we studied the contribution of autophagy, a lysosomal degradation pathway, for Cx43-hemichannel turnover by exposing cells to nutrient starvation, a condition known to trigger autophagic flux. Our data show that nutrient starvation results in a rapid decline of Cx43-protein levels, both as gap junctions and hemichannels, and of Cx43-hemichannel function, which could be partially rescued by Bafilomycin A1, an autophagy inhibitor acting at the level of the lysosomes.In conclusion, our study (i) identified key residues present in the CT9 region responsible for functional modulation of Cx43-hemichannels; (ii) elucidated the SH3-binding domain as a novel determinant underlying loop/tail interactions critical for Cx43-hemichannel function; and (iii) proposed autophagy as a turnover pathway of Cx43-hemichannels in particular in conditions of nutrient starvation.
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The cell division cycle is a crucial biological process, essential for development and survival of all organisms. In eukaryotes, the cell cycle is regulated by an intricate network of genes and proteins that controls the progress of the cycle. All of these interactions give rise to dynamical features such as bistability and time delay which aid in the coordination of cell cycle events. Understanding this complex interplay has been helped by mathematical models, which describe how the concentrations and activities of different components of this network change over time.In this thesis, we describe our results on different dynamical elements that play a role in the cell cycle. We have four different chapters with results. In the first chapter we investigate the role of time delay in the oscillations that drive the early embryonic cell cycle. This time delay acts between the important mitotic kinase Cdk1 and a protein complex called the APC/C. The delay has been measured experimentally, but its origin and exact role are not yet clear. In a simple model, we show how ultrasensitivity and delay together determine whether oscillations exist. Importantly, we illustrate that different implementations of the time delay may alter the conclusions. At the end of this chapter we describe a method to turn the ultrasensitive response into a bistable response, and briefly describe the implications of this method.In the second results chapter we show how such a bistable response curve can change dynamically in time. We use mitotic entry as a motivating example to show that bistable response curves can become time dependent if different compartments are introduced in an existing cell cycle model. We then explore the consequences of a changing bistable switch and show that it may provide robustness to cellular transitions and oscillations.The third results chapter starts from an experimental observation, namely that the timing of the early embryonic cell cycle has a particular dependence on temperature. We explore how this scaling can be explained by the dynamics of the cell cycle oscillator. For this, we make the reaction rates of two different cell cycle models dependent on temperature, and explore how different sensitivities of different rates can reproduce the observed scaling. This chapter is the report of work in progress.The final results chapter puts oscillators based on time delay and bistability in a spatial context. Such systems can produce traveling waves, and these waves are often generated by pacemakers: regions which oscillate faster than their surroundings. In biological systems, such waves can function to transmit information, and an important question is which elements of the oscillator and the pacemaker determine the speed of these waves. We answer this question using numerical simulation and analytical methods. In this way, we show that timescale separation is important for the speed, but only in the oscillator based on bistability. Moreover, we explain how the size and frequency difference of the pacemaker affect the speed of the waves, and the speed by which they permeate the rest of the medium. At the end of this chapter, we briefly explain what happens in a system where multiple pacemakers compete to entrain the medium.
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This project aspires 1. to demonstrate cancer cells dual addiction to anti-apoptotic Bcl-2 by exploiting the properties of BH3 mimetics and BH4-targeting tools 2. to screen and/or develop novel, in vivo applicable petidomimetics and TAT-IDPs peptide derivatives targeting Bcl-2 via its BH4-domain via a Celltox-TM Green Cytotoxicity Assay 3. to establish the effect of TAT-IDPs and derivatives on the survival and function of platelets from healthy volunteers and cancer patients.
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PP2A-related intellectual disability (ID) is a recently discovered syndrome, characterized by neurodevelopmental delay and various comorbidities. Thus far, causal mutations have been identified in the structural Aα and regulatory B56δ, -β and -γ subunits. In this thesis, we report 16 individuals with mutations in the catalytic Cα subunit, resulting in ID, often accompanied by seizures, behavioral problems and brain malformations. Biochemical analysis revealed divergent binding defects to both the structural A, and several regulatory B-type subunits. Some variants were thought to behave as dominant-negative to certain B-type holoenzymes, whereas others induced haploinsuffiency, often associated with a milder phenotype. In parallel, newly identified variants in the B56δ subunit were biochemically characterized and found to frequently induce binding deficiencies towards the PP2A core dimer, being the catalytic C and structural A subunits. Moreover, a set of selected B56δ variants showed significantly reduced interaction with several substrates, such as liprin-α1 and -α3, PRR14 and PRR14L, and phosphorylation of the Ser573 residue appeared to be dysregulated for four out of six tested B56δ mutations, suggesting a potential downstream effect on signaling. However, besides a, possibly artificial, inability to stabilize ERK Thr185/Tyr187 phosphorylation in an overexpression set-up and an induction of the mTOR pathway for the p.E420K variant, no general signaling defect could be clearly linked to these B56δ mutations. Finally, a mouse model heterozygously expressing the most common variant, B56δ p.E198K, was significantly smaller than its wild type counterpart, with no measurable effects on brain size. Furthermore, preliminary analysis using embryonic stem cells generated from this model showed a delayed cortical development. Together, these data provide substantial insight in PP2A-related ID's pathology, imperative in the further deciphering of causal mechanisms in light of potential therapeutic opportunities.
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Dynamic events of protein and lipid phosphorylation and dephosphorylation are involved virtually in all cellular signaling networks and transduction pathways. Kinases are the enzymes that catalyze phosphorylation reactions, while phosphatases are responsible for phosphate hydrolysis from the substrates. Defective or inappropriate activity of phosphatases contributes to the development of many human diseases, including cancer. PRL-3 is a plasma membrane-associated dual specificity phosphatase that exhibits a highly restricted and tightly regulated expression pattern. Additionally, PRL-3 is an emerging prognostic marker for cancer progression and a promising therapeutic target. Although in the last decade numerous clinical studies have extensively validated the causative role of PRL-3 in cancer metastasis, to date its substrate(s) and the direct downstream transduction pathways affected by PRL-3 overexpression are still elusive.The first part of this work concerns the in vitro characterization of PRL-3 phosphatase activity towards phosphopeptides and phosphoinositides. We show that PRL-3 does not present any activity against the library of phosphopeptides tested, while it robustly dephosphorylates the phosphoinositide PtdIns(4,5)P2. Moreover, our experimental results and molecular docking studies suggest that PRL-3 is a phosphatidylinositol 5-phosphatase. Finally, structure-activity relationship studies correlate the PRL-3 phosphatase activity toward PI(4,5)P2 with its ability to promote cell migration.The second part of this work concerns the characterization of PRL-3 oncogenic activity with respect to epithelial cell polarity. Using an organotypic 3D-culture system, in which epithelial cells form highly organized spherical cysts, we show that overexpression of PRL-3 significantly affects epithelial morphogenesis leading to the development of cysts with ectopic lumens. Moreover, we prove that a remnant organelle from the cytokinesis process, the post-mitotic midbody, is the first polarization signal during cyst development. Finally, we show that PRL-3 over- expression generates major defects in epithelial cell polarization by interfering with the fate of post-mitotic midbodies.In conclusion, we show that PRL-3 could be a new phosphoinositide phosphatase and that its overexpression affects epithelial cell polarization by altering post-mitotic midbody fate, suggesting a novel mechanism for epithelial tumorigenesis.
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