Recombinant Mouse Ubiquitin carboxyl-terminal hydrolase 17-like protein E (Usp17le)

What is the basic function of Usp17le in mouse cellular systems?

Usp17le functions as a deubiquitinating enzyme that removes conjugated ubiquitin from specific proteins to regulate different cellular processes including cell proliferation, cell cycle progression, apoptosis, and cell migration. Similar to other USP family members, it exhibits cysteine-type endopeptidase activity and cysteine-type deubiquitinase activity . It plays a particularly important role in the regulation of Ras activation and consequent signaling pathways by modulating RCE1, which is essential for Ras processing .

How does Usp17le differ structurally and functionally from other USP family members?

Usp17le belongs to the USP17 family, which is distinct from other USP families such as UCH (ubiquitin C-terminal hydrolase) family. While UCH members like UCH-L1 primarily process small C-terminal adducts of ubiquitin to generate monomeric ubiquitin , Usp17le more specifically targets ubiquitinated proteins involved in cell cycle regulation and signaling pathways. The USP17 family has unique structural characteristics not found in UCH proteins, with different substrate specificities and cellular localizations .

What are the known substrates of Usp17le?

Based on studies of human USP17 homologs, key substrates include RCE1 (Ras-converting enzyme 1), which regulates Ras activation , and CDC25A, a cell cycle regulator. USP17 deubiquitinates these proteins, preventing their degradation and thus affecting downstream signaling pathways. USP17 has also been shown to deubiquitinate SUDS3, a regulator of histone deacetylation, and influence the activation of Rho family GTPases including RAC1A, CDC42, and RHOA .

What expression systems are most effective for producing recombinant Usp17le?

Multiple expression systems have been used for recombinant Usp17 family proteins, with varying results:

• E. coli expression: Provides high yield but often results in inclusion bodies requiring refolding. E. coli is commonly used for initial characterization studies due to ease of manipulation .

• Yeast expression (P. pastoris): This system has been successful for related deubiquitinating enzymes like DmUCH, producing soluble, active enzyme at high yields (up to 210 mg/l). Although this data is for UCH rather than USP17 specifically, similar approaches may be applicable for Usp17le .

• Baculovirus and mammalian cell systems: These provide better post-translational modifications and folding environments, potentially resulting in more native-like protein, though at lower yields .

The choice depends on the specific research goals - E. coli for structural studies requiring high yields, and mammalian systems for functional studies requiring native folding and modifications.

What purification strategy is recommended for obtaining active recombinant Usp17le?

A multi-step purification strategy is recommended:

• Initial capture: Affinity chromatography using His-tag or other fusion tags (common constructs include C-terminal DDK/myc tags as seen in commercial plasmids ).

• Intermediate purification: Ion exchange chromatography to separate based on charge properties.

• Polishing: Size exclusion chromatography to obtain highly pure protein and separate monomeric from aggregated forms.

For active enzyme preparation, it's critical to:

• Include reducing agents (5 mM DTT) in buffers to maintain cysteine residues in the active site in reduced form

• Avoid multiple freeze-thaw cycles which can diminish enzymatic activity

• Consider adding 10% glycerol as a stabilizer in storage buffers

How can the deubiquitinating activity of recombinant Usp17le be measured in vitro?

Several established methods can be used:

• Fluorogenic substrate assay: Using Ub-AMC (ubiquitin with 7-amido-4-methylcoumarin) as substrate, which releases fluorescent AMC upon cleavage. This allows for kinetic measurements and determination of Km and kcat values .

• Ubiquitin-fusion protein cleavage assay: Similar to methods used for UCH enzymes, where cleavage of ubiquitin from a fusion partner can be monitored by SDS-PAGE. For example, the cleavage pattern of ubiquitin-magainin by UCH can be visualized on Tricine-SDS-PAGE .

• In vivo co-expression assay: Co-expressing Usp17le with AtUBQ10 (ubiquitin) in bacterial systems and detecting processing via Western blot with anti-ubiquitin antibodies .

A typical reaction buffer composition includes:

• 50 mM Tris-HCl pH 7.5

• 0.5 mM EDTA

• 5 mM DTT

• 0.1% BSA for stability

What controls should be included when assessing Usp17le enzymatic activity?

Essential controls include:

• Positive control: A well-characterized deubiquitinating enzyme like UCH-L3 or OsUBP6 (as used in plant studies) .

• Negative control: Either buffer alone or an inactive enzyme prepared by mutating the catalytic cysteine residue to serine (C→S mutation in the active site), similar to the OsUCH3 C96S mutant described in plant UCH studies .

• Inhibitor control: Include a specific DUB inhibitor such as Ub-VME (ubiquitin vinyl methyl ester) to confirm specificity of the observed activity .

• Substrate specificity control: Test activity on both ubiquitin and related molecules like NEDD8 to determine specificity, as some UCH enzymes like UCH-L1 can bind but not hydrolyze NEDD8 .

What are the optimal conditions for assessing Usp17le functional activity in cellular systems?

For cellular functional studies:

• Cell models: Murine cell lines like NIH3T3 or MEFs are appropriate for mouse Usp17le. For cancer-related studies, use relevant murine cancer cell lines with varying malignant potential .

• Expression approaches: Use either transient transfection with recombinant Usp17le or CRISPRa-based activation of endogenous Usp17le . Commercial plasmids like pCMV6-Entry with C-terminal myc-DDK tags are available for expression studies .

