USP15 Human

What is USP15 and what is its primary function in human cells?

USP15 (Ubiquitin carboxyl-terminal hydrolase 15) is an enzyme encoded by the USP15 gene located on human chromosome 12. It belongs to the ubiquitin-specific peptidase family of deubiquitinating enzymes (DUBs) . The primary function of USP15 is to remove ubiquitin molecules from protein substrates, thereby preventing their degradation through the ubiquitin-proteasome pathway.

Ubiquitin is a highly conserved protein involved in regulating intracellular protein breakdown, cell cycle regulation, and stress response. USP15 specifically mediates the disassembly of polyubiquitin chains that are released from degraded proteins . This deubiquitination activity allows USP15 to regulate protein stability and function across multiple cellular pathways.

Methodologically, researchers investigating USP15's basic function typically employ techniques such as immunoprecipitation followed by ubiquitin chain analysis, site-directed mutagenesis of catalytic residues, and substrate identification through mass spectrometry-based proteomics.

What signaling pathways does USP15 regulate in human cells?

USP15 plays crucial roles in multiple signaling pathways, with particularly well-characterized functions in the TGF-β signaling cascade. Through RNAi-mediated loss of function screening, USP15 was identified as a critical regulator of TGF-β signaling . It acts through several mechanisms:

• USP15 stabilizes the TGF-β receptor (TβRI) by deubiquitinating it, preventing its degradation

• It enhances the activity of downstream signal transducers, particularly R-SMADs

• USP15 forms a complex with SMAD7 and SMURF2, opposing SMURF2-mediated ubiquitination of TβRI when active TGF-β levels are low

Beyond TGF-β signaling, USP15 regulates multiple cellular processes including:

• Mitochondrial homeostasis

• Membrane trafficking from the endoplasmic reticulum

• Carcinogenesis

• Antiviral responses

Researchers studying USP15's role in signaling pathways typically employ receptor phosphorylation assays, SMAD nuclear translocation measurements, and reporter gene assays to quantify pathway activation.

How does USP15 influence RNA processing and splicing mechanisms?

USP15 has emerged as a key regulator of RNA metabolism through multiple mechanisms. Proteomic analyses have revealed that USP15 is implicated in RNA processing pathways . Specifically:

• USP15 regulates spliceosome assembly by deubiquitinating components of the spliceosome machinery

• It deubiquitinates terminal uridylyl transferase 1 (TUT1), which is critical for proper U6 snRNA processing

• The USP15-TUT1 interaction influences global RNA splicing patterns

In experimental models, loss of USP15 results in altered RNA splicing profiles. Mechanistically, USP15 interacts with SART3 (a U6 snRNA recycling factor) and TUT1, forming a functional complex that regulates U6 snRNA function . USP15 deubiquitinates TUT1, which is responsible for adding a short uridine tail to the 3' end of U6 snRNA. This uridylation is essential for recruiting LSm proteins and conferring proper functions to the U6 snRNP complex .

To study USP15's role in RNA processing, researchers utilize techniques such as RNA-seq, exon array analysis, in vitro splicing assays, and co-immunoprecipitation experiments to identify interacting RNA-processing factors.

What are the experimental approaches to study USP15's effect on RNA metabolism?

Researchers investigating USP15's role in RNA metabolism employ several methodological approaches:

• Genetic models: Generation of USP15 knockout mice and cell lines using CRISPR-Cas9 technology

• RNA-binding studies: RNA immunoprecipitation followed by sequencing (RIP-seq) to identify USP15-associated RNAs

• Splicing analysis: Exon arrays or RNA-seq to identify global changes in splicing patterns

• Protein interaction studies: Co-immunoprecipitation and proximity ligation assays to identify RNA processing factors that interact with USP15

• Subcellular localization: Immunofluorescence microscopy to determine USP15 localization in nuclear speckles or other RNA processing bodies

The knockout mouse model approach has proven particularly valuable, as seen in studies where Usp15^-/-^ mice exhibited altered expression of splicing factors and changes in global RNA splicing patterns . These models allow researchers to observe physiological consequences of USP15 deficiency in vivo, including effects on cerebellar function that may result from RNA processing defects.

What role does USP15 play in cerebellar maintenance and neurological function?

