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A proteomics approach to identify targets of the ubiquitin-like molecule Urm1 in Drosophila melanogaster
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
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. Vol. 12, no 9, article id e0185611
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-140904DOI: 10.1371/journal.pone.0185611ISI: 000411980300077PubMedID: 28953965OAI: oai:DiVA.org:umu-140904DiVA, id: diva2:1157751
Available from: 2017-11-16 Created: 2017-11-16 Last updated: 2018-06-09Bibliographically approved
In thesis
1. Function and targets of the Urm1/Uba4 conjugation machinery in Drosophila melanogaster
Open this publication in new window or tab >>Function and targets of the Urm1/Uba4 conjugation machinery in Drosophila melanogaster
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
Keywords
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:nbn:se:umu:diva-143187 (URN)978-91-7601-815-6 (ISBN)
Public defence
2018-01-26, hörsal E04, byggnad 6E, Norrlands Universitetsjukhus., Umeå, 13:00 (English)
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Supervisors
Available from: 2017-12-21 Created: 2017-12-19 Last updated: 2018-06-09Bibliographically approved

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