Function and targets of the Urm1/Uba4 conjugation machinery in Drosophila melanogaster

Behzad Khoshnood

Department of Molecular Biology Umeå 2017 This work is protected by the Swedish Copyright Legislation (Act 1960:729) Dissertation for PhD Umeå University Medical Dissertations New Series No. 1939 ISBN: 978-91-7601-815-6 ISSN: 0346-6612 Cover photo by Behzad Khoshnood, inspired by the art work from Nicolle Ragera from National Science Foundation of USA. The image belongs to public domain. Electronic version available at: http://umu.diva-portal.org/ Printed by: UmU print service, Umeå University Umeå, Sweden 2017

رهزگ دل من ز علم محروم نشد

کم ماند ز ارسار هک معلوم نشد

هفتاد و دو سال فکر رکدم شب و روز

معلومم شد هک چیه معلوم نشد

خیام

My heart was never deprived of the passion for science

There was little of the mysteries that I did not understand

Days and nights, my whole life I have contemplated

Only to realise how little I know

Omar Khayyam 1048-1131

Table of Contents

1. Abstract ...... iii 2. Papers included in this thesis ...... v 3. Abbreviations ...... vi 4. Introduction ...... 1 4.1. Drosophila melanogaster, serving science as a model system ...... 1 4.1.1 History ...... 1 4.1.2 Significance of using Drosophila as a model system ...... 3 4.2. Development and life cycle of Drosophila ...... 4 4.2.1 Embryonic development ...... 5 4.2.2 Larval development and metamorphosis...... 6 4.2.3 Eclosure and adult life ...... 7 4.3 Neuromuscular junctions (NMJs) ...... 7 4.3.1 Structure and classification of NMJs ...... 8 4.3.2 Activity and development of NMJs ...... 9 4.4 Posttranslational modification and UBLs ...... 10 4.4.1 Ubiquitin ...... 11 4.4.2 Ubiquitin-like modifiers (UBLs) ...... 12 4.5 Urm1 - Ubiquitin related modifier 1 ...... 13 4.5.1 Urm1 from evolutionary perspective ...... 13 4.5.2 Structure and Biochemistry ...... 14 4.5.3 Urm1 in tRNA modification ...... 15 4.5.4 Urm1 as a protein modifier ...... 17 4.6 Uba4 - The activating enzyme for Urm1 ...... 18 4.6.1 Structure and function of Uba4 ...... 19 4.7 Oxidative stress ...... 21 4.7.1 Molecular mechanisms behind oxidation and reduction ...... 21 4.8 The JNK pathway ...... 22 4.8.1 The JNK pathway in Drosophila ...... 23 4.8.2 The JNK pathway in stress responses and lifespan regulation ...... 25 4.9 Drosophila as a model to study Tax-mediated oncogenesis...... 26 4.9.1 Adult T-cell leukaemia/lymphoma ...... 26 4.9.2 HTLV-1 and Tax ...... 27 4.9.3 The NF-B pathway ...... 28 4.9.4 Posttranslational modification of Tax1 ...... 29 5. Results and Discussion ...... 31 5.1. Paper I ...... 31 Urm1: an essential regulator of JNK signaling and oxidative stress in Drosophila melanogaster...... 31 5.2. Paper II...... 35 A proteomics approach to identify targets of the ubiquitin-like molecule Urm1 in Drosophila melanogaster ...... 35

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5.3. Paper III ...... 36 The HTLV-1 oncoprotein Tax is modified by the Ubiquitin related modifier 1 ... 36 5.4. Paper IV ...... 38 Deciphering a novel role of the Urm1/Uba4 conjugation machinery for Neuromuscular Junction (NMJ) development in Drosophila melanogaster ...... 38 5.5. General discussion and future perspectives ...... 42 6. Acknowledgements ...... 43 7. References ...... 46

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1. Abstract

Posttranslational modification (PTM) of proteins is essential to maintain homeostasis and viability in all eukaryotic cells. Hence, besides the sequence and 3D folding of a polypeptide, modification by multiple types of PTMs, ranging from small molecular groups to entire protein modules, adds another layer of complexity to protein function and regulation. The ubiquitin-like modifiers (UBLs) are such a group of evolutionary conserved protein modifiers, which by covalently conjugating to target proteins can modulate the subcellular localization and activity of their targets. One example of such a UBL, is the Ubiquitin related modifier 1 (Urm1). Since its discovery in 2000, Urm1 has been depicted as a dual function protein, which besides acting as a PTM, in addition functions as a sulfur carrier during the thio-modification of a specific group of tRNAs. Due to this dual capacity, Urm1 is considered as the evolutionary ancestor of the entire UBL family. At present, it is well established that Urm1, with help of its dedicated E1 enzyme Uba4/MOCS3, conjugates to multiple target proteins (urmylation) and that Urm1 thus plays important roles in viability and the response against oxidative stress.

The aim of this thesis has been to, for the first time, investigate the role of Urm1 and Uba4 in a multicellular organism, utilising a multidisciplinary approach that integrates Drosophila genetics with classical biochemical assays and proteomics. In Paper I, we first characterized the Drosophila orthologues of Urm1 (CG33276) and Uba4 (CG13090), verified that they interact physically as well as genetically, and that they together can induce urmylation in the fly. By subsequently generating an Urm1 null Drosophila mutant (Urm1n123), we established that Urm1 is essential for viability and that flies lacking Urm1 are resistant to oxidative stress. Providing a molecular explanation for this phenotype, we demonstrated an involvement of Urm1 in the regulation of JNK signaling, including the transcription of the cytoprotective genes Jafrac1 and gstD1. Besides the resistance to oxidative stress, we have moreover (Manuscript IV) made an in- depth investigation of another phenotype displayed by Urm1n123 mutants, an overgrowth of third instar larval neuromuscular junctions (NMJs), a phenotype which is shared also with mutants lacking Uba4 (Uba4n29).

To increase the understanding of Urm1 in the fly, we next employed a proteomics- based approach to identify candidate Urm1 target proteins (Paper II). Using this strategy, we identified 79 Urm1-interacting proteins during three different stages of fly development. Of these, six was biochemically confirmed to interact covalently with Urm1, whereas one was found to be associated with Urm1 by non- covalent means. In Manuscript III, we additionally identified the virally encoded

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oncogene Tax as a target of Urm1, both in Drosophila tissues and mammalian cell lines. In this study, we established a strong correlation between Tax urmylation and subcellular localisation, and that Urm1 promoted a cytoplasmic accumulation and enhanced signalling activity of Tax, with implications for a potential role of Urm1 in Tax-induced oncogenesis.

Taken together, this thesis provides a basic understanding of the potential roles and targets of Urm1 in a multicellular organism. The four studies included cover different aspects of Urm1 function and clearly points towards a highly dynamic role of protein urmylation in fly development, as well as in adult life.

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2. Papers included in this thesis

I. Khoshnood, B., Dacklin, I. and Grabbe, C. (2016). Urm1: an essential regulator of JNK signaling and oxidative stress in Drosophila melanogaster. Cell Mol Life Science. 73:1939-1954.

II. Khoshnood, B., Dacklin, I. and Grabbe, C. (2017). A proteomics approach to identify targets of the ubiquitin-like molecule Urm1 in Drosophila melanogaster. PLoS One. 2017 Sep 27;12(9):e0185611.

III. Hleihel R*, Khoshnood B.*, Dacklin I, Omran H, Mouawad C, Dassouki Z, El-Sabban M, *Shirinian M, *Grabbe C and *Bazarbachi A. (2017) The HTLV-1 encoded oncoprotein Tax is modified by the Ubiquitin related modifier 1 (Urm1). Submitted manuscript.

IV. Khoshnood, B., Dacklin, I. and Grabbe, C. (2017). Deciphering a novel role of the Urm1/Uba4 conjugation machinery during neuromuscular junction (NMJ) development in Drosophila melanogaster. Manuscript.

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3. Abbreviations

AHP Alkyl hydroperoxide reductase AIDS Acquired immune deficiency syndrome ALDH Aldehyde dehydrogenases AP-1 Activator protein 1 aPKC Atypical protein kinase C AS2O3 Arsenic trioxide ATAC Ada two a containing ATF cAMP-dependent transcription factor ATG Autophagy-related protein ATL Adult T-cell leukaemia/lymphoma ATP Adenosine triphosphate cAMP Cyclic adenosine monophosphate CD Cluster of differentiation CDK Cyclin-dependent kinase CG Computed gene CNS Central nervous system CREB cAMP response element-binding protein CRISPR Clustered regularly interspaced short palindromic repeats dMKK Drosophila MAP kinase kinase DNA Deoxyribonucleic acid DTT Dithiothreitol DUB Deubiquitinating enzyme Eip71CD Ecdysone-induced protein 28/29 kd ELP Elongator protein ERK Extracellular signal-regulated kinases ESCRT Endosomal-sorting complex required for transport FAT HLA-F-adjacent transcript FUB Function of boundary GAL4 Galactose-responsive transcription factor 4 GFP Green fluorescent protein GG Glycine-glycine GILT1 Gamma-interferon-inducible lysosomal thiol reductase 1 GST Glutathione S-transferase

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GstD1 Glutathione S transferase D1 H2O2 Hydrogen peroxide HA Human influenza hemagglutinin HAM/TSP HTLV-1-associated myelopathy/tropical spastic paraparesis HCF Host cell factor Hep Hemipterous HIV Human immunodeficiency virus Hiwi Highwire Hsp Heat shock protein HTLV Human T-lymphotropic virus IFN Interferon IKK IκB kinase IL Interleukin ISG15 Interferon-induced 15 kda protein IκB Inhibitor of kappa B JNK c-Jun N-terminal kinase JNKK c-Jun N-terminal kinase kinase JNKKK c-Jun N-terminal kinase kinase kinase JNKKKK c-Jun N-terminal kinase kinase kinase kinase K Lysine LOF Loss of function MAPK Mitogen-activated protein kinase MBIP MAPK binding inhibitory protein MCM5 5-methoxycarbonylmethyl MCM5S2U 5-methoxycarbonylmethyl-2-thiouridine MEKK Mitogen-activated protein kinase kinase kinase MKP Map kinase phosphatase mM Millimolar MoaD Molybdenum cofactor biosynthesis protein D MoaE Molybdenum cofactor biosynthesis protein E MoCo Molybdenum cofactor MOCS Molybdenum cofactor synthesis MoeB Molybdenum cofactor biosynthesis protein B MPT Molybdopterin mRNA Messenger RNA MVB Multivesicular bodies

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NADPH Nicotinamide adenine dinucleotide phosphate NCS Nucleobase cation symporter NEDD8 Neural precursor cell expressed, developmentally down-regulated 8 NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells NLS Nuclear localization signal NMJ Neuromuscular junction NMR Nuclear magnetic resonance NQO1 Nad(p)h quinone dehydrogenase 1 Nrf2 Nuclear factor (erythroid-derived 2)-like 2 O2- Superoxide anion OH- Hydroxyl radical ORF Open reading frame pHgpX Phospholipid-hydroperoxide glutathione peroxidase PI3K Phosphatidylinositol-3 kinase pJNK PhosphoJNK PML Promyelocytic leukemia PRX Peroxiredoxin PTM Posttranslational modification RHD Rhodanese homology domain RING Really intresting new gene RNA Ribonucleic acid RNAi RNA interference RNF Ring finger protein ROS Reactive oxygen species S Sulfur SAMP Small archaeal modifier protein SAPK Stress-activated protein kinase SSR Subsynaptic reticulum SUMO Small ubiquitin-like modifier TAK Transforming growth factor beta-activated kinase TGFβ Transforming growth factor beta tRNA Transfer RNA U34 Uridine 34 UAS Upstream activation sequence UBA Ubiquitin activating enzyme UBC Ubiquitin conjugating enzyme

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UBL Ubiquitin like protein UFM Ubiquitin fold modifier URM Ubiquitin related modifier UV Ultra violet

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4. Introduction

4.1. Drosophila melanogaster, serving science as a model system

If we set world domination as the ultimate aim of life, there are only a few living organisms that can compete with the six-legged creatures that are found in most eco-systems on earth, the insects. Insects are the most diverse group of animals on earth, with a number of ~1.5 million species described to date and an estimated number of 10 quintillion individuals alive at any given time point (Larsen et al., 2017). As one of the most ancient groups of animals, their origin of evolution dates back to 479 million years ago (Misof et al., 2014).

Even though insects may be small and appear unimportant, they contribute significantly to human life in both good and bad ways. Insects can function as vectors for transferring human diseases such as malaria, but are at the same time indispensable for the pollination of fruits and nuts that are essential for our health and diet.

For more than a century, one specific species of insects has captured the attention of scientists. Heavily used as a model system in biological science, Drosophila melanogaster, in plain language known as the fruit fly, has significantly contributed to our understanding of cell and molecular processes, as well as genetics. D. melanogaster belongs to the “old-world” clade of the Sophophora subgenus within the Drosophilidae family (Van der linde et al., 2010), are roughly 2-2.5 mm in length, yellow-brown in colour with black stripes across the abdomen, and have brick read eyes. There are many reasons for why the fruit fly has become such a successful model system in research. Partly, it may be due to the rather fast and high reproduction rate of insects, compared to other animals, together with the low cost of maintaining and handling fly stocks. Moreover, the risk factors associated with handling mice or rats, namely biting or inter-species diseases, is virtually absent in the fly. In the following sections, I will describe more beneficial aspects of D. melanogaster as a model system and highlight the importance of Drosophila research in modern science.