• Functional readouts: Assess:

  • Cell proliferation (using trypan blue exclusion, MTT assay, or BrdU incorporation)

  • Cell migration (wound healing or Boyden chamber assays)

  • GTPase activation (pull-down assays for Ras, Rac, Rho)

  • Protein deubiquitination (immunoprecipitation followed by ubiquitin Western blot)

• Proper controls: Include both scrambled controls and catalytically inactive mutant Usp17le to distinguish enzymatic vs. scaffolding functions.

How does Usp17le regulate GTPase-mediated signaling pathways?

Usp17le regulates GTPase signaling through multiple mechanisms:

• Ras regulation: USP17 deubiquitinates RCE1, the enzyme responsible for processing the CAAX motif of Ras proteins. This affects Ras membrane localization and activation, consequently impacting downstream kinases like MEK and ERK .

• Rho GTPase control: Studies with human USP17 show that it regulates subcellular relocalization of Cdc42, Rac, and RhoA GTPases in response to chemokines, affecting cytoskeletal rearrangements and cell motility .

This makes Usp17le a critical regulator of both cell proliferation and migration pathways, with depletion of USP17 resulting in:

• Blocked chemokine-induced cell migration

• Inhibited cytoskeletal rearrangements

• Reduced membrane ruffling and lamellipodial formation

What is known about Usp17le's role in cancer models?

Research on USP17 family proteins in cancer has yielded complex, sometimes contradictory results:

• Tumor suppressor activity: USP17 suppresses tumorigenesis and tumor growth in some breast cancer models. Higher USP17 levels are detected in normal epithelial cells (MCF-10A) and less-malignant cells (MCF-7) compared to highly malignant MDA-MB-231 cells. Mechanistically, USP17 interacts with and deubiquitinates Asparaginyl endopeptidase (AEP), decreasing AEP protein levels and inhibiting breast cancer tumorigenesis through disruption of ERK signaling .

• Context-dependent roles: The function of USP17 appears to be cell-type and context-dependent, with evidence for both pro-oncogenic and tumor-suppressive roles depending on the cellular context and cancer type.

For researchers studying Usp17le in cancer models, it's essential to:

• Characterize Usp17le expression levels across different stages of tumor progression

• Examine both gain-of-function and loss-of-function phenotypes

• Identify tissue-specific substrates and interacting partners

• Correlate expression with clinical outcomes in relevant mouse models

What strategies can overcome stability and solubility challenges when working with recombinant Usp17le?

Deubiquitinating enzymes like Usp17le can present solubility and stability challenges. Consider these approaches:

• Fusion partners: Use solubility-enhancing fusion partners such as:

  • Thioredoxin (Trx)

  • Glutathione S-transferase (GST)

  • Maltose-binding protein (MBP)

  • SUMO tag

• Expression conditions optimization:

  • Lower induction temperature (16-18°C)

  • Reduced IPTG concentration (0.1-0.2 mM)

  • Co-expression with chaperones

• Buffer optimization:

  • Include 5-10% glycerol

  • Add reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol)

  • Test different pH ranges (7.0-8.5)

  • Include stabilizing additives (arginine, proline, non-detergent sulfobetaines)

• Storage considerations:

  • Store in small aliquots at -80°C

  • Include cryoprotectants like 50% glycerol for -20°C storage

  • Avoid repeated freeze-thaw cycles

How can researchers resolve contradictory findings regarding Usp17le's role in different experimental models?

When facing contradictory results about Usp17le function:

• Carefully consider cellular context:

  • Different cell types may express different Usp17le substrates

  • The ubiquitination status of targets may vary between cell types

  • Expression levels of interacting partners may influence function

• Methodological standardization:

  • Use consistent expression systems and protein preparation methods

  • Standardize activity assays and substrate concentrations

  • Employ both in vitro biochemical assays and cellular models

• Genetic background considerations:

  • Mouse strain variations may affect outcomes

  • Consider knockout/knockdown efficiency and specificity

  • Use rescue experiments with wildtype and mutant Usp17le

• Comprehensive substrate identification:

  • Perform proteome-wide analyses to identify all potential substrates

  • Validate key substrates across multiple experimental systems

  • Consider indirect effects through regulation of other deubiquitinating enzymes

What are emerging applications of recombinant Usp17le in studying ubiquitin homeostasis?

Recent research indicates potential applications in several areas:

• Ubiquitin pool regulation: Similar to UCH-L1's role in neurons, Usp17le may contribute to maintaining free ubiquitin pools in specific cellular contexts. Studying how Usp17le affects ubiquitin homeostasis could provide insights into cell-specific regulation of ubiquitin availability .

• Interplay with other ubiquitin-like modifiers: Investigating potential cross-regulation between ubiquitin and NEDD8 pathways, as some UCH enzymes can bind NEDD8 without hydrolyzing it .

• Stress response modulation: Examining how Usp17le activity changes under various cellular stresses, potentially revealing new roles in stress adaptation.

What novel methodologies are being developed to study Usp17le substrate specificity?

Cutting-edge approaches include:

• Activity-based probes: Development of Usp17le-specific activity-based probes to monitor enzyme activity in real-time within cellular contexts.

• PROTAC-based approaches: Using proteolysis-targeting chimeras to investigate the dynamics of Usp17le-mediated deubiquitination.

• Proximity labeling techniques: BioID or APEX2-based approaches to identify proteins in close proximity to Usp17le in living cells, potentially revealing new substrates and interacting partners.

• Ubiquitinome analysis: Combining Usp17le manipulation with proteome-wide ubiquitination profiling using mass spectrometry to comprehensively identify substrates.

• Structural biology advances: Cryo-EM and X-ray crystallography studies of Usp17le in complex with substrates to understand the molecular basis of specificity.