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

What is Ubiquitin carboxyl-terminal hydrolase 36 and what are its primary functions in Drosophila?

Ubiquitin carboxyl-terminal hydrolase 36 (Usp36) is a member of the ubiquitin-specific protease (USP) family that functions as a deubiquitinating enzyme (DUB), removing ubiquitin chains from target proteins. In Drosophila, Usp36 is also known by alternative names including deubiquitinating enzyme 36, protein scrawny, ubiquitin thioesterase 36, and ubiquitin-specific-processing protease 36 .

Usp36 plays critical roles in multiple cellular processes including:

• Nucleolar function and ribosome biogenesis

• Cell growth regulation

• Stem cell maintenance

• Autophagy activation

• Oxidative stress regulation

• Immunity signaling

• miRNA biogenesis

The enzyme primarily contributes to cellular homeostasis by preventing protein degradation through the removal of K48-linked ubiquitin chains from target proteins involved in these processes . Notably, it stabilizes nucleolar activity by deubiquitinating nucleophosmin/B23 and fibrillarin, while also regulating oxidative stress by stabilizing mitochondrial superoxide dismutase SOD2 .

What isoforms of Usp36 exist in Drosophila species and how do they differ functionally?

In Drosophila, the dUsp36 gene produces three main protein isoforms (B, C, and D) with distinct subcellular localizations and functions:

These isoforms share identical C-terminal regions containing the catalytic USP domain but differ in their N-terminal domains, which confer their specific localizations and functions . CRISPR-Cas9 generated isoform-specific mutations have demonstrated that the dUSP36-D nucleolar isoform plays the most significant role in growth regulation, with mutants displaying phenotypes similar to dMyc hypomorphic mutations .

How does Usp36 regulate growth signaling networks in Drosophila?

Usp36 serves as a crucial regulator of growth signaling networks in Drosophila through several interconnected mechanisms:

• MYC regulation: The nucleolar dUSP36-D isoform forms a complex with dMYC and AGO (an E3 ubiquitin ligase), stabilizing dMYC by counteracting AGO-mediated ubiquitination. This creates a positive feedback regulatory loop that enhances MYC-dependent transcription of growth-related genes .

• Hippo pathway modulation: Usp36 stabilizes YAP proteins by inhibiting their K48-linked polyubiquitination. As YAP is a key effector of the Hippo pathway, this stabilization promotes growth signaling activities .

• Ribosome biogenesis: Usp36 enhances protein synthesis capacity by regulating nucleolar processes including rRNA processing and ribosome assembly through its interactions with snoRNP components and RNA exoribonucleases .

• Oxidative stress management: By deubiquitinating SOD2 and regulating c-Myc, Usp36 influences cellular redox balance and metabolic homeostasis, which indirectly affects growth regulation .

This multi-faceted regulation positions Usp36 as a master coordinator of growth control in Drosophila development, integrating signals from various pathways into coherent growth responses.

What are the optimal storage and handling conditions for recombinant Drosophila Usp36?

Proper storage and handling of recombinant Drosophila Usp36 is critical for maintaining its enzymatic activity:

For reconstitution of lyophilized protein:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (optimally 50%) for long-term storage

• Aliquot to minimize freeze-thaw cycles

The stability of recombinant Usp36 is influenced by multiple factors including buffer composition, storage temperature, and the inherent stability of the protein structure itself . For experimental applications, it's recommended to validate enzymatic activity after prolonged storage.

What assays are recommended for validating Usp36 deubiquitinating activity?

Validating the enzymatic activity of recombinant Usp36 requires specific assays that measure its deubiquitinating function:

• Fluorogenic substrate assays:

  • Ubiquitin-AMC (7-amino-4-methylcoumarin) cleavage assay

  • Measure fluorescence release (excitation 380nm, emission 460nm) as a function of time

  • Calculate reaction kinetics (Km, Vmax) for quantitative comparison

• Di-ubiquitin or poly-ubiquitin chain cleavage assays:

  • Incubate Usp36 with different ubiquitin chain types (K48, K63, etc.)

  • Analyze reaction products by SDS-PAGE and western blotting

  • Determine chain-type specificity and processing rates

• Substrate-specific deubiquitination assays:

  • Incubate Usp36 with known ubiquitinated substrates (e.g., dMYC, SOD2)

  • Monitor decrease in substrate ubiquitination via western blotting

  • Compare results with catalytically inactive Usp36 controls

Essential controls should include:

• Positive control: commercial DUB with known activity

• Negative control: heat-inactivated Usp36 or catalytic mutant (C→S substitution in active site)

• Inhibitor control: pre-treatment with DUB inhibitors like ubiquitin-aldehyde or N-ethylmaleimide

These assays collectively provide a comprehensive assessment of Usp36 catalytic activity and substrate specificity.

