SUMO-Enriched Proteome for <i>Drosophila</i> Innate Immune Response

Handu, Mithila; Kaduskar, Bhagyashree; Ravindranathan, Ramya; Soory, Amarendranath; Giri, Ritika; Elango, Vijay Barathi; Gowda, Harsha; Ratnaparkhi, Girish S.

doi:10.1534/g3.115.020958

Cited by 33 publications

(44 citation statements)

References 94 publications

(159 reference statements)

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“…549 proteins (52%) of the proteins found in S2 cells were found previously SUMOylated in other Drosophila experiments done using different systems and developmental stages: Drosophila embryos, 37%37 and immune challenged S2 cells, 46%38 (Supplementary Fig. S1).…”

Section: Resultsmentioning

confidence: 82%

“…In the cytoplasm, represented functions include transport and trafficking (Short wing, Sw; VAMP-associated protein of 33 kDa orthologue A, Vap-33A) and protein folding (Tetratricopeptide repeat protein 2, Tpr2). Among those 20, 18 were previously identified as targets of SUMOylation (Supplementary Data S2)3738. In addition, the human homologues of 9 of them were also reported to be SUMOylated (UBE21, snRNP-U1-70K, Pont, CG7839, Sym, CG11123, Vap-33A, Nop60B and Adam), suggesting a conserved role for SUMOylation in the function of these proteins.…”

Section: Resultsmentioning

confidence: 98%

“…2B). Three of the validated proteins have been previously identified as SUMOylated proteins: Ultraspiracle (Usp), a nuclear receptor involved in steroid signalling46, the transcription factor Osa3747, the intermediate filament Lamin (Lam, also a key nuclear envelope component)38 and the eukaryotic initiation factor 4E (eIF-4E)48. One of the validated proteins was not previously described to be SUMOylated in Drosophila , the Glutathione S-transferase involved in axonogenesis Failed axon connections (Fax), and thus is the first Fax homolog to be identified as a target of SUMOylation.…”

Section: Resultsmentioning

confidence: 99%

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A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation

Pirone

Xolalpa

Sigurðsson

et al. 2017

Sci Rep

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Post-translational modification by ubiquitin and ubiquitin-like proteins (UbLs) is fundamental for maintaining protein homeostasis. Efficient isolation of UbL conjugates is hampered by multiple factors, including cost and specificity of reagents, removal of UbLs by proteases, distinguishing UbL conjugates from interactors, and low quantities of modified substrates. Here we describe bioUbLs, a comprehensive set of tools for studying modifications in Drosophila and mammals, based on multicistronic expression and in vivo biotinylation using the E. coli biotin protein ligase BirA. While the bioUbLs allow rapid validation of UbL conjugation for exogenous or endogenous proteins, the single vector approach can facilitate biotinylation of most proteins of interest. Purification under denaturing conditions inactivates deconjugating enzymes and stringent washes remove UbL interactors and non-specific background. We demonstrate the utility of the method in Drosophila cells and transgenic flies, identifying an extensive set of putative SUMOylated proteins in both cases. For mammalian cells, we show conjugation and localization for many different UbLs, with the identification of novel potential substrates for UFM1. Ease of use and the flexibility to modify existing vectors will make the bioUbL system a powerful complement to existing strategies for studying this important mode of protein regulation.

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Section: Resultsmentioning

confidence: 82%

Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation

Pirone

Xolalpa

Sigurðsson

et al. 2017

Sci Rep

View full text Add to dashboard Cite

show abstract

“…We were unable to detect changes in SUMOylation of candidate proteins MEF2, CaMKII, or CREB. More than 100 proteins in Drosophila have been identified to bind SUMO (Lehembre et al 2000;Sahota et al 2009;Abed et al 2011;Guruharsha et al 2011;Handu et al 2015) and further investigation of these and other SUMOylated proteins that are involved in synaptic plasticity in other organisms may shed light on the nature of this interaction. It also should be considered that the interaction between HDAC4 and Ubc9 could be independent of SUMOylation.…”

mentioning

confidence: 99%

Long-Term Memory inDrosophilaIs Influenced by Histone Deacetylase HDAC4 Interacting with SUMO-Conjugating Enzyme Ubc9

