Recombinant Dictyostelium discoideum Succinyl-CoA ligase [GDP-forming] subunit beta, mitochondrial (scsB), partial

How does Dictyostelium discoideum serve as an expression system for recombinant SCS?

D. discoideum has emerged as a promising eukaryotic host for heterologous recombinant protein expression with several advantages over traditional systems:

D. discoideum possesses the machinery required for orchestrating post-translational modifications similar to higher eukaryotes while being more economical and easier to maintain than mammalian cells .

What are the optimal methods for expressing recombinant scsB in Dictyostelium discoideum?

The expression of recombinant scsB in D. discoideum requires:

• Vector Selection: Extrachromosomal vectors containing a strong promoter (actin15 or discoidin) and appropriate selection markers (G418 or Blasticidin resistance)

• Transformation Protocol:

  • Electroporation method (optimal parameters: 0.85 kV/cm, 25 μF, 200 Ω)

  • Cell density: 1 × 10^7 cells/mL in electroporation buffer

  • Recovery: Immediate transfer to growth medium after pulse

  • Selection: Begin selection with appropriate antibiotic 24 hours post-transformation

• Culture Conditions:

  • Growth medium: HL5 medium with glucose (15.4 g/L)

  • Temperature: 22°C with shaking at 150 rpm

  • Growth phase: Maintain cells in exponential phase (1-8 × 10^6 cells/mL)

  • Induction: Some promoters may require induction with specific compounds

• Optimization Steps:

  • Codon optimization for D. discoideum (high A+T content in non-coding regions)

  • Addition of appropriate targeting sequences for mitochondrial localization

  • Fusion with secretory signals if secretion is desired

Research has shown that the G+C content of the scsB coding sequence (43.8%) is considerably higher than that of the 5' (14.8%) and 3' (13.3%) non-translated flanking sequences in related organisms, which should be considered when designing expression constructs .

What are the most effective purification strategies for recombinant scsB protein?

Purification of recombinant SCS from D. discoideum requires a multi-step approach:

• Cell Lysis Options:

  • Mechanical disruption (sonication, French press)

  • Gentle lysis using detergents for maintaining enzyme activity (1% Triton X-100)

  • Osmotic shock methods for mitochondrial fraction isolation

• Subcellular Fractionation:

  • Differential centrifugation to isolate mitochondria (10,000 × g for 15 min)

  • Further purification of mitochondrial fraction (Percoll gradient centrifugation)

• Affinity Purification:

  • His-tag purification using immobilized metal affinity chromatography

  • Nucleotide affinity columns (GDP/GTP-agarose)

• Additional Purification Steps:

  • Ion exchange chromatography (DEAE or Q-Sepharose)

  • Size exclusion chromatography to obtain pure heterodimeric complex

• Activity Preservation:

  • Include 10% glycerol and 2 mM DTT in all buffers

  • Maintain pH between 7.2-8.4 (optimal: pH 8.4)

  • Add protease inhibitors (PMSF, leupeptin, pepstatin)

Purified recombinant SCS can be stored in 3.2 M ammonium sulfate suspension for long-term stability .

How can the enzymatic activity of recombinant scsB be accurately measured?

Several methodologies exist for measuring SCS activity, each with specific advantages for different research questions:

• Forward Reaction (Succinyl-CoA → Succinate):

  • Coupled Assay with Pyruvate Kinase/Lactate Dehydrogenase:

    • Principle: GTP produced by SCS is used by pyruvate kinase to convert phosphoenolpyruvate to pyruvate, which is then reduced to lactate by lactate dehydrogenase with concomitant oxidation of NADH

    • Monitoring: Decrease in absorbance at 340 nm as NADH is oxidized

    • Buffer composition: 50 mM HEPES (pH 7.4), 10 mM MgCl₂, 0.2 mM succinyl-CoA, 1 mM GDP

• Reverse Reaction (Succinate → Succinyl-CoA):

  • DTNB (5,5'-dithiobis-2-nitrobenzoic acid) Assay:

    • Principle: Formation of thionitrobenzoate when CoA reacts with DTNB

    • Monitoring: Increase in absorbance at 412 nm

    • Buffer composition: 50 mM potassium phosphate (pH 8.4), 10 mM MgCl₂, 0.2 mM DTNB, 0.2 mM succinyl-CoA, 2 mM ATP or 1 mM GTP

• Standard Activity Definition:
  One unit of Succinyl-CoA synthetase activity is defined as the amount of enzyme required to release one μmole of succinyl-CoA from succinic acid (5.8 mM) per minute in the presence of NADH and Coenzyme A in Glycylglycine buffer (34 mM), pH 8.4 at 25°C .

