Recombinant Schizosaccharomyces pombe Uncharacterized oxidoreductase C16H5.14c (SPBC16H5.14c, SPBC21H7.08)

What is the genomic context of SPBC16H5.14c in Schizosaccharomyces pombe?

SPBC16H5.14c is a protein-coding gene in Schizosaccharomyces pombe (fission yeast) that encodes a putative dehydrogenase belonging to the DHRS (dehydrogenase/reductase) family . The gene is annotated in the S. pombe genome with Entrez Gene ID 2539946, and the corresponding mRNA transcript is documented as NM_001021841.2, which encodes the protein product NP_595933.1 . The gene is currently classified as an uncharacterized oxidoreductase, suggesting its enzymatic function has been predicted through sequence homology but has not been experimentally validated in detail.

What homologs of SPBC16H5.14c exist in other model organisms?

SPBC16H5.14c has homologs across multiple model organisms, suggesting evolutionary conservation of this dehydrogenase. Key orthologs include:

The conservation across fungi, mammals, and other eukaryotes suggests this protein family may serve a fundamental cellular function . When designing experiments to characterize SPBC16H5.14c, researchers should consider comparative analyses with these orthologs, especially the S. cerevisiae homolog, which may have more extensive functional characterization.

How can I express and purify recombinant SPBC16H5.14c for in vitro studies?

For recombinant expression of SPBC16H5.14c, researchers should consider a methodological approach similar to that used for other S. pombe proteins. Begin by cloning the complete SPBC16H5.14c ORF (NM_001021841.2) into an appropriate expression vector . For bacterial expression, pET series vectors with His-tags facilitate purification. When designing your expression construct:

• Include appropriate affinity tags (His6 or GST) for purification

• Consider codon optimization for the expression host

• Include protease cleavage sites for tag removal if needed for downstream applications

For purification, implement a multi-step approach:

• Initial capture using affinity chromatography (Ni-NTA for His-tagged proteins)

• Secondary purification using ion exchange chromatography

• Final polishing with size exclusion chromatography

For functional studies, ensure protein activity is preserved by testing different buffer conditions and including appropriate cofactors (likely NAD(P)H for dehydrogenases). Similar approaches have been successful for other S. pombe enzymes, such as recombinant Rad60, which was purified from E. coli for in vitro kinase assays .

How can I determine the substrate specificity of SPBC16H5.14c?

Determining substrate specificity for an uncharacterized oxidoreductase requires a systematic approach:

• Phylogenetic analysis: Compare SPBC16H5.14c with characterized DHRS family members across species to predict potential substrate classes . The SDR16C6 ortholog in mammals may provide initial insights.

• Screening methodology: Develop a high-throughput substrate screening assay using:

  • Spectrophotometric detection of NAD(P)H oxidation/reduction at 340 nm

  • Diverse substrate libraries including steroids, sugars, and xenobiotics

  • Activity-based protein profiling with chemical probes

• Metabolomics approach: Perform untargeted metabolomics comparing wild-type and SPBC16H5.14c deletion strains to identify accumulated or depleted metabolites.

• Structural predictions: Use AlphaFold2 or similar tools to predict the protein structure, focusing on the substrate binding pocket to narrow potential substrates based on size and chemical properties.

Record kinetic parameters (Km, kcat, kcat/Km) for each potential substrate to establish specificity profiles. Similar approaches have been successful in characterizing other oxidoreductases in fission yeast, though substrate identification for uncharacterized enzymes remains challenging.

What is the role of SPBC16H5.14c in DNA replication and repair pathways?

While SPBC16H5.14c is annotated as a dehydrogenase , its potential involvement in DNA metabolism should be investigated due to known connections between metabolic enzymes and genome stability in S. pombe. To explore this:

• Genetic interaction mapping: Create SPBC16H5.14c deletion strains and perform synthetic genetic array analysis to identify interactions with known DNA repair factors.

• DNA damage sensitivity assays: Test sensitivity of SPBC16H5.14c mutants to various DNA damaging agents (UV, MMS, HU) similar to studies with Rad60 .

• Localization studies: Create fluorescently tagged SPBC16H5.14c to monitor its subcellular localization during normal growth and after DNA damage, looking for nuclear relocalization patterns similar to those observed with Rad60 in response to replication stress .

• Chromatin association: Perform ChIP-seq to determine if SPBC16H5.14c associates with specific genomic regions, particularly during replication stress.

While direct evidence linking SPBC16H5.14c to DNA repair has not been established, the demonstrated role of other S. pombe proteins in regulating homologous recombination at stalled replication forks suggests that metabolic enzymes like SPBC16H5.14c could potentially influence these processes through redox regulation or metabolite production.

How does SPBC16H5.14c interact with the S. pombe proteome?

