Recombinant Mouse Inactive ubiquitin carboxyl-terminal hydrolase 44 (Usp44)

What is USP44 and what are its primary functions?

USP44 is a deubiquitinating enzyme belonging to the ubiquitin-specific proteases (USPs) family. It catalyzes the hydrolysis of isopeptide bonds between ubiquitin and substrate proteins, effectively removing ubiquitin molecules from target proteins . This process is crucial for regulating protein expression, conformation, localization, and function.

Primary functions of USP44 include:

• Regulating anaphase initiation during mitosis

• Modifying histone H2B at lysine 120

• Controlling chromosome segregation

• Maintaining genomic stability

• Modulating immune cell function, particularly in regulatory T cells

The inactive form of recombinant mouse USP44 retains structural properties but lacks enzymatic activity, making it valuable for control experiments and mechanistic studies of deubiquitination processes .

How is USP44 expression regulated in different tissues?

USP44 expression varies across different tissues and is subject to complex regulatory mechanisms:

• Transcriptional regulation: TGF-β signaling has been identified as a major inducer of USP44 expression. In T cells, activation with PMA and ionomycin can induce USP44 promoter activity .

• Tissue-specific expression: USP44 shows differential expression patterns across tissues, with notable expression in immune cells and varying levels in cancer tissues.

• Pathological regulation: In several cancer types, USP44 expression is dysregulated. For example, in hepatocellular carcinoma (HCC), reduced USP44 expression correlates with poor prognosis and more aggressive disease features .

• Cellular localization: USP44 primarily localizes to the nucleus, where it interacts with various substrates involved in chromatin modification and cell cycle regulation .

What are the common methods for detecting USP44 expression in mouse tissue samples?

Several established techniques can effectively detect and quantify USP44 expression:

• Western blotting: Using specific anti-USP44 antibodies to detect protein levels in tissue lysates. Typical molecular weight of mouse USP44 is approximately 80 kDa.

• Quantitative RT-PCR: For measuring USP44 mRNA expression levels, primers targeting conserved regions of the USP44 gene can be designed.

• Immunohistochemistry (IHC): As demonstrated in HCC studies using tissue microarrays, IHC can visualize USP44 expression patterns within intact tissue architecture .

• Immunofluorescence microscopy: Particularly useful for co-localization studies, such as confirming the nuclear localization of USP44 and its interaction with binding partners like Ku80 .

• Flow cytometry: For detecting USP44 in specific cell populations when combined with surface markers.

When analyzing results, it's important to include appropriate positive and negative controls and to validate findings using multiple detection methods.

What are the optimal conditions for producing and purifying recombinant mouse USP44?

For optimal production and purification of recombinant mouse USP44:

• Expression systems:

  • Mammalian expression systems (HEK293T cells) are preferred for maintaining proper folding and post-translational modifications

  • Baculovirus-insect cell systems can yield higher protein amounts while maintaining activity

• Tags and constructs:

  • N-terminal 6×His tag facilitates purification without interfering with catalytic activity

  • HA or FLAG tags are useful for immunoprecipitation experiments

  • For inactive USP44, site-directed mutagenesis of the catalytic cysteine residue is recommended

• Purification protocol:

  • Lysis in buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, protease inhibitors

  • Affinity chromatography using Ni-NTA for His-tagged proteins

  • Size exclusion chromatography to improve purity

  • Storage in buffer containing 10% glycerol at -80°C to maintain stability

• Activity verification:

  • Deubiquitination assays using fluorogenic ubiquitin substrates

  • Comparison with catalytically inactive mutant as negative control

For inactive USP44 specifically, maintaining proper folding while inactivating the catalytic site is crucial for experimental validity when used as controls.

How can I design knockout or knockdown experiments to study USP44 function in mouse models?

Effective strategies for USP44 loss-of-function studies include:

• CRISPR/Cas9 knockout:

  • Target conserved regions of the catalytic domain

  • Design multiple guide RNAs to enhance knockout efficiency

  • Verify knockout through both genomic sequencing and protein expression analysis

  • Multiple studies have used USP44 knockout in cell lines to study its effects on radiosensitivity and cell cycle regulation

• siRNA/shRNA knockdown:

  • For transient knockdown, siRNA targeting conserved regions of USP44 mRNA

  • For stable knockdown, lentiviral shRNA constructs can be employed

  • Multiple independent shRNA sequences should be tested to control for off-target effects

  • Knockdown efficiency should be verified by qRT-PCR and Western blot

• Mouse models:

  • Constitutive USP44 knockout mice have shown phenotypes related to aneuploidy and cancer susceptibility

  • Conditional knockout using Cre-loxP system allows tissue-specific deletion

  • Verify knockout efficiency in the specific tissue of interest

• Phenotypic analysis:

  • Cell cycle analysis using flow cytometry for H3S10 phosphorylation to assess G2/M arrest

  • Colony formation assays to assess cell survival and proliferation

  • In vivo tumor xenograft models to assess tumor growth and response to treatments like irradiation

What are the recommended assays for measuring USP44 deubiquitinase activity?