How should researchers design experiments to investigate isoform-specific functions of Usp36?

Investigating isoform-specific functions of Usp36 requires sophisticated experimental designs that can distinguish between the activities of dUSP36-B, dUSP36-C, and dUSP36-D:

• Genetic approaches:

  • CRISPR-Cas9 mutagenesis targeting isoform-specific exons

  • Generation of frameshift mutations in each isoform's unique N-terminal region

  • Analysis of mutants in trans over null alleles for clear phenotypic assessment

• Expression constructs:

  • Isoform-specific transgenes with appropriate tags for detection

  • Rescue experiments in isoform-specific mutant backgrounds

  • Structure-function analysis with domain deletion/mutation constructs

• Localization studies:

  • Fluorescent protein fusions to track subcellular distribution

  • Co-localization with compartment-specific markers

  • Live imaging to monitor dynamic localization changes

• Biochemical approaches:

  • Isoform-specific immunoprecipitation to identify interaction partners

  • Substrate specificity assays comparing deubiquitinating activity

  • SUMOylation promotion assays for nucleolar isoforms

• Phenotypic analysis:

  • Growth measurements at organismal and cellular levels

  • Tissue-specific effects using GAL4-UAS system

  • Quantitative assessment of downstream processes (ribosome biogenesis, etc.)

The comprehensive experimental design should verify results using multiple approaches and include appropriate controls for each technique employed.

What is known about Usp36's role in nucleolar function and ribosome biogenesis?

Usp36 serves as a master regulator of nucleolar function and ribosome biogenesis through multiple mechanisms:

• Promotion of snoRNP component SUMOylation: Usp36 enhances the SUMOylation of small nucleolar ribonucleoprotein (snoRNP) components including Nop58, Nhp2, Nop56, and DKC1. This post-translational modification increases their binding affinity to snoRNAs, which are critical for pre-rRNA processing .

• Regulation of RNA exoribonuclease activity: Usp36 interacts with EXOSC10, a component of the RNA exosome complex, facilitating its SUMOylation at Lysine 583. This modification is essential for EXOSC10 binding to pre-rRNAs and subsequent processing functions. Mutation of this lysine residue (K583R) fails to rectify rRNA processing deficiencies caused by EXOSC10 knockdown .

• RNA Polymerase I interaction: Usp36 has been implicated in regulating ribosome biogenesis through direct interaction with RNA polymerase I, the enzyme responsible for rDNA transcription .

The significance of these functions is evident in loss-of-function studies, where knockdown of Usp36 significantly impairs rRNA processing, translation, and ultimately cell growth. The nucleolar dUSP36-D isoform appears particularly critical for these functions, as dUsp36-D mutants display phenotypes similar to dMyc hypomorphic mutations, indicating a close functional relationship between nucleolar Usp36 activity and MYC-dependent growth regulation .

How does Usp36 regulate MYC stability and function in Drosophila?

Usp36 serves as a critical regulator of MYC stability and function in Drosophila through a sophisticated protein interaction network:

• Direct deubiquitination of dMYC: The nucleolar dUSP36-D isoform physically interacts with dMYC and removes ubiquitin modifications that would otherwise target dMYC for proteasomal degradation. This deubiquitination activity directly stabilizes dMYC protein levels .

• Antagonistic relationship with AGO: Usp36 forms a complex with both dMYC and AGO (an E3 ubiquitin ligase). Within this complex, AGO ubiquitinates dMYC, targeting it for degradation, while Usp36 counteracts this by removing the ubiquitin modifications. This creates a dynamic equilibrium controlling dMYC stability .

• Positive feedback regulatory loop: Evidence suggests that Usp36 and MYC form a positive feedback loop similar to that identified in human cells, where Usp36 stabilizes MYC, and MYC may in turn regulate Usp36 expression .

• Isoform specificity: The nucleolar dUSP36-D isoform plays the predominant role in dMYC regulation, with dUsp36-D mutants phenocopying dMyc hypomorphic mutations. This suggests compartmentalization of this regulatory function within the nucleolus .

• Nucleolar vs. nucleoplasmic regulation: While dUSP36-D primarily regulates nucleolar dMYC, other DUBs like PUF (puffyeye, orthologous to human USP34) may regulate dMYC in the nucleoplasm, creating a multi-layered control system .

The conservation of this regulatory mechanism between Drosophila and humans suggests its fundamental importance in growth control across species.

What methodological approaches are most effective for studying Usp36 substrate specificity?

