Conditional Degrons to Study Gene Functions During Saccharomyces cerevisiae Gametogenesis and Proliferation

Diploid cells of Saccharomyces cerevisiae can form stable spores to ensure survival under poor nutritional conditions. Sporulation is a coupled developmental program of meiotic divisions and spore formation. The latter process is initiated at onset of meiosis II at the spindle pole bodies (SPBs), the yeast centrosome equivalents. The SPBs are embedded in the nuclear envelope and duplicate twice during meiosis in a mostly conservative fashion. Thus, three generations of SPBs are present in meiosis II. The first SPB inherited from mitosis, the second formed in meiotic pro-phase and the two youngest SPBs generated prior to meiosis II. At the onset of meiosis II the cytoplasmic faces of the SPBs are modified by meiotic plaques. They serve as nucleation platform for the prospore membranes, which grow around the nuclear lobes and close after meiosis II spindle breakdown. The spore wall is then formed in the lumen of the double-layered prospore membrane. Finally, the former mother cell collapses and forms the spore-containing ascus. Cells are able to adjust the spore numbers according to the available nutrients by reducing meiotic plaque protein levels to generate asci with less than four spores. This regulation is facilitated by meiosis II spindle polarity, which directs meiotic plaque formation towards the younger SPBs. Yet, the underlying mechanisms are poorly understood, although this process significantly contributes to preservation of genetic variability and population fitness by ensuring encapsulation of non-sister chromosomes in asci with only two spores. Here, I developed different synthetic tools to study the role of the mitotic exit network (MEN) in meiotic spindle polarity and spore number control of S. cerevisiae. The MEN is a conserved signaling cascade essential for vegetative growth. It coordinates mitotic exit with genome segregation and cytokinesis and establishes mitotic spindly polarity in metaphase. However, the meiotic functions of this network are mainly unknown due to the lack of reliable methods for creation of meiosis-specific mutants of the mainly essential proteins of the MEN. To overcome this obstacle, I pursued two different approaches to control the abundance of a protein with sequences inducing conditional degradation (degrons). 1. I established a photo-sensitive degron module which combines the LOV2 photoreceptor domain of Arabidopsis thaliana phototropin 1 attached to a synthetic C-terminal degron. In the dark, this degron is sterically inaccessible. Upon blue-light illumination, structural rearrangements of the LOV2 domain lead to activation of the degron and degradation of the target protein it is fused to. 2. I improved an established system for protein destabilization, which employs tobacco etch virus (TEV) protease to activate a cryptic degron. Control of protease production by a meiosis-specific promoter has been used previously to study protein functions during sporulation. To develop a more efficient system, I followed two strategies in parallel: by directed evolution, I created a TEV protease variant with a higher substrate tolerance, allowing usage of stronger degrons. Independently, I combined transcriptional shut-off of the target gene upon initiation of meiosis with elevated protease levels during sporulation. The latter approach was used successfully to create meiosis-specific mutants of all core MEN components. I could demonstrate a role of the MEN in age-based selection of SPBs for meiotic plaque modification. Moreover, I found functional diversification of MEN components during sporulation. The upstream kinase Cdc15 is involved in regulation of meiotic plaque numbers and prospore membrane closure, while Cdc15 and the downstream kinase complexes consisting of Dbf2/20-Mob1 are all necessary for SPB selection at the onset of meiosis II. After the meiotic divisions, efficient genome inheritance requires Dbf2/20-Mob1 during subsequent spore wall formation. Together, these data reveal a developmental-specific plasticity of the signaling network. In contrast to mitosis, execution of meiosis does not require the MEN but faithful genome inheritance requires concerted action of different MEN components at distinct steps of spore formation

Programmable protein circuits in living cells

Synthetic protein-level circuits could enable engineering of powerful new cellular behaviors. Rational protein circuit design would be facilitated by a composable protein-protein regulation system in which individual protein components can regulate one another to create a variety of different circuit architectures. In this study, we show that engineered viral proteases can function as composable protein components, which can together implement a broad variety of circuit-level functions in mammalian cells. In this system, termed CHOMP (circuits of hacked orthogonal modular proteases), input proteases dock with and cleave target proteases to inhibit their function. These components can be connected to generate regulatory cascades, binary logic gates, and dynamic analog signal-processing functions. To demonstrate the utility of this system, we rationally designed a circuit that induces cell death in response to upstream activators of the Ras oncogene. Because CHOMP circuits can perform complex functions yet be encoded as single transcripts and delivered without genomic integration, they offer a scalable platform to facilitate protein circuit engineering for biotechnological applications