et al. 2016

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HDAC4 is a potent memory repressor with overexpression of wild type or a nuclear-restricted mutant resulting in memory deficits. Interestingly, reduction of HDAC4 also impairs memory via an as yet unknown mechanism. Although histone deacetylase family members are important mediators of epigenetic mechanisms in neurons, HDAC4 is predominantly cytoplasmic in the brain and there is increasing evidence for interactions with nonhistone proteins, suggesting HDAC4 has roles beyond transcriptional regulation.To that end, we performed a genetic interaction screen in Drosophila and identified 26 genes that interacted with HDAC4, including Ubc9, the sole SUMO E2-conjugating enzyme. RNA interference-induced reduction of Ubc9 in the adult brain impaired long-term memory in the courtship suppression assay, a Drosophila model of associative memory. We also demonstrate that HDAC4 and Ubc9 interact genetically during memory formation, opening new avenues for investigating the mechanisms through which HDAC4 regulates memory formation and other neurological processes.KEYWORDS histone deacetylase; SUMOylation; plasticity; neuron; memory T HE histone deacetylase HDAC4 is widely expressed in neurons throughout the brain (Darcy et al. 2010) and an increasing body of evidence indicates that HDAC4 plays important roles in neurological function Chen and Cepko 2009;Kim et al. 2012;Li et al. 2012;Sando et al. 2012;Sarkar et al. 2014). To that end, we recently demonstrated that in Drosophila, RNA interference (RNAi)-mediated knockdown of HDAC4 in the adult brain impairs long-term memory (LTM) in the courtship suppression assay, a model of associative memory (Fitzsimons et al. 2013). Similarly in humans, loss of one copy of HDAC4 correlates with brachydactyly mental retardation syndrome (BDMR), the neurological symptoms of which include intellectual disability and autism (Williams et al. 2010;Morris et al. 2012;Villavicencio-Lorini et al. 2013), and in mice, conditional knockout of HDAC4 in the brain results in impairments in hippocampal-dependent associative LTM . Despite this growing evidence of a critical role, the mechanism(s) through which HDAC4 positively influences LTM is unknown. This is in part because HDAC4 exists in both nuclear and cytoplasmic pools, and under basal conditions, the majority of HDAC4 is localized to the cytoplasm (Chawla et al. 2003;Darcy et al. 2010). Given the predominant nonnuclear localization, particularly the concentration of HDAC4 at dendritic spines (Darcy et al. 2010), we hypothesize that cytoplasmic HDAC4 is required in memory formation; however, the mechanisms through which HDAC4 acts outside the nucleus are unknown.The nuclear role of HDAC4 is less of an enigma. When in the nucleus, HDAC4 acts as a transcriptional repressor; although vertebrate HDAC4 is catalytically inactive as a histone deacetylase, rather it facilitates changes in gene expression through direct binding and inhibition of transcription factors such as MEF2 (Miska et al. 1999;Wang et al. 1999;Lu et al. 2000). As described abov...

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“…The cells (∼3×10³) were transiently co-transfected with pUAST-dICA69 FL , pUAST-dICA69 BAR , pUAST-dICA69 ICAC and actin-Gal4 (1 μg each) using Mirus TransIT transfection agent as described previously (Handu et al, 2015). For microscopy, S2R+ cells were spotted onto Concanavalin A (Sigma-Aldrich)-coated coverslips and imaged with a 63× objective.…”

Section: +mentioning

confidence: 99%

Regulation of neuromuscular junction organization by Rab2 and its effector ICA69 in Drosophila

et al. 2017

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The mechanisms underlying synaptic differentiation, which involves neuronal membrane and cytoskeletal remodeling, are not completely understood. We performed a targeted RNAi-mediated screen of Drosophila BAR-domain proteins and identified islet cell autoantigen 69 kDa (ICA69) as one of the key regulators of morphological differentiation of the larval neuromuscular junction (NMJ). We show that Drosophila ICA69 colocalizes with α-Spectrin at the NMJ. The conserved N-BAR domain of ICA69 deforms liposomes in vitro. Fulllength ICA69 and the ICAC but not the N-BAR domain of ICA69 induce filopodia in cultured cells. Consistent with its cytoskeleton regulatory role, ICA69 mutants show reduced α-Spectrin immunoreactivity at the larval NMJ. Manipulating levels of ICA69 or its interactor PICK1 alters the synaptic level of ionotropic glutamate receptors (iGluRs). Moreover, reducing PICK1 or Rab2 levels phenocopies ICA69 mutation. Interestingly, Rab2 regulates not only synaptic iGluR but also ICA69 levels. Thus, our data suggest that: (1) ICA69 regulates NMJ organization through a pathway that involves PICK1 and Rab2, and (2) Rab2 functions genetically upstream of ICA69 and regulates NMJ organization and targeting/ retention of iGluRs by regulating ICA69 levels.

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SUMO-Enriched Proteome for Drosophila Innate Immune Response

Cited by 33 publications

References 94 publications

A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation

A comprehensive platform for the analysis of ubiquitin-like protein modifications using in vivo biotinylation

Long-Term Memory inDrosophilaIs Influenced by Histone Deacetylase HDAC4 Interacting with SUMO-Conjugating Enzyme Ubc9

Regulation of neuromuscular junction organization by Rab2 and its effector ICA69 in Drosophila

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