• Optimal Assay Conditions:

  • Temperature: 25°C

  • pH: 8.4

  • Mg²⁺ concentration: 10 mM (essential cofactor)

The specific activity of purified recombinant prokaryotic SCS is approximately 9 U/mg at 25°C and pH 8.4 , which can serve as a reference for comparing D. discoideum-expressed recombinant enzyme activity.

What experimental approaches can be used to study allosteric regulation of scsB activity?

The study of allosteric regulation of SCS requires specialized techniques:

• Nucleotide-Binding Studies:

  • Equilibrium Dialysis:

    • Measures binding constants of GDP to the catalytic and allosteric sites

    • Can distinguish between high-affinity (catalytic) and low-affinity (allosteric) binding

  • Isothermal Titration Calorimetry (ITC):

    • Provides thermodynamic parameters of binding

    • Can detect multiple binding sites with different affinities

• Distinguishing Allosteric from Catalytic Effects:

  • Use of GDP[S] (guanosine 5'-[β-thio]diphosphate) as non-hydrolyzable analog

  • Comparison of phosphorylation patterns at different GDP concentrations:

    • Low concentrations (0.1-1 μM): Allosteric stimulation of α-subunit phosphorylation

    • High concentrations (100-1000 μM): Substrate-level dephosphorylation with GTP formation

• Dilution Experiments:

  • Non-covalent allosteric regulation is retained during purification

  • Stimulation of phosphorylation by low GDP concentrations is not diminished by dilution

• Gel-Filtration Analysis:

  • To determine if GDP binding alters the quaternary structure (α-β dimer)

  • SCS typically behaves as a non-interacting α-β dimer, regardless of GDP presence

• Phosphate Effect Quantification:

  • Pi can serve as an allosteric regulator of SCS

  • Measurement of SCS activity in presence of increasing Pi concentrations (1-10 mM)

  • Pi-induced activation increases SCS activity more than 2-fold

This table represents typical results showing Pi-induced activation of SCS activity in mitochondrial extracts .

What strategies can be employed for targeted mutagenesis of scsB to study structure-function relationships?

Several approaches can be used for mutagenesis of scsB:

• Site-Directed Mutagenesis:

  • QuikChange Method:

    • Design complementary primers containing desired mutation

    • PCR amplification generates mutated plasmid

    • DpnI digestion eliminates parental template

    • Key residues to target: catalytic histidine residue in α-subunit, nucleotide-binding site in β-subunit

  • Gibson Assembly:

    • Allows for multiple mutations simultaneously

    • Requires overlapping primers with mutations

• Domain Swapping:

  • Exchange domains between ADP-specific and GDP-specific β-subunits

  • Create chimeric proteins to identify determinants of nucleotide specificity

  • Example approach: Replace region 193-339 in scsB with corresponding region from SUCLA2

• Truncation Analysis:

  • Generate systematic deletions to identify minimal functional domains

  • C-terminal and N-terminal truncations to map functional regions

• Alanine Scanning:

  • Systematically replace conserved residues with alanine

  • Identify residues essential for catalysis, allosteric regulation, or subunit interaction

• Expression and Functional Testing:

  • Express mutated constructs in D. discoideum

  • Assess enzyme activity using standardized assays

  • Evaluate protein stability and complex formation

When designing mutations, consider that sequence comparisons show high conservation of the SCS β-subunit across species, with ~46-49% sequence identity between prokaryotic and eukaryotic forms .

How can gene knockout or knockdown techniques be applied to study scsB function in Dictyostelium discoideum?

Several genetic manipulation approaches are available:

• Homologous Recombination Knockout:

  • Design targeting vector containing selection marker (Blasticidin resistance)

  • Include 5' and 3' homology arms (1-2 kb each) flanking the selection marker

  • Transform D. discoideum by electroporation

  • Select transformants with appropriate antibiotic

  • Verify knockout by PCR, Southern blotting, and Western blotting

• REMI (Restriction Enzyme-Mediated Integration):

  • Linearize plasmid containing selection marker with restriction enzyme

  • Co-electroporate with the same restriction enzyme

  • Random integration creates insertional mutations

  • Identify mutants with altered phenotypes

  • Recover insertion sites by plasmid rescue

• RNA Interference:

  • Design hairpin RNAi constructs targeting scsB

  • Express under inducible promoters (e.g., discoidin)

  • Monitor knockdown efficiency by RT-qPCR and Western blotting

• CRISPR-Cas9 System:

  • Design sgRNAs targeting scsB exons

  • Express Cas9 and sgRNA from appropriate D. discoideum vectors

  • Screen for mutations by sequencing

  • Validate protein loss by Western blotting

• Phenotypic Characterization:

  • Growth rates in different carbon sources

  • Mitochondrial DNA content measurement

  • Cellular respiration analysis

  • Development progression analysis

Complete knockout of SCS genes often results in mitochondrial dysfunction and mtDNA depletion in mammalian models , which may affect D. discoideum development and multicellular stages.