For accurate measurement of USP44 deubiquitinase activity:

• Fluorogenic substrate assays:

  • Using ubiquitin-AMC (7-amino-4-methylcoumarin) as substrate

  • Measure fluorescence release as indicator of DUB activity

  • Include appropriate controls: positive control (active USP enzyme), negative control (catalytically inactive USP44)

• Ubiquitination status of known substrates:

  • Immunoprecipitation followed by ubiquitin immunoblotting

  • Analysis of specific substrates like FOXP3 or H2B has been documented

  • In vitro deubiquitination assays using purified components

• Cellular ubiquitination assays:

  • Transfect cells with USP44 and substrate along with HA-tagged ubiquitin

  • Immunoprecipitate the substrate and detect ubiquitination levels

  • This approach has been used to demonstrate that USP44 surprisingly increased polyubiquitination of Ku80

• Mass spectrometry-based approaches:

  • Quantitative proteomics to identify changes in ubiquitination patterns

  • Particularly useful for discovering novel substrates

  • Has been used successfully to identify Ku80 as a potential target of USP44

How does USP44 contribute to tumor suppression or promotion in different cancer types?

USP44 demonstrates context-dependent roles in cancer progression, functioning either as a tumor suppressor or oncogene depending on cancer type:

These diverse findings highlight the importance of cancer-type specific analysis when targeting USP44 for potential therapeutic applications.

What is the relationship between USP44 and DNA damage response pathways?

USP44 plays crucial roles in DNA damage response pathways:

• Regulation of DNA double-strand break (DSB) repair:

  • Influences non-homologous end joining (NHEJ) repair pathway

  • Interacts with and regulates Ku80, a key component of the NHEJ machinery

  • Mass spectrometry analysis identified Ku80 as a potential direct target of USP44

  • Surprisingly, USP44 promotes Ku80 degradation by enhancing its ubiquitination, contrary to its canonical deubiquitinase function

• Radiosensitization mechanism:

  • Overexpression of USP44 enhances radiosensitivity in NPC cells

  • Gene Set Enrichment Analysis shows that samples with low USP44 expression are enriched in radiation response pathways

  • USP44 overexpression significantly induces G2/M phase arrest and apoptosis after irradiation

  • The percentage of H3S10 phosphorylation-positive cells (a marker of G2/M arrest) increases after irradiation, which is affected by USP44 status

• Ubiquitin-proteasome pathway involvement:

  • USP44 promotes Ku80 degradation through the ubiquitin-proteasome pathway

  • Treatment with MG132 (proteasome inhibitor) reverses USP44-mediated destabilization of Ku80

  • USP44 increases polyubiquitination of Ku80, leading to its degradation

• In vivo confirmation:

  • Xenograft models show USP44 overexpression increases tumor sensitivity to irradiation

  • USP44 regulates TRIM25/Ku80 expression to inhibit cell proliferation and activate apoptosis

  • The radiosensitization effect of USP44 is almost completely rescued by knockout of TRIM25

How does USP44 regulate immune function, particularly in regulatory T cells?

USP44 plays important roles in immune regulation, particularly in Treg cells:

• Regulation of FOXP3 stability:

  • USP44 functions as a deubiquitinase for FOXP3, a master transcription factor for Treg cells

  • USP44 cooperates with USP7 to stabilize FOXP3 protein levels

  • By removing ubiquitin molecules from FOXP3, USP44 prevents its proteasomal degradation

• TGF-β mediated induction:

  • USP44 is upregulated via TGF-β signaling

  • TGF-β can induce USP44 promoter activity

  • This provides a mechanistic link between TGF-β signaling (important for Treg development) and FOXP3 stability

• Functional consequences in immune responses:

  • USP44 promotes Treg function during immune responses

  • This suggests potential roles in autoimmunity and cancer immunology

  • Modulating USP44 could potentially affect Treg stability and function in therapeutic contexts

• Experimental validation:

  • USP44 promoter-driven luciferase reporter assays confirm TGF-β-mediated upregulation

  • Activation with PMA and ionomycin can induce reporter activity

What are common pitfalls in USP44 research and how can they be addressed?