To characterize the protein-protein interaction network of SPBC16H5.14c:

• Affinity purification-mass spectrometry (AP-MS): Create strains expressing tagged SPBC16H5.14c (e.g., TAP-tagged or FLAG-tagged) and identify interacting proteins through co-immunoprecipitation followed by mass spectrometry.

• Yeast two-hybrid screening: Use SPBC16H5.14c as bait to screen an S. pombe cDNA library for interacting partners.

• BioID or APEX proximity labeling: Fuse SPBC16H5.14c with a proximity labeling enzyme to identify proteins in its vicinity in vivo.

• Co-expression analysis: Analyze transcriptomic data to identify genes with expression patterns that correlate with SPBC16H5.14c across different conditions.

Pay particular attention to potential interactions with known regulatory complexes in S. pombe. For instance, the Smc5/6 complex plays critical roles in DNA repair through homologous recombination , and understanding whether SPBC16H5.14c interacts with such complexes could provide insights into its cellular function.

How is SPBC16H5.14c regulated during cellular stress responses?

Investigating the regulation of SPBC16H5.14c during stress conditions:

• Transcriptional profiling: Analyze expression changes of SPBC16H5.14c during various stresses (oxidative, replicative, nutritional) using RT-qPCR or RNA-seq.

• Post-translational modification analysis: Identify potential phosphorylation, ubiquitination, or other modifications using mass spectrometry-based proteomics, particularly during stress responses. Consider whether SPBC16H5.14c might be regulated by checkpoint kinases like Cds1^Chk2^, which phosphorylates Rad60 during replication stress .

• Protein stability assessment: Create strains with epitope-tagged SPBC16H5.14c and monitor protein levels during various stress conditions using western blotting.

• Localization dynamics: Track subcellular localization changes under stress using fluorescence microscopy, similar to studies showing Rad60 dispersal from the nucleus in response to hydroxyurea treatment .

The regulation of SPBC16H5.14c might parallel other S. pombe proteins that show specific responses to replication stress. For example, Rad60 is phosphorylated by Cds1^Chk2^ at specific residues (T72 and S126) in response to hydroxyurea treatment, leading to its relocalization from the nucleus .

What approaches can be used to determine the three-dimensional structure of SPBC16H5.14c?

For structural characterization of SPBC16H5.14c:

• X-ray crystallography workflow:

  • Optimize recombinant expression to obtain milligram quantities of pure protein

  • Screen crystallization conditions systematically (sparse matrix screening)

  • Optimize crystal growth for diffraction quality

  • Consider co-crystallization with potential cofactors (NAD+/NADP+)

  • Collect diffraction data and solve the structure through molecular replacement using related DHRS family structures

• Cryo-EM approach:

  • Particularly valuable if SPBC16H5.14c forms larger complexes

  • Prepare grids with different protein concentrations and buffer conditions

  • Collect and process images using current single-particle analysis workflows

• NMR spectroscopy:

  • Express isotopically labeled protein (^15^N, ^13^C)

  • Record multidimensional spectra for backbone and side chain assignments

  • Determine solution structure through NOE distance restraints

• AlphaFold2 predictions:

  • Generate computational models as starting points for experimental validation

  • Use these models to guide mutational studies of predicted catalytic residues

Structural information will provide insights into the catalytic mechanism and substrate binding specificity, facilitating the functional characterization of this uncharacterized oxidoreductase.

How should I design genetic knockout or mutation experiments for SPBC16H5.14c?

When designing genetic manipulation experiments:

• Deletion strategy:

  • Use homologous recombination to replace SPBC16H5.14c with a selection marker

  • Verify deletion by PCR and sequencing of junction regions

  • Assess phenotypic effects under various growth conditions

• Point mutation design:

  • Identify conserved catalytic residues through alignment with characterized DHRS family members

  • Create targeted mutations of predicted active site residues

  • Consider creating phosphorylation-site mutants, similar to the T72A and S126A mutations created for Rad60

• Conditional alleles:

  • If SPBC16H5.14c proves essential, create temperature-sensitive or auxin-inducible degron alleles

  • Design repressible promoter constructs (e.g., nmt1) for controlled expression

• Fusion proteins:

  • Create C- and N-terminal epitope tags for immunodetection

  • Design fluorescent protein fusions for localization studies

  • Implement proximity labeling tags (BioID, APEX) for interaction studies

The experimental approach should include appropriate controls and validation steps, particularly verification of protein levels using western blotting. Similar methodologies have been successfully applied to study Rad60 function through site-directed mutagenesis of phosphorylation sites .

What omics approaches would be most informative for understanding SPBC16H5.14c function?

