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Function and targets of the Urm1/Uba4 conjugation machinery in Drosophila melanogaster
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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 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 localization, 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.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2017. , p. 63
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1939
Keyword [en]
Drosophila, Urm1, Uba4, MOCS3, Tax, HTLV, Posttranslational modification, ubiquitin-like modifiers, UBL, PTM, JNK, neuromuscular junctions, signaling
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-143187ISBN: 978-91-7601-815-6 (print)OAI: oai:DiVA.org:umu-143187DiVA, id: diva2:1167520
Public defence
2018-01-26, hörsal E04, byggnad 6E, Norrlands Universitetsjukhus., Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2017-12-21 Created: 2017-12-19 Last updated: 2018-01-04Bibliographically approved
List of papers
1. Urm1: an essential regulator of JNK signaling and oxidative stress in Drosophila melanogaster
Open this publication in new window or tab >>Urm1: an essential regulator of JNK signaling and oxidative stress in Drosophila melanogaster
2016 (English)In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 73, no 9, p. 1939-1954Article in journal (Refereed) Published
Abstract [en]

Ubiquitin-related modifier 1 (Urm1) is a ubiquitin-like molecule (UBL) with the dual capacity to act both as a sulphur carrier and posttranslational protein modifier. Here we characterize the Drosophila melanogaster homologues of Urm1 (CG33276) and its E1 activating enzyme Uba4 (CG13090), and show that they function together to induce protein urmylation in vivo. Urm1 conjugation to target proteins in general, and to the evolutionary conserved substrate Peroxiredoxin 5 (Prx5) specifically, is dependent on Uba4. A complete loss of Urm1 is lethal in flies, although a small number of adult zygotic Urm1 (n123) mutant escapers can be recovered. These escapers display a decreased general fitness and shortened lifespan, but in contrast to their S. cerevisiae counterparts, they are resistant to oxidative stress. Providing a molecular explanation, we demonstrate that cytoprotective JNK signaling is increased in Urm1 deficient animals. In agreement, molecular and genetic evidence suggest that elevated activity of the JNK downstream target genes Jafrac1 and gstD1 strongly contributes to the tolerance against oxidative stress displayed by Urm1 (n123) null mutants. In conclusion, Urm1 is a UBL that is involved in the regulation of JNK signaling and the response against oxidative stress in the fruit fly.

Keyword
Urm1, Drosophila, Oxidative stress, UBL, JNK pathway
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-120089 (URN)10.1007/s00018-015-2121-x (DOI)000374111800013 ()26715182 (PubMedID)
Available from: 2016-06-13 Created: 2016-05-09 Last updated: 2018-03-16Bibliographically approved
2. A proteomics approach to identify targets of the ubiquitin-like molecule Urm1 in Drosophila melanogaster
Open this publication in new window or tab >>A proteomics approach to identify targets of the ubiquitin-like molecule Urm1 in Drosophila melanogaster
2017 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 9, article id e0185611Article in journal (Refereed) Published
Abstract [en]

By covalently conjugating to target proteins, ubiquitin-like modifiers (UBLs) act as important regulators of target protein localization and activity, thereby playing a critical role in the orchestration of cellular biology. The most ancient and one of the least studied UBLs is Urm1, a dual-function protein that in parallel to performing similar functions as its prokaryotic ancestors in tRNA modification, also has adopted the capacity to conjugate to cellular proteins analogous to ubiquitin and other UBL modifiers. In order to increase the understanding of Urm1 and its role in multicellular organisms, we have used affinity purification followed by mass spectrometry to identify putative targets of Urm1 conjugation (urmylation) at three developmental stages of the Drosophila melanogaster lifecycle. Altogether we have recovered 79 Urm1-interacting proteins in Drosophila, which include the already established Urm1 binding partners Prx5 and Uba4, together with 77 candidate urmylation targets that are completely novel in the fly. Among these, the majority was exclusively identified during either embryogenesis, larval stages or in adult flies. We further present biochemical evidence that four of these proteins are covalently conjugated by Urm1, whereas the fifth verified Urm1-binding protein appears to interact with Urm1 via non-covalent means. Besides recapitulating the previously established roles of Urm1 in tRNA modification and during oxidative stress, functional clustering of the newly identified Urm1-associated proteins further positions Urm1 in protein networks that control other types of cellular stress, such as immunological threats and DNA damage. In addition, the functional characteristics of several of the candidate targets strongly match the phenotypes displayed by Urm1(n123) null animals, including embryonic lethality, reduced fertility and shortened lifespan. In conclusion, this identification of candidate targets of urmylation significantly increases the knowledge of Urm1 and presents an excellent starting point for unravelling the role of Urm1 in the context of a complex living organism.

