PSME3 Human

What is PSME3 and what are its alternative names in the scientific literature?

PSME3 (Proteasome Activator Complex Subunit 3) is a nuclear, homoheptameric protein that functions as a proteasome activator. In the scientific literature, it is also known by several alternative names including PA28γ, 11S REGγ, REG GAMMA, PA28g, and Ki nuclear autoantigen . The protein has a calculated and observed molecular weight of approximately 30 kDa and is encoded by the PSME3 gene (Gene ID: 10197) in humans .

What are the primary cellular functions of PSME3?

Key functions include:

• Regulation of nuclear organization, including nuclear speckles, Cajal bodies, and promyelocytic leukemia bodies

• Interaction with cell cycle regulators (p16, p19, p21) and transcriptional regulators (c-Myc, KLF2, SMURF2, NF-κB, LATS1/2)

• Promotion of MDM2-TP53 interaction, leading to ubiquitination-dependent proteasomal degradation of TP53

• Regulation of cell migration through interaction with the HSP90 co-chaperone NUDC

• Facilitation of myoblast differentiation in a proteasome-independent manner

What genomic techniques are effective for studying PSME3 chromatin interactions?

Cleavage Under Targets & Release Using Nuclease (CUT&RUN) has proven effective for investigating PSME3's association with chromatin. This technique revealed that PSME3 extensively associates with chromatin at over 5,000 distinct regions in cycling myoblasts, with a large majority of these peaks co-positive for the active promoter mark H3K4me3 . When studying dynamic chromatin binding across cell state transitions, researchers should consider:

• Performing CUT&RUN against endogenous PSME3 populations

• Comparing binding patterns at different stages (e.g., cycling cells vs. differentiated cells)

• Using mild formaldehyde fixation to reduce lability of transient chromatin interactions

• Analyzing binding preferences for different genomic regions (promoters, gene bodies, intergenic regions)

Research data demonstrated PSME3 shows a stronger preference for promoter-proximal regions (≤1kb) than H3K4me3, primarily at the expense of binding to gene bodies or intergenic regions .

What immunological methods are validated for detecting PSME3 in different experimental contexts?

PSME3 can be effectively detected using multiple immunological techniques with properly validated antibodies. Commercial antibodies such as 14907-1-AP have been validated for Western blotting (WB), immunohistochemistry (IHC), immunofluorescence/immunocytochemistry (IF/ICC), immunoprecipitation (IP), and ELISA applications .

Table 1: Validated Applications for PSME3 Detection

When designing experiments, researchers should follow specific protocols for each application to ensure optimal results, considering factors such as antibody dilution, incubation conditions, and appropriate positive and negative controls .

How does PSME3 regulate myoblast differentiation and what methodologies best elucidate this function?

PSME3 plays a critical role in myoblast differentiation through a proteasome-independent mechanism. Research methodologies to investigate this function include:

• Genomic binding analysis: CUT&RUN assays revealed PSME3 binds to highly active promoters in cycling myoblasts but becomes undetectable by the second day of myogenic differentiation, indicating dynamic chromatin association during differentiation .

• Protein interaction analysis: Techniques such as co-immunoprecipitation followed by mass spectrometry demonstrated that PSME3 interacts with the HSP90 co-chaperone NUDC, through which it may regulate cell migration rates, levels of cell adhesion-related proteins, and ultimately myogenesis .

• Functional assays: PSME3 depletion studies showed reduced efficiency of differentiation, likely related to alterations in cell migration. This can be assessed through:

  • Cell migration assays

  • Monitoring of cell adhesion-related protein levels

  • Visualization of myotube formation

• Proteasome dependence evaluation: Using proteasome inhibitors or PSME3 mutants incapable of proteasome interaction revealed that PSME3's role in differentiation occurs in a proteasome-independent manner .

The experimental evidence indicates that PSME3 likely influences myogenesis through regulation of cytoskeletal reorganization and cell mobility rather than through transcriptional regulation, as RNA sequencing showed little change in gene expression upon PSME3 depletion .