4.1.1 History

The very first steps of using Drosophila for research were taken in Thomas Hunt Morgan’s lab at Columbia University, the USA. His isolation of the first naturally

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occurring mutation, in the white gene locus in D. melanogaster (Morgan 1910), marks the clear starting point for decades of follow-up research. A few years later, Morgan and his three famous students, A. H. Sturtevant, C. B. Bridges and H. J. Muller, provided the first ground-breaking proof for the chromosomal inheritance theory, which was in agreement with Mendelian genetics (Sturtevant, 1913; Morgan, Sturtevant and Bridge, 1920)

One of the most important findings from those revolutionary years, was when Muller discovered that X-ray irradiation can induce mutations in the fly genome and thereby exploited as a tool in research. Following his presentation at the Fifth International Congress of Genetics in 1927, entitled “The problem of Genetic Modification”, he became one of the most respected scientists of the 20th century. The significance of Muller’s work was further acknowledged in 1946, when he similar to his mentor (T. H. Morgan), was awarded the Nobel Prize in Physiology or Medicine.

Throughout the years, many researchers have contributed to expanding the Drosophila research toolbox. The earliest example of these tools was based on the discovery that X-ray induced chromosomal inversions can suppress the crossover of genes (Muller, 1918). This finding resulted in the generation of the first balancer chromosome, today widely used to maintain recessive mutations in fly stocks (Bentley Glass, 1933; Araye and Sawamura, 2013). Another key event in the development of tools to study fly genetics, was the introduction of P elements for mutagenesis and transgenesis, reported in (Spradling and Rubin, 1982). By learning the skill of mobilizing non-autonomous P elements by introduction of an exogenous transposase (e.g. Δ2-3), Drosophila researchers got their hands on a multi-application gadget that could be employed for both chromosome engineering and gene manipulation (Ryder and Russell, 2003).

In 1993, Brand and Perrimon published a paper describing the UAS/GAL4 system, which revolutionized the Drosophila field yet again (Brand and Perrimon, 1993; Pan et al., 2009) (Figure 1). By integrating knowledge from yeast (S. cerevisiae) genetics into Drosophila, in which the GAL4 transcription factor induces transcription by binding to UAS (upstream activating sequence) sequences, the UAS/GAL4 system was launched as a technique to control gene expression in a tissue-specific manner. Since then, the UAS/GAL4 system has been applied in numerous different ways and today it offers scientists a methodology to perform sophisticated in vivo experiments. At present, there are numerous cell- and tissue-specific GAL4 drivers and an extensive amount of UAS transgenic constructs available to the fly community, which has resulted in that there nowadays are hardly any Drosophila labs that do not employ the UAS/GAL4 system in their research.

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Figure 1. The UAS/GAL4 system.

4.1.2 Significance of using Drosophila as a model system

I personally think that the modern era of Drosophila genetics began in the year 2000, when the entire genome of D. melanogaster was sequenced (Adams et al., 2000). This fascinating source of information revealed that the whole genome of Drosophila is composed of roughly 14 000 genes, compiled into only four chromosomes. Moreover, despite the lower complexity of the fly genome, flies still share many similarities with humans, such as genetic networks and physiological functions, as well as more complex behaviors such as memory, sleep and addiction. One important characteristic of the Drosophila genome, which has contributed to the success of the fruit fly as a model system, is that it in general contains a lower number of genes in each gene class, resulting in a low genetic redundancy. This feature makes the interpretation of mutant phenotypes more straightforward and simplifies the analysis of systemic loss-of-function (LOF) mutations.

In recent years, the introduction of the CRISPR/Cas system into fruit fly field (Yu et al., 2013; Gratz et al., 2015) has made life much easier for Drosophila researchers. This relatively easy and fast methodology to induce genetic manipulations, combined with the fast-result-scoring nature of the fruit fly, has made the Drosophila an interesting model to study almost all aspects of genetics and physiology.

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Despite its small genome, the adult fly is a fairly sophisticated and complex organism. The Drosophila displays many tissues that function in a similar manner as their mammalian counterparts, such as the heart, gut, and reproductive tract, as well as a complex and fascinating brain made of a remarkable number of more than 100,000 neurons. Together with the continuous verification of gene functions that are conserved from flies to humans, this encourages scientists to continue using flies as a model to study human biology and disease. Throughout the years, a large number of researchers have employed Drosophila to address complex scientific questions concerning immunity, drug development, and cancer, making it obvious that this little fruit fly is a potent model organism that has contributed a lot to our understanding of biological life.

4.2. Development and life cycle of Drosophila

Partly, the feasibility of using Drosophila as a model system comes from its rather fast development and high reproduction rate. The life cycle of the Drosophila starts with the fertilized egg, which after 24 hours of embryonic development hatch into a 1 mm long larvae. This larva is basically an eating machine that during five to six days of larval development increases its size 200 times. During this process the larva molts three times, marking the onset of three separate larval stages, the first, second and third instar stages (Church and Robertson, 1966). By the end of the third instar stage, the larva forms a pre-pupa and eventually a pupa (Figure 2), in which the fly goes through a complete metamorphosis. During metamorphosis, most larval tissues are degraded and the adult fly forms from information laid down in the larval imaginal discs. After 4-5 days of pupal development, the fly finally ecloses from the pupa and after a few hours of maturation, the adult fly is ready for mating and reproduction.

The life cycle and the distinctive stages of fly development strongly contributes to why Drosophila is such a powerful model system, since it offers multiple different angles of approach for scientific work, yet they are all Drosophila. For example, Drosophila embryos are excellent tools to answer developmental questions, while the larvae and adults can be used for physiological and behavioral investigations. In addition, there is plenty of potential to perform in vitro experiments in fly cell lines, such as the Drosophila Schneider (S2) cells.

Each cycle of development, from egg laying to maturation of the adult fly, takes roughly 10 days to complete (at 25C). It is also worth noting that a female fly can

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Figure 2. Life cycle of Drosophila melanogaster. produce up to 500 (Ashburner, Golic and Hawley, 2005) offspring during her life time. In spite of the rather short period of development, Drosophila melanogaster is still a very complex organism with diverse anatomical properties. The specific details regarding development of the nervous system, heart, muscles, digestive tract etc. of the fruit fly have been extensively described in multiple reviews (Jennings, 2011; McGurk, Berson and Bonini, 2015; Yaniv and Schuldiner, 2016; Liu and Jin, 2017; Piper and Partridge, 2017). However, I will below give a brief description of each period of the fly development.

4.2.1 Embryonic development

Within two hours after fertilization and 13 mitotic divisions, each Drosophila zygote contains roughly 6000 nuclei that share a single cytoplasm, thus forming a syncytium. Already upon egg-laying, the oocyte inherits a huge supply of mRNA molecules from the female. These “maternally contributed” mRNA molecules control protein expression in early embryonic stages, until the zygotic transcription starts to take over. Since there are no cell membranes separating the cells in the syncytial embryo, the maternal factors can freely diffuse in the syncytium, and thereby form morphogenic gradients. These gradients are

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responsible for setting up the polarity and provide cues for cellular identity in the early embryo, defined by the differential localization and concentration of these mRNA molecules. The Nobel Prize winning work of Christiane Nüsslein-Volhard and Eric Wieschaus, together with Edward B. Lewis, revolutionized the field of embryonic development. Together, they identified many genes important for early development, among which the morphogen-encoding genes that defines the anterior-posterior patterning, and the homeotic genes the orchestrates segmentation of the embryo, may be most important (Garcia-Bellido and Lewis, 1976; Nüsslein-volhard and Wieschaus, 1980; Lewis, 1982, 2007). In early embryos bicoid and hunchback mRNAs determine the formation of the head and thorax, while the protein products of nanos and caudal mRNAs are critical for formation of the abdominal segments (Zaffran, Robrini and Bertrand, 2014). After the first 13 divisions, each nuclei in the syncytial embryo becomes an individual cell (Mahowald 1963), whereupon gastrulation begins. Later, the embryonic ectoderm is divided into visible segments that will define the segmented structure of the adult insect (Levine, 2008).

The whole process of embryonic development takes around 24 hours at room temperature, and has been divided into 17 distinct stages (Campos-Ortega 1985). By the end of stage 17, the foundation of all critical organs required for larval development, such as body wall muscles, the intestinal tract, trachea and most importantly the nervous system, has formed.

4.2.2 Larval development and metamorphosis

In the final stage of embryogenesis (stage 17) the tracheal tree gets filled with air and 1-2 hours later the first instar larvae hatches out from the egg. The sole purpose of this embryo-sized wormlike creature is eating and growing, passing through the three instar larval stages.

Third instar larvae are heavily used in fly labs. Besides being semi-transparent, which facilitates the investigation of living animals under a fluorescent microscope, the internal organs are rather accessible and easy to dissect. Furthermore, the presence of fascinating tissues such as salivary glands with giant cells and polytene chromosomes, is a prime feature for researchers.

Another interesting feature of the larval stages is the presence of imaginal discs, the epithelial sac-like structures of dividing cells, which are the primordia of many of the major adult insect structures, such as wings, legs, halters, antennae and genitalia. Therefore, many of the morphological phenotypes of the adult fly have its origin in these tissues. When reaching a critical weight, associated with a distinctive pulse of the fly hormone Ecdysone, the third instar larvae crawls out

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of the food in search of a dry place to pupariate (Berreur et al., 1984). During the pupal stages, a majority of the adult structures are developed. Inside the pupa, the Drosophila undergoes a complete metamorphosis, where most of the larval tissues, except the central nervous system (CNS), are degraded and the insect is rebuilt following the instructions laid down in the imaginal discs (Campos- Ortega, 1997; Brody, 1999; Grumbling, 2006)

4.2.3 Eclosure and adult life

Adult fruit flies emerge from the pupal case after 3-4 days of pupal development. In the last step of morphogenesis, the wings are expanded and the fly acquires its final shape. The mean lifespan of an adult fruit fly is 2-3 months, when kept at room temperature and with sufficient supply of food (Helfand and Rogina, 2003). Apart from genetic factors, diet and food intake are critical determinants of fly longevity (Piper et al., 2011). However, adult life for a fruit fly is much more challenging out in nature, where it in order to survive needs to compete for resources and overcome environmental stressors and other threats, resulting in a significantly shorter life span.

4.3 Neuromuscular junctions (NMJs)

The Drosophila larval nervous system is an invaluable model to study the development and growth of neuromuscular junctions (NMJs), the insect equivalent to mammalian motor end plates. Simplicity aside, it is relatively easy to access the large, individually specified NMJs of a larvae in order to visualize or record their activity (Menon, Carrillo and Zinn, 2013). Similar to synapses in vertebrates, Drosophila NMJs contain glutamatergic synapses and exhibit functional plasticity, supporting the relevance of using Drosophila NMJs to understand neural development and function in general (Fernandes and Keshishian, 1999; Menon, Carrillo and Zinn, 2013).

The larval body is composed of three head segments, three thoracic segments (T1- T3) and eight abdominal segments (A1-A8). Of these, the larval locomotory system is formed by the body wall musculature in the thoracic and abdominal segments, together with the motor neurons that extends from the ventral nerve cord to innervate them (Kohsaka et al., 2012). There are 32 motor neurons in each abdominal hemisegment, each one innervating its specific target muscle. The rather stereotyped pattern of connectivity in the neuromuscular system is laid down already during embryogenesis, but the NMJs undergo extensive changes in size and morphology also during postembryonic development

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(Griffith and Budnik, 2006; Kohsaka et al., 2012; Menon, Carrillo and Zinn, 2013). In the following sections of this chapter I will briefly review the current knowledge about Drosophila NMJs. (Figure 3)

Figure 3. Neuromuscular anatomy of Drosophila third instar larvae, displaying the dissected body wall musculure (marked by phalloidin in green) and the motor neurons innervating them (marked by Cy3- conjugated anti-HRP antibodies in red). A close-up of the NMJ on muscle 4, abdominal segment 3, indicates the bouton morphology and active zones (marked by anti-Brp antibodies).

4.3.1 Structure and classification of NMJs

Each Drosophila NMJ consists of a stereotyped number of boutons (10-50 per muscle), i.e. points of contact between the motor neurons and the body wall musculature. The boutons are oval-shaped structures that house the synapses, which are concentrated at a number of neurotransmitter release sites, called active zones. In these active zones, proteins such as Bruchpilot (Brp) are specifically important for an effective organization and clustering of Ca2+ channels (Fouquet et al., 2009), and can also be used as markers for visualization of the active zones. On the postsynaptic side, another structural feature of the

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boutons can be found, the subsynaptic reticulum (SSR). The SSR is a highly folded specialized membrane domain that contains neurotransmitter receptors, ion channels, scaffolding proteins and postsynaptic signaling complexes. A key protein present in the SSRs is Discs large (Dlg), which is essential for controlling the shape, size and function of the synaptic structure. Loss of Dlg results in severe defects in SSR expansion (Lahey et al., 1994).

The Drosophila NMJ boutons are classified into three categories, type I, II and III, which differ in morphology and amount of SSR, as well as their type of neurotransmitter. Type I boutons are further divided into two classes, type Ib (big) and type Is (small), which primarily differ in size and the amount of Dlg that is accumulated on the post-synaptic side (Menon, Carrillo and Zinn, 2013).

