Recombinant Mouse 26S proteasome non-ATPase regulatory subunit 11 (Psmd11)

What is the molecular function of Psmd11 in the proteasome complex?

Psmd11 (26S proteasome non-ATPase regulatory subunit 11) functions as a non-ATPase subunit of the 19S regulator within the 26S proteasome complex. It participates in the ATP-dependent degradation of ubiquitinated proteins within a non-lysosomal pathway. Specifically, Psmd11 is a member of the proteasome subunit S9 family that can be phosphorylated by AMP-activated protein kinase. It serves as a critical component of the lid subcomplex of the 26S proteasome, which is involved in regulated protein degradation . In the proteasome complex, Psmd11 is particularly important for proteasome assembly, playing a key role in enhancing assembly of the 26S proteasome, which subsequently leads to higher proteasome activity .

How does Psmd11 expression vary across different cell types?

Based on single-cell analysis of the human homolog PSMD11, expression patterns vary significantly across different cellular subgroups. Research has revealed elevated expression in CD4 T cells, endothelial cells, epithelial cells, and macrophages, while showing comparatively lower expression in B cells, dendritic cells (DCs), and other myeloid cells . When designing experiments with mouse Psmd11, researchers should consider these cellular distribution patterns, as similar expression variations likely exist in murine models. This cell-specific expression suggests Psmd11 may play specialized roles in different cellular contexts and could influence experimental design when studying specific cell populations.

What is the relationship between Psmd11 and stem cell function?

Psmd11 has demonstrated a significant role in stem cell biology. High expression of Psmd11 in embryonic stem cells (ESCs) promotes enhanced assembly of the 26S proteasome, leading to higher proteasome activity that appears essential for maintaining pluripotency . Human embryonic stem cells exhibit high proteasome activity that correlates with increased levels of PSMD11, corresponding to increased assembly of the 26S/30S proteasome . Researchers working with mouse stem cell models should consider Psmd11's potential influence on stemness properties and proteasome-mediated protein turnover when designing experiments involving stem cell differentiation or maintenance.

What are effective methods for studying Psmd11's role in proteasome assembly?

To study Psmd11's role in proteasome assembly, researchers should implement a multi-faceted approach:

• Co-immunoprecipitation assays: Use anti-Psmd11 antibodies to pull down proteasome complexes, followed by immunoblotting for other proteasome subunits to assess assembly state.

• Native gel electrophoresis: Apply non-denaturing conditions to analyze intact proteasome complexes in wild-type versus Psmd11-altered samples.

• Sucrose gradient ultracentrifugation: Separate proteasome subcomplexes based on size and analyze fractions for Psmd11 and other components.

• CRISPR-Cas9 gene editing: Create conditional knockout or knockdown models to assess proteasome assembly in the absence or reduction of Psmd11.

• Fluorescence recovery after photobleaching (FRAP): Tag Psmd11 and other proteasome components with fluorescent proteins to study assembly dynamics in live cells.

When interpreting results, consider that Psmd11 is specifically required for proteasome assembly and plays a critical role in increased proteasome activity in certain cell states such as embryonic stem cells .

How can researchers effectively modulate Psmd11 expression in experimental models?

For precise modulation of Psmd11 expression in research models, consider these approaches:

For Overexpression:

• Lentiviral or adenoviral vectors carrying the Psmd11 gene under a strong promoter (CMV or EF1α)

• Transient transfection with expression plasmids in cell lines

• Transgenic mouse models with conditional Psmd11 overexpression using Cre-loxP systems

For Knockdown/Knockout:

• siRNA or shRNA targeting specific regions of Psmd11 mRNA

• CRISPR-Cas9-mediated knockout using guide RNAs targeting early exons

• Inducible knockdown systems (e.g., Tet-On/Off) for temporal control

Based on lung cancer research, overexpression of PSMD11 enhanced proliferation, migration, invasion, and tumor growth in the A549 cell line, while PSMD11 knockdown diminished these activities in PC9 cells . When designing similar experiments with mouse Psmd11, similar phenotypic assays should be considered with appropriate controls to accurately measure these parameters in response to expression changes.