What is the relationship between PSME3 and cell migration, and how can this be experimentally validated?

PSME3 regulates cell migration through interaction with the HSP90 co-chaperone NUDC and by maintaining levels of cell migration-related proteins. This relationship can be experimentally validated through several approaches:

• Protein-protein interaction studies: Co-immunoprecipitation experiments followed by western blotting or mass spectrometry can confirm direct interaction between PSME3 and NUDC .

• PSME3 depletion studies: Knockdown or knockout of PSME3 using siRNA, shRNA, or CRISPR-Cas9 techniques followed by:

  • Wound healing assays to measure migration rates

  • Time-lapse microscopy to track cell movement

  • Transwell migration assays to quantify migratory capacity

• Protein level analysis: Western blotting to monitor levels of migration-related proteins in PSME3-deficient versus control cells .

Research has shown that PSME3 depletion in C2C12 cells results in increased rates of cell migration. This is significant because proper balance of migration regulators is essential for myogenesis, suggesting that the migration alterations are functionally related to the reduced efficiency of differentiation observed in PSME3-depleted cells .

How is PSME3 implicated in cancer progression and what methodologies best capture its oncogenic mechanisms?

PSME3 is frequently overexpressed in various cancer cell lines and associated with accelerated cell division, metastatic potential, and reduced rates of apoptosis . Research methodologies to investigate its oncogenic mechanisms include:

• Expression analysis: Quantitative RT-PCR, western blotting, and immunohistochemistry to compare PSME3 levels between normal and cancerous tissues or cell lines .

• Functional studies:

  • Knockdown/overexpression studies to assess effects on proliferation, migration, and invasion

  • Cell cycle analysis using flow cytometry

  • Apoptosis assays to evaluate resistance to cell death

• Signaling pathway analysis: Western blotting and reporter assays to investigate how PSME3 affects cancer-related signaling pathways, including:

  • c-Myc-glycolysis signaling axis in pancreatic cancer

  • Wnt/β-catenin signaling in osteosarcoma

  • p53 pathways through modulation of MDM2-TP53 interaction

• In vivo studies: Xenograft models using PSME3-modulated cancer cells to assess tumor growth, invasiveness, and metastatic potential.

Published studies have demonstrated that REG γ (PSME3) knockdown suppresses proliferation by inducing apoptosis and cell cycle arrest in osteosarcoma, and that its oncogenic role is exerted by activating the Wnt/β-catenin signaling pathway . Additionally, PSME3 promotes pancreatic cancer growth via the c-Myc-glycolysis signaling axis .

What is the role of PSME3 in bone-related disorders, and what experimental approaches can elucidate its function in bone health?

PSME3 plays a crucial role in bone health and healing. Research has shown that suppression of PSME3 expression biases bone marrow stromal cells towards an adipogenic rather than osteogenic fate, and mice lacking PSME3 display corresponding bone-healing defects . Experimental approaches to investigate this function include:

• Differentiation assays: Compare osteogenic versus adipogenic differentiation potential of bone marrow stromal cells with normal or reduced PSME3 levels through:

  • Alkaline phosphatase staining

  • Alizarin red staining for mineralization

  • Oil Red O staining for lipid accumulation

  • Analysis of lineage-specific markers by qRT-PCR

• In vivo models: Generate PSME3 knockout or conditional knockout mice to study:

  • Bone development and homeostasis

  • Fracture healing processes

  • Microarchitecture analysis using micro-CT

  • Histomorphometric analysis

• Signaling pathway analysis: Investigate how PSME3 regulates osteogenic versus adipogenic fate decisions through:

  • Western blotting for key signaling molecules

  • Chromatin immunoprecipitation to identify regulatory targets

  • RNA-seq to identify global changes in gene expression profiles

The experimental data suggests that therapeutic strategies targeting PSME3 might be relevant for bone-related disorders, particularly in contexts where the balance between osteogenesis and adipogenesis is disrupted .

