PPP1R11 Human

What is PPP1R11 and what are its main functions in human cells?

PPP1R11 (also known as Inhibitor-3, HCGV, TCTE5, or Protein phosphatase inhibitor 3) serves dual molecular functions in human cells. It acts as an atypical E3 ubiquitin-protein ligase that targets specific proteins for proteasomal degradation, most notably Toll-like receptor 2 (TLR2) . Simultaneously, it functions as a potent inhibitor of protein phosphatase 1 (PP1), influencing various signaling cascades .

PPP1R11 contains RING finger domains characteristic of E3 ligases, enabling it to catalyze ubiquitination reactions. This capability has been confirmed through both in vitro and in vivo experiments demonstrating its auto-ubiquitination properties in the presence of ubiquitin, E1, and specific E2 conjugating enzymes such as UBE2R1 and UBE2D2 . The protein shows selective targeting, as studies indicate it doesn't affect other Toll-like receptors such as TLR3, TLR7, TLR8, or TLR9 .

Methodologically, researchers can assess PPP1R11 function through protein half-life studies, co-immunoprecipitation assays to detect protein-protein interactions, and in vitro ubiquitination assays using recombinant proteins.

How is PPP1R11 expression regulated in different tissues and cell types?

PPP1R11 expression shows contextual regulation in response to specific immune stimuli. In lung epithelial cells, treatment with the TLR2 ligand Pam3CSK4 upregulates PPP1R11 protein levels, coinciding with a gradual reduction in TLR2 protein levels after approximately 4-6 hours . This pattern suggests PPP1R11 participates in a negative feedback mechanism to prevent excessive inflammatory responses.

For researchers investigating tissue-specific expression, immunohistochemical analysis can be performed, as demonstrated in studies of human stomach tissue where PPP1R11 expression has been detected . Western blot analysis of cell lysates from various tissues and cell lines, particularly immune cells and epithelial cells, provides quantitative assessment of expression levels.

To study regulation mechanisms, researchers should consider time-course experiments following various stimuli, coupled with transcriptional and post-translational analysis methods.

How does PPP1R11 influence TLR2 signaling and inflammatory responses?

PPP1R11 functions as a critical negative regulator of TLR2 signaling through direct ubiquitination of TLR2 at lysine 754, targeting it for proteasomal degradation . This mechanism serves to attenuate inflammatory responses following TLR2 activation by gram-positive bacterial components.

In experimental models, ectopic expression of PPP1R11 decreases TLR2 protein levels in a dose-dependent manner without affecting TLR2 mRNA levels . Overexpression of PPP1R11 significantly reduces the half-life of TLR2, while PPP1R11 knockdown markedly increases TLR2's lifespan . This regulatory relationship has functional consequences for inflammatory responses, as PPP1R11 overexpression in murine lung epithelial cells significantly reduces Pam3CSK4-induced cytokine release .

Methodologically, researchers can investigate these effects through:

• In vitro ubiquitination assays with TLR2 as substrate

• Protein half-life studies following cycloheximide treatment

• Cytokine measurements after TLR2 ligand stimulation

• Bacterial clearance assays in cell culture and animal models

What is the role of PPP1R11 in T cell function and resistance to regulatory T cell suppression?

PPP1R11 plays a significant role in modulating T cell activation and susceptibility to regulatory T cell (Treg)-mediated suppression. Silencing of PPP1R11 in T cells renders them resistant to Treg-mediated suppression of TCR-induced cytokine expression, particularly affecting IL-2 and IFN-γ production .

Mechanistically, PPP1R11 functions as a negative regulator of T cell activation-induced cytokine expression. By inhibiting PP1, PPP1R11 influences various signaling pathways, potentially including phosphatidylinositol signaling, MAPK-AKT, and NF-κB pathways . When PPP1R11 is silenced, the inhibition on PP1 is released, leading to enhanced TCR-induced cytokine expression.

RNA sequencing analysis of PPP1R11-silenced T cells reveals that PPP1R11 differentially regulates not only specific T cell stimulation-induced cytokines but also other molecules and pathways in T cells . This suggests a broader role in shaping the T cell activation program beyond cytokine regulation.

