Recombinant Schizosaccharomyces pombe Patatin-like phospholipase domain-containing protein SPAC1786.01c (SPAC1786.01c, SPAC31G5.20c)

What is SPAC1786.01c and what role does it play in Schizosaccharomyces pombe?

SPAC1786.01c is a predicted triacylglycerol lipase found in the fission yeast Schizosaccharomyces pombe. It is a protein-coding gene that belongs to the patatin-like phospholipase family of proteins. The gene has been identified through genomic sequencing of S. pombe and is cataloged with the Entrez Gene ID 2542286 . As a predicted triacylglycerol lipase, SPAC1786.01c is likely involved in lipid metabolism pathways within the cell, particularly in the hydrolysis of ester bonds in triacylglycerols. The protein's function has been computationally predicted rather than experimentally validated in full detail, which is why it is often listed with the qualifier "predicted" in genomic databases and literature .

How does SPAC1786.01c compare to other patatin-like phospholipases across species?

The patatin-like phospholipase family is widely distributed across various organisms with diverse functions. When comparing SPAC1786.01c to patatin-like phospholipases in other species, notable similarities and differences emerge:

Notably, while S. pombe phospholipase B has been shown not to hydrolyze triglyceride , SPAC1786.01c is predicted to have triacylglycerol lipase activity, suggesting functional diversification within S. pombe phospholipases. Unlike bacterial patatin-like phospholipases such as CapV that may function in host-pathogen interactions , SPAC1786.01c likely plays primarily metabolic roles within the yeast cell.

What are the structural domains and catalytic sites of SPAC1786.01c?

SPAC1786.01c contains a patatin-like phospholipase domain (PNPLA), which is characterized by specific conserved motifs typical of this protein family. Though the exact structure of SPAC1786.01c has not been fully characterized in the provided search results, studies of related patatin-like phospholipases highlight critical structural elements likely present in this protein:

• A G-x-S-x-G catalytic motif containing the active site serine

• G-G-G-x-[K/R]-G and D-G-[A/G] motifs that contribute to the protein scaffold functionality

• An N-terminal PNPLA domain responsible for enzymatic activity

The importance of these structural elements is demonstrated in studies of homologous proteins like CapV, where mutations in the PNPLA domain significantly alter protein function, even when the catalytic site remains intact . This suggests that the protein scaffold plays crucial roles beyond direct catalytic activity, possibly in substrate recognition or protein-protein interactions.

How can researchers effectively express and purify recombinant SPAC1786.01c?

Expressing and purifying recombinant SPAC1786.01c requires careful consideration of expression systems and purification strategies tailored to this specific protein. Based on successful purification approaches for similar phospholipases from S. pombe, researchers should consider the following methodology:

What enzymatic assays are most suitable for characterizing SPAC1786.01c activity?

Characterizing the enzymatic activity of SPAC1786.01c requires assays tailored to its predicted triacylglycerol lipase function. Researchers should implement multiple complementary approaches:

• Substrate Specificity Assessment: Test activity against a panel of substrates including:

  • Various triacylglycerols with different fatty acid compositions

  • Mono- and diacylphospholipids

  • Phosphatidylinositol (which was preferentially hydrolyzed by S. pombe phospholipase B)

  • Lysophosphatidylcholine (to test for potential acyltransferase activity)

• pH and Temperature Profiles: Determine optimal conditions by assaying activity across:

  • pH range from 2.0-8.0 (with particular focus on acidic conditions based on related enzymes)

  • Temperature range from 20-50°C

  • Thermal stability assessment through pre-incubation at various temperatures

• Enzyme Kinetics Analysis: Calculate Km and Vmax values using:

  • Concentration gradients of preferred substrates

  • Time-course experiments to ensure linearity

  • Analysis of product formation using TLC, HPLC, or mass spectrometry

• Inhibition Studies: Characterize sensitivity to:

  • Metal ions (especially Fe²⁺ and Fe³⁺ which inhibit related enzymes)

  • Detergents at various concentrations

  • Specific lipase/phospholipase inhibitors

How can genetic manipulation approaches be used to study SPAC1786.01c function in vivo?

To elucidate the physiological roles of SPAC1786.01c in S. pombe, several genetic manipulation strategies can be employed:

• Gene Knockout/Deletion: Generate ΔSPAC1786.01c strains using homologous recombination techniques established for S. pombe . This approach allows researchers to:

  • Assess viability and growth under various conditions

  • Observe phenotypic changes in lipid profiles and cellular morphology

  • Evaluate stress responses and metabolic adaptation

• Site-Directed Mutagenesis: Create targeted mutations in conserved domains to identify critical residues for function. Priority targets include:

  • The catalytic serine in the G-x-S-x-G motif

  • Residues in the G-G-G-x-[K/R]-G and D-G-[A/G] motifs that may affect substrate binding

  • Similar to the Q329R mutation in CapV that dramatically alters function

• Fluorescent Protein Tagging: Fuse SPAC1786.01c with GFP or other fluorescent proteins to:

  • Determine subcellular localization

  • Monitor protein expression levels under different conditions

  • Track dynamic changes in response to stimuli

• Conditional Expression Systems: Implement regulatable promoters to control SPAC1786.01c expression:

  • Nmt1 promoter system (thiamine-repressible) for titrating expression levels

  • Study effects of overexpression, which may reveal functions masked at endogenous levels

• Tetrad Analysis: For examining genetic interactions, use S. pombe's amenability to tetrad analysis to:

  • Cross ΔSPAC1786.01c with other deletion strains

  • Identify synthetic lethal or synthetic sick interactions

  • Map genetic pathways involving SPAC1786.01c

What strategies can be used to analyze the lipid substrate profile of SPAC1786.01c?

