YOD1 Human

What is YOD1 and what is its primary function in human cells?

YOD1 (OTUD2) is a member of the OTU (ovarian tumor) deubiquitinase (DUB) family that plays critical roles in various cellular processes. The enzyme functions by removing ubiquitin modifications from target proteins, thereby regulating their stability, localization, or function. In mammalian cells, YOD1 facilitates protein quality control at the endoplasmic reticulum through the ER-associated protein degradation (ERAD) pathway, working in conjunction with Valosin-containing protein (VCP)/p97 . Its yeast homolog OTU1 acts as a cofactor of the hexameric AAA-ATPase Cdc48/p97 for protein processing . YOD1 has been identified as a critical regulator in multiple signaling pathways, including the Hippo pathway and IL-1-mediated inflammatory responses, positioning it as an important target for understanding cellular homeostasis mechanisms.

How is YOD1 expression and activity regulated in human cells?

YOD1 expression is regulated by multiple mechanisms, with microRNA-mediated control being particularly significant. Research has demonstrated that miR-21 regulates YOD1 levels in a cell-density-dependent manner, which consequently affects YAP/TAZ activity in the Hippo signaling pathway . This regulatory mechanism explains how YOD1 levels change in response to environmental cues such as cell density. Additionally, YOD1's interaction with key signaling proteins like TRAF6 is dynamically regulated by cellular stimuli. In unstimulated cells, YOD1 associates with TRAF6, but upon IL-1β stimulation, YOD1 dissociates from TRAF6, enabling TRAF6 auto-ubiquitination and downstream signaling events . This stimulus-dependent association represents a critical regulatory mechanism controlling YOD1's function in inflammatory signaling pathways.

How does YOD1 regulate the Hippo signaling pathway?

YOD1 serves as a positive regulator of YAP/TAZ in the Hippo signaling pathway, which controls organ size, cell proliferation, and tissue homeostasis. Mechanistically, YOD1 deubiquitinates ITCH, an E3 ligase that targets LATS kinases for degradation . By enhancing ITCH stability, YOD1 promotes the degradation of LATS1/2, which are key negative regulators of YAP/TAZ. Reduced LATS levels lead to decreased phosphorylation of YAP/TAZ, allowing these transcriptional co-activators to translocate to the nucleus and promote the expression of genes involved in cell proliferation and survival .

The significance of this regulatory mechanism has been demonstrated in vivo, where inducible expression of YOD1 in mouse liver enhanced hepatocyte proliferation and led to hepatomegaly (enlarged liver) in a YAP/TAZ-activity-dependent manner . This highlights YOD1's importance in regulating organ size through the Hippo pathway and suggests it may contribute to pathological conditions like cancer when dysregulated.

What is the role of YOD1 in IL-1 signaling and NF-κB activation?

YOD1 functions as a negative regulator of IL-1 signaling to NF-κB by competing with the adaptor protein p62/Sequestosome-1 for binding to TRAF6, a key ubiquitin ligase in this pathway . In unstimulated cells, YOD1 associates with the C-terminal TRAF homology domain of TRAF6, which is also the binding site for p62. This association prevents p62 from sequestering TRAF6 to cytosolic aggregates and thereby inhibits TRAF6 activation .

Upon IL-1β stimulation, YOD1 dissociates from TRAF6, allowing p62 to bind and facilitate TRAF6 auto-ubiquitination as well as substrate ubiquitination of NEMO/IKKγ, leading to IKK complex activation and subsequent NF-κB signaling . Experimental evidence supports this regulatory model, as YOD1 overexpression decreases IL-1-triggered IKK/NF-κB signaling and target gene induction, while YOD1 depletion augments these responses .

Interestingly, YOD1 appears to selectively regulate NF-κB signaling without significantly affecting IL-1-induced MAPK activation, which is also downstream of TRAF6 . This suggests that YOD1 specifically targets TRAF6/p62 complexes involved in NF-κB activation rather than all TRAF6-dependent pathways.

What genetic tools are available for studying YOD1 function in cellular models?

Several genetic tools have been developed to investigate YOD1 function in cellular models. CRISPR/Cas9 technology has been successfully employed to generate YOD1-deficient cell lines, providing valuable loss-of-function models. In published research, exon 4 of the YOD1 gene, which encodes almost the entire open reading frame, was deleted using flanking single guide RNAs together with Cas9, resulting in complete loss of YOD1 protein expression .