USP15 plays a crucial role in cerebellar maintenance and neurological function, with Usp15^-/-^ mice demonstrating significant cerebellar abnormalities and motor dysfunction. Several key findings highlight this role:

• USP15 is highly expressed in Purkinje cells of the cerebellum

• Loss of USP15 leads to motor defects, including tremor and abnormal hind limb clasping reflexes

• Usp15^-/-^ mice perform poorly in rotarod tests, indicating impaired motor coordination

• Anatomical analysis reveals impaired foliation in the cerebellar vermis at lobules crus 3 and 8

• Purkinje cells in Usp15^-/-^ mice show age-dependent size reduction and eventual neurodegeneration

Mechanistically, USP15 appears to maintain cerebellar integrity through proper RNA metabolism. Defects in the USP15-TUT1-U6 snRNA pathway result in aberrant RNA splicing, which can lead to the production of abnormal proteins that trigger ER stress and ultimately neurodegeneration. This establishes a novel mechanistic link between spliceosome dysfunction and ER stress-induced neurodegeneration .

For researchers studying USP15 in neurological contexts, behavioral testing (rotarod, grip strength), histological analysis of cerebellar architecture, and assessment of ER stress markers in Purkinje cells provide valuable experimental approaches.

How does USP15 deficiency lead to ER stress and neurodegeneration?

USP15 deficiency triggers a molecular cascade that connects RNA metabolism defects to ER stress and neurodegeneration:

• Loss of USP15 disrupts the USP15-TUT1-U6 snRNA pathway, leading to aberrant RNA splicing

• This results in the production of various mutant proteins generated by splicing errors

• Misfolded or structurally altered proteins accumulate in the ER, triggering ER stress

• Age-dependent increases in ER stress markers (BiP, CHOP) are observed in Usp15^-/-^ brains

• Prolonged ER stress ultimately leads to neuronal cell death, particularly affecting Purkinje cells

This mechanism is supported by experiments showing that knocking down TUT1 (a USP15 target) increases BiP expression, indicating enhanced ER stress . Additionally, specific targets identified through exon array analysis, such as Sparcl1 (a secreted protein from astrocytes) and Nrf1/Nfe2l1, may contribute to the observed phenotypes when their splicing is altered.

For researchers investigating this pathway, monitoring ER stress markers (BiP, CHOP, XBP1 splicing), analyzing protein aggregation, and assessing neuronal viability in USP15-deficient models are recommended experimental approaches.

What evidence supports USP15 as a cancer driver gene?

Several lines of evidence support USP15 as a potential cancer driver gene, particularly in pancreatic ductal adenocarcinoma (PDAC):

• In vivo CRISPR screens have identified USP15 as a novel driver of pancreatic cancer

• USP15 and SCAF1 (which functionally couples with USP15) mutations or copy number losses are observed in 31% of PDAC patients

• Mechanistically, loss of SCAF1 results in the formation of a truncated inactive USP15 isoform, functionally linking these two genes

• USP15 has been implicated in enhancing proliferation and migration in certain cellular contexts

The integrative approach combining human cancer genomics with mouse modeling has been particularly valuable in establishing USP15 as a cancer driver gene with potential prognostic and therapeutic implications .

For cancer researchers studying USP15, approaches including CRISPR screens, patient tumor sequencing, xenograft models, and assessment of cancer cell phenotypes (proliferation, migration, invasion) after USP15 modulation are recommended methodological strategies.

What are the mechanisms by which USP15 contributes to cancer pathogenesis?

USP15 contributes to cancer pathogenesis through several distinct mechanisms:

• TGF-β pathway modulation: USP15 enhances TGF-β signaling by stabilizing TGF-β receptor I (TβRI) and R-SMADs, potentially promoting epithelial-to-mesenchymal transition and cancer progression

• Fibroblast activation: USP15 enhances proliferation, migration, invasion, and collagen deposition in fibroblasts, potentially contributing to cancer-associated fibrosis and tumor microenvironment remodeling

• RNA processing dysregulation: Altered RNA splicing due to USP15 dysfunction may generate cancer-promoting splice variants of key regulatory proteins

• Functional interaction with SCAF1: Loss of SCAF1 results in truncated inactive USP15 isoforms, suggesting complex genetic interactions that may drive tumorigenesis

When investigating these mechanisms, researchers commonly employ techniques such as TGF-β reporter assays, fibroblast migration and invasion assays, RNA splicing analysis, and assessment of collagen deposition using techniques like Western blotting, qRT-PCR, and immunofluorescence microscopy.

How does USP15 influence fibroblast function and collagen deposition?