What are the proteasome-dependent versus proteasome-independent functions of PSME3, and how can researchers experimentally distinguish between them?

PSME3 functions through both proteasome-dependent and proteasome-independent mechanisms, with only a small portion of the PSME3 population associated with the core proteasome . To experimentally distinguish between these functions, researchers can employ several sophisticated approaches:

• Proteasome interaction-deficient mutants: Generate PSME3 mutants that cannot interact with the proteasome but retain other functions. Studies have shown that PSME3 can induce mitotic arrest, regulate p53 levels, and maintain global chromatin compaction even when prevented from interacting with the proteasome .

• Proteasome inhibition studies: Use specific proteasome inhibitors (e.g., MG132, bortezomib) to block proteasome function while examining PSME3-dependent processes. Indirubin-3'-monoxime has been identified as a proteasome inhibitor with therapeutic application in multiple myeloma .

• Protein degradation kinetics: Pulse-chase experiments to track protein turnover rates of PSME3 targets in the presence or absence of proteasome inhibitors.

• Structure-function analysis: Create domain-specific mutants to identify regions of PSME3 responsible for proteasome-dependent versus independent functions.

• Comparative proteomics: Use stable isotope labeling by amino acids in cell culture (SILAC) followed by mass spectrometry to identify proteins whose levels are affected by PSME3 in a proteasome-dependent or independent manner.

Research has demonstrated that PSME3 facilitates myoblast differentiation in a proteasome-independent manner, highlighting the importance of distinguishing between these dual functionalities when studying PSME3-related processes .

How does PSME3 interact with chromatin and what is its role in transcriptional regulation across different cell types?

Despite PSME3's association with chromatin, its precise role in transcriptional regulation appears complex and potentially context-dependent. Advanced methodologies to investigate this interaction include:

• Genome-wide binding studies: CUT&RUN and ChIP-seq analyses have revealed that PSME3 associates extensively with chromatin at over 5,000 distinct regions in cycling myoblasts, with peaks co-positive for the active promoter mark H3K4me3 . PSME3 shows a stronger preference for promoter-proximal regions than H3K4me3, primarily binding within 1kb of transcription start sites .

• Protein-protein interaction network analysis: PSME3 interacts with RPRD1A, a regulator of RNA polymerase II, suggesting a potential mechanism for its chromatin association . Techniques to explore this include:

  • Proximity labeling (BioID, APEX) to identify chromatin-associated interaction partners

  • Sequential ChIP to identify co-occupancy with other factors

  • High-resolution imaging to visualize nuclear localization patterns

• Transcriptomic analysis across differentiation: RNA-seq of PSME3-deficient cells at multiple stages of differentiation (cycling, confluent pre-differentiation, and differentiated cells) revealed no global changes in gene expression . This suggests that:

  • PSME3 may be primed to perform functions not engaged during normal differentiation

  • PSME3 might respond to specific cellular stresses rather than regulate basal transcription

  • PSME3's role may be post-transcriptional rather than directly affecting mRNA production

• Stress response studies: Investigate PSME3's role under conditions such as:

  • DNA damage (as PSME3 loss sensitizes cells to radiomimetic treatment)

  • Hyperosmotic stress

  • Other cellular insults that might activate PSME3-dependent pathways

The apparent paradox that PSME3 occupies highly active promoter regions despite having no apparent role in their regulation under normal conditions suggests sophisticated regulatory mechanisms that might be activated only under specific cellular contexts .

What is the significance of PSME3's interaction with the chaperone system, and how can this be methodologically investigated?