Studying Usp36 substrate specificity requires integrated methodological approaches combining biochemical, genetic, and proteomic techniques:

• Substrate identification:

  • Affinity purification coupled with mass spectrometry (AP-MS) to identify Usp36-interacting proteins

  • Stable isotope labeling by amino acids in cell culture (SILAC) combined with ubiquitin remnant profiling to identify proteins with altered ubiquitination in Usp36 mutants

  • Proximity-dependent biotin identification (BioID) using Usp36 as bait to identify proximal proteins in relevant compartments

• Physical interaction validation:

  • Co-immunoprecipitation experiments with endogenous or tagged proteins

  • Pull-down assays with recombinant proteins to test direct interactions

  • Bimolecular fluorescence complementation (BiFC) for in vivo interaction visualization

• Deubiquitination activity assessment:

  • In vitro deubiquitination assays comparing ubiquitination status of candidate proteins with wild-type vs. catalytically inactive Usp36

  • Ubiquitin chain type specificity analysis using linkage-specific antibodies or mass spectrometry

  • Cellular ubiquitination assays in Usp36-depleted vs. control cells

• Genetic validation:

  • Genetic interaction studies between Usp36 and putative substrate genes

  • Phenotypic rescue experiments with ubiquitination-resistant substrate variants

  • CRISPR-Cas9 mutagenesis of specific lysine residues in substrates

• Functional consequences:

  • Protein stability assays (cycloheximide chase) to monitor substrate half-life

  • Subcellular localization analysis to determine if deubiquitination affects protein trafficking

  • Activity assays specific to each substrate's function

These complementary approaches collectively provide robust evidence for substrate specificity while minimizing false positives that can arise from any single method.

What are the mechanisms underlying Usp36's dual role in deubiquitination and SUMOylation processes?

Usp36 demonstrates a remarkable dual enzymatic functionality in both deubiquitination and SUMOylation processes, representing a sophisticated mechanism for coordinating distinct post-translational modifications:

• Deubiquitinating activity:

  • Mediated by the canonical USP catalytic domain

  • Removes ubiquitin chains from target proteins (including dMYC, SOD2, and itself)

  • Prevents proteasomal degradation of substrates

  • Functions through the classical catalytic triad mechanism of USP family enzymes

• SUMO E3 ligase activity:

  • Facilitates SUMOylation through direct interaction with both SUMO2 and Ubc9 (the SUMO E2 conjugating enzyme)

  • Enhances SUMOylation of multiple nucleolar proteins including:

    • snoRNP components (Nop58, Nhp2, Nop56, DKC1)

    • RNA exosome component EXOSC10 (at Lysine 583)

    • microprocessor complex component DGCR8

The molecular architecture enabling this dual functionality likely involves:

• Distinct protein interaction domains for deubiquitination vs. SUMOylation activities

• Potential conformational changes regulating which activity predominates

• Compartment-specific regulation, particularly in the nucleolus

Regulatory mechanisms appear to modulate these activities based on cellular conditions. For example, hypoxia disrupts the Usp36-EXOSC10 interaction, leading to loss of EXOSC10 SUMOylation . This environmental responsiveness suggests that Usp36's dual functions may be differentially regulated based on cellular needs.

This dual functionality allows Usp36 to coordinate two distinct types of post-translational modifications, creating sophisticated regulatory circuits controlling critical cellular processes like ribosome biogenesis and miRNA processing.

How do environmental stressors modulate Usp36 activity and function?

Environmental stressors significantly modulate Usp36 activity and function through multiple regulatory mechanisms:

• Hypoxic stress response:

  • Disrupts the interaction between Usp36 and EXOSC10

  • Leads to loss of EXOSC10 SUMOylation

  • Potentially alters RNA exosome function and RNA processing

  • May represent an adaptive response to reduce energy-intensive processes like ribosome biogenesis during oxygen limitation

• Oxidative stress regulation:

  • Usp36 deubiquitinates SOD2 (superoxide dismutase) in mitochondria

  • Stabilizes SOD2, enhancing cellular antioxidant capacity

  • Impacts cellular respiration and metabolism

  • Creates a protective mechanism against reactive oxygen species damage

• Ischemic conditions:

  • Increases c-Myc protein levels, intensifying oxidative stress

  • Usp36 deubiquitinates c-Myc, inhibiting its degradation and enhancing c-Myc signaling

  • This relationship appears significant in acute kidney injury pathogenesis

  • Suggests Usp36 as a potential intervention target in ischemic conditions

• Nucleolar stress sensitivity:

  • Disruption of ribosome biogenesis reduces EXOSC10 SUMOylation

  • Indicates Usp36 activity responds to nucleolar integrity

  • May represent a feedback mechanism linking ribosome biogenesis status to Usp36 function

These stress-responsive functions position Usp36 as an important mediator of cellular adaptation to environmental challenges, coordinating post-translational modification landscapes to meet changing cellular requirements.

