PSMD10 Antibody

What is PSMD10 and what cellular functions does it serve?

PSMD10, also known as Gankyrin, is a non-ATPase subunit of the 26S proteasome that functions primarily as a chaperone protein. It contains 226 amino acids with a calculated molecular weight of 24 kDa, though it is typically observed at 24-28 kDa in experimental settings . PSMD10 interacts with target proteins to promote their degradation by the proteasome system .

Beyond its proteasomal functions, PSMD10 plays significant roles in autophagy regulation through interaction with autophagy-related proteins. Research demonstrates that PSMD10 homodimerization is critical for its interaction with ATG7 and ATG10, which are essential components of the autophagy machinery . Importantly, PSMD10 has been implicated in various pathological conditions, as it tends to be upregulated in cancer cells with RAS mutations, including lung cancers and adenocarcinomas .

What applications are validated for PSMD10 antibodies?

PSMD10 antibodies have been validated for multiple experimental applications with specific dilution recommendations:

Note: For IHC applications, antigen retrieval is suggested with TE buffer pH 9.0, with an alternative option of citrate buffer pH 6.0 . When using new sample types, it is recommended that researchers titrate the antibody in their specific testing system to obtain optimal results, as the optimal dilution can be sample-dependent .

How should PSMD10 antibodies be stored and handled for optimal performance?

For optimal performance of PSMD10 antibodies, storage conditions are critical. The antibody should be stored at -20°C, where it remains stable for one year after shipment . The antibody is typically supplied in PBS buffer containing 0.02% sodium azide and 50% glycerol at pH 7.3 .

For smaller quantities (20 μl sizes), the antibody may contain 0.1% BSA as a stabilizer . It's important to note that aliquoting is not necessary for -20°C storage, which simplifies laboratory handling procedures . When working with the antibody, standard safety precautions should be observed, particularly due to the presence of sodium azide in the storage buffer.

How does PSMD10 participate in autophagy regulation?

PSMD10 plays a crucial role in autophagy regulation through specific protein-protein interactions. Research has revealed that PSMD10 homodimerization is essential for its interaction with autophagy-related proteins ATG7 and ATG10, but not for its interaction with ATG12 . The interaction mechanism involves:

• The first ankyrin repeat of PSMD10 is critical for interaction with ATG7 and ATG10, as deletion of this region prevents these interactions .

• The last three ankyrin repeats of PSMD10 directly interact with ATG7, as demonstrated through photocrosslinking experiments .

• The three to five ankyrin repeats of PSMD10 are responsible for binding with ATG12 .

Studies using PSMD10 knockout cells have shown that PSMD10 is required for starvation- and LPS-induced autophagy . When the C4S mutant of PSMD10 (which fails to homodimerize) was introduced into these cells, it failed to enhance autophagy, indicating that homodimerization is essential for PSMD10's autophagy-promoting function . Importantly, experimental stabilization of PSMD10 C4S homodimers through chemical crosslinking or disulfide bonds restored its binding activity with ATG7 and enhanced autophagy in knockout cells .

What is the significance of PSMD10 homodimerization in cellular functions?

PSMD10 homodimerization serves as a critical regulatory mechanism with distinct functional implications:

• Autophagy regulation: Homodimerization of PSMD10 is essential for its interaction with ATG7 and ATG10, which are key components of the autophagy machinery . The PSMD10 C4S mutant, which fails to form homodimers, cannot interact with these proteins and thus cannot promote autophagy .

• Differential protein interactions: While homodimerization is required for PSMD10's interactions with ATG7 and ATG10, it is not necessary for interaction with ATG12 . This suggests that PSMD10 employs different binding mechanisms for different autophagy-related proteins.

• Proteasome function independence: The interaction of PSMD10 with the 26S proteasome AAA-ATPase subunit Rpt3 is not affected by its dimerization status, indicating that PSMD10 homodimerization may play important roles specifically in autophagy, but not in proteasome function .

Experimental validation has shown that stabilizing PSMD10 homodimers through chemical crosslinking or disulfide bonds can restore its autophagy-promoting function . This suggests potential therapeutic approaches targeting PSMD10 dimerization for modulating autophagy in disease states.

What experimental approaches can be used to study PSMD10 protein-protein interactions?