USP15 plays a significant role in fibroblast behavior and collagen production, particularly in the context of fibrotic disorders:

• USP15 enhances the proliferation, migration, invasion, and collagen deposition in hypertrophic scar-derived fibroblasts

• Mechanistically, USP15 appears to exert these effects by deubiquitinating TGF-β receptor I (TβRI)

• This stabilizes TβRI and enhances downstream TGF-β signaling, which is a major pro-fibrotic pathway

• Activation of TGF-β signaling leads to increased expression of fibrotic markers including α-SMA, COL1, and COL3

Researchers studying USP15 in fibrosis typically employ techniques such as:

• Fibroblast proliferation assays (Cell Counting Kit-8)

• Migration assays (scratch/wound healing)

• Invasion assays

• Collagen deposition quantification

• qRT-PCR and Western blotting for fibrotic markers

• Lentiviral-mediated knockdown or overexpression of USP15

These methodological approaches allow for detailed characterization of USP15's role in fibroblast function and fibrotic processes.

What is the relationship between USP15, TGF-β signaling, and fibrotic disorders?

The relationship between USP15, TGF-β signaling, and fibrosis forms a mechanistic axis central to many fibrotic disorders:

• USP15 deubiquitinates and stabilizes TGF-β receptor I (TβRI), preventing its degradation

• Enhanced TβRI stability leads to increased sensitivity to TGF-β ligands and prolonged signaling

• USP15 also forms a complex with SMAD7 and SMURF2, opposing SMURF2-mediated ubiquitination of TβRI

• Activated TGF-β signaling induces Smad2/3 phosphorylation and nuclear translocation

• This leads to transcriptional upregulation of pro-fibrotic genes including α-SMA, COL1, and COL3

• The resulting increased collagen production and myofibroblast differentiation contribute to fibrosis

In hypertrophic scarring, USP15 appears to be upregulated, suggesting a pathological role in excessive scarring and fibrosis . Understanding this pathway has significant implications for developing anti-fibrotic therapies targeting the USP15-TGF-β signaling axis.

For researchers investigating this relationship, monitoring TβRI ubiquitination status, Smad2/3 phosphorylation and nuclear localization, and fibrotic gene expression after USP15 modulation provides valuable mechanistic insights.

What are the optimal experimental approaches for studying USP15 deubiquitinase activity?

Researchers investigating USP15's deubiquitinase activity employ several specialized techniques:

• In vitro deubiquitination assays: Using purified USP15 protein and polyubiquitinated substrates to directly assess enzymatic activity

• Cell-based ubiquitination analysis:

  • Transfection of tagged ubiquitin and USP15 constructs

  • Treatment with proteasome inhibitors to prevent substrate degradation

  • Immunoprecipitation of specific substrates followed by ubiquitin Western blotting

  • Analysis of both K48-linked and K63-linked ubiquitin chains, as USP15 can deubiquitinate both types

• Catalytic mutant controls: Generation of catalytic-dead USP15 mutants by site-directed mutagenesis of key residues in the catalytic domain

• Substrate identification:

  • Co-immunoprecipitation followed by mass spectrometry

  • Proximity labeling techniques (BioID, APEX)

  • Comparative proteomics of wild-type vs. USP15-deficient cells

• Ubiquitin chain specificity analysis: Using ubiquitin chain-specific antibodies or mass spectrometry to determine which types of ubiquitin linkages USP15 preferentially cleaves

When studying specific substrates like TGF-β receptor I or TUT1, researchers should include appropriate controls and validation experiments to confirm direct deubiquitination by USP15 rather than indirect effects.

How can researchers effectively model USP15 deficiency or dysfunction in experimental systems?

Researchers have several options for modeling USP15 deficiency or dysfunction:

• Genetic knockout models:

  • Generation of Usp15^-/-^ mice using targeted gene disruption

  • CRISPR-Cas9-mediated knockout in cell lines

  • Conditional knockout systems (Cre-loxP) for tissue-specific USP15 deletion

• RNA interference approaches:

  • siRNA for transient USP15 knockdown

  • shRNA or lentiviral vectors for stable knockdown

  • Inducible knockdown systems for temporal control

• Pharmacological inhibition:

  • Small molecule inhibitors targeting USP15's catalytic activity

  • Evaluation of specificity using multiple DUB family members as controls

• Expression of dominant-negative constructs:

  • Catalytically inactive USP15 mutants

  • Truncated forms that interfere with endogenous USP15 function

• In vivo CRISPR screens:

  • Used successfully to identify USP15 as a cancer driver gene

  • Allows for high-throughput functional genomics in physiologically relevant contexts

When selecting a model system, researchers should consider the specific biological question, desired temporal control, and potential compensatory mechanisms. The generation of Usp15^-/-^ mice has provided valuable insights into USP15's role in RNA metabolism and cerebellar maintenance , while lentiviral approaches have been effective for studying USP15's role in fibroblast function .

