Recombinant Uncharacterized SURF1-like protein Mb2259 (Mb2259)

What is SURF1-like protein Mb2259 and how does it relate to human SURF1?

SURF1-like protein Mb2259 is structurally similar to the human SURF1 protein, which functions in the assembly of complex IV (cytochrome c oxidase or COX) in the mitochondrial respiratory chain. The human SURF1 protein is essential for oxidative phosphorylation, facilitating the conversion of energy from food into ATP through a series of reactions involving multiple protein complexes . Mb2259, though uncharacterized, likely shares functional domains with SURF1 that are involved in assembly factors for respiratory complexes, particularly in energy metabolism pathways.

What experimental approaches are recommended for initial characterization of uncharacterized SURF1-like proteins?

For initial characterization, researchers should employ a multifaceted approach:

• Sequence analysis and structural prediction: Compare the amino acid sequence with characterized SURF1 proteins using bioinformatics tools to identify conserved domains.

• Expression analysis: Determine tissue/condition-specific expression patterns.

• Subcellular localization: Use fluorescent tagging or cellular fractionation to determine where the protein localizes.

• Knockout/knockdown studies: Generate model systems lacking the protein to observe phenotypic effects.

• Complementation assays: Test if Mb2259 can rescue defects in SURF1-deficient yeast models (SHY1Δ) or other organisms .

What model systems are most appropriate for studying SURF1-like proteins?

Based on existing research, these model systems offer distinct advantages:

Yeast models have proven particularly valuable, as demonstrated by screens of approximately 2,500 drugs using SHY1 knockout strains to identify compounds that might restore mitochondrial function .

What expression systems yield optimal results for recombinant SURF1-like protein production?

For optimal recombinant expression of membrane proteins like SURF1-like Mb2259:

• Bacterial systems (E. coli): Use specialized strains like C41(DE3) or C43(DE3) designed for membrane protein expression with mild induction conditions (lower IPTG concentrations, 18-25°C).

• Yeast systems (P. pastoris): Provide proper membrane insertion and post-translational modifications.

• Insect cell systems: Baculovirus expression systems offer superior folding for complex membrane proteins.

Purification typically requires careful optimization of detergents for solubilization, with mild non-ionic detergents (DDM, LMNG) often proving most effective for maintaining protein stability and function.

How can researchers effectively measure the activity of recombinant SURF1-like proteins?

Measuring activity of SURF1-like proteins involves assessing their impact on respiratory complex assembly:

• Complex IV assembly assays: Monitor the formation of fully assembled complex IV using Blue Native PAGE.

• Cytochrome c oxidase activity assays: Measure enzymatic activity using spectrophotometric methods to track the oxidation of reduced cytochrome c.

• Oxygen consumption measurements: Use respirometry to quantify cellular oxygen consumption rates.

• ATP production assays: Assess the impact on cellular energy production.

For Mb2259 specifically, researchers should first establish whether it functions similarly to known SURF1 proteins by measuring these parameters in complementation studies using SURF1-deficient systems.

What are the critical factors for successful site-directed mutagenesis studies of SURF1-like proteins?

When designing site-directed mutagenesis experiments:

• Target selection: Focus on highly conserved residues between Mb2259 and characterized SURF1 proteins.

• Disease-relevant mutations: Consider introducing mutations analogous to those causing Leigh syndrome in human SURF1, such as those affecting splicing sites or creating premature stop codons .

• Transmembrane domain preservation: Avoid disrupting predicted transmembrane domains that could affect membrane insertion.

• Control mutations: Include mutations in non-conserved regions as controls.

• Validation methods: Verify expression levels and subcellular localization of mutant proteins to ensure observed effects are not due to protein degradation or mislocalization.

How can SURF1-like proteins be utilized in drug discovery for mitochondrial disorders?

Building on established methodologies:

• Yeast-based screening platforms: Develop growth assays using yeast lacking SHY1 (the yeast SURF1 ortholog) complemented with Mb2259 to screen for compounds that restore growth on non-fermentable carbon sources .

• Neural differentiation models: Utilize iPSC-derived neuronal models to evaluate drug candidates for improving neurogenesis and neuronal function .

• High-throughput bioenergetic assays: Measure oxygen consumption rates and ATP production in cellular models expressing Mb2259 variants.

A systematic approach has proven effective, as demonstrated by the Cure Mito Foundation's screening of 2,500 known drugs using yeast avatars of SURF1-deficient cells, which identified promising hit compounds for further investigation .

What are the challenges in interpreting conflicting data from different model systems when studying SURF1-like proteins?

Researchers face several challenges when integrating data across models:

• Species-specific functions: SURF1 functions may vary between species; SHY1Δ yeast show respiratory deficiency but animal models don't always recapitulate human Leigh syndrome neurological phenotypes .

• Tissue-specific effects: Neuronal cells are particularly vulnerable to SURF1 deficiency while other tissues may be less affected .

• Developmental timing: SURF1 deficiency impacts neurogenesis during development, which may not be captured in adult-onset models .

• Compensatory mechanisms: Different models may develop distinct compensatory pathways that mask phenotypes.

Integration strategies should include:

• Cross-validation across multiple model systems

• Cell type-specific analyses

• Developmental time course studies

• Rigorous controls for genetic background effects

How can researchers effectively study the interaction of SURF1-like proteins with complex IV assembly factors?

To characterize protein-protein interactions:

• Co-immunoprecipitation assays: Pull down Mb2259 and identify interacting partners by mass spectrometry.

• Proximity labeling methods: Use BioID or APEX2 fused to Mb2259 to identify neighboring proteins in the native cellular environment.

• Crosslinking mass spectrometry: Identify specific interaction sites between Mb2259 and assembly factors.

• Fluorescence resonance energy transfer (FRET): Monitor interactions in live cells.

