Recombinant Drosophila sechellia Ubiquitin carboxyl-terminal hydrolase 36 (Usp36), partial

What is the functional classification of Drosophila Usp36?

Drosophila Usp36 (Ubiquitin carboxyl-terminal hydrolase 36) belongs to the deubiquitinating enzyme (DUB) family. It functions primarily as an enzyme that hydrolyzes polyubiquitin at its C-terminal to release ubiquitin monomers . It is also known by several alternative names including Deubiquitinating enzyme 36, Protein scrawny, Ubiquitin thioesterase 36, and Ubiquitin-specific-processing protease 36 . The enzyme has an EC classification of 3.4.19.12, indicating its role in hydrolysis of peptide bonds at the C-terminal of ubiquitin .

What are the optimal storage conditions for recombinant Drosophila Usp36?

The shelf life and stability of recombinant Drosophila Usp36 depends on several factors including storage state, buffer composition, storage temperature, and the intrinsic stability of the protein itself. For optimal preservation, store the protein in liquid form at -20°C/-80°C for up to 6 months. If lyophilized, the shelf life extends to approximately 12 months at -20°C/-80°C . Avoid repeated freeze-thaw cycles as these can significantly degrade protein quality and functionality. For short-term use, working aliquots can be stored at 4°C for up to one week .

How should recombinant Drosophila Usp36 be reconstituted for experimental use?

Before opening, briefly centrifuge the vial to bring contents to the bottom. Reconstitute the protein in deionized sterile water to achieve a concentration between 0.1-1.0 mg/mL. For long-term storage after reconstitution, add glycerol to a final concentration of 5-50% and aliquot before storing at -20°C/-80°C . A standard glycerol concentration of 50% is recommended for optimal preservation of protein structure and function. This reconstitution protocol helps maintain protein stability while minimizing potential degradation during storage and experimental handling.

How does Usp36 influence developmental processes in Drosophila models?

Studies using Drosophila as a model system have demonstrated that Usp36 plays critical roles in developmental processes, particularly in eye development. Overexpression of dUCH (Drosophila homolog of UCH-L1, related to Usp36) in eye imaginal discs induces a rough eye phenotype in adults . This phenotype results partly from caspase-dependent apoptosis followed by compensatory proliferation. Importantly, Usp36 overexpression specifically impairs R7 photoreceptor cell differentiation, accompanied by reduced activation of extracellular-signal-regulated kinase (ERK) signals . This developmental impact appears to operate through down-regulation of the mitogen-activated protein kinase (MAPK) pathway, as demonstrated by rescue experiments with sevenless or Draf gene co-expression .

What is the relationship between Usp36 and the SUMOylation pathway?

Beyond its deubiquitinating activity, Usp36 has been shown to promote SUMOylation processes. In vitro studies demonstrate that Usp36 directly interacts with both Ubc9 (the SUMO E2 enzyme) and SUMO itself, particularly SUMO2/3-conjugated proteins and polymeric SUMO2 chains . The N-terminal region (amino acids 1-420) of Usp36 is sufficient for binding to both Ubc9 and SUMO2 . Functionally, recombinant Usp36 significantly enhances the SUMOylation of target proteins such as Nhp2 and Nop58 in a dose-dependent and time-dependent manner . This suggests Usp36 possesses a novel SUMO E3-like activity that may be mechanistically distinct from its deubiquitinating function.

What experimental methods are used to study Usp36 SUMOylation activity?

To investigate Usp36's SUMOylation activity, researchers employ in vitro SUMOylation assays using purified components. A typical reaction contains:

The SUMOylation reaction is conducted at appropriate temperature and time conditions, followed by analysis via SDS-PAGE and western blotting to detect SUMO-conjugated target proteins . Time-course experiments (examining SUMOylation at different time points) and dose-dependency studies (using varying concentrations of Usp36) provide insights into the kinetics and efficiency of Usp36-mediated SUMOylation .

What techniques can be used to study differential expression of Usp36 across Drosophila species?

To investigate differential expression of Usp36 across Drosophila species, researchers can employ several complementary approaches:

• RT-qPCR analysis: Quantifies mRNA expression levels of Usp36 in different species under controlled conditions

• Western blotting: Measures protein expression levels using species-cross-reactive antibodies

• RNA-seq: Provides genome-wide expression profiles to contextualize Usp36 expression within broader transcriptional networks

• Triple-hybrid crossing approach: Similar to techniques used in hybrid incompatibility studies , can help assess functional differences in Usp36 between species

• Immunohistochemistry: Evaluates tissue-specific expression patterns across development

For interspecies comparisons, standardized experimental conditions and appropriate normalization controls are essential for meaningful interpretation of expression differences.

