Recombinant Schizosaccharomyces pombe SPRY domain-containing protein C285.10c (SPCC285.10c)

What is SPCC285.10c and what domains does it contain?

SPCC285.10c is a protein encoded in the genome of the fission yeast Schizosaccharomyces pombe. The protein contains a SPRY (SPla and the Ryanodine Receptor) domain, which is a conserved structural module implicated in protein-protein interactions. The full-length protein consists of 382 amino acids, though recombinant variants often include only partial sequences that retain the functional SPRY domain .

The SPRY domain belongs to a family of evolutionarily ancient protein interaction modules found across diverse organisms from yeast to humans. These domains facilitate interactions with various protein partners involved in processes including immune regulation, RNA processing, and ubiquitination pathways .

How was SPCC285.10c initially identified and characterized?

SPCC285.10c was identified through large-scale proteomic studies of S. pombe that collectively detected numerous regulatory proteins, including 45 kinases, 20 transcriptional regulators, and 21 mitochondrial proteins. The protein was discovered alongside other SPRY domain-containing proteins, which are overrepresented in proteomic datasets due to their typically higher mRNA levels and protein abundance relative to other proteins.

Initial characterization involved sequence analysis and domain identification, revealing homology to other SPRY domain-containing proteins across species. The protein is cataloged in major biological databases with the following identifiers:

• KEGG: spo:SPCC285.10c

• STRING: 4896.SPCC285.10c.1

What is the evolutionary relationship between SPRY and B30.2 domains?

The evolutionary relationship between SPRY and B30.2 domains is complex and historically has caused some confusion in nomenclature. SPRY domains are evolutionarily ancient and can be identified in organisms across animals, plants, and fungi, including S. pombe . In contrast, B30.2 domains represent a more recent evolutionary adaptation that emerged in vertebrates.

The B30.2 domain is actually a composite structure formed by the combination of a SPRY domain with an additional domain called PRY . This fusion appears to have occurred after the emergence of vertebrates, as B30.2 domains have only been identified in species with adaptive immune systems, including humans, mice, chickens, and Xenopus .

The sequence homology between SPRY and B30.2 domains includes nine conserved residues comprising two minimal motifs separated by up to 80 amino acids . This limited but significant conservation confirms their distant evolutionary relationship.

What are the recommended expression systems for recombinant SPCC285.10c production?

For structural studies requiring high purity and proper folding, consider:

• Bacterial expression (E. coli BL21) for simple purification protocols, though potential issues with solubility may arise with the full-length protein

• Yeast expression systems (P. pastoris or S. cerevisiae) for improved protein folding when the complete SPRY domain functionality is required

• Insect cell expression for complex studies requiring post-translational modifications

Optimization parameters to consider include:

• Induction temperature (typically lowered to 16-18°C for improved folding)

• IPTG concentration (0.1-0.5 mM for bacterial systems)

• Fusion tags (His-tag commonly used, but MBP or GST may improve solubility)

What experimental approaches are most effective for studying SPCC285.10c protein interactions?

Given that SPRY domains function primarily as protein-protein interaction modules, several complementary approaches are recommended for investigating SPCC285.10c interactions:

In vitro approaches:

• Pull-down assays using recombinant His-tagged SPCC285.10c as bait protein

• Surface plasmon resonance (SPR) for quantitative binding kinetics

• Isothermal titration calorimetry (ITC) for thermodynamic characterization

• ELISA-based interaction studies, where recombinant SPCC285.10c is already employed in commercial kits

In vivo approaches:

• Yeast two-hybrid screening to identify novel interaction partners

• Co-immunoprecipitation from S. pombe lysates followed by mass spectrometry

• Proximity-based labeling methods (BioID or APEX) to capture transient interactions

• Fluorescence microscopy with tagged proteins to confirm co-localization

When designing interaction studies, it's critical to consider that SPCC285.10c was identified alongside numerous regulatory proteins in S. pombe, including kinases and transcriptional regulators, suggesting potential functional associations with these protein classes.