4.3.2 Activity and development of NMJs

As indicated earlier, the development of the fly NMJs begins during embryogenesis (stage 13-15). Throughout the development, however, there are three major stages of synaptic refinement. The first synaptic refinement occurs in late embryonic stages, when misplaced synapses on non-target muscles are eliminated (Keshishian et al., 1993; Keshishian, Chang and Jarecki, 1994; Jarecki and Keshishian, 1995; Carrillo et al., 2010). The next refinement occurs during larval development, primarily to compensate for the tremendous increase in muscle size. During this period, boutons are continuously added to (and removed from) the NMJs, meanwhile the number of active zones at each bouton are also increasing up to tenfold (Atwood, Govind and Wu, 1993; Davis, Schuster and Goodman, 1996). The last round of synapse refinement takes place during metamorphosis, where dismantling of synapses occurs via signaling pathways instructed by postsynaptic side (Liu et al., 2010).

Over the past decades a large set of genes, including spastin, futsch and shaggy, have been characterized as important for the regulation of synaptic targeting, growth and refinement of Drosophila NMJs (Menon, Carrillo and Zinn, 2013; Vonhoff and Keshishian, 2017). Among these, one group of genes contribute to the regulation of NMJ growth by specifically altering the neuronal terminal cytoskeleton and positively regulating the bouton number. A few examples of such genes are futsch (Roos et al., 2000), which acts through atypical protein kinase C (aPKC) (Ruiz-Canada et al., 2004) as well as arrow (arr) and dishevelled (dsh), which act through the Wnt signaling pathway (Miech et al., 2008). In contrast, genes such as the E3 ubiquitin ligase highwire (hiw) have been shown to negatively regulate NMJ growth, resulting in a NMJ overgrowth phenotype in LOF mutants (Wan et al., 2000; DiAntonio et al., 2001; Wu, 2005).

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One major signaling pathway involved in the regulation of synaptic growth is the Jun N-terminal kinase (JNK) pathway (Sanyal et al., 2002, 2003; Collins et al., 2006). In fact, research has shown that the underlying reason for the NMJ overgrowth displayed by hiw mutants is a hyperactivation of the AP-1 heterodimeric transcription factor, via the JNK pathway (Collins et al., 2006; Shen and Ganetzky, 2009) resulting in a transcriptional upregulation of a panel of downstream genes that affect synaptic growth and activity. Complementary work by Etter and co-workers (Etter et al., 2005) has in addition identified a list of genes that are transcriptionally upregulated in neurons with hyperactive JNK signaling. Because of the significance of the JNK pathway in oxidative stress, lifespan and neuromuscular junction development, I have dedicated chapter 4.8 of this thesis to discuss the JNK signaling in more detail.

4.4 Posttranslational modification and UBLs

Posttranslational modification (PTM) is a molecular event, in which amino-acid residues of a protein can be modified, often resulting in a change in the target protein, such as altered structural conformation or subcellular localization, increased or decreased activity, or alteration of protein stability (Prabakaran et al., 2012). To rapidly change the properties of target proteins by the addition of PTMs, is a powerful tool for regulating biological processes in all organisms across evolution, from bacteria to humans (Grangeasse, Stülke and Mijakovic, 2015). While some prevalent PTMs such as phosphorylation and ubiquitination are dynamic and reversible, others are thought to be irreversible or more accurately put, “not known to be reversible” and possess limited processing capacity (Prabakaran et al., 2012). For example, the anchoring of signal- transduction molecules such as Src and Ras to the cytoplasmic side of the plasma membrane is facilitated by the covalent attachment of lipid groups, which are not readily reversible (Berg et al., 2002).

To date, more than 200 types of PTMs has been identified (Prabakaran et al., 2012), which all rely on one or more enzymes to catalyse the attachment of the modifying agent to the target protein. Based on the nature of the modifying element, there are several kinds of PTMs. Firstly, target proteins can be modified by the attachment of a small molecular moiety, which occurs in the case of phosphorylation and acetylation. In the example of protein phosphorylation, the balance in the activity of kinases and phosphatases regulates the addition (and removal) of a phosphate group to the target protein, often resulting in a conformational and functional change of the target (Cieśla, Fraczyk and Rode, 2011). Besides small chemical groups, proteins can also be modified by entire

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polypeptides, such as ubiquitin or any of the ubiquitin-like proteins (UBLs) (Schwartz and Hochstrasser, 2003; Prabakaran et al., 2012).

4.4.1 Ubiquitin

The most common type of polypeptide-based posttranslational modification is ubiquitination. Ubiquitin is a small protein, composed of 76 amino acids (roughly 8.5 kDa), which form a β-grasp fold structure. Besides this globular fold, the most characteristic structural feature of ubiquitin is the glycine-glycine (GG) motif used for conjugation in the C-terminal tail, together with the conserved lysine residues that can be used to build ubiquitin chains, namely K6, K11, K27, K29, K33, K48 and K63 (in addition to the starting methionine (M1)). Ubiquitin is highly conserved among eukaryotic species (yeast and human ubiquitin share 96% sequence identity), and is in addition a very stable protein that can withstand a large variety of different pH values and temperatures (Vijay-Kumar et al., 1985; Wilkinson, 2005; Kimura and Tanaka, 2010; van der Veen and Ploegh, 2012). Ubiquitin was first described in 1975 (Goldstein et al., 1975) and since then there have been thousands of studies linking ubiquitination to a wide range of cellular processes, such as gene expression, DNA repair, protein degradation and apoptosis (Schwartz and Hochstrasser, 2003).

Ubiquitination (also called ubiquitylation) is the process in which ubiquitin is covalently attached to a target protein via a cascade of enzymatic activity, driven by ATP hydrolysis. From a mechanistic point of view, the transfer of ubiquitin to its substrate is initiated by an E1 activating enzyme. Following activation, ubiquitin is transferred onto an E2 enzyme (ubiquitin conjugating enzyme). The subsequent and final step in the cascade is handled by an E3 enzyme (ubiquitin ligase), whereby the target protein and the conjugating enzyme are recruited to the E3, to enable conjugation of ubiquitin to a specific lysine residue in the target protein (Glickman and Ciechanover, 2002). Considering that ubiquitination is a reversible process, a fourth class of enzymes can be added to the ubiquitin machinery, the deubiquitinating enzymes (DUBs). DUBs release the modified protein from ubiquitin, by catalysing the breakage of the peptide bond between ubiquitin and the ubiquitinated protein (Wilkinson, 2000).

One of the most extensively studied outcomes of ubiquitination is the proteasomal degradation of target proteins. Most commonly, the targeting of proteins for degradation by the proteasome requires a modification of the target by an ubiquitin chain, in which multiple ubiquitins are linked to each other via covalent conjugation of the K48 residue in one ubiquitin, to the carboxyl group of G76 in the adjacent ubiquitin molecule in the chain. Besides these branched K48- linked chains, ubiquitin chains with other linkages, as well as modification by one

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single ubiquitin moiety (monoubiquitination), can instead of instructing protein degradation, translate into several different scenarios for the target. One common example is the monoubiquitination of histone proteins, which is associated with chromatin remodeling and transcriptional regulation (Busch and Goldknopf, 1981; Briggs et al., 2002). Another important function of ubiquitination is the sorting of cargo proteins along the endocytic pathway, which is predominantly regulated by monoubiquitination and K63-linked ubiquitin chains, but may also involve other linkages such as K11, K29 and K48 chains (Shields and Piper, 2011). During endocytic sorting, ubiquitin is involved in most steps, from the initial internalization of the cargo, to the sorting into multivesicular bodies (MVBs) through activation of the endosomal-sorting complex required for transport (ESCRT) machinery (Teo, Veprintsev and Williams, 2004).

The ubiquitin system is fairly well conserved throughout evolution, but the composition of E1, E2 and E3 enzymes differ between species. However, in general terms, most eukaryotes display hundreds of E3 encoding genes, whereas the number of E2s is considerably lower and E1 often exists as a single gene (Hicke, Schubert and Hill, 2005; Petroski and Deshaies, 2005).

4.4.2 Ubiquitin-like modifiers (UBLs)

The ubiquitin-like proteins (UBLs) is a family of small globular proteins that similar to ubiquitin displays the structural feature of a β-grasp superfold and C- terminal GG-motif. To date the list of identified UBLs includes Smt3 (SUMO-1, - 2, -3, -4), NEDD8, FUB1, FAT10, ISG15, Atg12, Atg8, UFM1 and Urm1 (Hochstrasser, 2009; Swatek and Komander, 2016; Cappadocia and Lima, 2017).

Among the UBLs, SUMO is the most well studied modifier, which also seems to affect the largest number of target proteins, compared with the rest of UBL family members (Meulmeester and Melchior, 2008). In spite of only 18% sequence identity with ubiquitin, the three-dimensional structures of SUMO and ubiquitin are highly similar (Bayer et al., 1998). SUMO is primarily recognized for its role in nuclear functions including transcriptional regulation, nuclear transport and genome integrity, but is also involved in cytoplasmic signal transduction pathways (Rodríguez, 2014).

The UBL that shares the highest sequence identity with ubiquitin (approximately 60%) is NEDD8. NEDD8 conjugation shares the similar mechanistic features as ubiquitin, involving the employment of NEDD8-specific E1 (Uba3), E2 (Ubc12) and E3 (ROC1) enzymes (Kumar, Yoshida and Noda, 1993). Modification by NEDD8, referred to as neddylation, has been shown to regulate the

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multifunctional transcription factor NF-κB and in this way affect immune responses and apoptotic cell death (Kawakami et al., 2001).

During the past decades, essential functions have been ascribed also to many of the other UBLs, such as Atg8 and Atg12 in autophagy (Hanada and Ohsumi, 2005), as well as Fat10 in the regulation of the cell cycle (Ren et al., 2006; Lukasiak et al., 2008; Merbl et al., 2013). Nevertheless, the depth of our understanding of UBLs is still very shallow and requires further research.

4.5 Urm1 - Ubiquitin related modifier 1

Ubiquitin related modifier 1 (Urm1) is one of the least studied of the UBLs. Urm1 has been established to be the most ancient member in the UBL family and is therefore considered as the evolutionary origin of polypeptide-based posttranslational modifications (Iyer, Burroughs and Aravind, 2006; Pedrioli, Leidel and Hofmann, 2008).

The study that led to the identification of Urm1 was performed by Furukawa and co-workers, in which Urm1 was characterized as a protein modifier based on a search of the yeast genome for proteins with sequence similarity to prokaryotic sulfur carriers. This study further identified Uba4 as the specific E1 activating enzyme for Urm1 in S. cerevisiae (Furukawa et al., 2000) and established Urm1/Uba4 as the fifth protein conjugation system in yeast. Further studies revealed that Urm1 has inherited the sulfur transfer activity from its prokaryotic ancestors (Huang, Lu and Bystrom, 2008; Nakai, Nakai and Hayashi, 2008; Schlieker et al., 2008; Leidel et al., 2009; Noma, Sakaguchi and Suzuki, 2009), and thus reinforced the conclusion that Urm1 is indeed the “molecular fossil” that links the ancient UBL progenitors to eukaryotic protein modifiers (Iyer, Burroughs and Aravind, 2006; Schlieker et al., 2008).

4.5.1 Urm1 from evolutionary perspective

Similar to other members of the UBL family, Urm1 has the ability to form conjugates with other proteins. However, upon identification, it was noticed that S. cerevisiae Urm1p shares ~40% sequence identity with the E.coli proteins of MoaD and ThiS, which are involved in the biosynthesis of molybdopeterin and thiamine, respectively. (Furukawa et al., 2000; Goehring, Rivers and Sprague, 2003a). These early studies of Urm1 in yeast linked Urm1 to nutrient sensing, budding, rapamycin hypersensitivity and impaired growth at high temperatures (Furukawa et al., 2000; Goehring et al., 2003; Goehring, Rivers and Sprague,

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2003a). A few years later, Urm1 was in addition demonstrated to function in the modification of cytosolic tRNAs, specifically displaying a key role as a sulfur carrier in tRNA thiolation (Huang, Lu and Bystrom, 2008; Nakai, Nakai and Hayashi, 2008; Schlieker et al., 2008; Leidel et al., 2009; Noma, Sakaguchi and Suzuki, 2009). Apart from its role during cytokinesis in HeLa cells (Schlieker et al., 2008), Urm1 has in recent years primarily been considered as a protein important for the cellular response against oxidative stress (A. S. Goehring, Rivers and Sprague, 2003; Goehring, Rivers and Sprague, 2003b; Van der Veen et al., 2011).

Multiple species of archaea, which phylogenetically are placed closer to eukaryotes than prokaryotes, also express UBL proteins that display a dual ability to function in tRNA thiolation and protein conjugation (Humbard et al., 2010; Miranda et al., 2011; Ranjan et al., 2011; Anjum et al., 2015), suggesting that the dual functionality of UBLs initially appeared in archaea. However, this theory was recently challenged by the discovery that the UBL TtuB in prokaryotic T. thermophiles, which in addition to acting as a sulfur carrier in tRNA modification, also has the capacity to conjugate to target proteins (Shigi, 2012; Maupin-Furlow, 2014).