What techniques are recommended for investigating Psmd11's interaction with the cuproptosis pathway?

To study Psmd11's involvement in cuproptosis, a copper ion-induced form of regulated cell death, implement these specialized techniques:

• Copper chelation/supplementation assays: Treat cells with copper chelators (e.g., tetrathiomolybdate) or CuCl₂ while monitoring Psmd11 expression and proteasome activity.

• Co-expression analysis: Measure correlation between Psmd11 and known cuproptosis genes (DLAT, DLD, PDHA1) using qRT-PCR or Western blotting under varying copper conditions .

• Mitochondrial function assessment: Since cuproptosis is linked to mitochondrial metabolism, measure oxygen consumption rate, ATP production, and mitochondrial membrane potential in cells with modified Psmd11 expression.

• Lipidomics profiling: Analyze changes in lipoylated proteins, which are targets in cuproptosis, in response to Psmd11 manipulation.

• Cell death assays: Quantify cell death using Annexin V/PI staining and flow cytometry following copper treatment in cells with normal versus altered Psmd11 expression.

Research has revealed that expression of cuproptosis genes (DLAT, DLD, and PDHA1) positively correlates with PSMD11 expression (P<0.001) , suggesting functional relationships worth investigating in mouse models.

How should researchers design experiments to study Psmd11's role in tumor progression?

When investigating Psmd11's role in tumor progression, implement a comprehensive experimental design:

In vitro approaches:

• Compare Psmd11 expression between normal and cancer cell lines using Western blotting and qRT-PCR

• Perform proliferation assays (MTS/MTT, EdU incorporation) after Psmd11 modulation

• Conduct migration and invasion assays (wound healing, transwell) with Psmd11 overexpression or knockdown

• Assess colony formation capacity in soft agar following Psmd11 alterations

In vivo approaches:

• Generate xenograft models using cells with stable Psmd11 overexpression or knockdown

• Implement orthotopic mouse models to study cancer progression in native tissue environments

• Use inducible expression systems to study temporal effects of Psmd11 modulation

• Analyze tumor samples for correlations between Psmd11 expression and clinical parameters

Human PSMD11 studies have demonstrated that overexpression enhanced proliferation, migration, invasion, and tumor growth in lung carcinoma cell line A549, while knockdown reduced these properties in PC9 cells . Similar functional readouts should be applied when studying mouse Psmd11 in cancer models, with careful attention to cell-type specific effects.

What are the connections between Psmd11 and immune regulation in cancer models?

Psmd11 has emerging roles in immune regulation that are particularly relevant for cancer immunology research:

• Immune cell infiltration analysis: Perform flow cytometry or immunohistochemistry to quantify immune cell populations (particularly MDSCs and T cell subtypes) in tumors with varying Psmd11 expression levels.

• Immune checkpoint correlation: Analyze expression of immune checkpoint molecules (PD-1, PD-L1, CTLA-4) in relation to Psmd11 expression using multiplex immunofluorescence or mass cytometry.

• T cell co-culture assays: Set up cancer cell-T cell co-cultures to evaluate how Psmd11 expression affects T cell activation, proliferation, and cytokine production.

• Cytokine profiling: Measure pro- and anti-inflammatory cytokines in the tumor microenvironment in response to Psmd11 modulation.

PSMD11 expression has been positively correlated with myeloid-derived suppressor cells (MDSCs) infiltration and increased expression of immunosuppressive molecules . Additionally, PSMD11 shows strong positive correlations with T helper 2 cells, gamma-delta T cells, and T regulatory cells, while exhibiting negative correlations with B cells, mast cells, and CD8+ T cells , suggesting complex immune regulatory functions that should be explored in mouse models.