What human diseases are associated with USP15 dysfunction?

USP15 dysfunction has been implicated in several human diseases:

• Cancer:

  • USP15 and SCAF1 mutations or copy number losses are observed in 31% of pancreatic ductal adenocarcinoma patients

  • USP15 may function as a cancer driver gene with prognostic implications

• Neurological disorders:

  • USP15 is expressed in both neurons and glial cells

  • Aberrant USP15 functions are associated with neuroinflammation

  • USP15 dysfunction has been linked to neurodegenerative disorders including ataxia and Parkinson's disease

• Fibrotic disorders:

  • USP15 enhances fibroblast proliferation, migration, and collagen deposition

  • It is implicated in hypertrophic scarring through TGF-β pathway enhancement

• Embryonic development:

  • Usp15^-/-^ mice are born at lower frequencies than predicted by Mendelian ratios, suggesting a role in embryogenesis

The multifaceted roles of USP15 across different cellular processes make it a potential contributor to various pathological conditions, particularly those involving aberrant TGF-β signaling, RNA processing defects, or protein homeostasis disruption.

What are the therapeutic implications of targeting USP15?

The diverse roles of USP15 in disease pathology suggest several therapeutic opportunities:

Therapeutic development targeting USP15 would need to consider:

• Specificity among DUB family members

• Potential off-target effects due to USP15's multiple roles

• Tissue-specific delivery to avoid unintended consequences in tissues where USP15 plays protective roles

• Appropriate patient selection based on molecular profiling

Research in this area requires careful validation of USP15's role in specific disease contexts before proceeding to therapeutic development.

What are the current gaps in USP15 research and future research priorities?

Despite significant advances in understanding USP15 biology, several important knowledge gaps remain:

• Comprehensive substrate identification:

  • Beyond TGF-β receptors and TUT1, many USP15 substrates likely remain unidentified

  • Unbiased proteome-wide approaches are needed to fully characterize USP15's substrate landscape

• Tissue-specific functions:

  • USP15 may have different roles in different tissues

  • Tissue-specific conditional knockout models could help elucidate these functions

• Regulatory mechanisms:

  • How USP15 activity itself is regulated remains poorly understood

  • Post-translational modifications of USP15 that control its function need further investigation

• Structural insights:

  • Detailed structural studies of USP15 in complex with substrates would facilitate drug development

  • Understanding binding interfaces could aid in developing specific inhibitors

• Clinical correlations:

  • More comprehensive analysis of USP15 expression, mutation, or dysfunction across human diseases

  • Establishment of USP15 as a biomarker for disease prognosis or treatment response

Future research priorities should include:

• Developing more specific tools to modulate USP15 activity

• Establishing the full extent of USP15's role in RNA metabolism

• Clarifying the mechanistic links between USP15, ER stress, and neurodegeneration

• Exploring USP15 as a therapeutic target in fibrosis and cancer

Collaborative approaches combining structural biology, proteomics, genetics, and disease modeling will be essential to address these knowledge gaps.

How do USP15's functions integrate across different cellular processes?

USP15 functions as a central integrator across multiple cellular processes:

• Signaling and RNA metabolism integration:

  • USP15 regulates both TGF-β signaling and RNA processing

  • This suggests potential crosstalk between these pathways, where alterations in one may affect the other

• Protein homeostasis and ER stress:

  • USP15's role in deubiquitination influences protein stability

  • Its impact on RNA splicing affects protein synthesis

  • Together, these functions contribute to protein homeostasis, with dysfunction leading to ER stress

• Development and disease:

  • USP15's role in embryogenesis suggests fundamental developmental functions

  • The same pathways contribute to disease processes in adulthood, including neurodegeneration and cancer

• Cellular stress responses:

  • USP15 appears to function in multiple stress response pathways

  • This positions it as a potential integrator of cellular responses to various stressors

A comprehensive model of USP15 function requires consideration of these integrated roles. For example, USP15's impact on cerebellar maintenance likely involves both its direct effects on neuronal protein homeostasis and its influence on RNA processing, which together determine cellular fitness and stress responses over time.