Recombinant Spermophilus tridecemlineatus 26S protease regulatory subunit 10B (PSMC6)

What is the structural role of PSMC6 in the 26S proteasome?

PSMC6 (also known as RPT4 in yeast) is one of the 19S regulatory particle triple-A ATPase (RPT) subunits of the proteasome. Structurally, the RPT ring anchors the 19S regulatory particle to the 20S core particle, forming the complete 26S proteasome. PSMC6 serves as an intermediary bridge that facilitates substrate processing from recognition to entry into the hydrolysis chamber .

As a proteasomal AAA+ ATPase molecular motor, PSMC6 contributes to the RPT ring that releases energy through ATP hydrolysis, generating mechanical tension used to:

• Stimulate deubiquitinase (DUB) Rpn11 activity

• Drive substrate protein unfolding and translocation

• Direct terminal conformational changes that open the α-ring gated channel

Notably, PSMC6 is the only RPT subunit without core particle insertion, suggesting it may function as a pivotal anchor for flexible bolstering between the two interfaces in the highly dynamic 26S proteasome mechanism .

What gene ontology processes is PSMC6 associated with?

PSMC6 participates in multiple biological processes according to Gene Ontology annotations:

These processes highlight PSMC6's multifunctional role in cellular homeostasis, particularly in energy-dependent protein degradation and cell cycle regulation .

What are the optimal conditions for expressing recombinant Spermophilus tridecemlineatus PSMC6?

While the search results don't provide species-specific expression conditions for S. tridecemlineatus PSMC6, researchers typically apply standard recombinant protein expression methodologies with considerations for proteasomal proteins:

• Expression Systems:

  • Bacterial systems (E. coli BL21(DE3)) for isolated PSMC6

  • Mammalian cell lines (HEK293T, COS-7) for studies requiring proper folding and post-translational modifications

  • Insect cell systems (Sf9, Hi5) for higher eukaryotic processing

• Expression Optimization:

  • Temperature: Lower temperatures (16-20°C) often increase solubility of proteasomal proteins

  • Induction: IPTG concentration 0.1-0.5mM for bacterial systems; doxycycline for mammalian inducible systems

  • Co-expression: Consider co-expressing with proteasome assembly chaperones to improve folding

• Purification Strategy:

  • Affinity tags (His, GST, FLAG) positioned to avoid interference with ATPase function

  • Size exclusion chromatography for isolation of monomeric vs. assembled forms

  • ATP/Mg²⁺ presence in buffers to maintain native conformation

The choice of expression system should align with the experimental objectives, whether studying isolated PSMC6 or its integration into functional proteasome complexes.

What molecular techniques are effective for studying PSMC6 genetic variations?

Based on methodologies applied to related proteasome genes, several techniques have proven effective for studying genetic variations in PSMC6:

• Genotyping Approaches:

  • PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism): Useful for identifying species-specific substitutions, as demonstrated in the analysis of Spermophilus species

  • SNP (Single Nucleotide Polymorphism) analysis: Effective for detecting polymorphisms, as shown in studies of PSMA6 and PSMC6 variations in multiple sclerosis

• Sequencing Methodologies:

  • Direct sequencing of PCR products using standard Sanger sequencing

  • Next-generation sequencing for high-throughput variant identification

  • Analysis of chromatograms using software such as Lasergene 11 SeqMan package for manual editing and verification

• Haplotype Analysis:

  • Phasing of diploid markers based on alleles found in homozygotes

  • Multiple alignment and trimming of dangling ends for comparative analysis

When analyzing sequence data, researchers should employ alignment algorithms such as MUSCLE in MEGA X and encode heterozygous positions where two overlapping peaks are consistently observed in chromatograms .

How is PSMC6 expression altered in neurodegenerative disorders?