Another model organism in which Urm1 has been studied is the plant Arabidopsis thaliana, which encodes two genes that are considered as orthologues of Urm1; Urm11 and Urm12, both activated by the ubiquitin- activating enzyme-like protein Cnx5 (Nakai et al., 2012). Urm11 and Urm12 are highly similar to each other and exhibit 55% and 54% sequence identity to human Urm1, respectively. Interestingly, also in plants, loss of the Urm1 results in severe organ development defects, as well as impaired modification of tRNAs (Nakai et al., 2012).

4.5.2 Structure and Biochemistry

Urm1 displays the typical UBL β-grasp structural fold and conserved C-terminal tail that ends with the characteristic GG-motif (Pedrioli, Leidel and Hofmann, 2008; Wang et al., 2011) In 2006, the structural properties of Urm1 in Mus musculus (AAH26994.1) was studied in detail by NMR spectroscopy. Similar to MoaD, ThiS and ubiquitin, M. musculus Urm1 was indeed found to exhibit the conserved β-grasp topology and GG-motif, however, it showed a stronger sequence identity to its prokaryotic orthologous (14%-22%), as compared with the other UBLs (6%-14%) (Singh et al., 2005). Hence, this structural analysis of Urm1 yet again supported the hypothesis that eukaryotic UBLs have evolved, via Urm1, from sulfur carrier proteins in prokaryotes.

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Urm1 is a dual function protein. On one hand it functions as a protein modifier by covalently conjugating to target proteins, and on the other hand it has a well- defined role as a sulfur carrier during tRNA modification. Nevertheless, these two processes show striking similarities, primarily since the initial step in both pathways involves activation of Urm1 by acyl adenylation of its C-terminus and formation of a complex with Uba4. The subsequent steps however, are different, as shown in Figure 4 and described in detail below. Following activation, Urm1 can either be utilized as a sulfur carrier in the thiolation of lysine, glutamine or glutamate tRNA, or conjugate to cellular target proteins.

4.5.3 Urm1 in tRNA modification

Between the years 2008 and 2009 a chain of independent publications linked Urm1 to tRNA modification (Huang, Lu and Bystrom, 2008; Nakai, Nakai and Hayashi, 2008; Schlieker et al., 2008; Leidel et al., 2009; Noma, Sakaguchi and Suzuki, 2009). tRNA molecules contain several non-canonical nucleotides that are under control of posttranscriptional modifications, which affect the folding, stability and turnover of tRNAs (Wang, Yan and Guan, 2007; Agris, 2008). These modified nucleotides can also regulate the interaction between mRNAs and the ribosome scaffold, and therefore alter the effectiveness of protein translation (Ghosh, 1976; Bjork et al., 2007; Johansson et al., 2008).

One of the best characterized tRNA modifications is the modification of the wobble uridine 34 (U34) in the tRNA molecules tRNALys (UUU), tRNAGlu (UUC) and tRNAGln (UUG) (Gustilo, Vendeix and Agris, 2008; Phizicky and Hopper, 2010; Laxman et al., 2013). In general, there are two sets of 5 modifications that target U34. These include mcm U34, where a methoxy- carbonyl-methyl group is added to position 5 of U34, an event that often is followed by a second modification, namely the replacement of the oxygen at 5 2 position 2 by a sulfur atom, resulting in mcm s U34 (Laxman et al., 2013). However, it should be noted that these conserved modifications can also exist separately on their own (Yarian et al., 2002; Chen, Huang, Eliasson, et al., 2011). Modification by mcm5 is mediated by the Elongator protein (ELP) complex together with the methyltransferase Trm9p and its associated protein Trm112p (Kalhor and Clarke, 2003; Huang, 2005; Begley et al., 2007; Chen, Huang, Anderson, et al., 2011), whereas thiolation is achieved by a different set of proteins, including Nfs1, Tum1, Ncs2, Ncs6, Uba4 and Urm1. In the latter pathway, Urm1 functions as a sulfur carrier, which transfers sulfur derived from a cysteine in the sulfur donor protein Nfs1, onto U34 (Goehring, 2003; Nakai et al., 2004; Nakai, Nakai and Hayashi, 2008; Schlieker et al., 2008; Leidel et al., 2009; Noma, Sakaguchi and Suzuki, 2009; Juedes et al., 2016). Specifically, the sulfur rely that leads to tRNA thiolation begins with the transfer of a sulfur atom

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Figure 4. Urm1 is involved in UBL conjugation, as well as sulfur rely in tRNA modification. from a cysteine residue in the desulfurase enzyme Nfs1 onto Uba4, which occurs with help of the sulfur transferase Tum1. Next, Uba4 forms a complex with Urm1, leading to the activation of Urm1. Following a reduction of the acyl-disulfide bond between Urm1 and Uba4, the thiolase enzymes Ncs2 and Ncs6 orchestrate the transfer of sulfur onto the specific tRNA. (Figure 4)

It is evident that U34 modifications modulate translation by enhancing the codon-anticodon interaction and facilitating the mRNA-tRNA translocation on the ribosome, allowing the next codon to move into the decoding center. Importantly, these modifications allow the translation of AGA and AGG codons during DNA damage (Yarian et al., 2002; Murphy et al., 2004; Phelps et al., 2004; Begley et al., 2007). In recent years, it has further been shown that nutrient availability, in particular of sulfur containing resources, affect tRNA thiolation, and that the levels of sulfur-containing amino acids is a determinative factor for 2 the occurrence of s U34 modifications (Laxman et al., 2013).

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4.5.4 Urm1 as a protein modifier

Even though Urm1 shares minimal sequence similarity with ubiquitin, it can similar to ubiquitin attach to and modify other proteins in a process known as urmylation (Furukawa et al., 2000; Goehring, Rivers and Sprague, 2003; Van der Veen et al., 2011). As indicated earlier, several of the mechanistic properties of UBL conjugation are comparable to the events that occur during the biosynthesis of sulfur-containing cofactors (Rudolph et al., 2001). The first protein to be identified as a binding partner of Urm1 was Uba4, picked up in a yeast-2-hybrid assay (Furukawa et al., 2000).

A few years after the Urm1-Uba4 conjugation machinery was discovered in yeast, Alkyl hydroxide reductase 1 (Ahp1p) was pinpointed as the first bona fide target of urmylation. Besides providing evidence for a covalent conjugation of Urm1 to Ahp1, this study demonstrated that apart from temperature sensitivity, the urmylation pathway displays anti-oxidant roles (A. S. Goehring et al., 2003). In subsequent reports, it was further confirmed that following exposure to oxidative stress agents such as H2O2 and diamide, yeast cells show a striking elevation of Urm1 conjugation (Van der Veen et al., 2011).

As demonstrated by Furukawa et al already in 2000, the interaction between Urm1 and its activating enzyme Uba4 is sensitive to the presence of reducing agents such as DTT, suggesting that a thioester bond is linking the two proteins. However, this conclusion was later challenged by Schmitz and co-workers, who demonstrated the presence of a thiocarboxylate at the C-terminus of Urm1p, thus implicating that the Urm1-Uba4 interaction is more likely mediated via a disulfide bridge (Schmitz et al., 2008).

To date there are no reports on any Urm1 specific E2 or E3 enzymes. However, given the presence of an RHD domain in Uba4, it has been proposed that Uba4 may function as an E1-E2 hybrid protein (Hochstrasser, 2000). This idea is supported also by the identification of an acyl-disulfide bond between Urm1 and the active-site cysteine in Uba4, which is located in the RHD domain (Schmitz et al., 2008). In keeping with this hypothesis, acyl-disulfide bonds are frequently observed between E2 conjugating enzymes and their corresponding UBLs (Pedrioli, Leidel and Hofmann, 2008).

For a long time Uba4 and Ahp1 were the only known Urm1-associated proteins. However, by employing a proteomics approach, Van der Veen et al., identified 21 novel Urm1 targets in HeLa cells, which were conjugated by Urm1 in response to oxidative stress. Importantly, this study suggested a variety of different molecular processes that putatively could be affected by urmylation, including nuclear

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transport, apoptosis, DNA repair, and ubiquitination (Van der Veen et al., 2011). Another important contribution to our current knowledge about the targets of urmylation originates from the archaea field, where Humbard and co-workers have identified a rather extensive number of target proteins in H. volcanii, including proteins involved in Ubl-/S- chemistry, stress responses and metabolism, as well as DNA replication, transcription, translation and mRNA processing (Humbard et al., 2010). Interestingly, this study indicates that of the two UBLs present in H. volcanii, SAMP1 targets proteins for degradation in proteasomes and that SAMP2, similar to ubiquitin, can form chains and possibly compete with SAMP1 (Humbard et al., 2010). Similarly, the Urm1 orthologue in S. acidocaldarius has also been found to utilize urmylation to induce proteasomal degradation of its targets (Anjum et al., 2015).

It is estimated that in a yeast cell under normal conditions, ∼60% of Urm1 can be found it its thiocarboxylated form. This suggests that Urm1 activation is necessary but not sufficient for its conjugation, and therefore that urmylation most likely requires some type of stimulation (Petroski, Salvesen and Wolf, 2011). It is not clear to what extent target proteins are modified by Urm1, yet according to the analogy with other UBLs, and the example of Ahp1, only a small amount of the total population of the target proteins is likely to undergo modification at a given time point (Van der Veen et al., 2011).

4.6 Uba4 - The activating enzyme for Urm1

The mechanistic similarities between UBL conjugation and the biosynthesis of sulfur-containing co-factors originates in the initial step of the two processes, which begins with adenylation of the C-terminal glycine of the UBL molecule. In both prokaryotes and eukaryotes, this event is handled by proteins in the E1-like- enzyme superfamily (Burroughs, Iyer and Aravind, 2009). The activity of Urm1, whether it is acting as a sulfur carrier or a UBL, is dependent on its dedicated activating enzyme Uba4, known as MOCS3 (Molybdenum Synthesis Cofactor 3) in mammals (Furukawa et al., 2000).

Uba4 is a highly conserved protein and its orthologues are universally present in phylogeny, from ancient prokaryotes to man. In parallel to the suggestion that Urm1 and the UBL superfamily originated from MoaD and ThiS, the proteins MoeB and ThiF were put forward as the evolutionary progenitors of the E1 superfamily (Iyer, Burroughs and Aravind, 2006). Besides acting as the Urm1- specific E1-activating enzyme, human MOCS3 in addition plays a well- characterized role in the biosynthesis of the molybdenum co-factor (MoCo),

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which is required for the activity of a group of enzymes called molybdoenzymes (Schwarz and Mendel, 2006).

4.6.1 Structure and function of Uba4

Structurally, Uba4 consists of two specific domains, an E1-like domain and a rhodanese homology domain (RHD), located in the N-terminal and C-terminal part of the protein, respectively. Even though Uba4 resembles the bacterial proteins ThiF and MoeB, the sequence similarity shared between these prokaryotic ancestors and human MOCS3 is limited to the N-terminus of the protein, where the E1 domain is located (Andreas Matthies, Manfred Nimtz and Silke Leimkühler, 2005). Nevertheless, within the eukarya domain, the Uba4/MOCS3 is highly conserved, as indicated by a sequence identity of 36% shared between yeast Uba4 and human MOCS3 (Schmitz et al., 2008). Another critical structural characteristic of Uba4/MOCS3 is the presence of two strictly conserved cysteine residues within the conserved domains. These catalytic cysteines are essentially the active sites for the enzymatic activity of Uba4 (Krepinsky and Leimkühler, 2007; Schmitz et al., 2008). The C-terminal RHD domain displays rhodanese activity. Rhodaneses are sulfur carrier enzymes that catalyse the transfer of sulfane (thiosulfoxide) sulfur atoms from thiosulfate to cyanide, and can be found as extensions to other proteins, as single-domain proteins or as tandem repeats (Bordo and Bork, 2002; Schmitz et al., 2008). Sulfane sulfurs function in co-factor synthesis, tRNA sulfuration, enzyme activity modulation and regulation of the redox environment in the cell (Toohey and Cooper, 2014).

The dual functions of Uba4 are mediated via two distinct pathways (Figure 5). Firstly, by activating Urm1, Uba4 initiates protein urmylation, as well as tRNA modification. Secondly, Uba4 can also contribute to the biosynthesis of MoCo (molybdenum co-factor), by activating another UBL protein, Mocs2A. Interestingly, there is a clear homologue of Mocs2A also in the D. melanogaster genome, CG42503-R), which displays 40% amino acid sequence identity to human Mocs2A. While Urm1 and Uba4 have shown to be essential for invasive vegetation and budding in yeast, S. cerevisiae is in fact one of few organisms that does not employ molybdoenzymes, and therefore the role of Mocs2A and MoCo can be disregarded in yeast. In contrast, human MOCS3 has retained the ability to activate both Urm1 and Mocs2A (Schmitz et al., 2008).

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Figure 5. Molecular mechanism underlying the dual activities of Uba4, mediated via interaction with Urm1 and Mocs2A, respectively.

In humans, loss of MOCS3 is strongly implicated in the aetiology of the rare disease molybdenum co-factor deficiency, an inborn condition that also is associated with the loss of other key enzymes in the MoCo biosynthesis pathway, namely MOCS1, and MOCS2 (in addition to MOCS3). Patients suffering from molybdenum co-factor deficiency are severely affected during the neonatal period and display abnormalities such as seizures and skeletal dysmorphic features. In most cases the disease causes mortality within a few months after birth (Atwal and Scaglia, 2016; Huijmans et al., 2017).