For gain-of-function studies, lentiviral expression systems have been utilized to introduce wild-type YOD1 or catalytically inactive YOD1 C160S mutants into cells . Additionally, doxycycline-inducible systems based on the tTR-KRAB hybrid protein have enabled controlled expression of YOD1 variants or shRNAs for YOD1 knockdown . These tools provide researchers with flexible options for manipulating YOD1 levels and activity in various cellular contexts.

When using these genetic tools, it's important to note that clonal selection of cells may introduce heterogeneity in terms of cell proliferation, gene induction, and signaling responses, as observed with YOD1-knockout HeLa cells . Therefore, using lentiviral reconstitution followed by FACS sorting to obtain homogenous cell populations may be preferable for comparing effects within individual knockout clones.

What biochemical methods are most effective for analyzing YOD1 interactions and deubiquitinating activity?

For studying YOD1 protein-protein interactions, co-immunoprecipitation (Co-IP) assays have proven effective in detecting associations with key partners like TRAF6 . These assays can reveal dynamic changes in protein interactions in response to stimuli, such as the dissociation of YOD1 from TRAF6 following IL-1β treatment. When designing Co-IP experiments, researchers should consider both endogenous protein interactions and those involving overexpressed tagged proteins, as overexpression may affect the physiological relevance of the results.

To assess YOD1's deubiquitinating activity, in vitro deubiquitination assays using purified components can determine substrate specificity and catalytic efficiency. Additionally, cellular ubiquitination assays examining the ubiquitination status of potential substrates (e.g., ITCH) or downstream effectors (e.g., NEMO/IKKγ) in the presence or absence of YOD1 can provide insights into its function in vivo . The catalytic activity of YOD1 can be specifically interrogated by comparing the effects of wild-type YOD1 with those of the catalytically inactive C160S mutant.

For monitoring YOD1's impact on signaling pathways, Western blotting to detect phosphorylation and degradation of pathway components (e.g., IκBα), as well as electrophoretic mobility shift assays (EMSAs) to assess NF-κB DNA binding, have been successfully employed . These approaches, combined with quantitative RT-PCR to measure expression of target genes, provide comprehensive analysis of YOD1's functional effects.

What is the evidence linking YOD1 to cancer development and progression?

Emerging evidence suggests YOD1 may play significant roles in cancer development and progression, particularly through its regulation of the Hippo pathway. A strong correlation between YOD1 and YAP expression has been observed in liver cancer patients, suggesting potentially coordinated functions in hepatocarcinogenesis . This clinical observation is supported by experimental evidence from a transgenic mouse model where inducible expression of human YOD1 in mouse liver enhanced hepatocyte proliferation and led to hepatomegaly in a YAP/TAZ-activity-dependent manner .

The mechanistic link between YOD1 and cancer likely involves its positive regulation of YAP/TAZ, which are known oncogenic factors in multiple cancer types. By deubiquitinating ITCH and enhancing its stability, YOD1 reduces LATS levels, leading to increased YAP/TAZ activity and subsequent promotion of cell proliferation and survival genes . This regulatory circuit could contribute to uncontrolled cell growth and tumorigenesis when dysregulated.

While research directly linking YOD1 to human cancers is still emerging, the functional connections to established oncogenic pathways position YOD1 as a potential therapeutic target in cancer treatment strategies.

How might YOD1 contribute to inflammatory disorders through its regulation of IL-1 signaling?

YOD1's role as a negative regulator of IL-1-induced NF-κB signaling suggests it may influence inflammatory conditions where this pathway is dysregulated. By controlling TRAF6-dependent ubiquitination events through competition with p62, YOD1 serves as a molecular brake on IL-1 signaling to NF-κB . Defects in this regulatory mechanism could potentially contribute to excessive or prolonged inflammatory responses observed in various disorders.

Unlike other negative regulators of this pathway such as CYLD and A20, which act as negative feedback regulators terminating post-inductive TRAF6 activity, YOD1 appears to function during the early phase of the IL-1 response by controlling the accessibility of p62 to TRAF6 . This temporal regulation may be critical for appropriate inflammatory responses, as early NF-κB signaling and gene induction are increased upon YOD1 depletion .

Interestingly, YOD1 selectively affects NF-κB signaling without significantly influencing IL-1-induced MAPK activation, suggesting it specifically targets TRAF6/p62 complexes involved in NF-κB activation rather than all TRAF6-dependent pathways . This selective regulation could make YOD1 a more precise therapeutic target for inflammatory conditions where NF-κB activity is pathogenic.

How does YOD1 function in the context of other deubiquitinases regulating the same pathways?