PSMC6 shows significant expression changes in neurodegenerative disorders, particularly in Alzheimer's Disease (AD):

• Expression Pattern in AD:

  • PSMC6 expression decreases monotonically with disease severity

  • Downregulation occurs across all affected brain regions compared to controls

• Functional Implications:

  • Decreased PSMC6 indicates reduced efficiency of ATP-dependent 26S proteasomal degradation

  • PSMC6 appears more closely associated with AD than other proteasome activators like PSME1

  • Suggests that the 19S activator complex (containing PSMC6) is particularly relevant to AD pathology

• Coordination with α-ring Subunits:

  • The coherence between PSMC6 and α-ring subunits (PSMA family) serves as a potential marker for AD

  • Down-regulation of both PSMC6 and α-ring expression with limited deviation between them correlates with significant AD risk

  • In AD progression, active proteasomes enhance degradation efficiency through improved coordination, compensating for reduced total proteasome numbers

These findings highlight the potential of PSMC6 as both a biomarker and therapeutic target in neurodegenerative disorders characterized by protein aggregation.

What is the relationship between PSMC6 genetic variations and autoimmune diseases?

While the search results don't directly address PSMC6 genetic variations in autoimmune diseases, related proteasome genes show significant associations:

• Multiple Sclerosis Associations:

  • Polymorphisms in proteasome genes PSMA6 and PSMC6 have been implicated in various autoimmune conditions including juvenile idiopathic arthritis, asthma, and type 1 diabetes mellitus

  • The PSMA6-rs1048990 polymorphism shows potential as an independent marker for prognosis of interferon-β therapy response in multiple sclerosis

• Genotype-Phenotype Correlations:

  • Single- and multi-loci variations in PSMA6, PSMC6, and PSMA3 proteasome genes contribute to multiple sclerosis risk in certain populations

  • These genetic variations may provide insight into disease pathogenesis and offer perspectives for future pharmaceutical interventions

• Mechanisms of Impact:

  • Proteasome genetic variations may affect protein degradation efficiency

  • Altered processing of autoantigens may influence immune tolerance

  • Changes in inflammatory signaling pathways dependent on proteasome function

Researchers investigating autoimmune associations should consider both single polymorphisms and haplotype analyses to capture the complex genetic architecture underlying these conditions.

How does PSMC6 interact with other proteasome subunits to regulate gate opening?

PSMC6 plays a sophisticated role in proteasome gate regulation through several mechanisms:

• Energy Transduction:

  • As part of the RPT ring, PSMC6 contributes to ATP hydrolysis that powers conformational changes

  • These changes propagate through the complex to control the opening of the α-ring gate channel

• Structural Uniqueness:

  • Unlike other RPT subunits, PSMC6 lacks direct core particle insertion sites

  • This unique property suggests it may function as a specialized anchor that provides flexible support between the 19S and 20S interfaces in the dynamic 26S proteasome

• Coordination with α-ring:

  • PSMC6 and α-ring subunits demonstrate coordinated expression patterns that correlate with proteasome function

  • Their coherent regulation represents a potential functional marker for proteasome activity

• Pathway Integration:

  • PSMC6 is involved in multiple cellular processes including cell cycle regulation, DNA damage response, and RNA metabolism

  • This multi-pathway involvement suggests PSMC6 may integrate various cellular signals to modulate proteasome activity

Advanced research should focus on the structural dynamics between PSMC6 and other subunits using techniques such as cryo-electron microscopy, hydrogen-deuterium exchange mass spectrometry, or FRET-based approaches to capture the conformational changes during substrate processing.

What methodological considerations are important when designing CRISPR-Cas9 experiments targeting PSMC6?