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4.7 Oxidative stress

Oxygen molecules are essential for aerobic life. In eukaryotic cells, the major consumption of oxygen occurs in mitochondria, during the production of ATP in the cellular respiration pathway. An unavoidable by-product of oxygen metabolism in different cellular compartments is the generation of reactive − oxygen species (ROS), such as superoxide anions (O2 ), hydrogen peroxide − (H2O2) and hydroxyl radicals (OH ). Production of ROS can also be induced by environmental stressors such as molecular pollutants, UV radiation and hyperthermia (Finkel and Holbrook, 2000; Taylor and Pouyssegur, 2007; Zaher et al., 2007).

Accumulation of ROS leads to oxygen toxicity (e.g. oxidative stress) and hence, it is critical for every cell to be able to counteract oxidative stress in order to survive. Excessive ROS can directly damage cellular macromolecules, such as DNA, proteins and lipids, which may result in cell cycle arrest and ultimately cell death (Cacciuttolo et al., 1993). On a larger scale, in humans, inadequate responses to oxidative stress contributes to ageing, as well as the initiation and progression of disease, such as cancer and diabetes (Zhang, Wang and Su, 2009; Le Lay et al., 2014)

4.7.1 Molecular mechanisms behind oxidation and reduction

Over the course of evolution, cells have developed several different strategies to defend themselves against oxidative stress. One of the most important families of anti-oxidant proteins are the peroxiredoxins, which act by catalyzing the reduction of hydrogen peroxide to water. The peroxiredoxins (Prxs) are one of the most abundant protein families and are, based on the number and position of Cys residues that participate in catalyzing reduction, historically classified into three groups, including the; 1) Typical 2-Cys Prxs, 2) Atypical 2-Cys Prxs, and the 3) 1- Cys Prxs (Sue, Ho and Kim, 2005).

Another mechanism of counteracting oxidative stress is the upregulation of cytoprotective genes, including glutathione S-transferases (GSTs), aldehyde dehydrogenases (ALDH) and NAD(P)H:quinone oxido-reductases (NQO1) (Taylor and Pouyssegur, 2007; Jones, 2008; Zhang, Wang and Su, 2009). These proteins function by maintaining a cellular buffer of redox proteins such as glutathione, which detoxify ROS through a non-enzymatic mechanism (Le Lay et al., 2014).

Several signalling pathways collaborate in order to induce the appropriate responses needed to enable a cell to withstand oxidative stress. Among these the

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JNK, p38 MAPK, p53, NF-κB and Nrf2 pathways have been recognized as most important. The activity of these pathways is by no means specific to oxidation- reduction responses, as they are known to have crucial roles in the response also to other stressors, as well as in normal development, growth and metabolism. It is also needless to say that the nature and duration of the stress inducer, together with the cell type, are also important factors that determine the degree of activation and the outcome of these pathways (Finkel and Holbrook, 2000).

Drosophila has been extensively used in research to understand the mechanisms and cellular defense programs against oxidative stress. One of the most commonly used ROS inducers in Drosophila labs is paraquat (1,1′dimethyl-4- 4′bipyridynium dichloride), a quaternary nitrogen herbicide, that is highly toxic to humans and animals (Arking et al., 1991; Wang, Bohmann and Jasper, 2003). The toxicity of paraquat is caused by the generation of ROS, such as hydroxyl radicals and hydrogen peroxide, which may result in acute poisoning, multiple- organ failure and ultimately death (Suntres, 2002; Sittipunt, 2005; Raghu et al., 2013). In flies, paraquat exposure leads to locomotor defects and shortened lifespan, and is on the cellular level associated with increased levels of apoptotic death and activation of the JNK pathway (Peng et al., 2004; Klintworth et al., 2007; Jordan et al., 2012).

4.8 The JNK pathway

Stress-activated protein kinases (SAPKs) are evolutionary conserved signaling molecules that enable living organisms to respond to intrinsic and extrinsic stimuli in the form of stress. One of the most versatile and ubiquitous stress sensors, is the c-Jun N-terminal kinase (JNK) signaling pathway, which is triggered by a wide range of environmental insults including UV radiation, inflammatory cytokines, DNA damage, heat stress, bacterial antigens, and perhaps most importantly, ROS (Biteau et al., 2011).

Classified as one of the mitogen-activated protein kinase (MAPK) pathways, the JNK pathway is a pleiotropic signaling cascade with well-established roles in the response to stress signals, as well as proliferation, apoptosis, motility, metabolism and DNA repair (Biteau et al., 2011). Therefore, it is no surprise that aberrant activity of JNK signaling is involved in human pathogenesis, linked to multiple birth defects and diseases such as neurodegeneration, chronic inflammation and cancer (Biteau et al., 2011).

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As the name indicates, JNK activity results in the phosphorylation of c-Jun, specifically at residues Ser63 and Ser73, located in the N-terminal part of the protein (Behrens, Sibilia and Wagner, 1999). Together with Fos, Maf and ATF, c- Jun contributes to the formation of the major JNK-induced AP-1 heterodimeric transcription factor complex, which upon phosphorylation mediates gene- specific transcriptional control (Behrens, Sibilia and Wagner, 1999; Shaulian and Karin, 2001; Johnson and Nakamura, 2007).

The canonical JNK pathway is built on a chain of phosphorylation events that is triggered by a stimulus-induced, selective activation of a JNK kinase kinase (JNKKK), which phosphorylates a JNK kinase (JNKK) that subsequently induces phosphorylation of JNK. Following activation, JNK in turn affects its downstream targets, the c-Jun and Fos components of the AP-1 complex, as well as the transcription factor Forkhead Box O (FoxO), subsequently triggering a chain of events that lead to changes in the cellular transcriptome (Johnson and Nakamura, 2007; Weston and Davis, 2007) (Figure 6).

4.8.1 The JNK pathway in Drosophila

In contrast to vertebrates, where the components of the JNK pathway are represented by a rather large set of genes, the low redundancy in the fly genome renders a Drosophila JNK pathway that is far less complex and much easier to study (Biteau et al., 2011). In mammals, there are three JNK proteins, encoded by three separate genes (Bsk1, Bsk2 and Bsk3), whereas in flies there is only one known JNK homologue, basket (bsk), which regulates the fly AP-1 complex, consisting of Jra (dJun) and Kayak (dFos). Upstream of Drosophila Bsk, there is one major JNK kinase, Hemipterous (Hep), together with the less-characterized JNKK, dMKK4 (Davis, 2000; Boutros, Agaisse and Perrimon, 2002; Chen, White and Cobb, 2002; Geuking et al., 2009). In Drosophila, the AP-1 complex have been shown to regulate the transcription of a battery of stress-associated genes, as exemplified by hsp68, gstD1, fer1HCH, and mtnA (Wang, Bohmann and Jasper, 2003). Another important target gene of the fly AP-1 complex is puckered (puc), a phosphatase that targets JNK itself and thereby downregulates its activity in a negative transcriptional feedback loop (Martín-Blanco et al., 1998; McEwen, 2005). Despite its relative simplicity, JNK signaling in Drosophila remains highly diverse and context-dependent, involved in an extensive array of physiological events, such as embryonic development, immunity, stress responses and longevity (Riesgo-Escovar et al., 1996; Sluss et al., 1996).

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Figure 6. The JNK pathway in Drosophila.

Mutation in any of the genes encoding the major components of the JNK pathway (e.g. hep, bsk, kay, jra and even puc), severely affects embryonic development in Drosophila. Commonly, they all cause lethality due to a disruption of dorsal closure, pinpointing that a strictly regulated JNK signaling is required for proper development of the fly (Martín-Blanco et al., 1998; Agnès, Suzanne and Noselli, 1999; Noselli and Agnès, 1999; Stronach and Perrimon, 1999; McEwen, 2005). Furthermore, there have been many additional studies, in which critical roles of the JNK pathway have been reported in pupal thorax closure, endoderm patterning, wing patterning and eye development (Kockel, Homsy and Bohmann, 2001). All these functions are clearly in agreement with the established role of JNK signaling in vertebrates, including cell migration, wound healing, and bone morphogenesis, as well as neuronal cell death and differentiation, strongly indicating an evolutionary conserved role for the JNK pathway in development (Martin, 2004).

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As briefly mentioned previously in the NMJ growth context, JNK signaling is involved in the formation of neuronal circuits in fruit fly. In third instar larvae, neuronal AP-1 positively regulates synapse formation and growth (e.g. bouton count), as well as synaptic strength (Sanyal et al., 2002; Etter et al., 2005). Hence, simultaneous neuronal overexpression of Jun and Fos results in an increased bouton count, whereas expression of dominant negative variants of the proteins induces the reciprocal effect. In agreement with being a downstream event of JNK activity, this neuronal function of AP-1 is antagonized by the expression of Puc (Sanyal et al., 2002). Molecularly, the outcome of neuronal hyperactivation is a transcriptional upregulation of multiple downstream genes (Etter et al., 2005), and even if the biological importance of these have not been determined, it is obvious that many rapid changes in gene expression occurs during the refinement of NMJs (Zhang et al., 2016).

Interestingly, JNK activity at NMJs has previously been shown to be regulated by the ubiquitin system, where the E3 ubiquitin ligase Highwire (Hiw) targets the JNKKK Wallenda (Wnd) for ubiquitination and subsequent degradation in the proteasome (Collins et al., 2006). As a result, highwire (hiw) loss-of-function mutants display an enhanced JNK/AP-1 activation, resulting in a significant NMJ overgrowth (Collins et al., 2006; Shen and Ganetzky, 2009). Altogether, it can be concluded that increased activity of the JNK pathway triggers NMJ overgrowth, regardless of the underlying cause. In support of this theory, a rather recent study demonstrated a role of oxidative-stress-induced JNK activity in promoting NMJ growth (Milton et al., 2011).

4.8.2 The JNK pathway in stress responses and lifespan regulation

According to the “free radical theory of aging” (Harman, 1956), an organism’s lifespan is determined by its genetic ability to counteract oxidative stress. Consequently, it is common that the signal transduction pathways that promote protection of cellular macromolecules during oxidative stress, also positively influence the lifespan of an animal (Nathan and Ding, 2010; Sykiotis and Bohmann, 2010; Biteau et al., 2011). In agreement with its role as a key player in the network of cytoprotective genes, the JNK pathway mediates protection against oxidative damage, as well as increased lifespan, which at least in part is due to the expression of anti-oxidant proteins (Wang, Bohmann and Jasper, 2003, 2005). This is particularly important in neuronal cells, where the generation of ROS is high and the levels of anti-oxidant proteins are low (Lin and Beal, 2006).

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As discussed earlier, peroxiredoxins have important functions in the cellular response against oxidative stress and play a crucial role in ROS reduction. It is therefore important to note that one of the prominent targets that is upregulated in response to JNK activation is the peroxiredoxin Jafrac1 (also known as PrxII). Indeed, Jafrac1 overexpression has been shown both to increase the resistance against oxidative stress and extend lifespan in flies, whereas Jafrac1 knockdown by RNAi results in reciprocal phenotypes (Lee et al., 2009).

4.9 Drosophila as a model to study Tax-mediated oncogenesis

When the sequencing of the human genome was completed, the similarities between the human and D. melanogaster genomes strengthened the role of the fruit fly as a model system for understanding human biology and disease, as well as for the development of drugs. Importantly, it is estimated that approximately 75% of the disease-related genes in humans, have functional orthologues in Drosophila (Reiter et al., 2001; Pandey and Nichols, 2011). In recent years, many researchers have employed Drosophila to address questions in the cancer research field, extensively described in multiple reviews (Potter, Turenchalk and Xu, 2000; Halder and Mills, 2011; Miles, Dyson and Walker, 2011). Similar to these studies, we utilized the fruit fly in the third study (submitted manuscript) included in this thesis, to increase the understanding of how the viral oncogene Tax1 is regulated by posttranslational modifications (e.g. Urm1) in Adult T-cell leukemia/lymphoma (ATL).

4.9.1 Adult T-cell leukaemia/lymphoma

Adult T-cell leukemia/lymphoma (ATL) is a rare and often aggressive T-cell lymphoproliferative malignancy that can be found in the blood (leukemia), lymph nodes (lymphoma), skin, and multiple other tissues of the human body (Matutes, 2006). ATL has been linked to infection by the Human T-cell lymphotropic virus type 1 (HTLV-1), however, only 5-10% of the HTLV-1-infected individuals develop ATL. It is also worth noting that the T-lymphocyte transformation commonly occurs after 20-40 years of latency (Kannian and Green, 2010), suggesting that a multistep process is likely to underlie the disease (Barmak et al., 2003; Hasegawa et al., 2006; Matsuoka and Jeang, 2007). ATL almost exclusively affects adults, with a median age of onset in the mid-sixties and no gender preferences (Matutes, 2006). According to present classification systems, ATL exists in four clinical forms, including acute, chronic and smouldering ATL, as well as lymphoma.

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Among these, the acute form is the most severe and also the most common type, representing almost 65% of the total number of ATL cases. (Aouba et al., 2004; Matutes, 2006)

4.9.2 HTLV-1 and Tax

HTLV-1 is a retrovirus that can be transmitted through sexual contact, blood transfusions, and from mother to child via breast-feeding (Edlich, Arnette and Williams, 2000; Matsuoka, 2003; Ohsugi and Koito, 2007). At present, it is estimated that nearly 10 to 20 million people around the world are infected with HTLV-1, predominantly in areas including Japan, the Caribbean, Africa, South America and certain areas in the Middle East and Eastern Europe (Barmak et al., 2003; Van Dooren et al., 2007; Rafatpanah et al., 2011).