Researchers frequently encounter several challenges when studying USP44:

• Protein stability issues:

  • USP44 protein can be unstable during purification and storage

  • Solution: Include protease inhibitors in all buffers; store with glycerol at -80°C; avoid repeated freeze-thaw cycles

• Specificity of commercial antibodies:

  • Variable quality of anti-USP44 antibodies can lead to inconsistent results

  • Solution: Validate antibodies using positive controls (overexpression) and negative controls (knockout); use multiple antibodies targeting different epitopes

• Catalytic activity variability:

  • Recombinant USP44 may show inconsistent enzymatic activity

  • Solution: Always include appropriate positive and negative controls in deubiquitination assays; use freshly prepared protein when possible

• Functional redundancy with other DUBs:

  • Other deubiquitinases may compensate for USP44 loss in knockout models

  • Solution: Consider double knockout approaches targeting related DUBs; perform rescue experiments to confirm specificity

• Context-dependent functions:

  • USP44 shows opposing roles in different cancer types

  • Solution: Always specify cell type and disease context; avoid broad generalizations about USP44 function

• Non-canonical functions:

  • USP44 can promote ubiquitination of some targets (e.g., Ku80) rather than removing ubiquitin

  • Solution: Verify both ubiquitination and deubiquitination activities; consider indirect mechanisms through interaction partners

How can I reconcile contradictory findings about USP44 function in the literature?

When facing contradictory findings about USP44 in the literature:

What are promising therapeutic applications targeting USP44 in cancer and immune disorders?

Several promising therapeutic applications targeting USP44 are emerging:

• Cancer therapy approaches:

  • Radiotherapy sensitization: Modulating USP44 expression could enhance the effectiveness of radiotherapy in specific cancers like NPC

  • Cancer type-specific targeting: Inhibiting USP44 in cancers where it acts as an oncogene (glioma, prostate cancer) while enhancing it in cancers where it functions as a tumor suppressor (HCC)

  • Combination therapy: Targeting the USP44-TRIM25-Ku80 axis in combination with DNA-damaging agents could provide synergistic effects

• Immune modulation strategies:

  • Treg function regulation: Targeting USP44 could modulate Treg stability and function in autoimmune disorders

  • TGF-β pathway intervention: USP44 represents a downstream effector of TGF-β signaling, offering potential for more specific immune modulation than direct TGF-β targeting

  • Cancer immunotherapy: Modulating USP44 in Tregs could potentially enhance cancer immunotherapy approaches

• Development of specific USP44 modulators:

  • Small molecule inhibitors targeting the catalytic domain

  • Protein-protein interaction disruptors targeting specific USP44-substrate interactions

  • Gene therapy approaches to modulate USP44 expression in specific tissues

• Potential biomarker applications:

  • USP44 expression levels as prognostic markers in HCC and other cancers

  • Monitoring USP44 pathway activity to predict therapy response

Despite these promising directions, several challenges remain, including developing selective USP44 modulators and understanding the exact mechanisms of substrate specificity .

What are the key unresolved questions about USP44 biology that require further investigation?

Several critical aspects of USP44 biology remain poorly understood:

• Substrate recognition mechanisms:

  • How does USP44 achieve specificity for each substrate?

  • What determines whether USP44 promotes deubiquitination versus ubiquitination of targets?

  • What conformational changes in USP44 affect its substrate specificity?

• Regulatory mechanisms:

  • Beyond TGF-β, what other signaling pathways regulate USP44 expression and activity?

  • How is USP44 itself regulated post-translationally?

  • What determines USP44's subcellular localization and how does this affect function?

• Contextual function determinants:

  • Why does USP44 function as a tumor suppressor in some cancers but as an oncogene in others?

  • What molecular factors determine these context-specific functions?

  • How do genetic and epigenetic backgrounds influence USP44 activity?

• Therapeutic targeting considerations:

  • How can USP44 be selectively targeted without affecting other DUBs?

  • What are potential off-target effects of USP44 modulation?

  • Could resistance mechanisms develop against USP44-targeted therapies?

• Physiological functions:

  • What is the full spectrum of USP44's physiological roles beyond the currently known functions?

  • How does USP44 contribute to normal development and tissue homeostasis?

  • What compensatory mechanisms exist when USP44 function is lost?

Addressing these questions will require integrated approaches combining structural biology, proteomics, genetic models, and clinical studies to fully elucidate USP44's complex biology and therapeutic potential.