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

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

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

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

Compiled list of the Urm1-interacting proteins in <i>Drosophila</i> embryos, larvae and adults, identified by mass spectrometry.

<p>Compiled list of the Urm1-interacting proteins in <i>Drosophila</i> embryos, larvae and adults, identified by mass spectrometry.</p

Functional characterization of the newly identified candidate targets of Urm1 conjugation in <i>Drosophila</i>.

<p><b>A.</b> Subcellular distribution of the proteins identified as Urm1-binding partners by mass spectrometry, depicting an accumulation of candidate targets of urmylation in the cytoplasmic compartment and/or associated with distinct membrane-bound organelles. <b>B.</b> Venn diagram illustrating the amount of unique versus shared Urm1-interacting proteins in the developmental stages investigated; embryos, larvae and adults. <b>C.</b> Functional clustering of the Urm1-associated proteins established using the DAVID database, suggesting that Urm1 most likely displays its most important functions in oxidation-reduction processes and tRNA modification. Enrichment scores of >3.0 were considered as meaningful. <b>D.</b> A functional classification of the Urm1-binding proteins based on gene ontology (GO) classification suggests that Urm1 may be involved in multiple different biological processes.</p

Protein-protein interaction analysis by STRING clusters Urm1 conjugation targets in multiple distinctive functional networks.

<p>STRING analysis of the Urm1-intercting partners identified by mass spectrometry, depicting functional networks where Urm1 may be involved in embryos (A), larvae (B) and adult flies (C). During all developmental stages, Urm1 appears to be associated with networks of proteins that regulate oxidation-reduction processes, tRNA modification, immune responses, mRNA processing, translation and protein folding, as well as cytoskeletal dynamics. In embryos, Urm1 is additionally linked to a protein network which is involved in chromatin remodeling.</p

Strategy for identification of Urm1 conjugation targets in <i>Drosophila</i> embryos, larvae and adults <i>in vivo</i>.

<p><b>A.</b> Schematic representation of the rationale for identifying Urm1-binding proteins and thereby candidate targets of urmylation during three critical stages of <i>Drosophila</i> development; embryogenesis, late larval stages and adulthood. Shortly, Flag-Urm1 associated proteins were enriched by immunoprecipitation with Flag M2 magnetic beads, which subsequently were subjected to on-bead trypsin digestion, followed by mass spectrometry and identification by standard bioinformatics analysis. <b>B.</b> Western blot illustrating the distribution of candidate Urm1 targets in embryos (left panel), larvae (middle panel) and adults (right panel), respectively. Urm1-interacting proteins were captured in the presence of NEM by Flag M2 immunoprecipitation, using magnetic beads, resolved under denaturing conditions by SDS-PAGE and detected by anti-Urm1 antibodies (* depicts endoge^◆nous Urm1 expressed in control <i>Actin5C>w</i>^<i>1118</i> samples and indicates the unconjugated Flag-Urm1 fusion protein). Input represents 30 μg of the total lysate of the indicated genotypes.</p

<i>In vivo</i> confirmation of the binding of Urm1 to a panel of target proteins identified by mass spectrometry.

<p>Confirmation of a physical interaction between Urm1 and five of the newly identified candidate targets of urmylation. Using the UAS/GAL4-system, Flag-Urm1 was either expressed alone, or co-expressed separately with HA-Ciao1, HA-MsrA/Eip71CD, GFP-GILT1 or GFP-Crammer, respectively, under control of the Actin5C-GAL5 driver. In protein lysates from the resulting flies, the interaction between Urm1 and the candidate target proteins were subsequently analyzed by immunoprecipitation, performed in in the presence of NEM. By immunoblotting Flag-Urm1 immunoprecipitates with anti-Jafrac1, anti-HA or anti-GFP antibodies, an interaction could thereby be verified between Flag-Urm1 and endogenous <i>Drosophila</i> Jafrac1 (A), HA-tagged Ciao1 (B), HA-tagged MsrA/Eip71CD (C), GFP-tagged GILT1 (D) and GFP-tagged Crammer (E). When comparing the molecular weights of the candidate Urm1 target proteins following immunoprecipitation, a size shift of ~15 kDa could be observed for Jafrac1 (A), HA-Ciao1 (B), HA- MsrA/Eip71CD (C) and GFP-GILT1 (D) as compared with protein lysate controls, which is in agreement with the covalent conjugation of one Flag-Urm1 moiety (e.g. target protein urmylation). In contrast, GFP-Crammer displayed the same molecular weight in both Flag-Urm1 immunoprecipitates and crude fly lysates, depicting a non-covalent mode of interaction, which is sensitive to denaturing conditions (E). Input represents 30 μg of the total lysate of the indicated genotypes.</p

Identification of Urm1-binding proteins by mass spectrometry.