How can SPCC285.10c be incorporated into functional genomics studies in S. pombe?

SPCC285.10c can be incorporated into functional genomics studies through several approaches:

Gene deletion/modification strategies:

• CRISPR-Cas9 targeted deletion to generate knockout strains

• Epitope tagging at the endogenous locus for immunoprecipitation studies

• Promoter replacement for conditional expression

Integrative approaches:

• Integration into protein interaction network prediction models specific to S. pombe

• Correlation with datasets from genome-wide screens, such as the one that identified 61 genes promoting retrotransposon integration

• Phenotypic profiling across stress conditions, as SPRY-containing proteins in S. pombe have been associated with contractile ring assembly and cytokinesis

A comprehensive approach would combine these methods with proteomic data to place SPCC285.10c in its functional context within fission yeast cellular processes.

What is the potential role of SPCC285.10c in retrotransposon biology?

Recent research suggests potential involvement of SPCC285.10c in retrotransposon biology, though direct evidence remains limited. Studies on host factors that promote retrotransposon integration in S. pombe have identified numerous genes involved in this process . While SPCC285.10c was not specifically highlighted in these screens, other SPRY domain-containing proteins have been implicated in retroviral defense mechanisms.

In particular, TRIM5α, which contains both SPRY and B30.2 domains, is known to restrict retroviral replication in mammals . The evolutionary pattern of B30.2 domains, which incorporate SPRY, suggests selection pressure related to immune defense against retroviruses .

Experimental approaches to investigate SPCC285.10c's potential role could include:

• Gene deletion followed by transposition assays using the Tf1 retrotransposon in S. pombe

• Analysis of physical interactions between SPCC285.10c and retrotransposon components

• Comparative analysis with other SPRY domain proteins with established roles in retroviral defense

How does the structure of SPCC285.10c contribute to its functional specificity?

The three-dimensional structure of SPCC285.10c has not been fully resolved, presenting an opportunity for structural biology investigations. Based on knowledge of other SPRY domains, several structural features likely contribute to functional specificity:

Key structural elements:

• SPRY domains typically adopt a β-sandwich fold with two β-sheets

• The variable loops connecting these core elements often determine binding specificity

• Conserved motifs within the SPRY domain (including the two minimal motifs mentioned previously) form the structural scaffold

For experimental determination of SPCC285.10c structure, researchers should consider:

• X-ray crystallography of the isolated SPRY domain, which may crystallize more readily than the full-length protein

• Cryo-EM for analyzing larger complexes formed with interaction partners

• Computational modeling based on homologous SPRY domain structures

Understanding structural determinants of specificity would provide insight into how this evolutionarily ancient domain maintains distinct functions across different proteins and species.

What are the methodological challenges in analyzing post-translational modifications of SPCC285.10c?

Analysis of post-translational modifications (PTMs) in SPCC285.10c presents several technical challenges:

Experimental challenges:

• The typically low abundance of the native protein in S. pombe

• Potential loss of modifications during recombinant expression

• The transient nature of some modifications (e.g., phosphorylation in signaling cascades)

Recommended methodological approaches:

• Enrichment strategies:

  • Tandem affinity purification of tagged endogenous protein

  • Phospho-peptide enrichment using TiO₂ or IMAC

  • Ubiquitin remnant profiling for detecting ubiquitination sites

• Mass spectrometry approaches:

  • Data-dependent acquisition for discovery proteomics

  • Parallel reaction monitoring for targeted analysis of specific modifications

  • Top-down proteomics for intact protein analysis with modifications

• Confirmation methods:

  • Site-specific antibodies against predicted modification sites

  • Mutation of putative modification sites followed by functional assays

  • In vitro modification assays with purified modifying enzymes

The presence of SPCC285.10c in proteomic datasets alongside numerous kinases suggests potential regulation by phosphorylation, making this a promising area for investigation.