Integrated omics strategies to elucidate SPBC16H5.14c function:

• Transcriptomics:

  • Compare gene expression profiles between wild-type and SPBC16H5.14c deletion or overexpression strains

  • Analyze expression changes under various stress conditions

  • Identify co-regulated genes that may function in the same pathway

• Proteomics:

  • Quantitative proteomics to identify proteins affected by SPBC16H5.14c manipulation

  • Phosphoproteomics to detect signaling changes

  • Interaction proteomics using affinity purification-mass spectrometry

• Metabolomics:

  • Targeted analysis of potential substrates based on DHRS family activity

  • Untargeted profiling to identify altered metabolites in mutant strains

  • Flux analysis using isotope-labeled precursors

• Functional genomics:

  • Synthetic genetic array analysis to identify genetic interactions

  • Chemical-genetic profiling to identify conditions affecting SPBC16H5.14c mutants

  • CRISPR-based screens to identify genes with related functions

For each approach, design experiments with appropriate biological replicates, controls, and statistical analysis methods. The integration of multiple omics datasets will provide a systems-level understanding of SPBC16H5.14c function within the cellular network.

How does SPBC16H5.14c compare with its homologs in other fungal species?

Comparative analysis reveals evolutionary insights about SPBC16H5.14c:

• Sequence conservation:

  • Perform multiple sequence alignments of SPBC16H5.14c with homologs from other fungi, including S. cerevisiae (YDL114W), Neurospora crassa (NCU06845), and Magnaporthe oryzae (MGG_12752)

  • Identify conserved domains, particularly catalytic residues characteristic of the DHRS family

  • Calculate sequence identity and similarity percentages across species

• Synteny analysis:

  • Examine conservation of gene neighborhood and chromosomal location

  • Identify patterns of genome rearrangement that may indicate functional specialization

• Functional comparison:

  • Evaluate whether the homologs in other species have been functionally characterized

  • Determine if cross-species complementation (e.g., expressing SPBC16H5.14c in S. cerevisiae YDL114W mutants) can restore function

• Structural comparison:

  • Compare predicted protein structures across fungal species

  • Identify conserved and divergent structural features that may relate to substrate specificity

This comparative approach provides evolutionary context for understanding the function of SPBC16H5.14c and may suggest conserved biological roles across fungal species.

How can I establish functional correlations between SPBC16H5.14c and its mammalian homologs?

To establish functional relationships with mammalian homologs:

• Expression in mammalian systems:

  • Clone and express SPBC16H5.14c in mammalian cell lines

  • Compare subcellular localization with mammalian SDR16C6 proteins

  • Assess whether SPBC16H5.14c can complement defects in mammalian SDR16C6-deficient cells

• Substrate conservation:

  • Test whether SPBC16H5.14c can utilize the same substrates as mammalian homologs

  • Compare enzyme kinetics and specificity constants between fungal and mammalian proteins

• Regulatory conservation:

  • Determine if SPBC16H5.14c responds to the same regulatory signals as mammalian homologs

  • Investigate whether mammalian checkpoint kinases can phosphorylate SPBC16H5.14c similar to how Cds1^Chk2^ phosphorylates Rad60 in S. pombe

• Interactome comparison:

  • Compare protein-protein interaction networks between SPBC16H5.14c and mammalian homologs

  • Identify conserved interaction partners that may suggest shared functional pathways

Understanding these relationships may provide insights into the evolution of dehydrogenase functions and potential applications in biomedical research.

What are the key unanswered questions about SPBC16H5.14c function?

Several critical questions remain to be addressed:

• What is the native substrate of SPBC16H5.14c, and what metabolic pathway does it participate in?

• Is SPBC16H5.14c activity regulated in response to cellular stresses, similar to other S. pombe proteins like Rad60 ?

• Does SPBC16H5.14c have moonlighting functions beyond its predicted oxidoreductase activity?

• How does SPBC16H5.14c contribute to cellular redox homeostasis in S. pombe?

• Are there disease-relevant insights that can be gained from studying this enzyme and its mammalian homologs?

Addressing these questions will require integrated approaches combining biochemistry, genetics, and systems biology. The findings may have broader implications for understanding redox regulation in eukaryotic cells and potentially inform studies of related human enzymes.

How might SPBC16H5.14c research contribute to understanding fundamental cellular processes?

Research on SPBC16H5.14c may illuminate:

• Metabolic regulation: Understanding how oxidoreductases like SPBC16H5.14c contribute to cellular redox balance and metabolic adaptation.

• Stress responses: Determining whether SPBC16H5.14c is regulated during stress responses, similar to how Rad60 is regulated by Cds1^Chk2^ phosphorylation during replication stress .

• Evolutionary conservation: Providing insights into conserved metabolic functions between fungi and mammals, potentially identifying fundamental cellular processes.

• Protein-protein interactions: Elucidating how metabolic enzymes may interact with other cellular machinery, possibly including DNA repair complexes like Smc5/6, which has been shown to interact with other proteins during homologous recombination in S. pombe .

• Translational relevance: Findings from SPBC16H5.14c studies may inform research on human dehydrogenases implicated in disease processes, particularly those in the SDR16C family .