<p><b>A, C, E.</b> Heat maps depicting a correlation analysis of the mass spectrometry results obtained from the two control <i>Actin5C>w</i>^<i>1118</i> replicate reads (Control replicate 1 and 2), versus the two Flag-Urm1 rescued <i>Urm1</i>^<i>n123</i> <i>Drosophila</i> replicate reads (Rescue replicate 1 and 2) for embryos (A), larvae (C) and adults (E), respectively. For all developmental stages, the two control and rescue replicates show high similarity, respectively, with a clear distinction between the controls versus the rescue samples. The color key code represents the Pearson correlation coefficient of the two replicates, where 1 depicts 100% similarity between the two replicates and 0 depicts no correlation between the samples. <b>B, D, F.</b> Volcano plots illustrating the magnitude of differential distribution (log2 fold-change) of the signal intensity between the Flag-Urm1 rescue and the control samples, together with the adjusted p-value for embryonic (B), larval (D) and adult (F) samples, respectively. Red dots depict peptides that displayed a log2 fold-change of less than 1.3 and a high adjusted p-value, when comparing the rescue and control samples. Blue dots represent peptides that did not show any significant difference between the rescue and the control samples, with a lower adjusted p-value. Green dots pinpoint peptides that demonstrated a low adjusted p-value and were enriched in the rescue samples to a minimum of 1.3 log2 fold-change, as compared with the control samples. The green dots represent the peptides that were finally singled out as Urm1-interacting proteins, with the cut-off marked by a dashed yellow line.</p

Characterization and frequency of antibiotic resistance related to membrane porin and efflux pump genes among Acinetobacter baumannii strains obtained from burn patients in Tehran, Iran

Objective: To explore the characterization and frequency of antibiotic resistance related to membrane porin and efflux pump genes among Acinetobacter baumannii (A. baumannii) strains obtained from burn patients in Tehran, Iran. Methods: In this cross-sectional descriptive study, 100 strains of A. baumannii isolated from burn patients visiting teaching hospitals of Tehran were collected from January 2016 to November 2017. After A. baumannii strains were confirmed, antimicrobial susceptibility testing was done via Kirby-Bauer disc diffusion method according to the Clinical and Laboratory Standards Institute guidelines. PCR amplification was performed for detection of β -lactamase adeR, OprD, adeS genes among A. baumannii strains. Results: All isolates (100%) were resistant to ceftazidime, cefotaxime, cefepime, ciprofloxacin, and piperacillin, and most isolates indicated high resistance (95%-97%) to meropenem, imipenem, gentamicin, ceftriaxone, trimethoprim-sulfamethoxazole, piperacillin-tazobactam, amikacin, and tetracycline. The most effective antibiotic against A. baumannii isolates was colistin (97% sensitivity), followed by tigecycline. The frequency of OprD, adeS, and adeR genes were 98%, 91%, and 77%, respectively. Conclusions: This study shows that the majority of A. baumannii isolates are highly resistant to the antibiotics most commonly used in burn patients. Also, high distribution of OprD and adeRS genes may be responsible for the observed resistances among A. baumannii isolates that demonstrate the possible role of both efflux pumps in simultaneous of carbapenemase production during antibiotic resistance

Single-cell multimodal analysis in a case with reduced penetrance of Progranulin-Frontotemporal Dementia

We identified an autosomal dominant progranulin mutation carrier without symptoms of dementia in her lifetime (Reduced Penetrance Mutation Carrier, RedPenMC). This resistance to develop expected pathology presents a unique opportunity to interrogate neurodegenerative mechanisms. We performed multimodal single-nuclei analyses of post-mortem frontal cortex from RedPenMC, including transcriptomics and global levels of chromatin marks. RedPenMC had an increased ratio of GRN-expressing microglia, higher levels of activating histone mark H3k4me3 in microglia and lower levels of the repressive chromatin marks H3k9me1 and H3k9me3 in the frontal cortex than her affected mutation carrier son and evidence of higher protein levels of progranulin in both plasma and brain homogenates. Although the study is limited to one case, the results support that restoring brain progranulin levels may be sufficient to escape neurodegeneration and FTD. In addition to previously identified modifier genes, it is possible that epigenetic marks may contribute to the increased progranulin expression in cases of reduced penetrance. These findings may stimulate similar follow-up studies and new therapeutic approaches