Recombinant Schizosaccharomyces pombe Uncharacterized oxidoreductase C663.06c (SPCC663.06c)

What is SPCC663.06c and what is its primary function in S. pombe?

SPCC663.06c is a gene in Schizosaccharomyces pombe (fission yeast) that encodes a putative short-chain dehydrogenase with oxidoreductase activity. Based on sequence analysis and functional studies, it likely belongs to the short-chain dehydrogenase/reductase (SDR) superfamily. While its precise function remains to be fully characterized, expression analyses suggest it plays a role in cellular stress responses, particularly under oxidative stress conditions. Gene expression studies show that SPCC663.06c is upregulated in certain genetic backgrounds, including the dbl2Δ mutant, suggesting it may have a role in cellular pathways regulated by Dbl2, which is involved in DNA repair and gene silencing mechanisms .

What protein family does SPCC663.06c belong to and what are its predicted domains?

SPCC663.06c likely belongs to the short-chain dehydrogenase/reductase (SDR) family, similar to its paralog SPCC663.09c. SDRs typically contain a conserved Rossmann-fold NAD(P)H-binding domain that facilitates cofactor binding and a catalytic tetrad (Tyr-Lys-Ser/Thr-Asn) responsible for its enzymatic activity. These proteins generally catalyze NAD(P)H-dependent oxidoreduction reactions involving various substrates such as alcohols, sugars, and steroids. The conserved domains in SPCC663.06c would likely include the NAD(P)H-binding motif at the N-terminus and the catalytic site residues positioned appropriately in the three-dimensional structure to facilitate electron transfer during redox reactions.

How is SPCC663.06c regulated in response to different stress conditions?

Expression analyses indicate that SPCC663.06c responds to cellular stress conditions. In studies examining gene repression mechanisms, SPCC663.06c showed elevated expression in the dbl2Δ mutant background . This suggests that Dbl2, which is involved in DNA repair and gene silencing, normally contributes (directly or indirectly) to repression of SPCC663.06c expression. Based on patterns observed with similar oxidoreductases in S. pombe, SPCC663.06c may be regulated as part of stress response networks. It might show expression patterns similar to SPCC663.09c, which exhibits upregulation under oxidative stress (+1.1 log2 fold change), iron deprivation (+1.7 log2 fold change), and zinc deficiency (+2.9 log2 fold change).

What are the predicted structural features of SPCC663.06c?

While detailed structural information specific to SPCC663.06c is not directly provided in the search results, we can infer its likely structural characteristics based on its classification as a short-chain dehydrogenase. The protein likely adopts a Rossmann fold structure, which is characteristic of dehydrogenases that utilize NAD(P)H as a cofactor. This fold consists of a central beta-sheet flanked by alpha-helices, creating a binding pocket for the nucleotide cofactor. The catalytic site would contain the conserved catalytic tetrad (Tyr-Lys-Ser/Thr-Asn) positioned to facilitate proton transfer during catalysis. The substrate binding site would likely be adjacent to the cofactor binding site, positioned to allow electron transfer between the substrate and NAD(P)H.

What experimental approaches are most effective for characterizing SPCC663.06c function?

To characterize SPCC663.06c function effectively, multiple complementary approaches should be employed:

Genetic Manipulation:

• Generate a SPCC663.06c deletion strain using homologous recombination techniques established for S. pombe

• Create point mutants targeting the predicted catalytic residues to assess their importance for function

• Construct conditional expression systems (e.g., using nmt1 promoter) for controlled overexpression studies

Expression Analysis:

• Perform quantitative PCR to measure transcript levels under various stress conditions

• Use Northern blotting with [α-32P] dCTP-labeled probes containing the complete ORF, similar to techniques used for related genes

• Conduct RNA-seq analysis comparing wild-type and mutant strains, following protocols similar to those described for dbl2Δ studies

Protein Characterization:

• Express recombinant SPCC663.06c with affinity tags for purification

• Perform in vitro enzymatic assays with potential substrates, monitoring NAD(P)H oxidation/reduction spectrophotometrically

• Conduct substrate screening using metabolite libraries to identify physiological substrates

Interaction Studies:

• Perform chromatin immunoprecipitation to identify potential interactions with transcription factors such as Pap1

• Use co-immunoprecipitation followed by mass spectrometry to identify protein interaction partners

• Conduct genetic interaction screens to identify functional relationships with other stress response genes

How does SPCC663.06c contribute to the oxidative stress response network in S. pombe?