How can recombinant scsB be used to study mitochondrial disease models?

Recombinant scsB can serve as a valuable tool for mitochondrial disease research:

• Complementation Studies:

  • Express wild-type or mutant scsB in patient fibroblasts

  • Assess rescue of enzyme activity and cellular phenotypes

  • Example methodology:

    • Transduce patient fibroblasts with scsB-lentivirus

    • Select stable expressors using Blasticidin (5 μg/mL) for 10 days

    • Assess protein expression by Western blot

    • Measure SCS enzyme activity

    • Evaluate mtDNA content and cellular respiration

• Structure-Function Analysis of Disease Mutations:

  • Introduce equivalent disease-causing mutations into scsB

  • Analyze effects on:

    • Protein stability and complex formation

    • Catalytic activity

    • Allosteric regulation

    • mtDNA maintenance

• Mitochondrial DNA Depletion Models:

  • SCS deficiency causes mtDNA depletion in humans

  • Study relationship between SCS activity and mtDNA levels

  • Quantify mtDNA by qPCR relative to nuclear DNA

• Cellular Respiration Analysis:

  • Measure oxygen consumption rate (OCR)

  • Assess specific complex activities in SCS-deficient cells

  • Compare respiratory parameters with and without SCS restoration

Data from complementation studies in human cells show that:

These values represent typical results observed in SUCLG1-deficient human fibroblasts before and after complementation with wild-type SUCLG1 .

What considerations are important when designing experiments to study the role of scsB in the TCA cycle versus its non-canonical functions?

Recent research has revealed that SCS may have functions beyond its classical role in the TCA cycle:

• Experimental Design Considerations:

  • Subcellular Fractionation:

    • Carefully separate mitochondrial and cytosolic fractions

    • Use appropriate markers to confirm fraction purity (e.g., citrate synthase for mitochondria)

    • Assess distribution of SCS between compartments

  • Metabolic Flux Analysis:

    • Use 13C-labeled substrates to trace metabolic pathways

    • Compare flux through TCA cycle versus alternative pathways

    • Measure incorporation into succinate, succinyl-CoA, and related metabolites

  • Protein Interaction Studies:

    • Immunoprecipitation to identify interaction partners

    • Proximity labeling techniques (BioID, APEX)

    • Yeast two-hybrid screening

• Non-TCA Cycle Functions to Investigate:

  • Redox Balance Regulation:

    • Assess protein expression of redox-scavenging enzymes

    • Measure cellular ROS levels with and without scsB

    • Evaluate stress granule formation

  • Regulation of Protein Succinylation:

    • Global protein succinylation analysis by mass spectrometry

    • Correlation between SCS activity and protein succinylation patterns

    • Functional consequences of altered succinylation

  • mtDNA Maintenance:

    • Mechanism connecting SCS to mtDNA levels

    • Role in nucleotide metabolism

    • Potential physical interactions with mtDNA maintenance proteins

• Technical Approaches:

  • Multiomics Integration:

    • Combine proteomics, metabolomics, and transcriptomics data

    • Network analysis to identify affected pathways

    • Validation of key nodes using genetic manipulation

  • Advanced Microscopy:

    • Super-resolution imaging to localize SCS within mitochondria

    • Live-cell imaging to track dynamics during stress responses

    • FRET/FLIM to detect protein-protein interactions

Recent findings in cancer cells indicate that SUCLA2 (the ADP-forming β-subunit) promotes stress granule formation and redox balance independently of its role in the TCA cycle . Similar non-canonical functions may exist for the GDP-forming β-subunit (scsB) in D. discoideum.

How does scsB expression and activity change during Dictyostelium's transition from unicellular to multicellular stages?