Several sophisticated experimental approaches have been employed to study PSMD10 protein-protein interactions, which can serve as methodological guidelines for researchers:

• Pairwise chemical crosslinking: This approach uses genetically incorporated proximity-enabled unnatural amino acids (Uaas) that react with specific amino acids like Cys and Lys when they are in close proximity . This method offers high specificity and stability for detecting intermolecular proximities in living cells, overcoming the limitations of traditional disulfide bond formation techniques .

• Immunoprecipitation and co-immunoprecipitation: These techniques have been successfully used to detect interactions between PSMD10 and proteins such as ATG7, ATG10, ATG12, and the proteasome subunit Rpt3 . For example, researchers demonstrated that PSMD10 co-immunoprecipitates with ATG10 and ATG12 in living cells .

• Pulldown assays: These assays have been employed to study how PSMD10 interacts with the 26S proteasome components .

• Photocrosslinking: This method has been used to demonstrate that the last three ankyrin repeats of PSMD10 directly interact with ATG7 .

• Mutagenesis studies: Creation of specific mutants (such as PSMD10 C4S mutant) has allowed researchers to identify critical residues and domains required for PSMD10 interactions and functions .

These approaches, when combined with cellular and biochemical assays, provide a comprehensive toolkit for investigating PSMD10's role in protein-protein interaction networks.

How is PSMD10 implicated in cancer pathogenesis?

PSMD10 has significant implications in cancer pathogenesis through multiple mechanisms:

• Upregulation in RAS-mutated cancers: PSMD10 tends to be upregulated in cancer cells with RAS mutations, including lung cancers and adenocarcinomas at the protein level . This association suggests a potential role in RAS-driven oncogenesis.

• Involvement in cholangiocarcinoma: Studies using the Clonorchis sinensis-induced hamster cholangiocarcinoma (CCA) model have investigated PSMD10 expression alongside other oncogenes (CDK4) and tumor suppressors (p53 and RB) . This suggests PSMD10 may contribute to the development or progression of bile duct cancers.

• Potential impact on tumor suppressor pathways: Given that PSMD10 functions as a chaperone that can promote protein degradation via the proteasome , its overexpression could potentially lead to enhanced degradation of tumor suppressor proteins, contributing to cancer development.

The mechanisms by which PSMD10 contributes to cancer pathogenesis warrant further investigation, particularly regarding its interactions with established oncogenes and tumor suppressors in different cancer types. Researchers should consider PSMD10 as a potential biomarker or therapeutic target in cancers with specific genetic alterations, especially those with RAS mutations.

What methods can be used to study PSMD10 in disease models?

To investigate PSMD10 in disease models, researchers can employ several methodological approaches:

• Protein expression analysis: Western blotting is an effective method to quantify PSMD10 protein levels in disease models. Protocols typically involve separation of proteins (40 μg/lane) by SDS-PAGE (8-12%), transfer to PVDF membranes, blocking in 5% non-fat milk, and probing with appropriate antibodies . Relative quantitative analysis can be performed using image analysis software such as ImageJ .

• Knockout and rescue experiments: Generation of PSMD10 knockout cells using CRISPR/Cas9 technology provides a valuable model to study PSMD10 function in disease contexts . Rescue experiments with wild-type or mutant PSMD10 (such as the C4S mutant) can help elucidate specific functional domains important in disease processes .

• Immunohistochemistry: PSMD10 antibodies can be used for IHC studies in tissue samples from disease models or patient specimens . For optimal results with PSMD10 antibodies, antigen retrieval with TE buffer (pH 9.0) or citrate buffer (pH 6.0) is recommended .

• Protein interaction studies: Co-immunoprecipitation, pulldown assays, and crosslinking techniques can be employed to investigate how PSMD10 interactions with other proteins may be altered in disease states . These approaches can help identify disease-specific interaction partners or altered binding properties.

• Animal models: Studies have successfully employed animal models, such as the hamster cholangiocarcinoma model, to investigate PSMD10 in cancer development . These models allow for in vivo analysis of PSMD10 function in disease progression.

How can researchers optimize Western blot protocols for PSMD10 detection?

Optimizing Western blot protocols for PSMD10 detection requires attention to several technical details:

• Sample preparation and loading: PSMD10 has a calculated molecular weight of 226 aa, 24 kDa, but is typically observed at 24-28 kDa on Western blots . Using adequate protein concentration determination methods like BCA protein assay is recommended, with standard loading of approximately 40 μg protein per lane .