SPCC663.06c likely contributes to the oxidative stress response network in S. pombe through multiple mechanisms:

Redox Homeostasis:
SPCC663.06c may function similar to SPCC663.09c in maintaining redox balance during oxidative stress. It could participate in NAD(P)H regeneration systems that are crucial for detoxifying reactive oxygen species (ROS). Its oxidoreductase activity might directly reduce oxidized cellular components or regenerate reduced forms of other antioxidant molecules.

Transcriptional Regulation:
The gene appears to be repressed under normal conditions through mechanisms involving Dbl2 . Under stress conditions, this repression may be relieved, allowing increased expression. It may be co-regulated with other stress response genes, potentially as part of the Core Environmental Stress Response (CESR) program in S. pombe, similar to SPCC663.09c.

Integration with Known Antioxidant Systems:
S. pombe utilizes multiple systems for hydrogen peroxide detoxification, with Tpx1 (thioredoxin peroxidase) serving as the primary scavenger during normal aerobic growth, while catalase (Ctt1) becomes crucial when peroxide levels increase . SPCC663.06c may function in conjunction with these systems or provide redundancy under specific conditions.

Interaction with Stress Response Regulators:
The transcription factor Pap1 is a key regulator of oxidative stress responses in S. pombe . Expression of SPCC663.06c might be regulated by Pap1, either directly or indirectly, as part of a coordinated stress response. This could be investigated through chromatin immunoprecipitation experiments using protocols similar to those described for other Pap1 targets .

What genetic interactions has SPCC663.06c been shown to participate in?

Genetic interaction studies have revealed several important relationships between SPCC663.06c and other regulatory factors:

Interaction with Dbl2:
SPCC663.06c shows increased expression in dbl2Δ mutants, suggesting that Dbl2 normally contributes to its repression. Dbl2 is involved in DNA repair and gene silencing mechanisms, indicating SPCC663.06c may be subject to chromatin-based regulation .

Interactions with Chromatin Regulators:
The expression pattern of SPCC663.06c has been analyzed in various mutant backgrounds affecting chromatin structure and gene silencing. When the dbl2Δ mutation was combined with mutations in chromatin regulators, complex effects on SPCC663.06c expression were observed:

• In dbl2Δclr6-1 double mutants, SPCC663.06c showed an additive increase in transcript levels compared to either single mutant, suggesting Clr6 (a histone deacetylase) and Dbl2 repress SPCC663.06c through different pathways

• Expression patterns in other double mutants involving dbl2Δ with mutations in HIRA complex components (hip1Δ, hip3Δ, slm9Δ) showed various effects, indicating complex regulatory relationships

This data suggests SPCC663.06c is regulated by multiple chromatin-modifying factors and silencing pathways, positioning it within a complex gene regulatory network in S. pombe.

What are the recommended protocols for expressing and purifying recombinant SPCC663.06c?

For successful expression and purification of recombinant SPCC663.06c, the following protocol is recommended:

Expression Systems:

• Bacterial Expression: Use E. coli BL21(DE3) with a pET vector system containing a 6xHis-tag or GST-tag for affinity purification

• Yeast Expression: Consider homologous expression in S. pombe using pREP vectors with nmt1 promoter for native-like folding

• Insect Cell Expression: For higher eukaryotic post-translational modifications, baculovirus expression system may be advantageous

Optimization Parameters:

Purification Strategy:

• Harvest cells and lyse using sonication or cell disruption in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT, and protease inhibitors

• Perform affinity chromatography using Ni-NTA (for His-tagged protein) or glutathione resin (for GST-tagged protein)

• Include size exclusion chromatography step to remove aggregates and ensure homogeneity

• Consider ion exchange chromatography for further purification if needed

Stability Considerations:

• Include reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) to prevent oxidation of catalytic cysteine residues

• Add 10-20% glycerol to storage buffer to prevent freeze-thaw damage

• Store purified protein at -80°C in small aliquots to avoid repeated freeze-thaw cycles

• Consider adding NAD(P)H cofactor (0.1-0.5 mM) to stabilize protein structure during storage

How can researchers investigate the catalytic mechanism of SPCC663.06c?