What validation steps are critical when working with recombinant mouse Psmd11?

When using recombinant mouse Psmd11 in research, implement these rigorous validation steps:

• Protein integrity verification: Perform SDS-PAGE and Western blotting with Psmd11-specific antibodies to confirm full-length protein and absence of degradation products.

• Functional activity assessment: Measure proteasome assembly and activity using fluorogenic substrates (e.g., Suc-LLVY-AMC) in systems with recombinant Psmd11 compared to controls.

• Post-translational modification analysis: Check for phosphorylation status using phospho-specific antibodies or mass spectrometry, as Psmd11 is known to be regulated by phosphorylation.

• Interaction verification: Confirm binding to known proteasome partners using pull-down assays or surface plasmon resonance.

• Cellular localization: Use immunofluorescence to verify proper subcellular localization of recombinant Psmd11 when introduced into cells.

• Endotoxin testing: For in vivo applications, ensure preparation is endotoxin-free using LAL or recombinant Factor C assays.

These validation steps ensure that experimental outcomes truly reflect Psmd11 biology rather than artifacts from improperly processed recombinant protein.

How can researchers address variability in Psmd11 expression across different experimental systems?

To manage variability in Psmd11 expression across experimental systems, implement these standardization approaches:

• Reference gene selection: Carefully validate reference genes for normalization in qRT-PCR; avoid using single reference genes.

• Cell passage control: Maintain consistent cell passage numbers when comparing Psmd11 expression across experiments.

• Multiple detection methods: Use complementary techniques (qRT-PCR, Western blot, immunofluorescence) to confirm expression changes.

• Internal controls: Include positive and negative controls in each experiment to calibrate expression measurements.

• Standardized reporting: Document culture conditions, cell densities, and harvest protocols that may influence Psmd11 expression.

• Synchronization protocols: When relevant, synchronize cells to account for cell cycle-dependent variations in Psmd11 expression.

How do findings from mouse Psmd11 studies translate to human PSMD11 research?

When translating findings between mouse Psmd11 and human PSMD11 studies, consider these comparison strategies:

• Sequence homology analysis: Perform detailed sequence alignment between mouse and human proteins to identify conserved functional domains and potential species-specific differences.

• Functional conservation testing: Compare biochemical activities of mouse and human proteins in parallel assays to establish degree of functional conservation.

• Expression pattern mapping: Create comparative expression maps across tissues and developmental stages between species.

• Cross-species validation: Validate key findings from mouse models in human cell lines or patient-derived samples when possible.

• Animal model selection: Consider humanized mouse models for certain applications where human-specific interactions are critical.

Human PSMD11 has demonstrated significant roles in cancer progression and prognosis, especially in lung adenocarcinoma where it correlates with clinical characteristics including age, disease stage, and survival . When designing mouse studies aimed at translational applications, these clinical correlations should inform experimental endpoints and analyses.

What are the methodological approaches for studying Psmd11 as a potential therapeutic target?

To investigate Psmd11 as a therapeutic target, implement these methodological approaches:

• Small molecule screening: Develop high-throughput assays to identify compounds that modulate Psmd11 function or expression.

• Structure-based drug design: Use crystallographic data or homology models to design compounds targeting specific Psmd11 functional domains.

• PROTAC development: Design proteolysis-targeting chimeras to selectively degrade Psmd11 in disease models.

• Combination therapy assessment: Test Psmd11 inhibitors in combination with established therapies (chemotherapy, immunotherapy) to identify synergistic effects.

• Biomarker identification: Develop companion biomarkers to predict sensitivity to Psmd11-targeting interventions.

• Resistance mechanism investigation: Study potential mechanisms of resistance to Psmd11 inhibition through long-term exposure experiments.

Research indicates that PSMD11 has the potential to be a novel therapeutic target and sensitive biomarker for patients with lung adenocarcinoma . Similar therapeutic potential should be explored in mouse models of cancer and other diseases where proteasome function is implicated.