Studying scsB during Dictyostelium development requires specialized approaches:

• Expression Profile Analysis:

  • Temporal Expression Patterns:

    • Collect cells at different developmental stages (0, 4, 8, 12, 16, 20, 24 hours)

    • Extract RNA for RT-qPCR or RNA-seq analysis

    • Quantify protein levels by Western blotting

    • Compare with other metabolic enzymes as references

  • Spatial Expression:

    • In situ hybridization to localize scsB mRNA in multicellular structures

    • Immunohistochemistry using anti-SCS antibodies

    • Reporter constructs (scsB promoter driving GFP expression)

• Cell-Type Specific Analysis:

  • Separate prestalk and prespore cells by density gradient centrifugation

  • Isolate specific cell populations using cell-type specific markers

  • Compare enzyme activity and expression levels between cell types

• Metabolic Remodeling:

  • Monitor changes in TCA cycle flux during development

  • Assess alternations in GDP/GTP versus ADP/ATP utilization

  • Measure activities of both GDP-forming and ADP-forming SCS isoforms

• Functional Analysis:

  • Create developmental stage-specific knockdowns using inducible systems

  • Assess impact on multicellular morphogenesis

  • Evaluate energy metabolism during development

The developmental cycle of D. discoideum involves significant metabolic remodeling, including changes in cAMP signaling . These signaling pathways may influence SCS activity through allosteric regulation, similar to what has been observed in other organisms .

What methodologies can determine if scsB plays a role in cAMP-mediated signaling during Dictyostelium development?

Several approaches can investigate connections between scsB and cAMP signaling:

• Biochemical Interaction Studies:

  • cAMP Binding Assays:

    • Test direct binding of cAMP to purified SCS

    • Measure changes in enzyme activity with varying cAMP concentrations

    • Compare effects of cAMP and cGMP on SCS activity

  • Phosphorylation Studies:

    • Assess if PKA (cAMP-dependent protein kinase) phosphorylates SCS

    • Determine phosphorylation sites by mass spectrometry

    • Create phosphomimetic and phosphodeficient mutants

• Genetic Interaction Analysis:

  • Generate double mutants with cAMP signaling components

  • Test epistatic relationships between scsB and:

    • Adenylyl cyclases (ACA, ACB, ACG)

    • cAMP phosphodiesterases (PdsA, RegA)

    • PKA catalytic and regulatory subunits

• Developmental Phenotype Analysis:

  • Monitor development under various conditions:

    • Normal development on non-nutrient agar

    • Development with exogenous cAMP pulses

    • Development under various stresses (osmotic, oxidative)

  • Compare wild-type, scsB mutants, and complemented strains

• cAMP-Related Signal Transduction:

  • Measure changes in intracellular cAMP levels

  • Monitor PKA activity using phospho-specific antibodies

  • Assess expression of cAMP-regulated genes by RT-qPCR

Evidence from D. discoideum research shows that cAMP and cGMP signaling are crucial during development, with cGMP being a more potent activator of certain pathways . It would be valuable to investigate whether GDP-forming SCS (encoded by scsB) responds differently to these cyclic nucleotides compared to ADP-forming SCS.

What innovative technologies can enhance the study of recombinant scsB structure, dynamics, and interactions?

Advanced technologies offer new opportunities for scsB research:

• Structural Biology Approaches:

  • Cryo-Electron Microscopy:

    • Near-atomic resolution structures of SCS complexes

    • Visualization of conformational changes upon substrate binding

    • Complex formation with interaction partners

  • Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

    • Map dynamic regions and conformational changes

    • Identify allosteric communication networks

    • Characterize effects of mutations on protein dynamics

  • AlphaFold2 and Molecular Dynamics Simulations:

    • Predict structures of D. discoideum-specific variants

    • Simulate substrate binding and catalytic events

    • Model effects of disease-related mutations

• Single-Molecule Techniques:

  • FRET (Förster Resonance Energy Transfer):

    • Monitor real-time conformational changes

    • Detect transient interactions with other proteins

    • Observe catalytic cycle at single-molecule level

  • Optical Tweezers and AFM:

    • Measure mechanical properties and stability

    • Force-dependent unfolding and refolding

• Advanced Imaging:

  • Super-Resolution Microscopy:

    • Nanoscale localization within mitochondria

    • Co-localization with mtDNA nucleoids

    • Redistribution during cellular stress

  • FIB-SEM and Cryo-Electron Tomography:

    • 3D visualization in cellular context

    • Integration with mitochondrial ultrastructure

    • Spatial organization in relation to other TCA cycle enzymes

• High-Throughput Approaches:

  • Deep Mutational Scanning:

    • Comprehensive mutation libraries

    • Functional impact of thousands of variants

    • Identification of regulatory hotspots

  • Chemogenomics and Chemical Biology:

    • Specific inhibitors or activators of GDP-form

    • Activity-based protein profiling

    • Covalent ligands for mechanistic studies

How can microarray and genomic technologies be applied to study the systemic effects of scsB manipulation in Dictyostelium?