How can recombinant Usp36 be used to investigate protein-protein interactions in Drosophila development?

Recombinant Usp36 serves as a valuable tool for investigating protein-protein interactions through several methodological approaches:

• Pull-down assays: GST-tagged recombinant Usp36 can be used to identify binding partners from Drosophila cell or tissue lysates. This approach successfully identified interactions between Usp36 and components of the SUMOylation machinery, including Ubc9 and SUMO2 .

• Co-immunoprecipitation studies: Using tagged recombinant Usp36 in combination with potential interacting proteins expressed in cellular systems to verify direct interactions.

• Yeast two-hybrid screening: Recombinant Usp36 can serve as bait to screen for novel interacting partners.

• Biolayer interferometry or surface plasmon resonance: These techniques enable measurement of binding kinetics between Usp36 and potential partners.

• Crosslinking mass spectrometry: Identifies interaction interfaces between Usp36 and binding partners at amino acid resolution.

When designing these experiments, it's important to consider that the N-terminal region (amino acids 1-420) of Usp36 has been identified as sufficient for interaction with components of the SUMOylation machinery , suggesting this domain may be particularly important for protein-protein interactions.

What mechanisms explain the dual role of Usp36 in deubiquitination and SUMOylation pathways?

The seemingly contradictory dual functionality of Usp36 in both deubiquitination and SUMOylation represents an intriguing research question. Several mechanistic hypotheses can be proposed:

• Domain-specific functions: Different domains within Usp36 may mediate distinct functions - the catalytic domain handling deubiquitination while the N-terminal domain (aa 1-420) promotes SUMOylation through interactions with Ubc9 and SUMO2/3 .

• Substrate-dependent activity switching: Conformational changes induced by different binding partners may activate either the deubiquitinating or SUMO-promoting function.

• Regulatory crosstalk: Usp36 may serve as a regulatory node between ubiquitination and SUMOylation pathways, potentially coordinating these post-translational modifications on shared substrates.

• Context-dependent functionality: Cellular conditions, developmental timing, or tissue-specific factors may determine which function predominates.

To investigate these mechanisms, research approaches could include structure-function studies with domain-specific mutations, identification of regulatory post-translational modifications on Usp36 itself, and temporal analysis of Usp36 activity during development.

How does Usp36 overexpression affect MAPK signaling pathway components in Drosophila eye development?

Overexpression of dUCH (a Drosophila homolog related to Usp36) in Drosophila eye imaginal discs leads to impaired R7 photoreceptor cell differentiation through down-regulation of the MAPK pathway . This is evidenced by reduced activation of extracellular-signal-regulated kinase signals in affected tissues. The mechanism appears to involve multiple levels of the signaling cascade, as the rough eye phenotype can be rescued by co-expression of either the sevenless gene (receptor tyrosine kinase) or the Draf gene (downstream component of the MAPK cascade) .

A proposed experimental workflow to investigate this relationship further would include:

• Quantitative phospho-proteomics: To identify specific phosphorylation changes in MAPK pathway components upon Usp36 overexpression.

• Epistasis analysis: Testing genetic interactions between Usp36 and various components of the MAPK pathway to determine points of intersection.

• Real-time signaling assays: Using FRET-based biosensors to monitor MAPK activation dynamics in live tissues with varying Usp36 levels.

• Proteasome activity assessment: Given Usp36's deubiquitinating function, measuring changes in proteasome activity to determine if stabilization of negative regulators of MAPK signaling contributes to pathway inhibition.

• SUMOylation analysis of MAPK components: Investigating whether Usp36-mediated SUMOylation of MAPK pathway components might contribute to signaling inhibition.

What are common challenges in expressing and purifying recombinant Drosophila Usp36, and how can they be addressed?

Expression and purification of recombinant Drosophila Usp36 present several challenges:

• Protein solubility issues:

  • Challenge: Full-length Usp36 may exhibit poor solubility due to its size and complex domain structure.

  • Solution: Express functional domains separately, optimize buffer conditions (adding glycerol, non-ionic detergents, or appropriate salt concentrations), or use solubility-enhancing tags such as SUMO or MBP.