To elucidate the catalytic mechanism of SPCC663.06c, researchers should employ a multi-faceted approach:

Structural Analysis:

• Determine the three-dimensional structure using X-ray crystallography or cryo-EM, ideally with bound cofactor and/or substrate

• Perform molecular docking studies to predict substrate binding modes

• Use homology modeling based on related oxidoreductases with known structures if experimental structures are challenging to obtain

Site-Directed Mutagenesis:

• Generate point mutations of predicted catalytic residues (likely a Tyr-Lys-Ser/Thr-Asn tetrad based on SDR family patterns)

• Create mutations in the predicted NAD(P)H binding site to assess cofactor specificity

• Measure activity of mutants to establish structure-function relationships

Enzyme Kinetics:

• Determine steady-state kinetic parameters (kcat, KM) for putative substrates

• Perform pH-dependence studies to identify catalytic residues based on pKa profiles

• Conduct isotope effects studies using deuterated substrates to probe rate-limiting steps

Spectroscopic Analysis:

• Use stopped-flow spectroscopy to measure pre-steady-state kinetics and identify reaction intermediates

• Employ fluorescence spectroscopy to monitor protein conformational changes upon substrate/cofactor binding

• Utilize NMR spectroscopy to examine substrate binding and protein dynamics

Computational Approaches:

• Perform quantum mechanics/molecular mechanics (QM/MM) simulations to model the reaction mechanism

• Use molecular dynamics simulations to investigate protein dynamics during catalysis

• Apply bioinformatic analyses to identify conserved catalytic motifs across related oxidoreductases

What are the best assays to measure SPCC663.06c oxidoreductase activity?

Several complementary assays can be employed to measure SPCC663.06c oxidoreductase activity:

Spectrophotometric Assays:

• NAD(P)H Oxidation/Reduction: Monitor the change in absorbance at 340 nm corresponding to NAD(P)H oxidation or NAD(P)+ reduction

• Coupled Enzyme Assays: Use auxiliary enzymes to couple SPCC663.06c activity to a measurable spectrophotometric change

• Tetrazolium Salt Reduction: Employ MTT or NBT as artificial electron acceptors that form colored formazan products upon reduction

Chromatographic Methods:

• HPLC Analysis: Separate and quantify substrates and products to directly measure conversion rates

• LC-MS/MS: Provide more sensitive detection and identification of reaction products, particularly useful for unknown substrate screening

Polarographic Methods:

• Oxygen Electrode: If the reaction involves oxygen consumption or production, an oxygen electrode can directly measure changes in oxygen concentration

• Hydrogen Peroxide Detection: For reactions producing H2O2, use specific electrodes or coupled assays with peroxidase

Substrate Screening Approaches:

Assay Optimization Parameters:

• Buffer composition: Test multiple buffers (phosphate, Tris, HEPES) at pH range 6.0-8.5

• Temperature range: 25-37°C

• Cofactor concentration: 0.1-1.0 mM NAD(P)H

• Substrate concentration: Typically 0.1-10 mM depending on expected KM

• Enzyme concentration: Adjusted to achieve linear reaction rates

How can I generate and validate a SPCC663.06c deletion strain?

Creating and validating a SPCC663.06c deletion strain requires a systematic approach:

Deletion Strategy:

• PCR-Based Gene Targeting: Construct a deletion cassette containing a selectable marker (e.g., kanMX6, hphMX6, or ura4+) flanked by homology regions (~80-100 bp) to sequences upstream and downstream of SPCC663.06c

• Transformation: Transform S. pombe cells using lithium acetate method with PEG and carrier DNA, or electroporation for higher efficiency

• Selection: Plate transformants on appropriate selective media and incubate at 30°C for 3-5 days

Validation Methods:

• PCR Verification:

  • Design primers outside the targeted region and within the marker

  • Use colony PCR or genomic DNA extraction followed by PCR

  • Verify both integration junctions (5' and 3')

• Southern Blot Analysis:

  • Digest genomic DNA with appropriate restriction enzymes

  • Probe with sequences specific to SPCC663.06c and the marker

  • Confirm the expected band size shifts between wild-type and deletion strains

• RT-PCR or RNA-seq:

  • Extract total RNA using protocols similar to those described in the literature

  • Confirm absence of SPCC663.06c transcript

  • Use cDNA synthesis followed by PCR or RNA-seq analysis

• Phenotypic Characterization:

  • Compare growth rates under standard conditions

  • Test sensitivity to various stressors (oxidative stress, heat shock, nutrient limitation)

  • Compare with phenotypes of related oxidoreductase mutants

• Complementation Test:

  • Reintroduce wild-type SPCC663.06c on a plasmid

  • Verify restoration of any observed phenotypes

  • This confirms that phenotypes are due to SPCC663.06c deletion rather than secondary mutations

What transcriptomic approaches can reveal the role of SPCC663.06c in cellular stress responses?