System-level approaches offer comprehensive insights:

• Transcriptomic Analysis:

  • RNA-Seq Comparison:

    • Wild-type vs. scsB mutants at different developmental stages

    • Identify differentially expressed genes

    • Pathway enrichment analysis

    • Co-expression network construction

  • Time-Course Analysis:

    • Capture dynamic changes during development

    • Identify early vs. late-responding genes

    • Temporal clustering to find functionally related genes

• Proteomic Approaches:

  • Quantitative Proteomics:

    • SILAC or TMT labeling for comparative analysis

    • Post-translational modification profiling (phosphorylation, succinylation)

    • Protein complex analysis by BN-PAGE followed by MS

  • Spatial Proteomics:

    • APEX2 or BioID proximity labeling

    • Subcellular fractionation combined with proteomics

    • In situ spatial proteomics using laser microdissection

• Metabolomic Profiling:

  • Targeted Metabolomics:

    • Focus on TCA cycle intermediates

    • Nucleotide pools (ATP/GTP/GDP/ADP ratios)

    • Coenzyme A derivatives

  • Untargeted Approaches:

    • Global metabolite changes

    • Stable isotope labeling to track metabolic flux

    • Integration with transcriptomic and proteomic data

• Computational Integration:

  • Multi-Omics Data Integration:

    • Correlation networks across data types

    • Causal inference from time-series data

    • Machine learning to identify key regulatory nodes

  • Genome-Scale Metabolic Modeling:

    • Incorporate scsB activity constraints

    • Predict metabolic flux distributions

    • Identify synthetic lethal interactions

Example from published research shows microarray analysis identified genes (gapA and rtoA) induced by hyperosmotic stress in D. discoideum that are dependent on STAT transcription factors . Similar approaches could reveal genes whose expression is linked to scsB activity or mitochondrial energy metabolism.

How does D. discoideum scsB compare with SCS variants from other organisms in terms of structure, function, and regulation?

Comparative analysis provides evolutionary insights:

• Sequence and Structure Comparison:

• Nucleotide Specificity Determinants:

  • ATP-specific β-subunits contain distinctive sequence motifs

  • GDP-specific β-subunits have unique residues in nucleotide-binding domain

  • Non-specific prokaryotic enzymes show intermediate features

  • Chimeric constructs can help identify specificity-determining regions

• Regulatory Mechanisms:

  • Allosteric Regulation:

    • Low GDP concentrations (0.1-1 μM) stimulate phosphorylation

    • High GDP concentrations (100-1000 μM) lead to dephosphorylation

    • Pi binding activates enzyme activity >2-fold

    • Compare regulation patterns across species

  • Post-Translational Modifications:

    • Histidine phosphorylation of α-subunit

    • Succinylation patterns

    • Species-specific modifications

• Subcellular Localization:

  • Mitochondrial in eukaryotes

  • Hydrogenosomal in anaerobic eukaryotes

  • Cytosolic in some prokaryotes

  • Targeting signals vary across species

The β-subunit of SCS shows distinct tissue distribution patterns in mammals, with ATP-specific form predominant in tissues with high oxidative capacity (heart, muscle) and GDP-specific form in biosynthetic tissues (liver) .

What are the methodological challenges in comparing enzyme activities between different expression systems, and how can they be addressed?

Accurate cross-system comparisons require careful controls:

• Standardization Challenges:

  • Activity Normalization:

    • Different specific activities in different expression systems

    • Various host-specific post-translational modifications

    • Impact of purification methods on activity retention

  • Enzyme Stability Differences:

    • Temperature sensitivity variations

    • Buffer composition effects

    • Storage condition optimization

• Methodology Harmonization:

  • Assay Standardization:

    • Use identical assay conditions across systems

    • Include internal standards in each assay

    • Measure activity using multiple complementary methods

  • Purification Protocol Alignment:

    • Apply identical purification schemes when possible

    • Include recovery controls at each step

    • Consider native versus tagged protein comparisons

• Expression System Variables:

  • Host-Specific Factors:

    • Chaperone availability and folding environments

    • Codon usage optimization requirements

    • Post-translational modification differences

  • Control Measurements:

    • Express identical constructs in different hosts

    • Include well-characterized control enzymes

    • Measure intrinsic SCS activity in host systems

• Advanced Analytical Approaches:

  • Structural Comparisons:

    • CD spectroscopy for secondary structure

    • Thermal shift assays for stability

    • Limited proteolysis for domain organization

  • Kinetic Parameter Analysis:

    • Compare Km, Vmax, and kcat across systems

    • Determine substrate specificity profiles

    • Measure allosteric effects quantitatively

When comparing prokaryotic versus D. discoideum-expressed SCS, consider that the optimal conditions for bacterial SCS activity are pH 8.4 at 25°C, which may differ for the eukaryotic enzyme .