For T cell researchers, methodological approaches should include:

• siRNA-mediated silencing of PPP1R11 in primary human T cells

• T cell-Treg co-culture suppression assays

• Cytokine gene and protein expression analysis

• Global transcriptome analysis through RNA sequencing

• Targeted analysis of TCR signaling pathways

• PP1 activity assays

What are the optimal techniques for detecting and quantifying PPP1R11 protein expression?

For reliable detection and quantification of PPP1R11 protein expression, researchers should employ multiple complementary techniques:

Western Blotting: Using validated antibodies such as rabbit polyclonal antibodies against PPP1R11 that have been confirmed for specificity . Optimal dilutions (e.g., 1/1000) should be determined for each antibody and cell type. HepG2 cell lysates have been successfully used as positive controls .

Immunohistochemistry: For tissue samples, formalin-fixed, paraffin-embedded tissues can be analyzed using anti-PPP1R11 antibodies at appropriate dilutions (e.g., 1/100) . This allows for spatial localization of PPP1R11 expression within tissue architecture.

Quantitative Mass Spectrometry: For absolute quantification and phosphorylation state analysis, targeted proteomics approaches can be employed, particularly relevant for studying the relationship between PPP1R11 phosphorylation and function.

When analyzing samples from patients or experimental models, it's crucial to establish appropriate normalization controls and consider the temporal dynamics of PPP1R11 expression, especially following stimuli like TLR2 ligands that can induce its expression .

What genetic approaches are most effective for manipulating PPP1R11 expression in research models?

Multiple genetic approaches have proven effective for manipulating PPP1R11 expression, each with specific advantages depending on the research question:

RNA Interference: siRNA-mediated silencing has been successfully used in primary human T cells to study PPP1R11 function . This approach offers transient knockdown suitable for short-term experiments.

Lentiviral Gene Transfer: For both overexpression and shRNA-mediated knockdown, lentiviral vectors have been effectively employed in both cell culture and in vivo mouse models . This approach allows for stable manipulation of PPP1R11 expression and has been successfully used to study its role in S. aureus infection models.

CRISPR-Cas9 Gene Editing: CRISPR-Cas9 technology has been used to introduce frameshifts in the Ppp1r11 gene in murine lung epithelial cells, creating knockout cell lines . This method allows for complete elimination of PPP1R11 expression for studying loss-of-function phenotypes.

For optimal results, researchers should:

• Confirm knockdown/overexpression efficiency at both mRNA and protein levels

• Include appropriate controls (scramble siRNA, empty vectors)

• Consider potential off-target effects, especially for RNAi approaches

• Assess the stability of genetic manipulation over time

• Validate phenotypes with multiple independent approaches

How does the E3 ligase activity of PPP1R11 specifically target TLR2 for ubiquitination?

PPP1R11 functions as a genuine RING finger E3 ligase that specifically targets TLR2 for ubiquitination at lysine 754, leading to its proteasomal degradation . The specificity of this interaction is demonstrated by PPP1R11's inability to alter protein levels of other Toll-like receptors including TLR3, TLR7, TLR8, and TLR9 .

The E3 ligase activity of PPP1R11 depends on its RING finger domains, which utilize specific cysteine and histidine residues to interact with zinc, forming the structural elements required for activity . Mutation studies of key residues within these RING finger domains have demonstrated loss-of-function in TLR2 degradation, confirming their critical role .

PPP1R11 exhibits auto-ubiquitination capabilities characteristic of RING E3 ligases, and preferentially interacts with specific E2 conjugating enzymes, particularly UBE2R1 and UBE2D2 . These interactions have been demonstrated through both in vitro binding assays and cellular co-immunoprecipitation.

For researchers investigating this mechanism, methodology should include:

• In vitro ubiquitination assays with purified components

• Mutagenesis of key residues in both PPP1R11 and TLR2

• Co-immunoprecipitation to confirm protein-protein interactions

• Proteasome inhibition studies to confirm degradation pathway

• Mass spectrometry to identify ubiquitination sites

What are the molecular mechanisms by which PPP1R11 inhibits protein phosphatase 1 (PP1) activity?