What therapeutic potential exists in targeting Usp36 for disease intervention?

Emerging research highlights several promising avenues for therapeutic targeting of Usp36 in disease intervention:

• Cancer therapy potential:

  • Usp36 stabilizes YAP proteins by inhibiting their K48-linked polyubiquitination

  • Positively influences Hippo/YAP signaling activity in esophageal squamous cell carcinoma (ESCC)

  • Targeting Usp36 expression or function may provide a novel therapeutic approach for ESCC patients

  • Could potentially disrupt the growth-promoting effects of aberrant YAP signaling

• Modulation of MYC-driven diseases:

  • The USP36/MYC positive feedback regulatory loop appears conserved between humans and Drosophila

  • Disrupting this loop could potentially downregulate MYC activity in MYC-dependent cancers

  • More selective than direct MYC inhibition, which has proven challenging in clinical settings

  • Provides an indirect approach to targeting this critical oncogene

• Ischemia-reperfusion injury protection:

  • Usp36 regulates SOD2 and c-Myc in the context of oxidative stress

  • Modulating Usp36 could potentially mitigate oxidative damage in ischemia-reperfusion scenarios

  • Particularly relevant for acute kidney injury and other ischemic conditions

  • Represents a novel intervention target distinct from traditional antioxidant approaches

• Inflammatory disease applications:

  • Known roles of Usp36 in immunity signaling suggest potential in inflammatory conditions

  • Deubiquitination of immune pathway components could be therapeutically modulated

  • May offer more selective intervention than broad immunosuppression

What implications do evolutionary studies of Usp36 have for understanding its function in higher organisms?

Evolutionary studies of Usp36 across species provide critical insights into its fundamental functions and adaptations in higher organisms:

• Conservation of core mechanisms:

  • The USP36/MYC regulatory loop identified in both Drosophila and humans indicates an ancient and essential growth control mechanism

  • Suggests therapeutic approaches targeting this interaction may be broadly applicable across species

• Isoform specialization:

  • The evolution of specialized isoforms with distinct subcellular localizations represents a sophisticated regulatory adaptation

  • Understanding this compartmentalization in Drosophila provides a framework for investigating similar specialization in mammalian systems

• Substrate relationship evolution:

  • The co-evolution of Usp36 with substrates like MYC and components of the SUMOylation machinery illuminates how protein interaction networks evolve

  • Reveals both constraints (maintenance of critical interactions) and flexibility (acquisition of new substrates) in DUB-substrate relationships

• Nucleolar function conservation:

  • The consistent role of Usp36 in nucleolar processes across species highlights the fundamental importance of DUB activity in ribosome biogenesis

  • Points to the nucleolus as a critical regulatory compartment for protein modification and stability control

• Disease relevance:

  • Evolutionary conservation helps distinguish essential functions (more likely to be conserved) from species-specific adaptations

  • Guides the selection of disease-relevant targets within the Usp36 interaction network

  • Informs the development of model systems for studying Usp36-related pathologies

These evolutionary insights not only enhance our understanding of Usp36 biology but also inform translational research approaches seeking to leverage this knowledge for therapeutic applications.

How can comparative studies between D. grimshawi and D. pseudoobscura Usp36 inform recombinant protein design?

Comparative studies between D. grimshawi and D. pseudoobscura Usp36 can significantly inform the design of optimized recombinant proteins for research applications:

• Domain optimization:

  • Identification of highly conserved regions essential for catalytic activity

  • Recognition of variable regions that might be dispensable for core functions

  • Design of chimeric constructs incorporating the most stable elements from each species

  • Potential for creating minimal functional domains with enhanced expression and stability

• Solubility enhancement:

  • Comparative analysis of surface-exposed residues that differ between species

  • Identification of naturally occurring substitutions that enhance solubility

  • Strategic mutation of problematic residues based on inter-species comparison

  • Development of expression constructs with optimized physiochemical properties

• Expression system selection:

  • Analysis of codon usage bias in different Drosophila species

  • Optimization of codon selection for the chosen expression system (e.g., yeast)

  • Incorporation of species-specific post-translational modification sites if functionally relevant

  • Selection of appropriate purification tags based on structural predictions from multiple species

• Functional validation strategies:

  • Design of activity assays based on conserved substrates

  • Identification of species-specific substrates for specialized applications

  • Development of standardized assay conditions applicable across Usp36 orthologs

  • Creation of negative controls using catalytically inactive mutants informed by evolutionary conservation

This comparative approach leverages natural evolutionary experiments to identify optimal properties for recombinant protein design, potentially yielding more stable, active, and experimentally tractable Usp36 constructs for research applications.