• Split reporter assays: Use split GFP or luciferase constructs to confirm specific interactions.

For validating functional relevance, researchers should:

• Perform temporal analysis to determine the sequence of interactions during complex IV assembly

• Test the effect of disease-associated mutations on these interactions

• Develop in vitro reconstitution assays to test direct interactions

How does SURF1 deficiency affect neuronal differentiation and what research models best capture these effects?

SURF1 deficiency profoundly impacts neuronal differentiation through multiple mechanisms:

• Disrupted neural progenitor cell (NPC) bioenergetics: Defects occur at the NPC stage, compromising their neurogenic potential .

• Impaired neuronal branching: SURF1-deficient neurons show reduced branching complexity .

• Altered mitochondrial dynamics: Increased mitochondrial motility and reduced stationary mitochondria in developing neurons .

• Transcriptional reconfiguration: Suppression of neuronal differentiation genes with failure to downregulate pluripotency and progenitor markers .

The most effective research models include:

• Patient-derived iPSCs differentiated into neurons, which recapitulate the neuronal maturation defects

• Cerebral organoids, which demonstrate the three-dimensional tissue organization defects with disrupted neuronal layering and reduced cortical thickness

• CRISPR/Cas9-corrected isogenic controls, which confirm the specificity of observed phenotypes to SURF1 mutations

What techniques provide the most accurate assessment of mitochondrial function in SURF1-deficient models?

For comprehensive mitochondrial assessment:

Multiple techniques should be employed in parallel, as SURF1 deficiency can impact various aspects of mitochondrial function beyond just complex IV activity.

What are the latest approaches for genetic correction of SURF1 mutations in cellular models?

Current genetic correction strategies include:

• CRISPR/Cas9 biallelic correction: Successfully employed to repair point mutations in patient-derived iPSCs, restoring SURF1 expression and function .

• Gene augmentation: Introduction of wild-type SURF1 can improve NPC bioenergetics and promote neurogenesis even without correcting the underlying mutation .

• Base editing: Newer CRISPR approaches allow for precise nucleotide changes without double-strand breaks.

• Prime editing: Enables targeted insertions, deletions, and all possible base-to-base conversions without requiring donor DNA templates.

Research indicates that correction at the neural progenitor stage may be sufficient to improve neuronal differentiation and function, suggesting a potential therapeutic window before permanent neurological damage occurs .

How can findings from SURF1-like protein research inform therapeutic strategies for Leigh syndrome?

Research on SURF1 and related proteins provides several therapeutic directions:

• Gene therapy approaches: SURF1 gene augmentation has demonstrated efficacy in cellular models, improving NPC bioenergetics and promoting neurogenesis .

• Drug repurposing: Screening efforts using yeast models have identified compounds that may bypass or compensate for SURF1 deficiency .

• Metabolic bypasses: Compounds that provide alternative energy sources for neurons could potentially alleviate the bioenergetic deficits.

• Developmental timing considerations: Interventions targeting neural progenitor stage may be most effective, suggesting early therapeutic intervention is critical .

The identification of neurogenesis as a central pathological mechanism in SURF1-related Leigh syndrome represents a paradigm shift from viewing it as purely a bioenergetic disorder to recognizing its developmental component .

What ethical and methodological considerations should researchers address when using patient-derived samples for SURF1 research?

Researchers working with patient samples must address:

• Informed consent: Ensure comprehensive consent for derivation of iPSCs and their differentiation into various cell types.

• Return of results: Establish protocols for handling incidental findings.

• Data sharing: Balance open science principles with patient privacy.

• Genetic manipulation: Consider ethical implications of genetic correction in patient-derived cells.

• Patient diversity: Include samples from diverse genetic backgrounds to ensure findings are broadly applicable.

Methodologically, researchers should:

• Generate multiple clonal lines per patient to account for clone-to-clone variability

• Include isogenic controls via CRISPR correction to isolate the effects of SURF1 mutations

• Validate findings across multiple patient lines with different SURF1 mutations

• Consider the impact of epigenetic memory in iPSC-derived models

What emerging technologies might advance our understanding of uncharacterized SURF1-like proteins?

Cutting-edge approaches for future research include:

• Cryo-electron microscopy: Resolve the structure of SURF1-like proteins and their interactions with assembly factors.

• Single-cell multi-omics: Investigate cell-type-specific responses to SURF1 deficiency.

• Human brain organoids with vascularization: Develop more mature models of neuronal networks affected by SURF1 deficiency.

• In vivo gene editing: Test therapeutic approaches in relevant animal models.

• Systems biology approaches: Integrate transcriptomic, proteomic, and metabolomic data to identify key regulatory nodes in SURF1-deficient cells.

• AI-driven drug discovery: Apply machine learning to identify novel therapeutic compounds targeting SURF1-like protein pathways.

These technologies could help bridge the gap between basic research and clinical applications, particularly in understanding why neurons are especially vulnerable to SURF1 deficiency.

How might researchers address the challenge of patent restrictions when developing experimental tools for SURF1 research?

When navigating patent considerations:

• Understand the legal landscape: The research exemption for patents is more limited than commonly believed; using patented technology as part of a broader research program is NOT necessarily exempted .

• Material Transfer Agreements (MTAs): Negotiate clear terms for research use of patented materials or methods.

• Alternative approaches: Develop novel methods that achieve similar experimental goals without infringing on patents.

• Collaborative arrangements: Partner with patent holders to advance research with clear agreements on intellectual property.

• Open science initiatives: Participate in consortia that establish pre-competitive open access to research tools.

Researchers should consult with institutional intellectual property offices before developing tools that might be covered by existing patents, as the 2002 Madey v. Duke University case clarified that academic research is not automatically exempt from patent infringement .