HTLV-1 can be detected in cells from virtually all ATL patients and besides causing ATL, HTLV-1 infection has in addition been associated with the neuroinflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (Shimoyama, 1991; Shembade, 2010; Shirinian et al., 2013). To date, four types of HTLV have been identified, numerically named as HTLV-1 to HTLV-4. The most publicly known member of the group is HTLV-3, which was later renamed as Human Immunodeficiency Virus (HIV), the virus that causes AIDS. HTLV-1 and HTLV-2 share approximately 70% genome homology and structural similarity, yet they are completely different in terms of pathogenesis and disease manifestation (Roucoux and Murphy, 2004; Calattini et al., 2005; Matsuoka and Jeang, 2007; Mahieux and Gessain, 2009).

The RNA genome of HTLV-1 is composed of 9032 nucleotides, encoding the structural proteins of the viral core particle (Gag, Env, and Pol), as well as the proteins of the retroviral enzymatic machinery (reverse transcriptase, integrase and protease). In addition, the HTLV-1 genome displays a pX cluster with five open reading frames (ORFs) that can produce accessory proteins such as Tax, p12I, p27I, p13II, and p30II, through alternative splicing (Cereseto et al., 1997; Grant et al., 2002; Azran, Schavinsky-Khrapunsky and Aboud, 2004).

The most extensively studied protein of HTLV-1 is Tax (e.g. Tax-1). Tax is a 40 kDa protein, which is both necessary and sufficient for tumor formation (Shembade, 2010). However, the Tax protein encoded by HTLV-2, Tax-2, does not show any of the oncogenic characteristics of Tax. From a molecular perspective, expression of Tax disrupts the regulation of hundreds of cellular genes, among which proto-oncogenes, cytokines, growth factor receptors, cyclin- dependent kinases (CDKs), inhibitors of CDKs, DNA repair machineries and cell adhesion molecules can be found (Ng et al., 2001; Grassmann, Aboud and Jeang,

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2005). The pleiotropic function of Tax originates from its wide range of direct interactions with cellular proteins, many of which are involved in signal transduction pathways. These include the PI3K/Akt, NF-B, MAPK, TGFβ and CREB (cyclic AMP responsive element binding)/ATF (activating transcription factor) pathways, as well as multiple small G proteins that regulate cytoskeletal organization and chemotaxis (Shirinian et al., 2013). Among these, one of the most important signaling pathways that are directly influenced by Tax, is the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-B) pathway (Suzuki et al., 1996; Neuveut et al., 1998; Haller et al., 2002; Wu et al., 2004).

4.9.3 The NF-B pathway

In mammals there are five members in the NF-κB protein family, including RelA (p65), RelB, c-Rel, p50/p105 (NF-κB1) and p52/p100 (NF-κB2) (Napetschnig and Wu, 2013). By forming homo- and heterodimeric complexes in response to a variety of stimuli, the NF-κB proteins utilize their Rel homology domains to bind DNA and regulate gene expression (Napetschnig and Wu, 2013). A constitutive activation of NF-κB has been reported in many types of human cancers, including ATL (Sun and Yamaoka, 2005). Activity of NF-κB is essential for the regulation and maintenance of the immune system (Hayden and Ghosh, 2011), skeletal system (Novack, 2011), and epithelial tissues (Wullaert, Bonnet and Pasparakis, 2011). At the cellular level, NF-κB functions to promote cell survival, differentiation, and proliferation (Hayden and Ghosh, 2012). In Drosophila, many studies have linked the NF-κB pathway to multiple events during development, as well as functions in the immune system (Minakhina and Steward, 2006; Hayden and Ghosh, 2012).

In the absence of activation, NF-κB proteins are kept inactive in the cytoplasm by binding to a protein of the IκB family, which masks the nuclear localization signal (NLS) of NF-κB (Napetschnig and Wu, 2013). NF-κB is activated following phosphorylation of the IκB by the IKK complex (consisting of IKKα, IKKβ and IKKγ/NEMO). Phosphorylation of IκB enables the subsequent ubiquitination and proteasomal degradation of IκB, resulting in a release and translocation of NF-κB into the nucleus, where it functions as a transcriptional regulator (Chen, 2005).

Tax has been shown to interact with several of the NF-κB proteins, including RelA, p50, and p52 (Sun and Yamaoka, 2005), however, the key event in Tax- mediated activation of NF-κB, is the binding of Tax to IKKγ/NEMO, one of the three components of the IKK complex. Following the binding of Tax to the IKK complex, Tax recruits kinases that normally act upstream of the IKK complex, such as TAK1 (TGF-β activating kinase 1) and MEKK1 (MAPK/ERK kinase kinase

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1) to the complex. These kinases phosphorylate and activate IKKα and IKKβ, resulting in IκB degradation and nuclear translocation of NF-κB, independently of external stimuli (Chu et al., 1999; Harhaj and Sun, 1999; Harhaj et al., 2000; Xiao, Harhaj and Sun, 2000; Xiao et al., 2001; Harhaj, Sun and Harhaj, 2006; Wu and Sun, 2007). In addition, Tax has been demonstrated to interact directly with and activate the kinase activity of IKKα and IKKβ, independent of upstream kinases (Chu et al., 1998). Studies of Tax in transgenic mouse models support the finding that Tax maintains these oncogenic properties in vivo (Kwon et al., 2005; Hasegawa et al., 2006; El Hajj et al., 2012). Recently, it was further shown by Shirinian et al. that tissue specific expression of Tax in Drosophila plasmatocytes (resembling mammalian leukocytes) and the compound eye, results in increased plasmatocyte proliferation and ommatidial perturbation, respectively, leading to the formation of a “rough-eye” phenotype (Shirinian et al., 2015). Interestingly, the Tax-induced rough eye phenotype was found to be dependent on Kenny, the Drosophila orthologue of IKKγ/NEMO, which is in keeping with the previous finding that Tax activation of NF-κB is inhibited in T-cells deficient of IKKγ/NEMO (Harhaj et al., 2000).

4.9.4 Posttranslational modification of Tax1

Tax is modified by a number of different posttranslational modifications, including phosphorylation, acetylation, O-GlcNAcylation, ubiquitination, and sumoylation. These modifications are important for Tax-mediated activation of NF-κB signaling, inhibition of DNA repair, repression of the tumor suppressor p53 and cell cycle control (Shembade, 2010; Shirinian et al., 2013).

Tax undergoes phosphorylation on multiple serine and threonine residues, of which at least phosphorylation of Ser300 or Ser301 is necessary for the translocation of Tax into nuclear bodies, as well as the activation of gene expression via NF-κB (Bex et al., 1999; Durkin et al., 2006). Further boosting the activity of Tax, ubiquitination has been demonstrated to result in a cytoplasmic retention of Tax, which is essential for the activation of IKKs and NF-κB signaling (Shembade, 2010). Primarily, Tax is conjugated by K63-linked polyubiquitin chains that enhances Tax activity (Chiari et al., 2004; Shembade et al., 2007; Kfoury et al., 2008), but it can also be modified by K48-linked chains, which in contrast induce proteasomal degradation of Tax (Kfoury et al., 2008). In addition to ubiquitin, Tax is also targeted by sumoylation, which results in a nuclear retention of Tax and formation of promyelocytic leukemia nuclear bodies (PML bodies) (Ariumi et al., 2003). Interestingly, the sites targeted by sumoylation and ubiquitination partly overlap, indicating that a cross-talk between different posttranslational modifications may determine the fate of Tax in the cell (Lamsoul et al., 2005). In line with this hypothesis, Tax degradation was recently

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associated with PML-dependent sumoylation, followed by ubiquitination mediated by the ubiquitin E3 ligase RING finger 4 (RNF4) (Dassouki 2015).

Although there has been intense research on ATL, the prognosis and therapeutic options are still very poor. A recent study of the available treatments and outcomes among 1594 ATL patients reported that the median survival of acute, lymphoma, chronic, and smoldering ATL subtypes were 8.3, 10.6, 31.5, and 55.0 months, respectively. This clearly illustrates that despite recent advances, the current therapeutic options for the acute and lymphoma types of ATL are still very poor (Katsuya et al., 2015; Watanabe, 2017). Currently ATL patients are commonly treated with conventional chemotherapy, monoclonal antibodies (against CD2, CD4, CD52, IL-2 and transferrin receptors), hematopoietic stem cell (HSC) transplantation and anti-viral therapy methods (Bazarbachi et al., 2011). However, our collaborators in Lebanon have demonstrated that treatment with a combination of arsenic trioxide (As2O3) and interferon (IFN)-α can induce cell-cycle arrest and apoptosis in HTLV-1–infected and freshly isolated leukemia cells from ATL patients (Bazarbachi et al., 1999; El-Sabban et al., 2000; Kchour et al., 2009; Dassouki et al., 2015). The arsenic/IFN treatment have yielded promising results in mouse model of ATL (El Hajj et al., 2010) and seems to act via a rapid shut-off of the NF-κB pathway and a delayed shut-off of cell cycle– associated genes, in addition to triggering Tax degradation in the proteasome (Mahieux et al., 2001; Nasr et al., 2003; Wu et al., 2016).

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5. Results and Discussion

The seed for the concept of employing model organisms to understand biological life was planted in the middle of the 19th century, by the work of scientists like Charles Darwin and Gregor Mendel. Without doubt, the work on model organisms have led to significant advancements in science and the understanding of human biology. Taking these principles into account, the idea of taking the knowledge from one organism (“a model organism”) and applying it on another (humans) is the foundation of the work that has been done in this thesis.

The protein Urm1 and its specific E1 activating enzyme Uba4 lie at the heart of the four papers/manuscripts included in this thesis. In the first paper, we provide the first identification and characterization of Urm1 in Drosophila. While confirming the identity of the annotated gene CG33276 as the fly homologue of Urm1, this paper also provides a new perspective into the potential regulatory roles of Urm1 in cellular signaling pathways (e.g. the JNK pathway). In the second paper, we aimed at expanding the knowledge about the urmylation landscape, by identifying novel targets of Urm1 in three developmental stages of the fly. The work in the third paper takes the concept of urmylation to a new level, by establishing a putative novel role for Urm1 in Tax-mediated oncogenesis. Finally, in the fourth paper we have investigated the function of Urm1 and Uba4 in the development and growth of Drosophila NMJs.

5.1. Paper I

Urm1: an essential regulator of JNK signaling and oxidative stress in Drosophila melanogaster.

After the discovery of Urm1 by Furukawa and co-workers in 2000 (Furukawa et al., 2000), a majority of the studies on Urm1 was performed in S. cerevisiae or mammalian cells, without any insights into the role of Urm1 in a multicellular organism. By analyzing the Drosophila genome, we found that it encodes one single clear Urm1 homologue, the annotated gene CG33276, located on the third chromosome (66A8). Drosophila Urm1 shares a striking 66% sequence identity with its human counterpart and 37% sequence identity with the S. cerevisiae Urm1p. The similarity is not limited to the sequence, as all the characteristics of fly Urm1, including size, predicted structural folding and C-terminal GG-motif are similar to the yeast and human homologues.

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Having the right set of tools is a necessity for performing high quality research. Therefore, the early years of this project was dedicated to building a toolbox for studying Urm1 in flies. This included generation of an Urm1 specific antibody, transgenic flies expressing tagged (Flag-, GFP-, HA-) wildtype Urm1, or Urm1 lacking the capacity to conjugate to target proteins (Urm1GG), and most importantly a fly strain deficient of the Urm1 locus (the Urm1n123 null allele).

One of the main benefits of analyzing protein function in a multicellular organism, such as Drosophila, compared to yeast, is the diversity of specified tissues and developmental stages. While the yeast model provides less complexity and facilitates conclusions to be made, the fact that a certain protein can display different expression levels and perform different functions in specific tissues and/or developmental stages, fails to be addressed. A clear illustration of the latter is the apparent dynamics of Urm1 activity in flies, where there is a dramatic difference in both the expression level and urmylation pattern of an early embryo, compared to adult flies. Besides this temporal regulation of Urm1 expression and activity, it is also clear that Urm1 is differentially expressed in different tissues and at specific developmental stages. This is exemplified by the relatively high levels of Urm1 that can be found in the wing discs of third instar larvae, compared with the salivary glands, where it is hardly detectable (illustrated in Manuscript III). Such diversity in expression can possibly translate into a stage- and/or tissue-specificity of Urm1 targeting, which is highlighted in the second paper.

When discussing a posttranslational protein conjugation system, the first question that comes to mind is the subject of protein modification itself. Similar to findings in S. cerevisiae (Furukawa et al., 2000; Goehring, Rivers and Sprague, 2003; Goehring, Rivers and Sprague George F., 2003), we showed in Paper I that Urm1 interacts with Uba4, and that co-expression of Urm1 and Uba4 can induce urmlyation in Drosophila tissues. Importantly, we also demonstrated that urmylation in flies is dependent on Uba4, since RNAi-mediated knockdown of Uba4 resulted in significantly reduced levels of target protein urmylation.