Place, publisher, year, edition, pages
PUBLIC LIBRARY SCIENCE, 2017
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-140904 (URN)10.1371/journal.pone.0185611 (DOI)000411980300077 ()28953965 (PubMedID)
Available from: 2017-11-16 Created: 2017-11-16 Last updated: 2017-12-19Bibliographically approved
3. The HTLV-1 oncoprotein Tax is modified by the Ubiquitin related modifier 1 (Urm1)
Open this publication in new window or tab >>The HTLV-1 oncoprotein Tax is modified by the Ubiquitin related modifier 1 (Urm1)
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: Adult T-cell leukemia/lymphoma (ATL) is an aggressive malignancy secondary to chronic Human T-cell Lymphotropic Virus 1 (HTLV-1) infection, triggered by the virally encoded oncoprotein Tax. The transforming activity and subcellular localization of Tax is strongly influenced by posttranslational modifications, among which ubiquitylation and SUMOylation have been identified as key regulators of the nuclear/cytoplasmic shuttling of Tax, as well as its ability to activate NF-κB signaling.

Results: Adding to the complex posttranslational modification landscape of Tax, we here demonstrate that Tax also interacts with the Ubiquitin-related modifier 1 (Urm1). Conjugation of Urm1 to Tax results in a redistribution of Tax to the cytoplasm, potentially contributing to oncogenic Tax-induced NF-ĸB activation. Utilizing a tax-transgenic Drosophila model, we show that the Urm1-dependent subcellular targeting of Tax is evolutionary conserved, and that the presence of Urm1 is strongly correlated with the transcriptional output of Diptericin, an antimicrobial peptide and established downstream target of NF-B in flies.

Conclusions: These data put forward Urm1 as a novel Tax modifier that modulates its oncogenic activity and hence represents a potential novel target for developing new strategies for treating ATL.

Keyword
ATL, HTLV-1, Tax, Urm1, NF-B, oncogenesis
National Category
Cancer and Oncology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-143183 (URN)
Available from: 2017-12-19 Created: 2017-12-19 Last updated: 2017-12-19
4. Deciphering a novel role of the Urm1/Uba4 conjugation machinery for Neuromuscular Junction (NMJ) development in Drosophila melanogaster
Open this publication in new window or tab >>Deciphering a novel role of the Urm1/Uba4 conjugation machinery for Neuromuscular Junction (NMJ) development in Drosophila melanogaster
(English)Manuscript (preprint) (Other academic)
Abstract [en]

In Drosophila melanogaster, development of the part of the peripheral nervous system that is dedicated to orchestrate locomotion, relies on an intricate interplay between the motor neurons that emanate from the ventral nerve cord, and the body wall musculature that they are destined to innervate. A rather large array of cytoskeletal and signaling proteins are implicated in the formation and growth of the synapses, where motor neurons contact the musculature, specifically at structures known as neuromuscular junctions (NMJs). Master regulators of NMJ development and growth include the WNT, BMP/TGFβ, MAPK, PI3K and JNK signaling pathways. Here we describe a novel role of the ubiquitin-like molecule (UBL) Urm1 (Ubiquitin-related modifier 1) in the regulation of NMJ formation. Specifically, we show that Drosophila Urm1n123 null mutants, as well as flies deficient of the dedicated Urm1 E1 activating enzyme Uba4, Uba4n29, display a significant NMJ overgrowth in third instar larvae, and that Urm1 and Uba4 interact genetically in this process. By utilizing the UAS/GAL4 system, we further provide evidence that Urm1 appears to act in both pre- and post-synaptic tissues, but rescue experiments emphasize a primary role of Urm1 in presynaptic motor neurons. In keeping with the previously established link between loss of Urm1 and excessive activation of JNK signaling, we can conclude that there is a strong genetic interaction between Urm1 and Drosophila JNK pathway components also during NMJ formation. Taken together, we have identified a role of the Urm1/Uba4 conjugation machinery in the regulation of NMJ development and growth, putatively induced by enhanced pre-synaptic activation of the JNK pathway.

National Category
Biochemistry and Molecular Biology Neurology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-143186 (URN)
Available from: 2017-12-19 Created: 2017-12-19 Last updated: 2017-12-19

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