• Maintaining enzymatic activity:

  • Challenge: Preserving the catalytic activity of Usp36 during purification.

  • Solution: Include protease inhibitors and reducing agents in all buffers, minimize purification time, and validate activity with functional assays post-purification.

• Protein yield optimization:

  • Challenge: Low expression levels in heterologous systems.

  • Solution: Consider yeast expression systems (as used for related proteins ), optimize codon usage for the expression host, and fine-tune induction conditions.

• Preventing aggregation during storage:

  • Challenge: Protein aggregation during freeze-thaw cycles.

  • Solution: Add glycerol (5-50%) to storage buffer and prepare single-use aliquots to avoid repeated freeze-thaw cycles .

• Confirming protein quality:

  • Challenge: Ensuring proper folding and function.

  • Solution: Employ multiple quality control methods including SDS-PAGE (>85% purity standard ), activity assays, and thermal shift assays to confirm protein stability.

How can researchers design conclusive experiments to distinguish between Usp36's deubiquitinating and SUMOylation-promoting activities?

To experimentally separate Usp36's dual functions, researchers should consider:

• Structure-guided mutagenesis:

  • Create point mutations in the catalytic site for deubiquitination without affecting the N-terminal domain (aa 1-420) that interacts with SUMOylation machinery .

  • Generate truncation constructs separating the N-terminal region from the catalytic domain.

• Substrate-specific assays:

  • For deubiquitination: Use fluorogenic ubiquitin substrates to measure DUB activity.

  • For SUMOylation: Employ in vitro SUMOylation assays with recombinant components as described previously .

• Temporal separation experiments:

  • Use temperature-sensitive or chemically-inducible Usp36 variants to activate one function at a time.

  • Monitor the dynamics of both activities in response to developmental cues or cellular stresses.

• Targeted interactome analysis:

  • Identify protein interactions specific to each function through BioID or proximity labeling approaches.

  • Map the interactome changes under conditions that favor either deubiquitination or SUMOylation.

• Comparative analysis across species:

  • Examine whether the dual functionality is conserved across Drosophila species, which could indicate evolutionary significance.

Through these approaches, researchers can dissect the molecular mechanisms underlying Usp36's multifunctional nature and determine whether these activities are independent or coordinated.

What is the potential role of Usp36 in Drosophila sechellia hybrid incompatibility?

While direct evidence linking Usp36 to hybrid incompatibility in Drosophila sechellia is not established in the provided literature, this represents an intriguing research direction. The D. simulans clade (including D. sechellia) exhibits resistance to hybrid rescue mechanisms that work in other Drosophila species , suggesting species-specific genetic interactions. Future research could investigate:

• Whether Usp36 variants differ between D. sechellia and other Drosophila species in ways that affect hybrid viability.

• If Usp36-mediated regulation of developmental pathways (such as MAPK signaling ) contributes to species-specific developmental incompatibilities.

• Whether the dual functionality of Usp36 in deubiquitination and SUMOylation evolved differently in D. sechellia compared to other species.

• If Usp36 interacts genetically with known hybrid incompatibility loci such as Lhr or the newly identified Satyr locus .

The triple-hybrid cross approach developed for mapping hybrid incompatibility loci could be adapted to investigate these questions specifically for Usp36.

How might advances in CRISPR-Cas9 technology enhance the study of Usp36 function across Drosophila species?

CRISPR-Cas9 technology offers transformative approaches for studying Usp36 function:

• Precise genomic editing:

  • Introduction of species-specific Usp36 variants into common genetic backgrounds

  • Creation of domain-specific mutations to separate deubiquitinating and SUMOylation functions

  • Generation of tagged endogenous Usp36 for in vivo localization and interaction studies

• Transcriptional modulation:

  • CRISPRa/CRISPRi systems to achieve tissue-specific up- or down-regulation

  • Temporal control of Usp36 expression to study developmental roles

• Cross-species functional analysis:

  • Replacement of endogenous Usp36 with orthologs from different Drosophila species

  • Creation of chimeric Usp36 proteins with domains from different species

• High-throughput screening:

  • CRISPR screens to identify genetic interactors of Usp36

  • Parallel analysis of Usp36 function across multiple genetic backgrounds

• In vivo dynamics:

  • Integration of fluorescent reporters to monitor Usp36 activity in real-time

  • Optogenetic control of Usp36 function to assess temporal requirements

These approaches would significantly enhance our understanding of Usp36's species-specific functions and evolutionary significance within the Drosophila genus.