PPP1R11 functions as a potent inhibitor of protein phosphatase 1 (PP1), influencing various signaling cascades . As a regulatory subunit of the PP1 holoenzyme, PPP1R11 modulates the substrate specificity and activity of PP1 catalytic subunits.

PP1 exists in more than 650 different complexes arising from combinations with various subunits and PP1-interacting proteins (PIPs) . While PP1 catalytic subunits themselves have relatively low substrate specificity compared to kinases, their interactions with regulatory subunits like PPP1R11 provide context-specific regulation.

When PPP1R11 is silenced, the inhibitory effect on PP1 is released, resulting in enhanced PP1 activity. This has been shown to augment TCR-induced cytokine expression in T cells , suggesting that PP1 plays a positive role in TCR signaling pathways that is normally restrained by PPP1R11.

Research approaches to study this mechanism should include:

• PP1 activity assays following PPP1R11 manipulation

• Structural studies of PPP1R11-PP1 interaction

• Identification of PP1 substrates affected by PPP1R11 inhibition

• Analysis of phosphorylation dynamics of key signaling molecules

• Targeted investigation of phosphatidylinositol signaling, MAPK-AKT, and NF-κB pathways previously implicated in PPP1R11 function

How do the dual functions of PPP1R11 as an E3 ligase and phosphatase inhibitor integrate in cellular signaling networks?

PPP1R11's dual roles as both an E3 ubiquitin ligase and a PP1 inhibitor represent an intriguing case of functional convergence in a single protein. The integration of these functions likely enables PPP1R11 to coordinate complex cellular responses through both proteasomal degradation of targets and modulation of phosphorylation-dependent signaling.

In immune responses, these dual functions may operate in parallel pathways or in sequence. For example, in TLR2 signaling, PPP1R11 directly targets the receptor for degradation , while in T cell activation, it modulates signaling through inhibition of PP1 . Whether these mechanisms cross-talk or operate independently in different cellular contexts remains an open research question.

Global transcriptome analyses through RNA sequencing of PPP1R11-silenced T cells have revealed that PPP1R11 regulates multiple pathways beyond cytokine expression . These may include phosphatidylinositol signaling, MAPK-AKT, and NF-κB pathways, suggesting broad influence on cellular signaling networks.

To investigate this integration, researchers should employ:

• Systems biology approaches including proteomics, phosphoproteomics, and network analysis

• Temporal studies examining the sequence of ubiquitination and phosphorylation events

• Simultaneous monitoring of both E3 ligase and phosphatase inhibitor functions

• Domain-specific mutations that selectively disable one function while preserving the other

• Integration of data from multiple cell types and stimulation conditions

What is the clinical relevance of PPP1R11 in bacterial infections, particularly S. aureus?

PPP1R11 demonstrates significant clinical relevance in bacterial infections, particularly those caused by gram-positive bacteria like Staphylococcus aureus. A study of white blood cell samples from patients with S. aureus infections revealed a negative correlation between PPP1R11 and TLR2 levels, suggesting that during infection, TLR2 levels may be attenuated in part by PPP1R11 expression . This correlation was not observed in control patients without infection, indicating that this regulatory mechanism becomes active specifically during the infectious process.

In experimental mouse models, manipulation of PPP1R11 levels significantly impacts both inflammatory responses and bacterial clearance. Lentiviral gene transfer of PPP1R11 reduced lung inflammation but increased bacterial loads in multiple organs following S. aureus challenge . Conversely, PPP1R11 knockdown increased lung inflammation but enhanced bacterial clearance .

For translational researchers, approaches should include:

• Analysis of PPP1R11 and TLR2 levels in patient samples across various infection states

• Correlation of PPP1R11/TLR2 ratio with clinical outcomes and disease severity

• Investigation of genetic variations in PPP1R11 that may influence susceptibility to bacterial infections

• Exploration of targeted interventions that modulate PPP1R11 activity to enhance bacterial clearance while preventing excessive inflammation

How might targeting PPP1R11 be utilized for immunomodulatory therapeutic approaches?