• Gel percentage selection: Given PSMD10's molecular weight, 10-12% SDS-PAGE gels are suitable for optimal resolution in the 24-28 kDa range .

• Primary antibody dilution: The recommended dilution range for PSMD10 antibody in Western blot applications is 1:500-1:2000 . Researchers should titrate the antibody within this range for their specific experimental system to determine the optimal concentration that provides the best signal-to-noise ratio.

• Validation in multiple cell lines: PSMD10 antibodies have been validated in multiple cell lines including A549, HEK-293, HeLa, K-562, and PC-3 cells . When working with different cell types, preliminary validation experiments are advisable.

• Controls: Appropriate positive controls should be included, such as cell lines known to express PSMD10 (e.g., A549, HEK-293, HeLa cells) . For studies examining PSMD10 in disease contexts, PSMD10 knockout cells can serve as valuable negative controls .

• Detection system: Enhanced chemiluminescence reagents have been successfully used for PSMD10 detection . The choice of HRP-conjugated secondary antibody should match the host species of the primary antibody (e.g., anti-rabbit for rabbit polyclonal PSMD10 antibodies) .

What are the critical considerations for interpreting PSMD10 expression in autophagy studies?

When interpreting PSMD10 expression data in autophagy studies, researchers should consider several critical factors:

• PSMD10 dimerization status: PSMD10 homodimerization is essential for its interaction with key autophagy proteins ATG7 and ATG10 . Therefore, assessing not only total PSMD10 levels but also its dimerization status is crucial for understanding its functional impact on autophagy.

• Interaction with autophagy proteins: The interaction of PSMD10 with ATG7, ATG10, and ATG12 should be evaluated, as these interactions are differentially regulated and have distinct functional implications . Co-immunoprecipitation or crosslinking experiments can be employed to assess these interactions.

• Experimental manipulation considerations: When using genetic approaches to modulate PSMD10 (knockout, knockdown, or overexpression), researchers should verify that observed autophagy phenotypes are specific to PSMD10 and not due to off-target effects. Rescue experiments with wild-type or mutant PSMD10 (e.g., C4S mutant) can help establish specificity .

• Context-dependency: PSMD10's role in autophagy may vary depending on cell type, stress conditions, or disease context. For example, pathogens like EPEC can modulate PSMD10 function to block host autophagic responses . These contextual factors should be considered when interpreting experimental results.

• Integrated analysis: PSMD10 functions in both the proteasome system and autophagy . Therefore, integrated analysis of both pathways provides a more comprehensive understanding of PSMD10's role in cellular homeostasis. Researchers should assess how perturbations in one pathway might affect the other.

How can researchers differentiate between PSMD10's proteasomal and autophagy-related functions?

Differentiating between PSMD10's dual roles in proteasomal and autophagy pathways requires specific experimental strategies:

• Domain-specific mutant analysis: Different domains of PSMD10 mediate distinct functions. For example, the first ankyrin repeat is critical for interaction with ATG7 and ATG10, while the PSMD10 C4S mutant fails to interact with ATG7 and ATG10 but maintains interaction with the proteasome subunit Rpt3 . Using domain-specific mutants allows researchers to selectively disrupt one function while preserving the other.

• Dimerization-focused experiments: PSMD10 homodimerization is important for its autophagy-related functions but not for its proteasome-related functions . Experimental approaches that specifically target dimerization (such as the C4S mutation or chemical crosslinking) can help distinguish between these functions.

• Pathway-specific readouts: Employing specific readouts for proteasome function (e.g., proteasome activity assays, ubiquitinated protein accumulation) and autophagy (e.g., LC3 puncta formation, p62 degradation, ATG protein interactions) allows researchers to assess the differential impact of PSMD10 perturbations on each pathway.

• Temporal analysis: Monitoring the dynamics of PSMD10 interactions with proteasome components versus autophagy-related proteins under different conditions (e.g., starvation, LPS stimulation) can reveal context-dependent functional shifts.

• Subcellular localization studies: Investigating the subcellular localization of PSMD10 may provide insights into its functional roles, as interaction with NleE and PSMD10 was detected only in the cytoplasm despite NleE being present in both cytoplasm and nucleus .

By implementing these strategies, researchers can effectively distinguish between and separately analyze PSMD10's contributions to proteasomal and autophagic processes.