PSME3 interacts with components of the cellular chaperone system, notably the HSP90 co-chaperone NUDC, which mediates cargo transfer between HSP70 and HSP90 . The methodological investigation of this interaction and its significance requires sophisticated approaches:

• Protein-protein interaction mapping:

  • Co-immunoprecipitation followed by mass spectrometry

  • Proximity labeling techniques (BioID, APEX)

  • Fluorescence resonance energy transfer (FRET) to detect direct interactions

  • Yeast two-hybrid screening to identify specific interaction domains

• Functional analysis of the PSME3-NUDC axis:

  • NUDC knockdown/knockout studies to assess effects on PSME3-dependent functions

  • Double depletion experiments to identify epistatic relationships

  • Rescue experiments with NUDC mutants to identify critical domains

• Chaperone-mediated protein quality control assessment:

  • Analysis of protein folding and stability in PSME3-deficient cells

  • Investigation of BAG2, which is known to associate with both HSP70 and PSME3 under stress conditions

  • Examination of protein aggregation under normal and stress conditions

• Temperature-dependent studies: Research has shown that cold temperature extends longevity and prevents disease-related protein aggregation through PA28γ (PSME3)-induced proteasomes . Methodologies include:

  • Exposure of cells/organisms to different temperature conditions

  • Measurement of proteasome activity and protein aggregation

  • Analysis of lifespan and disease markers in model organisms

The interaction between PSME3 and the chaperone system represents an important intersection between protein quality control pathways and may explain PSME3's diverse cellular functions, particularly under stress conditions .

How can single-cell technologies advance our understanding of PSME3 function in heterogeneous cell populations?

Single-cell technologies offer unprecedented opportunities to understand PSME3 function in heterogeneous cell populations during development, differentiation, and disease progression. Methodological approaches include:

• Single-cell RNA-seq (scRNA-seq):

  • Profile PSME3 expression across different cell types and states

  • Identify cell populations where PSME3 is differentially expressed

  • Correlate PSME3 expression with cell-type specific gene signatures

• Single-cell ATAC-seq (scATAC-seq):

  • Map chromatin accessibility in PSME3-high versus PSME3-low cells

  • Identify regulatory elements potentially influenced by PSME3

  • Integrate with scRNA-seq data to correlate accessibility with expression

• Single-cell CUT&Tag or CUT&RUN:

  • Map PSME3 binding at single-cell resolution

  • Identify cell-type specific binding patterns

  • Correlate binding with cellular states during differentiation

• Spatial transcriptomics:

  • Map PSME3 expression in tissue contexts

  • Correlate expression with cellular microenvironments

  • Identify spatial relationships between PSME3-expressing cells

Given PSME3's role in differentiation of myoblasts and T cells, single-cell approaches could reveal how PSME3 functions in subpopulations during lineage commitment and terminal differentiation processes .

What is the potential of PSME3 as a therapeutic target, and what preclinical models are most appropriate for validation?

PSME3's involvement in multiple cellular processes, including cancer progression, differentiation, and stress response, makes it a potential therapeutic target. Methodological approaches for preclinical validation include:

• Cancer models:

  • Patient-derived xenografts from cancers with PSME3 overexpression

  • Genetically engineered mouse models with tissue-specific PSME3 alterations

  • Cell line panels representing different cancer types to assess specificity

• Drug discovery approaches:

  • High-throughput screening for PSME3 inhibitors

  • Structure-based drug design targeting PSME3's proteasome-binding site

  • Peptide inhibitors disrupting specific protein-protein interactions

• Combination therapy assessment:

  • Testing PSME3 inhibitors with proteasome inhibitors

  • Evaluating synergy with DNA-damaging agents, given PSME3's role in DNA damage response

  • Combining with HSP90 inhibitors to disrupt the PSME3-NUDC-HSP90 axis

• Disease-specific models:

  • Bone fracture models to evaluate PSME3 modulation for bone healing

  • T cell differentiation models to assess immunomodulatory potential

  • Neurodegenerative disease models to test effects on protein aggregation

Recent research showing that PSME3 (PA28γ)-induced proteasomes can prevent disease-related protein aggregation suggests potential applications beyond cancer, particularly in neurodegenerative diseases characterized by protein aggregation .