The interplay between YOD1 and other deubiquitinases (DUBs) regulating the same pathways presents an intriguing area of investigation. Within the IL-1 signaling pathway, multiple DUBs including CYLD and A20 act as negative regulators but through distinct mechanisms and at different points in the signaling cascade . While CYLD and A20 function as negative feedback regulators that terminate post-inductive TRAF6 activity through catalytic or non-catalytic mechanisms respectively, YOD1 operates earlier in the signaling cascade by controlling the association between TRAF6 and p62 in unstimulated cells .

This temporal and mechanistic diversity suggests these DUBs may act in a concerted manner at different steps of the pathway, potentially providing redundancy and robust control of inflammatory signaling. Indeed, research suggests an interdependency among these negative regulators that can potentially act as a fail-safe mechanism, compensating for the loss of one another . This concept is supported by observations that enhanced signaling upon loss of YOD1 may be partially impeded by CYLD during an IL-1 response, as p62 also recruits CYLD at later stages .

Understanding these complex interactions among different DUBs will require sophisticated experimental approaches, including the generation of double or triple knockout models and temporal analyses of signaling dynamics.

What are the contradictions regarding the catalytic versus non-catalytic functions of YOD1 in different cellular contexts?

An intriguing aspect of YOD1 biology is the apparent dichotomy between its catalytic and non-catalytic functions in different cellular contexts. While YOD1's deubiquitinating activity is essential for its function in the ERAD pathway and Hippo signaling, evidence suggests its regulation of TRAF6 in IL-1 signaling occurs through a non-catalytic mechanism .

In the Hippo pathway, YOD1 deubiquitinates ITCH, an E3 ligase, enhancing its stability and leading to reduced levels of LATS kinases . This catalytic activity ultimately promotes YAP/TAZ activity and cell proliferation. In contrast, YOD1's inhibition of TRAF6 in the IL-1 pathway appears to involve competitive binding with p62 rather than deubiquitination of TRAF6 or other pathway components . This is supported by observations that YOD1 abolishes the sequestration of TRAF6 to cytosolic p62 aggregates by a non-catalytic mechanism .

This dual functionality raises important questions about how YOD1's catalytic and scaffolding roles are regulated in different cellular contexts. Structural studies and domain-specific mutants could help dissect these distinct functions and identify the molecular determinants that direct YOD1 toward specific modes of action in different signaling pathways.

What emerging technologies could advance our understanding of YOD1 regulation and function?

Several cutting-edge technologies hold promise for deepening our understanding of YOD1 biology. Proximity-based labeling methods such as BioID or APEX could help map the complete interactome of YOD1 in different cellular compartments and under various stimulation conditions. These approaches would provide a more comprehensive view of YOD1's functional networks beyond the currently known interactions with TRAF6 and ITCH.

Advanced proteomics approaches, particularly ubiquitinome analysis, could identify the full spectrum of YOD1 substrates and reveal how its deubiquitinating activity reshapes the cellular ubiquitin landscape under different conditions. This would help clarify YOD1's catalytic targets and potentially resolve some of the contradictions regarding its catalytic versus non-catalytic functions.

CRISPR-based screens could identify synthetic lethal interactions or functional dependencies involving YOD1, potentially revealing new therapeutic opportunities in diseases where YOD1 is dysregulated. Additionally, single-cell analyses examining YOD1 expression and activity across diverse cell types and disease states could provide insights into cell-type-specific functions and heterogeneity in YOD1-dependent responses.

How should researchers approach studying the tissue-specific roles of YOD1 in human physiology and pathology?

Investigating tissue-specific roles of YOD1 requires integrated approaches spanning multiple model systems. Conditional knockout mouse models with tissue-specific YOD1 deletion would allow examination of its function in specific organs without developmental confounding factors. These models would be particularly valuable for studying YOD1's role in the liver, where it has been implicated in hepatocyte proliferation and potentially hepatocarcinogenesis .

For translational relevance, patient-derived organoids or induced pluripotent stem cell (iPSC) models could be employed to study YOD1 function in a human genetic background. These systems allow manipulation of YOD1 expression or activity in a physiologically relevant context and can recapitulate tissue-specific aspects of disease.

Correlative studies examining YOD1 expression, localization, and activity in human tissue samples across different pathological conditions will be essential for validating findings from model systems. The observed correlation between YOD1 and YAP expression in liver cancer patients provides a starting point , but comprehensive analyses across multiple cancer types and inflammatory disorders are needed to fully understand YOD1's clinical relevance.

Integration of these approaches with systems biology methods to model YOD1's role in complex signaling networks will help predict context-dependent functions and identify potential therapeutic opportunities for targeting YOD1 in disease.