PPP1R11 represents a promising target for immunomodulatory therapeutic approaches based on its dual roles in regulating TLR2-mediated inflammation and T cell responses. Its position as a regulatory node in multiple immune pathways offers several potential intervention strategies:

For Bacterial Infections: Temporary inhibition of PPP1R11 could enhance TLR2 signaling and improve bacterial clearance, particularly for gram-positive pathogens like S. aureus . This approach might be valuable in cases where bacterial persistence is problematic, though careful management of the resulting enhanced inflammation would be necessary.

For Autoimmune Conditions: Enhancing PPP1R11 activity might help suppress excessive T cell activation, as PPP1R11 has been shown to be a negative regulator of T cell activation-induced cytokine expression . This could potentially reduce pathological inflammation in autoimmune contexts.

For Cancer Immunotherapy: Inhibition of PPP1R11 could potentially render effector T cells more resistant to suppression by regulatory T cells, enhancing anti-tumor immune responses . This approach might complement existing checkpoint inhibitor therapies.

Therapeutic targeting strategies could include:

• Small molecule inhibitors or activators of PPP1R11's E3 ligase activity

• Peptide-based inhibitors of PPP1R11-PP1 interaction

• Targeted degradation approaches like PROTACs specifically designed for PPP1R11

• Localized gene therapy to modulate PPP1R11 expression in specific tissues

What are the major technical challenges in studying PPP1R11 and how can they be addressed?

Researchers face several technical challenges when studying PPP1R11:

Protein Expression and Purification: Full-length PPP1R11 with proper folding and E3 ligase activity can be difficult to express and purify in bacterial systems. Alternative approaches include:

• Using mammalian or insect cell expression systems

• Expressing functional domains separately

• Employing fusion tags that enhance solubility without compromising activity

Antibody Specificity: Ensuring antibody specificity for PPP1R11 is critical, especially given potential cross-reactivity with other phosphatase inhibitors or RING E3 ligases. Researchers should:

• Validate antibodies using PPP1R11 knockout controls

• Test multiple antibodies targeting different epitopes

• Consider using epitope-tagged versions in experimental systems

Temporal Dynamics: PPP1R11 expression and activity appears to be highly dynamic, particularly following immune stimulation . Capturing these dynamics requires:

• Carefully designed time-course experiments

• Synchronization of cellular responses

• Live-cell imaging approaches with fluorescently tagged PPP1R11

Distinguishing Dual Functions: Separating PPP1R11's E3 ligase activity from its phosphatase inhibitor function presents a significant challenge. Approaches include:

• Domain-specific mutations that selectively disable one function

• In vitro assays that specifically measure each activity

• Rescue experiments with selective activity variants

What are the most promising future research directions for understanding PPP1R11 function?

Several promising research directions could significantly advance our understanding of PPP1R11 function:

Expanded Target Identification: While TLR2 has been identified as a key ubiquitination target , PPP1R11 likely has additional substrates that remain to be discovered. Approaches include:

• Proteome-wide ubiquitination profiling following PPP1R11 manipulation

• Proximity labeling techniques to identify interaction partners

• Targeted screening of candidate substrates in relevant immune pathways

Regulatory Mechanisms: Understanding how PPP1R11 itself is regulated could reveal additional control points in immune signaling. Investigation areas include:

• Post-translational modifications of PPP1R11

• Transcriptional and epigenetic regulation

• Protein-protein interactions that modulate PPP1R11 activity

Tissue-Specific Functions: While much research has focused on lung epithelial cells and T cells , PPP1R11 may have distinct functions in other tissues and cell types. Future studies should explore:

• Cell type-specific knockout models

• Single-cell analysis approaches

• Organ-specific functions in development and disease

Translational Applications: Developing PPP1R11-targeted therapeutics represents an exciting frontier. Research should focus on:

• High-throughput screening for small molecule modulators

• Structure-based drug design targeting specific functional domains

• Delivery systems for tissue-specific targeting

• Biomarker development for patient stratification