Already from the early years, urmylation has been associated with oxidative stress, given the identification of the anti-oxidant protein Ahp1 as a the first identified target of Urm1, and the accumulation of urmylated targets detected in response to exposure to diamide or H2O2 by Van der Veen et al. in 2011 (Van der Veen et al., 2011). In agreement, we found Urm1 to physically interact with the fly homologue of Ahp1, Peroxiredoxin 5 (Prx5), an interaction which seems to be further boosted in response to paraquat-induced oxidative stress (to levels easily detectable by Western blot). (Michalak, Orr and Radyuk, 2008; Radyuk et al., 2009). This suggests that in non-stressed conditions, only a small portion of Prx5 is urmylated, whereas during oxidative stress the levels of urmylated Prx5 is elevated. This finding was further supported in the second paper, where Prx5 was

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identified among the targets of urmylation. Interestingly, Prx5 displays a cytoprotective role against oxidative stress and apoptosis, and thereby promotes longevity in flies.

A homozygous loss of function mutation in Urm1 (Urm1n123) is predominantly lethal in flies, however, there appears to be certain factors that can contribute to support the survival and completion of development into adulthood of at least a small percentage of the mutant population. Analyzing the survival graph for Urm1n123 mutants, it is noticeable that roughly 30% of the eggs do not hatch into first instar larvae. This is especially interesting, since there is a high level of Urm1 activity during embryogenesis. In situ hybridization analysis of Urm1 mRNA suggests that Urm1 is highly maternally contributed, which most likely enables the survival of Urm1n123 animals. In agreement, if the maternal contribution of Urm1 is removed through genetic approaches, the survival rates dramatically drop to zero, with mutant eggs displaying severe early embryonic developmental defects. Furthermore, in a mixed population of heterozygous Urm1n123 flies, it is rare to come across the Urm1n123 mutant flies. However, if the mutant animals are sorted and kept separately from the first instar stage, their survival rate increase, which may indicate that homozygous mutants fail to compete in an environment with heterozygous siblings. The small population of escapers that eventually eclose into adults are valuable animals, since we can employ them to investigate the effects of Urm1 loss of function (LOF) in adult life. Homozygous Urm1n123 escapers have short life span, are slightly smaller and skinnier than wild type flies, and display a distinctive female sterility.

It was against our expectations to find that Urm1n123 mutant flies were not sensitive, but instead more tolerant to paraquat-induced oxidative stress. Despite confirming the evolutionary phenomenon of Urm1 conjugation to Prx5, we were unable to establish any biological proof for a genetic interaction between Prx5 and Urm1, which could explain the stress resistance. Arguably, loss of Urm1 is even beneficial for the stress sensitivity displayed by Prx5 null mutants (Figure 7), suggesting an involvement of a parallel pathway in oxidative stress tolerance.

Elevation of pJNK in Urm1n123 LOF clones is an unmistakable sign of JNK pathway hyperactivation in the absence of Urm1 (Glise, Bourbon and Noselli, 1995; Stronach, 2005). This was further reinforced by an increased nuclear accumulation of dJun, as well as upregulation of proteins that are transcriptionally regulated by JNK, such as gstD1 and Jafrac1. This increased JNK activity is a plausible explanation for the oxidative stress resistance in Urm1n123 flies, but also raises an important question. JNK activity has been associated with an enhanced longevity in flies (Wang, Bohmann and Jasper, 2003, 2005), then why is the lifespan shorter in Urm1n123 mutants? There are several possible explanations and answers, like interference of Urm1 with other

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signaling cascades, or activation of the pro-apoptotic role of the JNK pathway (Igaki, 2009) in older adult flies, which could contribute to shortening the lifespan and induce premature aging in Urm1n123 mutants, but the exact answers remain unknown.

Figure 7. Loss of Urm1 enhances the tolerance of Prx5 mutants to oxidative stress and increases their survival rate.

Other important questions in Paper I addresses the role of Urm1 in the regulation of the JNK pathway, and whether any of the major components in the JNK pathway are direct targets of urmylation. In our investigations, we have not managed to prove any urmylation events in the pathway, but this may very well be due to technical limitations and the absence of proper tools. There is published data from other labs that may suggest Urm1 to function as a sulfur carrier in the JNK pathway, since two established inhibitors of JNK signaling, Puckered and MKP4, both are influenced by thiolation (Saitoh et al., 1998) and the cellular redox status (Cavigelli et al., 1996; Kamata et al., 2005; Sun et al., 2008). Hence, the exact molecular mechanism linking the JNK pathway and Urm1 remains to be revealed.

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5.2. Paper II

A proteomics approach to identify targets of the ubiquitin-like molecule Urm1 in Drosophila melanogaster

To unravel potential Urm1 interacting regulators of the JNK pathway was certainly a catalyst, but not the main motivation to perform the proteomics analysis to find putative Urm1 targets. One of the ultimate goals of studying a protein modification system, is to know the identity of the modified targets, since they pinpoint the biological processes in which the modifier may be involved and represent starting points for future studies. For Urm1 specifically, prior to our study, the interacting network was quite small and not that many studies had aimed at expanding it. Nevertheless, efforts by Goehring et al., Van der Veen et al., and Humbard et. al. (reviewed in chapter, 4.5) should be recognized for their pioneering work to discover target proteins of Urm1 (Goehring, Rivers and Sprague, 2003; Humbard et al., 2010; Van der Veen et al., 2011). Hence, to overall increase the knowledge about the targets of urmylation, and to specifically identify Urm1-interacting proteins in a multicellular organism and at different developmental stages, we utilized a proteomics-based approach to decipher candidate targets of urmylation in Drosophila.

Since Paper II does not aim at making any functional conclusions regarding the biological role of the interaction of Urm1 with the identified binding partners, the take home massage from this paper is essentially a list of 79 Urm1-interacting proteins, identified specifically at three developmental stages of Drosophila development. A wide range of diversity in molecular processes is covered by these candidate Urm1 targets, such as oxidative stress, tRNA modification, aging, DNA damage response, and immunity. Importantly, among these, we have confirmed five proteins, including Jafrac1, Ciao1, MsrA/EIP71CD, GILT1 and Crammer, in addition to the previously studied Uba4 and Prx5, to interact physically with Urm1. Interestingly, while the majority of these targets were found to behave as urmylation targets that display a size shift following conjugation to Urm1, we discovered that one of these Urm1-interacting proteins, Crammer, showed a non- covalent interaction with Urm1. In contrast to the bona fide urmylation targets, there is no size shift of Crammer on Western blots following immuneprecipitation together with Urm1, suggesting a mode of binding that is sensitive to denaturing conditions (i.e. a non-covalent interaction).

In light of the identification of novel Urm1-associated proteins in this paper, we found more clues regarding the potential connection between JNK signaling and Urm1. For instance, among the putative urmylation targets, we found Sac1, which

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displays a reported role as a repressor of JNK pathway activation in flies (Wei et al., 2003). Another protein that is interesting to highlight in this regard is Par-1, a kinase with a regulatory function in the Wnt signaling pathway, and further known to also actively block JNK signaling by acting as a kinase switch for the downstream responses mediated by the JNK activator, Dishevelled (Dsh) (Sun et al., 2001).

As the nature and functionality of many of the targets are adequately discussed in the paper, I will not go through them one by one. Still, I think that some key findings should be mentioned here. Except for the six targets that were found in all stages (Uba4, PHGPx, Prx5, MsrA/Eip71CD, CG8078 and GILT1), the majority of the identified proteins were stage-specific. However, considering that the samples were extracted from whole animals, the possibility of having other Urm1- associated proteins that are not predicted by our study cannot be excluded. Other unanswered questions that can be raised in this aspect is firstly which of the proteins that are posttranslationally modified by Urm1 and which ones that interact with Urm1 by other means, and secondly in which tissues all these interactions occur. Likewise, another important consideration is to assess which amount of the target proteins that is modified by and/or interact with Urm1. There are currently no studies addressing this issue, however, based on the general analogy of UBLs, it is to be expected that only a small percentage of the targets is modified. Anyhow, at least for one of the urmylation targets, Prx5, we have noticed a change in modification status following oxidative stress, emphasizing that signaling events or environmental clues most likely have the potential to induce protein urmylation.

5.3. Paper III

The HTLV-1 oncoprotein Tax is modified by the Ubiquitin related modifier 1

Studies on Tax and HTLV-1 have a long history. Since 1977, when the first clinical cases of ATL were reported (Takatsuki, 2005), many researchers have dedicated their time and effort to understand the disease and its underlying molecular mechanisms. As discussed earlier in the introduction (chapter 4.9.2), the causative agent that triggers ATL, the virally encoded oncoprotein, Tax, is at the crossroad of protein modifications.

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In Manuscript III of this thesis, we contribute to the understanding of ATL, by adding yet another molecule to the list of PTMs that modify Tax, namely Urm1. By collaborating together with established researchers in the field, Prof. Ali Bazarbachi and Dr. Margret Shirinian at the American University of Beirut (Lebanon), we discovered that Urm1 conjugates to Tax in HeLa cells, specifically targeting lysine residues 4-8 in Tax. By following up these results with studies in Drosophila, we could further confirm that Urm1 and Tax interact physically also in fly tissues (in vivo), indicating that urmylation of Tax is evolutionary conserved from fly to man.

Given that Tax is known to be modified by ubiquitin and SUMO and that modification by these UBLs strongly affect the subcellular targeting of Tax, we next investigated whether Urm1 also influence the localization of Tax. When consequently co-expressing Tax together with excessive levels of Urm1, it was obvious that Urm1 strongly promotes a cytoplasmic localization of Tax, in HeLa cells as well as in vivo in Drosophila salivary glands. Interestingly, this cytoplasmic accumulation of Tax was also observed upon expression of Urm1 in HuT-102 cells, which are HTLV-1 positive cells derived from an ATL patient. Further supporting a role of Urm1 in the subcellular targeting of Tax, we found that a reduction of Urm1 levels by RNA interference in Drosophila, reciprocally triggered a nuclear accumulation of Tax.

One of the most important cellular pathways that have been depicted as responsible for driving the oncogenesis downstream of Tax, is the NF-B pathway. Similar to the modification of Tax by ubiquitin and SUMO, we have in this manuscript established that also the presence of Urm1 affects the ability of Tax to activate NF-B signaling. Specifically, co-overexpression of Urm1 and Tax results in enhanced activity of NF-κB signaling, as indicated by increased levels of Diptericin mRNA, an antimicrobial peptide and classical downstream target of NF-B in Drosophila. This finding further emphasizes a regulatory role of urmylation, not only in Tax localization, but also for the oncogenic capacity of Tax.

Considering that the same lysine residues that are targeted by ubiquitin and SUMO, also appear to be targeted by Urm1, one possible explanation for the differential localization of Tax may be the result of a competition between Urm1, ubiquitin and SUMO for these lysine residues in Tax (Lamsoul et al., 2005; Nasr et al., 2006). The level of Urm1, ubiquitin and SUMO could thus be a determinative factor for ATL, since the localization, oncogenic activity and also Tax degradation most likely is strongly dependent on the balance between these three UBLs.

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Taken together, the Tax-Urm1 interaction seems to represent an important event in the regulatory network that orchestrates the oncogenic properties of Tax. These preliminary results provide an excellent starting point for further investigations about the regulation of Tax by urmylation and its involvement in ATL.

5.4. Paper IV

Deciphering a novel role of the Urm1/Uba4 conjugation machinery for Neuromuscular Junction (NMJ) development in Drosophila melanogaster

In Paper I, we established a close link between Urm1 and activation of the JNK pathway in adult flies, focusing on its role in the response against oxidative stress. However, this does not rule out that Urm1 could potentially also affect JNK signaling in other developmental stages. The majority of Urm1n123 mutant animals die in late pupal stages, which provides an opportunity to study homozygous mutants during larval development. At first glance, Urm1n123 mutant larvae do not show any obvious developmental defects up until pupariation, however, a closer look at the neuromuscular junctions (NMJs) of third instar larvae, reveals a striking overgrowth phenotype.

Similar to the deficiency of Urm1 protein in Urm1n123 mutants, RNAi-mediated knockdown of Urm1 in both pre- and post-synaptic tissues also results in comparable NMJ phenotypes (utilizing OK371-GAL4 and 24B-GAL4, respectively). Attempt to rescue the NMJ overgrowth of Urm1n123 mutants by overexpressing Urm1 in pre- or post-synaptic tissues, does indeed lead to a partial rescue, confirming that this phenotype is specifically caused by Urm1, and is not the result of a background genetic alteration. In agreement, Urm1rv164 larvae display completely normal NMJs. This finding is particularly interesting, as rescue attempts employing the ubiquitous GAL4 driver Actin-5C, fails to rescue the NMJ overgrowth phenotype in Urm1n123 mutants, suggesting a high tissue- specificity of Urm1 activities that is not satisfactory fulfilled by Actin-5C during NMJ growth.

Based on our previous studies in adult flies, we were keen to investigate whether the JNK pathway is involved also in causing the NMJ overgrowth phenotype in Urm1n123 larvae. These investigations were further prompted by the multitude of papers that depict an important role of JNK signaling during NMJ growth

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(reviewed in the introduction). Our initial results in this regard clearly indicated that the JNK pathway is involved in triggering the NMJ overgrowth caused by the absence of Urm1, since downregulation of JNK activity in synaptic tissue can (at least partially) suppress the phenotype induced by Urm1 knockdown. This further confirms the genetic interaction between Urm1 and the JNK pathway and also suggests that NMJ growth regulation by Urm1 occurs via regulation of the JNK pathway.