Programmable protein circuits in living cells

Synthetic protein-level circuits could enable engineering of powerful new cellular behaviors. Rational protein circuit design would be facilitated by a composable protein-protein regulation system in which individual protein components can regulate one another to create a variety of different circuit architectures. In this study, we show that engineered viral proteases can function as composable protein components, which can together implement a broad variety of circuit-level functions in mammalian cells. In this system, termed CHOMP (circuits of hacked orthogonal modular proteases), input proteases dock with and cleave target proteases to inhibit their function. These components can be connected to generate regulatory cascades, binary logic gates, and dynamic analog signal-processing functions. To demonstrate the utility of this system, we rationally designed a circuit that induces cell death in response to upstream activators of the Ras oncogene. Because CHOMP circuits can perform complex functions yet be encoded as single transcripts and delivered without genomic integration, they offer a scalable platform to facilitate protein circuit engineering for biotechnological applications

Photocaged amino acids for photoinducible protein-protein interactions in Caenorhabditis elegans

Natural proteins are polymers of the twenty canonical amino acids. The twenty canonical amino acids have a variety of structures, and while sufficient to build the observed versatility of protein functions, they represent a miniscule portion of the possible amino acid structural space. The rest of this space is occupied by noncanonical amino acids (ncAAs). ncAAs can be designed to impart novel chemistries to proteins for specific tasks and can be incorporated into proteins in vivo by the method of Genetic Code Expansion (GCE). Photocaged amino acids are a variety of ncAA that can install photocontrol over incorporated protein function. Photocaged amino acids consist of a canonical amino acid structure conjugated with a photosensitive aromatic moiety called a caging group that is removed by illumination with 365 nm light. The caging group can prevent protein function, but allows function to be regained upon its removal by 365 nm illumination. In the nematode worm Caenorhabditis elegans, photocaged amino acids have been used for single-cell activation of CRE recombinase in induce gene expression and caspase-8 to induce apoptosis. In this thesis, I explore the application of photocaged amino acids to control protein-protein interactions in C. elegans. Photocaged protein interactions could be used to install photocontrol over protein localisation, which is a viable strategy for controlling protein activity in the case that activity can’t be controlled by the photocaging of a catalytic residue. In Chapter 1, I give a general introduction to the thesis. I begin with a background on translation, that process which is central to GCE. I describe translation’s key components – aminoacyl-tRNA synthesases, tRNAs, ribosomes, and the degenerate genetic code – and how each has been adapted for the incorporation of ncAAs. Finally, the model organism C. elegans is detailed: its history, traits, and uses, particularly in the context of GCE with photocaged amino acids. In Chapter 2, photocaged amino acids are used to install photocontrol over nanobody/GFP interactions. Nanobodies are small, single domain antibodies that bind targets with high specificity and strength, and are efficiently expressed in eukaryotic cytoplasm. Two anti-GFP nanobodies have been identified to have tyrosine residues that appear integral to GFP binding. Substitution of these tyrosine residues with photocaged tyrosines does not break GFP binding, and therefore fails to achieve photoinducibility. I use computational alanine scanning to identify other nanobody residues that when mutated could attenuate GFP binding to achieve photoinducibility. These mutations in combination with photocaging were found to achieve photoinducible GFP binding. Chapter 3 describes an alternate approach for using photocaged amino acids to control protein interactions. When the SpyTag and SpyCatcher proteins interact, an isopeptide bond is formed between them. Replacing one of the catalytic lysine residues on SpyCatcher with a photocaged lysine could install photocontrol over the interaction between these proteins. Preliminary results suggest photoinducible binding is achieved by this photocaging, but substantial work is required to reduce background synthesis of non-photocaged SpyCatcher. In Chapter 4, a potential application of photocaged interactions for photoinducible protein degradation is investigated. Ubiquitin ligase proteins such as TIR-1 have been used to induce ubiquitination and subsequent degradation of proteins in C. elegans. Preliminary results suggest that an autoinhibited TIR-1 could be activated by TEV protease. A photocaged TEV protease has been reported in mammalian cells, and could provide a route to photoactivation of the autoinhibited TIR-1 for photoinducible protein degradation