When designing CRISPR-Cas9 experiments targeting PSMC6, researchers should consider several critical factors:

• Essentiality Assessment:

  • PSMC6 is essential for proteasome function and complete knockout may be lethal to cells

  • Consider conditional knockout systems (e.g., Cre-loxP, tet-inducible) or partial knockdown approaches

• Guide RNA Design:

  • Target regions that distinguish PSMC6 from other ATPase family members to prevent off-target effects

  • Avoid functional domains crucial for ATP binding and hydrolysis if the goal is to create viable mutants

  • Prioritize regions with species-specific variations if working with S. tridecemlineatus PSMC6

• Functional Domain Considerations:

  • For structure-function studies, create point mutations in key residues:

    • Walker A/B motifs for ATP binding/hydrolysis

    • Arginine fingers for inter-subunit communication

    • C-terminal regions involved in gate opening

• Verification Strategies:

  • Confirm editing through sequencing and protein expression analysis

  • Assess functional consequences with proteasome activity assays

  • Evaluate phenotypic effects on cellular processes known to involve PSMC6 (cell cycle, protein degradation)

• Alternative Approaches:

  • Consider base editing or prime editing for precise point mutations

  • Use homology-directed repair (HDR) templates for introducing tags or reporter constructs

Given PSMC6's essential nature, researchers should extensively validate guide RNAs and consider the use of rescue constructs to confirm phenotype specificity.

How conserved is PSMC6 across different Spermophilus species and what implications does this have for research models?

While the search results don't provide direct comparative data for PSMC6 across Spermophilus species, we can draw insights from related information:

• Interspecies Hybridization Impact:

  • Spermophilus species show evidence of hybridization events that have left traces on their genomes

  • This suggests that functional genes, including those involved in essential cellular processes like the proteasome, may show hybridization-related variations

• Conservation Patterns:

  • Proteasome components generally show high conservation due to their essential cellular functions

  • Sequence divergence tends to be higher in regions not directly involved in core functions such as ATP binding or subunit interactions

• Research Model Implications:

  • When using S. tridecemlineatus as a model, researchers should verify PSMC6 sequence identity compared to target species

  • Species-specific variations may affect antibody reactivity, protein-protein interactions, or regulatory mechanisms

  • For cross-species comparisons, focus on conserved functional domains rather than variable regions

• Molecular Marker Considerations:

  • Species-specific variations in PSMC6 could serve as molecular markers for evolutionary studies

  • Researchers working with hybridization zones should consider how PSMC6 variants might influence proteasome function and cellular fitness

Comparative studies across Spermophilus species would benefit from phylogenetic analysis of PSMC6 sequences to understand the evolutionary pressures on this essential proteasomal component.

What unique features of Spermophilus tridecemlineatus PSMC6 make it valuable for specific research applications?

While specific information about unique features of S. tridecemlineatus PSMC6 is not provided in the search results, several aspects make this species potentially valuable for proteasome research:

• Hibernation Adaptation:

  • As a hibernating species, S. tridecemlineatus undergoes dramatic physiological changes including regulated protein synthesis and degradation

  • PSMC6's role in the proteasome may include adaptations that facilitate cellular maintenance during torpor and arousal cycles

  • These adaptations could provide insights into regulated proteostasis under metabolic stress

• Temperature Sensitivity:

  • The proteasome must function across a range of body temperatures in hibernating species

  • S. tridecemlineatus PSMC6 may possess structural adaptations that maintain ATPase activity at lower temperatures

  • This could be valuable for understanding temperature-dependent protein degradation mechanisms

• Comparative Research Value:

  • Comparing S. tridecemlineatus PSMC6 with non-hibernating mammals could reveal adaptations in proteasome regulation

  • Such comparisons may identify novel therapeutic approaches for conditions involving proteostasis disruption

  • The functional conservation or divergence of PSMC6 across species can inform evolutionary studies of essential cellular machinery

• Model System Applications:

  • S. tridecemlineatus serves as a natural model for studying:

    • Neuroprotection mechanisms (relevant to neurodegenerative disorders)

    • Metabolic regulation (relevant to metabolic diseases)

    • Ischemia resistance (relevant to cardiovascular research)

  • PSMC6's function in these contexts may reveal novel regulatory mechanisms

Researchers leveraging S. tridecemlineatus PSMC6 should consider these potential adaptations when designing experiments and interpreting results in comparison to other model systems.