In light of previously discussed role of Par-1 as a negative regulator of the JNK pathway (Sun et al., 2001), one of the potential candidates that could explain the NMJ overgrowth in Urm1n123 mutants, is Par-1. Par-1 is a kinase that regulates the synaptic targeting of Dlg through phosphorylation, and thereby affects NMJ growth. Loss of par-1 leads to increased synapse formation, together with an abnormal synaptic ultrastructure and increased synaptic transmission, whereas overexpression of Par-1 has the opposite effects (Zhang et al., 2007).

Arguably, since the function of Urm1 is dependent on its activating enzyme, Uba4, it is equally important to characterize Uba4 and its interaction with Urm1, as it is to investigate Urm1 itself. Similar to our research on Urm1, we started our investigations of Uba4 by generating the required tools (including the mutant strain Uba4n29) and confirmed its genetic and physical interaction with Urm1 (Papers I, II).

Similar to the Urm1n123 strain, Uba4n29 mutants exhibit a NMJ overgrowth phenotype. However, the structural characteristics of the NMJs in Uba4n29 animals are slightly different from those displayed by Urm1 deficient flies, particularly in terms of synapse branching. This can possibly be the result of a different genetic background, or a demonstration that although Urm1 and Uba4 are acting in the same pathway, there are also other pathways (e.g. MoCo biosynthesis) that are affected by lack of Uba4, which may also affect the synaptic structure.

What makes the Uba4n29 mutants a very interesting strain, is the unusually long extension of the larval stages, which is a hallmark for a disturbed hormonal regulation. Considering the striking prolongation of the larval development up to 22 days after egg laying, the list of candidate mechanisms, which may be affected by the absence of Uba4 is rather short. Several hormones and neuropeptides are known to be involved in the regulation of developmental timing in Drosophila, but the master regulator of the events leading to metamorphosis is evidently the steroid hormone ecdysone (Marchal et al., 2010; Yamanaka, Rewitz and O’Connor, 2013). We still do not know if Uba4 is associated with and/or affects the ecdysone pathway, but it is indeed highly tempting to speculate that the explanation for the disturbed timing of development in Uba4n29 mutants is a

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dysfunctional regulation of ecdysone signaling. This hypothesis is to some degree supported by the identification of ecdysone-induced peptides among the Urm1 target candidates described in paper II. However, since Urm1n123 mutants do not show any prolonged larval phenotype and survive quite well in the third instar larval stage, it is unlikely that the Urm1/Uba4 system directly interacts with the ecdysone pathway and may suggest that the influence on ecdysone regulation could be indirect.

Another possible explanation for the prolonged larval stage and lethality in Uba4n29 mutants, may be an involvement of Uba4 in one or several of the major signaling pathways that are important for sulfur metabolism. In this regard, it is important to remember that Uba4 is a dual function protein, which besides activating the Urm1 machinery, also is involved in MoCo biosynthesis. In the MoCo biosynthesis pathway Uba4 interacts with another UBL protein, Mocs2A (Chowdhury et al., 2012). Here again, a sulfur rely system is involved, as follows. The MoCo pathway is initiated by the Molybdopterin (MPT) synthase, which catalyzes the conversion of precursor Z to molybdopterin, a MoCo precursor, by donating sulfur to form a dithiolene group and thereby coordinating the molybdenum atom. Importantly, MoaD is the sulfur donor used during the formation of the dithioloene group in molybdopterin (Rizzi and Schindelin, 2002). MoCo is essential for the function of multiple metabolic enzymes in the xanthine oxidase and sulfite oxidase families (Hille, Nishino and Bittner, 2011). In the absence of MoCo, these enzymes cannot function, leading to an accumulation of toxic chemicals, including sulfite, S-sulfocysteine, xanthine, and hypoxanthine, and ultimately early natal lethality. It is tempting to speculate that a similar cause may underlie the lethality in Uba4n29 mutants.

Similar to the prokaryotic ancestors, mammalian MPT synthases are heterotetramers, consisting of two MoaE and two MoaD subunits (Schwarz and Mendel, 2006; Daniels et al., 2008). In Drosophila, the annotated genes CG42503 and CG10238 encode the small (Mocs2A) and large (Mocs2B) subunits of Mocs2 (dMoaE), respectively. We have generated Mocs2A mutant strains in the lab, but due to lack of time these flies have not been studied in detail. However, Mocs2A null mutants appear to be homozygous viable as adults, but fail to be established as a homozygous mutant line, due to sterility.

The Drosophila counterpart of Mocs2B, CG10238, contains a C-terminal sequence that codes for a MAPK upstream kinase (MUK)-binding inhibitory protein (MBIP) (Suganuma et al., 2010). Interestingly, it was recently shown that dMoaE (Mocs2) acts as a negative regulator of the JNK cascade, mediated via its function as a subunit of the ATAC (Ada two A containing) histone acetyltransferase complex. The ATAC complex directly interacts with the JNK pathway, by acting as a transcriptional cofactor for Jun (Suganuma et al., 2008,

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2010; Wang et al., 2008). This raises the possibility of a putative crosstalk between the Urm1/Uba4 machinery and Uba4/Mocs2 complex involved in MoCo biosynthesis, potentially offering a link between Urm1 and the JNK pathway, via the ATAC complex. In support of this theory, one of the components of the ATAC complex, HCF, has also popped up as an Urm1-interacting protein in our proteomics study (Paper II). Nevertheless, making any detailed conclusions about the relationship between the Urm1 pathway and developmental phenotypes such as JNK-dependent NMJ overgrowth requires further studies.

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5.5. General discussion and future perspectives

In this final part of the discussion, I would like to present my personal perspective on my doctoral project and highlight some reflections and theories that may lie outside the scope of the thesis work.

In the past six years, my understanding of the Urm1 field has led me to believe that there is a huge gap between the different ideas that people in the field express. On one side, there is one group of researchers who tend to argue that Urm1 is only involved in tRNA modification, and that any protein modification function merely is a result of the hyperactive nature of activated Urm1. Hence, these people suggest that protein modification by Urm1, if anything, is a very primitive system that was later specialized in the form of ubiquitin and other UBLs. This may in fact actually be a probable theory, considering that Urm1 is indeed a “molecular fossil”, phylogenetically placed between ancient sulfur carriers and eukaryotic UBLs. On the other side, a second group of researchers including our laboratory, instead believe that Urm1 truly is a dual function protein, which in addition to the sulfur transfer activity displayed during tRNA modification, has acquired the capacity to modify cellular proteins, thereby suggesting that several diverse molecular functions are regulated by urmylation. This hypothesis is supported by our work considering the tissue and developmental-stage specificity of Urm1 interacting proteins, as well as the genetic interaction of Urm1 with one of the major signaling pathways in the cell, the JNK pathway.

Another theory that I would like to put forward here regards the JNK pathway and how the Urm1/Uba4 machinery interacts with it. Considering that the two UBL proteins Urm1 and Mocs2A both interact with Uba4, one hypothesis could be that there exists a competitive relationship between them. Lack of Urm1 could in that case potentially shift the balance towards increased levels of Mocs2A bound to Uba4 and therefore a lower degree of ATAC complex assembly, which could lead to the elimination of a key inhibitory mechanism of the JNK pathway. I do not have much time left as a PhD student, but I sincerely hope that future research will uncover the nature of the interaction and functions of the Urm1/Uba4/Mocs2A cellular machinery and reveal the molecular mechanism behind how it affects the JNK pathway.

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6. Acknowledgements

Twelve years ago, when I decided to study cell and molecular biology at the university, I set my goal to become a successful scientist. Those years are long gone and now I have realized that becoming a scientist is a long journey and that I am just at the beginning of the road, yet that there are checkpoints and subaims to be reached along the way. Now when I am reaching one of these checkpoints in my scientific career, I would like to take the time and thank the people who have supported me during these years.

On top of the list is of course my kind supervisor, Dr. Caroline Grabbe. Thank you for believing in me and giving me the opportunity to work in your lab. I have always looked at you as a wise person, an intellectual boss, a good friend and a role model. You are one of the most precise and organized people that I have ever met. In the past 6 years you have literally raised me as a PhD student and I have learned a lot from you. I wish you the best of luck in the future.

Our research group was a very small with only three people in the meetings for the most part, however, having a speedster technical expert in our lab have boosted our ability to perform experiments and do good research. Ingrid, I have been really lucky to have someone like you as a colleague. Publishing the papers included in this thesis would not have been possible without your effort. Thank you for always being nice and helpful.

I would like to mention our former lab member, Henrik Lindehell, for his work that contributed to generate the Mocs2A mutants, even if they were never really studied in detail. I wish you good luck with your own PhD studies.

Next, I would like to thank our collaborators, Dr. Margret Shirinian and Prof. Ali Bazarbachi, and all the people who contributed to the work of the Tax paper in Lebanon.

Thanks also to my co-supervisor, Prof. Ruth Palmer, for participating in my seminar committee multiple times even when she was away in Gothenburg. Also thank you for giving me the chance to start working as a master student in your lab back in 2011. The current and former members of the Palmer lab, Yasuo, George, Kathrin and Patricia. I honesty miss the crowded fly room and the journal clubs back in those days, when you were still at the Department of Molecular biology. I wish you the best of luck in your experiments and a successful career in Gothenburg (or wherever you will end up).

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To Fredrik my very good colleague and friend. I have enjoyed your fellowship in the past seven years. Thank you for helping me with fly crosses. We did not get to finish those experiments, but the results show that you are a reliable person and a compassionate friend. I wish you a successful career, and who knows, maybe someday we will publish our own collaborative paper.

To the Åsa Rasmuson-Lestander lab, thanks for sharing the lab and office space with us, especially you Sa, always friendly and kind. Thank you for all the times that you shared your fly lines and antibodies with me, as well as fly food and protocols….!! Always notifying me when there was cake in the kitchen. Working alongside you was truly fun.

Next I want to thank the people in the Department of Molecular biology:

To Gunilla Jäger for helping us with the tRNA data and HPLC, the Fly food and Media facility, who have a critical role in catalyzing the speed of research at the department. To Johnny Stenman, for handling the orders, deliveries, posts and literally solving any possible technical problem in the department, to Marek Wilczynski for computer and IT support and finally the current and former administration staff, Maria, Jannike, Patrik and Lina.

When I joined the Department of Molecular biology in 2011, I became a part of a small but great community within the department, the fly floor. I think that our common goal in fly floor is to prove that the Drosophila model system still has its greatest days ahead. This being said, I would like to thank the fly people for nearly seven years of friendliness.

Thanks also to the Epicon groups for the weekly journal clubs, which gave me the chance to learn more about epigenetics and chromosomes. Prof. Jan Larsson and Prof. Dan Hultmark for always smiling and agreeing to join my seminar committee. Respectable Fly researchers Yuri Schwartz and Per Stenberg for interesting scientific discussions in journal clubs and my dear fly floor collogues, Jens-Ola, Inez, Anna-Mia, Masha, Tatiana, Alexander, Jana, Juan, Edvin and Jacob, for friendly interactions in these years.

During my years here in Umeå, I have also made some valuable friendships with some of my colleagues. One of them is my good friend Aman, one of the coolest guys on the planet. Thank you for all of your ideas and support regarding the bioinformatics. I guess that soon it will be your turn to write your PhD thesis, so best of luck to you. My fellow poker buddy, Isaac, thank you for all the fly food support, protocol advice and the brief scientific discussions that we have had here and there. For you too, I wish the best of luck with your PhD thesis. One of my very first friends in Umeå, Marie-Line, thank you for more than seven years of

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friendship, I hope that you and Hasan and baby Tomas stay happy forever. Also, I wish you too a good luck with your PhD studies.

Last but not least, I would like to thank the fellow colleagues with whom I had the pleasure to share the teaching and also everyone else who I failed to mention here.

یس رد انتها الزم میدونم هک چند خطی رو هم هب افرسی بنو م ربای کسانی هک وجودشون هب زندگی من معنا میده و هدف و نهایت تمام

تالش اهم خوشحالیه این زعزیاهن.

پدر و مارد زعزیم، هن فقط نوشتن این اپیان ان،هم، هک تمام موفقیت اهی زندگی من ب خارط از خود گذشتگی شماست. وقت ی هب نوشتن این

لی متن رسیدم متوهج شدم هک اقیانوسی از رحف ربای گفتن و هب اندازه ی ردیایی بی رکان، د ل ربای سپاسگ زار بودن دارم پ س

م همه ی ان گفته اهم رو رد این ممهه لاله یکنم. رمسی، بابت همه چیز.

شبخ مم سخ تلخ زرهای زعزیم، تو امید و خو تی رو ربای من معنا رکدی. ازت نونم بابت اینکه رد تمام تی اه و ی اه و شادی اه و غم اه

مم هس حمایتم رکدی و سختیه رغبت رو ربام آسون رکدی. ازت نونم بابت اینکه تی.

مث بهنام جان، با داشتن رباردی ل تو، من رهزگ احساس تنهایی نکردم. رمسی از اینکه پشتواهن ی من بودی و جور نبودن من رد

کنار لانواده رو هب دوش کشیدی.

صم م رد نهایت یماهن رتین سپاس اه رو تقدیم هب شما دوستان و زندی کانی یکنمهک هچ رد رغب ت و هچ رد وطن من روهمراهی رکدید و تنور دوستی

و معرفت رو رگم نگه داشتید.

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