Comprehensive transcriptomic approaches can elucidate SPCC663.06c's role in stress responses:

RNA-seq Analysis:

• Experimental Design:

  • Compare wild-type vs. SPCC663.06c deletion strains

  • Test multiple stress conditions (oxidative, heat, nutritional)

  • Include appropriate time courses (e.g., 0, 15, 30, 60 minutes post-stress)

  • Use four biological replicates per condition as done in similar studies

• Sample Preparation:

  • Grow cultures to exponential phase (OD595 = 0.5-0.55)

  • Extract total RNA using established protocols for S. pombe

  • Verify RNA quality using NanoDrop spectrophotometry and Bioanalyzer

  • Prepare libraries following TruSeq Stranded Total RNA protocols with ribosomal RNA depletion

• Data Analysis Pipeline:

  • Perform quality control using FastQC and trim adapters with Trimmomatic

  • Align reads to S. pombe genome using HISAT2 or STAR

  • Quantify gene expression using featureCounts or HTSeq

  • Conduct differential expression analysis with DESeq2 or edgeR

  • Perform GO term enrichment and pathway analysis

Targeted Expression Analysis:

• RT-qPCR for Key Genes:

  • Design primers for known stress response genes, particularly those in the Core Environmental Stress Response

  • Include genes co-regulated with other oxidoreductases such as glutathione S-transferases and thioredoxin peroxidases

  • Analyze transcription factor binding sites in promoters of differentially expressed genes

• Northern Blot Analysis:

  • Use [α-32P] dCTP-labeled probes for specific stress response genes

  • Include time course experiments to capture transient responses

  • Compare expression patterns with other oxidoreductase mutants

Chromatin Immunoprecipitation (ChIP):

• ChIP-seq for Transcription Factors:

  • Focus on stress-responsive transcription factors like Pap1

  • Perform as described in previous studies

  • Compare binding patterns between wild-type and SPCC663.06c deletion strains

• ChIP-qPCR for Specific Loci:

  • Target promoter regions of differentially expressed genes

  • Analyze histone modifications at these loci

  • Investigate potential changes in chromatin structure

How does SPCC663.06c expression change in different genetic backgrounds?

Studies have revealed complex patterns of SPCC663.06c expression in different genetic backgrounds:

Observed Expression Patterns:
SPCC663.06c shows differential expression across various mutant strains affecting chromatin structure and gene silencing. The gene appears to be regulated by multiple pathways and shows distinct expression patterns in different genetic backgrounds .

Key Genetic Interactions:

Methodological Approaches for Further Investigation:

• Quantitative RT-PCR:

  • Generate a panel of single and double mutants affecting chromatin regulation

  • Measure SPCC663.06c expression levels under standard and stress conditions

  • Use appropriate housekeeping genes for normalization

• RNA-seq Analysis:

  • Perform transcriptome-wide analysis of various mutant backgrounds

  • Cluster genes with similar expression patterns to identify co-regulated networks

  • Compare with ChIP-seq data to correlate with changes in chromatin structure

• Promoter Studies:

  • Clone the SPCC663.06c promoter region into reporter constructs

  • Introduce these constructs into different genetic backgrounds

  • Identify cis-regulatory elements responsible for differential expression

• Epigenetic Analysis:

  • Perform ChIP assays to examine histone modifications at the SPCC663.06c locus

  • Analyze DNA methylation patterns if applicable

  • Investigate chromatin accessibility using ATAC-seq or DNase-seq

What is the role of SPCC663.06c in relation to other S. pombe H2O2 scavenging systems?

While specific information about SPCC663.06c's role in H2O2 scavenging is limited, its function can be investigated in the context of known S. pombe peroxide detoxification systems:

Known H2O2 Scavenging Systems in S. pombe:
S. pombe employs several key enzymes for hydrogen peroxide detoxification, with distinct roles:

• Tpx1 (Thioredoxin Peroxidase):

  • Primary scavenger during normal aerobic growth

  • High sensitivity to H2O2 and cellular abundance

  • Essential for aerobic growth on solid media

• Catalase (Ctt1):

  • Secondary barrier when peroxide levels increase

  • Constitutively expressed in trr1Δ cells in a Pap1-dependent manner

  • Can compensate for Tpx1 deficiency under certain conditions

• Gpx1 (Glutathione Peroxidase):

  • Minor role in peroxide detoxification

  • Primarily involved when extracellular peroxides are added

Experimental Approaches to Determine SPCC663.06c's Role:

• Genetic Interaction Studies:

  • Generate double and triple mutants combining SPCC663.06c deletion with mutations in known H2O2 scavenging enzymes (Δtpx1, Δctt1, Δgpx1)