Programming Bacteria With Light—Sensors and Applications in Synthetic Biology

Photo-receptors are widely present in both prokaryotic and eukaryotic cells, which serves as the foundation of tuning cell behaviors with light. While practices in eukaryotic cells have been relatively established, trials in bacterial cells have only been emerging in the past few years. A number of light sensors have been engineered in bacteria cells and most of them fall into the categories of two-component and one-component systems. Such a sensor toolbox has enabled practices in controlling synthetic circuits at the level of transcription and protein activity which is a major topic in synthetic biology, according to the central dogma. Additionally, engineered light sensors and practices of tuning synthetic circuits have served as a foundation for achieving light based real-time feedback control. Here, we review programming bacteria cells with light, introducing engineered light sensors in bacteria and their applications, including tuning synthetic circuits and achieving feedback controls over microbial cell culture

The Potential of Proteolytic Chimeras as Pharmacological Tools and Therapeutic Agents

The induction of protein degradation in a highly selective and efficient way by means of druggable molecules is known as targeted protein degradation (TPD). TPD emerged in the literature as a revolutionary idea: a heterobifunctional chimera with the capacity of creating an interaction between a protein of interest (POI) and a E3 ubiquitin ligase will induce a process of events in the POI, including ubiquitination, targeting to the proteasome, proteolysis and functional silencing, acting as a sort of degradative knockdown. With this programmed protein degradation, toxic and disease-causing proteins could be depleted from cells with potentially effective low drug doses. The proof-of-principle validation of this hypothesis in many studies has made the TPD strategy become a new attractive paradigm for the development of therapies for the treatment of multiple unmet diseases. Indeed, since the initial protacs (Proteolysis targeting chimeras) were posited in the 2000s, the TPD field has expanded extraordinarily, developing innovative chemistry and exploiting multiple degradation approaches. In this article, we review the breakthroughs and recent novel concepts in this highly active discipline

Analysis of SUMO-protein modification and its effect on cellular processes

Conjugation of SUMO (small ubiquitin-related modifier) to a substrate protein is a posttranslational modification, which can have various consequences on the substrate’s activity, localization and stability. Structurally very similar to ubiquitin, SUMO is attached to a substrate in a similar enzymatic cascade, but in contrast, SUMO is not an immediate degradation signal for the substrate. However, ULS (ubiquitin ligases for sumoylated proteins) negatively regulate the abundance of SUMO conjugates by ubiquitylation which in turn leads to proteasomal degradation. Additionally, the desumoylating enzymes, in S. cerevisiae primarily Ulp2, negatively control the length of SUMO chains. Preliminary experiments from our laboratory have demonstrated that a failure in the control of sumoylation causes mitochondrial fragmentation. Dnm1 is the key enzyme for mitochondrial fission and it is known that sumoylation regulates the activity of its human ortholog Drp1. Motivated by these reports, a starting question was whether Dnm1 is sumoylated as well. In order to answer this question, various methods for the analysis of SUMO conjugates were established and applied. One of these methods was the application of cleavage-resistant SUMO variants, carrying the mutation Q95P, for the stabilization of these conjugates. Furthermore an optimized protocol for denaturing purification of SUMO and ubiquitin conjugates was established. In the following, this purification method was the basis for an approach to identify sumoylation sites by the application of the mass spectrometry optimized variant 8H-Smt3-KallR-I96R. In addition to identifying new SUMO substrates (for example the (Na+, K+)/H+ antiporter Vnx1), this analysis revealed N-terminal modification of SUMO, presumably by ubiquitin. From a more physiological point of view, the effect of the SUMO system on Dnm1 localization was investigated. Generation of a Ulp2 variant with a low-temperature degron and application of an improved mtGFP construct revealed that fragmentation of mitochondria occurs in the course of one day at restrictive temperature and is therefore probably a consequence of the accumulation of sumoylated proteins. Furthermore, it was discovered that specifically in the strain dnm1∆ fzo1∆, defective in mitochondrial dynamics, the absence of ULS leads to an increase in the formation of petite colonies. This in turn suggests that in particular in the absence of the mitochondrial quality control systems of fission and fusion, ULS are important to ensure respiratory competence. Regarding Dnm1 itself, a ubiquitylation of this protein could be demonstrated. However, at the same time under the applied conditions, a sumoylation of Dnm1 seems to be unlikely. Cdc11 could be successfully employed as a known SUMO substrate which confirmed the general capability of the applied methods. In agreement with the detection of ubiquitylated forms, it was found that Dnm1 is degraded via the ubiquitin-proteasome system. Furthermore, ethanol was discovered as a treatment that leads to degradation of Dnm1. Summarized, the methods established in the course of this study will be useful tools for the analysis of SUMO conjugates, and the finding of ubiquitylated Dnm1 could be a starting point for further analysis of this modification in respect to mitochondrial dynamics