How can SPCC285.10c research inform our understanding of SPRY domain proteins in higher eukaryotes?

Research on SPCC285.10c provides valuable insights into the fundamental functions of SPRY domain proteins that can be extrapolated to homologs in higher eukaryotes:

Comparative aspects:

• SPRY domains are found in 11 distinct protein families in humans, including ryanodine receptors (RyRs), DDX1, hnRNPs, HERC1, and RanBPM

• The functional diversity of these proteins ranges from RNA metabolism to calcium signaling and developmental regulation

• The evolutionary conservation of core SPRY domain structure facilitates comparative studies

S. pombe offers several advantages as a model system:

• Genetic tractability allowing precise manipulation

• Cellular complexity closer to higher eukaryotes than S. cerevisiae

• Ability to study SPRY domain functions in isolation from B30.2 domains

Translational research approaches could include:

• Complementation studies where human SPRY domain proteins are expressed in S. pombe SPCC285.10c deletion strains

• Identification of conserved interaction networks across species

• Development of high-throughput screens in S. pombe to identify modulators of SPRY domain function with potential relevance to human disease

What can we learn from comparing SPCC285.10c to other SPRY domain-containing proteins in S. pombe?

Comparative analysis of SPCC285.10c with other SPRY domain-containing proteins in S. pombe can reveal functional specialization and redundancy:

Comparative genomic approaches:

• Phylogenetic analysis of all SPRY domain proteins in the S. pombe genome

• Analysis of gene expression patterns across different conditions and developmental stages

• Systematic deletion of multiple SPRY domain genes to identify synthetic phenotypes

Functional comparison:

• Localization studies to determine subcellular distribution patterns

• Interactome mapping to identify shared versus specific interaction partners

• Phenotypic profiling under various stress conditions to identify condition-specific roles

This comparative approach would help distinguish between SPRY domain functions that are protein-specific versus those that represent core functionalities of the domain itself.

What emerging technologies could advance our understanding of SPCC285.10c function?

Several cutting-edge technologies hold promise for elucidating SPCC285.10c function:

CRISPR screening approaches:

• Base editing for introducing point mutations in conserved SPRY domain residues

• CRISPRi/CRISPRa for modulating expression levels without complete deletion

• CRISPR-based tagging at endogenous loci for live-cell imaging

Single-cell technologies:

• Single-cell RNA-seq to detect cell-to-cell variation in SPCC285.10c expression

• Single-molecule imaging to track SPCC285.10c dynamics in living cells

• Mass cytometry for correlating SPCC285.10c levels with cellular phenotypes

Structural biology advances:

• AlphaFold2 and related AI approaches for structure prediction

• Integrative structural biology combining multiple experimental datasets

• Hydrogen-deuterium exchange mass spectrometry to map protein-protein interaction surfaces

Spatial transcriptomics and proteomics:

• Methods to correlate SPCC285.10c localization with local translation or protein complexes

• Proximity labeling approaches to map the spatial organization of SPCC285.10c and its partners

What are the critical unanswered questions regarding SPCC285.10c that warrant investigation?

Despite the information available, several fundamental questions about SPCC285.10c remain unanswered:

• Physiological function: What is the primary biological role of SPCC285.10c in S. pombe, and how does it contribute to cellular fitness?

• Regulation: How is SPCC285.10c expression and activity regulated in response to cellular stimuli or stress conditions?

• Structural determinants of specificity: Which residues within the SPRY domain determine specific protein-protein interactions?

• Evolutionary pressures: What selective pressures have maintained SPCC285.10c in the S. pombe genome throughout evolution?

• Integration into cellular networks: How does SPCC285.10c fit into broader protein interaction networks and signaling pathways?

• Potential as a model: Can SPCC285.10c serve as a simplified model for understanding more complex SPRY domain functions in multicellular organisms?

Addressing these questions will require integrative approaches combining genetics, biochemistry, structural biology, and systems biology perspectives.