A comprehensive understanding of SPAC1786.01c's substrate profile requires multiple analytical approaches:

• Lipidomic Analysis: Compare lipid profiles between wild-type and ΔSPAC1786.01c strains using:

  • Thin-layer chromatography (TLC) for preliminary screening

  • Liquid chromatography-mass spectrometry (LC-MS) for detailed profiling

  • Quantification of triacylglycerols, phospholipids, and other potential substrates

• In Vitro Enzyme Assays with Diverse Substrates:

  • Test purified recombinant SPAC1786.01c against various lipid substrates

  • Employ radiolabeled substrates for highest sensitivity

  • Analyze reaction products via TLC, HPLC, or mass spectrometry

• Competition Assays:

  • Use mixtures of potential substrates to determine preference

  • Calculate relative rates to establish hierarchy of substrate specificity

The lack of triglyceride hydrolysis activity observed in S. pombe phospholipase B contrasts with the predicted function of SPAC1786.01c as a triacylglycerol lipase , making this comparative analysis particularly important for understanding the functional diversity of lipases in this organism.

How can structural studies enhance our understanding of SPAC1786.01c function?

Structural biology approaches can provide critical insights into SPAC1786.01c's mechanism of action:

• X-ray Crystallography:

  • Crystallize purified recombinant SPAC1786.01c alone and with inhibitors

  • Determine high-resolution structures to identify catalytic residues

  • Compare with structures of related enzymes to identify unique features

• Cryo-Electron Microscopy (Cryo-EM):

  • Useful if crystallization proves challenging

  • Particularly valuable if SPAC1786.01c forms larger complexes

  • May reveal dynamic conformational states

• Molecular Modeling and Simulations:

  • Generate homology models based on related structures

  • Perform molecular dynamics simulations to predict substrate binding

  • Design rational mutations based on structural predictions

• Small-Angle X-ray Scattering (SAXS):

  • Obtain low-resolution envelope structures in solution

  • Study conformational changes upon substrate binding

  • Complement crystallographic data for dynamic regions

The findings that G-G-G-x-[K/R]-G and D-G-[A/G] motifs in the PNPLA domain are required for function in patatin-like phospholipases, independent of the catalytic G-x-S-x-G motif , highlight the importance of structural studies in understanding these enzymes beyond their catalytic mechanisms.

What approaches can be used to investigate potential protein-protein interactions of SPAC1786.01c?

Understanding SPAC1786.01c's interaction network is essential for placing it in cellular pathways:

• Affinity Purification Coupled with Mass Spectrometry (AP-MS):

  • Express tagged SPAC1786.01c in S. pombe

  • Isolate protein complexes under native conditions

  • Identify interacting partners via mass spectrometry

• Yeast Two-Hybrid Screening:

  • Use SPAC1786.01c as bait to screen S. pombe cDNA libraries

  • Identify direct binary interactions

  • Validate with targeted assays

• Proximity-Dependent Biotin Identification (BioID):

  • Fuse SPAC1786.01c to a biotin ligase

  • Identify proteins in close proximity in vivo

  • Detect transient or weak interactions missed by other methods

• Co-Immunoprecipitation (Co-IP):

  • Use antibodies against SPAC1786.01c or potential partners

  • Validate specific interactions identified by high-throughput methods

  • Examine interactions under different cellular conditions

• Fluorescence Resonance Energy Transfer (FRET) or Bimolecular Fluorescence Complementation (BiFC):

  • Visualize interactions in living cells

  • Map subcellular locations of interactions

  • Study dynamics of complex formation

How does SPAC1786.01c contribute to lipid metabolism in S. pombe?

SPAC1786.01c likely plays a significant role in S. pombe lipid metabolism, particularly in lipid turnover and homeostasis. As a predicted triacylglycerol lipase , it may contribute to:

• Energy Mobilization: Releasing fatty acids from stored triacylglycerols during periods of nutrient limitation

• Membrane Remodeling: Participating in phospholipid turnover and membrane composition adjustment in response to environmental changes

• Lipid Signaling: Generating lipid mediators through its enzymatic activity that may function in cellular signaling pathways

The observation that S. pombe phospholipase B demonstrates acyltransferase activity, forming diacylphosphatidylcholine from lysophosphatidylcholine , suggests that SPAC1786.01c might also have dual hydrolase/transferase activities, potentially participating in lipid remodeling rather than simple degradation.

What is the evolutionary significance of patatin-like phospholipases across species?

Patatin-like phospholipases represent an evolutionarily conserved protein family with diverse functions across species:

• Functional Diversity: These proteins serve various roles across kingdoms:

  • Storage proteins in plants (e.g., potato tubers)

  • Lipid metabolism enzymes in mammals

  • Potential virulence factors in bacteria

  • Metabolic enzymes in fungi

• Structural Conservation: Despite functional divergence, the core PNPLA domain structure remains conserved, suggesting fundamental importance in lipid metabolism across evolution

• Specialized Adaptations: The presence of homologs in diverse fungi (Neurospora crassa, Magnaporthe oryzae) suggests specialized adaptations to each organism's ecological niche

The contrasting functions of patatin-like phospholipases across species—from storage proteins in plants to lipid metabolism in mammals to bacterial virulence factors —highlight how evolution has repurposed this ancient protein scaffold for diverse biological roles.