  • Assess growth under aerobic conditions and with exogenous H2O2

  • Determine if SPCC663.06c provides redundancy or has specialized functions

• H2O2 Sensitivity Assays:

  • Compare survival of wild-type and SPCC663.06c deletion strains under various H2O2 concentrations

  • Measure intracellular ROS levels using fluorescent probes (e.g., DCFDA, CellROX)

  • Determine IC50 values for H2O2 in different genetic backgrounds

• Biochemical Characterization:

  • Test recombinant SPCC663.06c for direct H2O2 scavenging activity

  • Investigate potential roles in NADPH regeneration for other antioxidant systems

  • Examine interactions with components of known H2O2 detoxification pathways

• Transcriptional Regulation:

  • Analyze expression patterns of SPCC663.06c in response to oxidative stress

  • Determine if it's regulated by stress-responsive transcription factors like Pap1

  • Compare with expression patterns of known H2O2 scavenging enzymes

What are the current knowledge gaps and future research directions for SPCC663.06c?

Despite progress in understanding SPCC663.06c, significant knowledge gaps remain that present opportunities for future research:

Structural Characterization:
The three-dimensional structure of SPCC663.06c remains unresolved. Determining its structure through X-ray crystallography or cryo-EM would provide crucial insights into substrate specificity and catalytic mechanism. Comparative structural analysis with related oxidoreductases could reveal functional adaptations specific to S. pombe.

Physiological Substrates:
The natural substrates of SPCC663.06c remain unknown. Comprehensive metabolomic profiling comparing wild-type and deletion strains could identify physiological substrates. Untargeted metabolomics approaches before and after oxidative stress challenges may reveal substrate candidates that change in abundance or modification state.

Regulatory Networks:
While SPCC663.06c shows differential expression in various genetic backgrounds, the complete regulatory network controlling its expression remains to be elucidated . Systematic analysis of transcription factor binding, chromatin modifications, and genetic interactions would provide a more comprehensive understanding of its regulation.

Integration with Stress Response Pathways:
How SPCC663.06c interfaces with established stress response pathways, particularly the H2O2 scavenging systems and the Core Environmental Stress Response, requires further investigation. Genetic interaction studies and phenotypic analyses under various stress conditions would help position SPCC663.06c within these networks.

Evolutionary Conservation:
Comparative genomic and functional analyses across fungal species could reveal the evolutionary conservation and divergence of SPCC663.06c. This could provide insights into its fundamental importance and potential specialization in S. pombe.

Future Research Priorities:

• Develop specific antibodies against SPCC663.06c for localization and interaction studies

• Perform systematic substrate screening using metabolite libraries

• Conduct high-throughput genetic interaction screens to position SPCC663.06c in cellular networks

• Investigate potential roles in specialized metabolic pathways unique to fission yeast

• Explore connections between SPCC663.06c function and chromatin regulation, given its interactions with silencing factors

How does research on SPCC663.06c contribute to our broader understanding of oxidoreductases in eukaryotic systems?

Research on SPCC663.06c contributes significantly to our understanding of eukaryotic oxidoreductases in several key ways:

Model System Advantages:
S. pombe serves as an excellent model for studying conserved eukaryotic processes. Insights from SPCC663.06c can be extrapolated to understand oxidoreductase functions in more complex eukaryotes, including humans. The genetic tractability of fission yeast allows for sophisticated experimental approaches that may be challenging in higher organisms.

Stress Response Integration:
Understanding how SPCC663.06c functions within the broader stress response network provides insights into how eukaryotic cells coordinate multiple enzymatic systems during stress adaptation. This knowledge has implications for understanding stress response mechanisms in diverse eukaryotes and their evolution.

Metabolic Network Regulation:
Oxidoreductases like SPCC663.06c play crucial roles in metabolic adaptations to changing environments. Research on these enzymes helps elucidate how metabolic networks are reconfigured during stress responses and how redox homeostasis is maintained across different cellular compartments.

Chromatin-Metabolism Connections:
The interactions between SPCC663.06c expression and chromatin regulators highlight the emerging understanding of connections between metabolism and epigenetic regulation . This research area is increasingly recognized as important in diverse biological contexts, from development to disease.

Therapeutic Applications:
Understanding the function and regulation of oxidoreductases like SPCC663.06c can inform therapeutic strategies for conditions involving oxidative stress or metabolic dysregulation. While S. pombe is an experimental model, the principles discovered may apply to human health and disease contexts.