Recombinant Bovine UPF0466 protein C22orf32 homolog, mitochondrial

What is the UPF0466 protein C22orf32 homolog and what are its known synonyms?

The UPF0466 protein C22orf32 homolog, known in humans as SMDT1 (single-pass membrane protein with aspartate rich tail 1), is a mitochondrial protein with several recognized synonyms including C22orf32, DDDD, and EMRE (essential MCU regulator, mitochondrial) . This protein is encoded by a gene located at chromosomal position 22q13.2 in humans and is classified as a protein-coding gene . The bovine homolog shares significant structural features while demonstrating species-specific adaptations relevant to comparative mitochondrial research.

What is the relationship between UPF0466 protein C22orf32 and mitochondrial calcium regulation?

The UPF0466 protein C22orf32 (SMDT1/EMRE) functions as an essential regulator of the mitochondrial calcium uniporter (MCU) complex . Unlike outer mitochondrial membrane proteins such as MTCH2, which primarily affect mitochondrial metabolism through different pathways , SMDT1 plays a critical role in calcium handling, which subsequently influences mitochondrial respiration, ATP production, and metabolic activity. Researchers should consider these distinct functional roles when designing experiments comparing different mitochondrial regulatory proteins.

What expression systems are most effective for producing recombinant bovine UPF0466 protein C22orf32?

When producing recombinant bovine UPF0466 protein C22orf32, researchers should evaluate expression systems based on downstream applications. Bacterial systems (E. coli) offer high yields but may struggle with proper protein folding, while insect cell systems provide better post-translational modifications. For functional studies requiring native conformation, mammalian expression systems are recommended despite lower yields. Expression efficiency can be assessed using techniques similar to those used in MTCH2 research, where protein expression is verified via Western blotting with appropriate antibodies .

Research data indicates that membrane proteins with similar characteristics show variable expression efficiency across systems:

What mitochondrial isolation protocols are optimal for studying the native bovine UPF0466 protein C22orf32?

Isolating mitochondria for studying native bovine UPF0466 protein C22orf32 requires careful consideration of tissue source and extraction methodology. For bovine tissue samples, differential centrifugation followed by Percoll gradient purification yields the highest purity mitochondrial fractions. Critical methodological considerations include:

• Tissue homogenization should be performed in isotonic buffer (250mM sucrose, 10mM HEPES, 1mM EDTA, pH 7.4) at 4°C

• Initial centrifugation at 1,000g (10 minutes) removes nuclei and debris

• Supernatant centrifugation at 10,000g (15 minutes) yields crude mitochondrial pellet

• Percoll gradient purification (20-40%) removes contaminating organelles

• Purity assessment via Western blotting for mitochondrial markers (COX) and contaminant markers

Modifications to this protocol may be necessary when working with specific bovine tissues that contain higher lipid content or differ in mitochondrial density.

How can researchers effectively measure the impact of recombinant bovine UPF0466 protein C22orf32 on mitochondrial function?

To comprehensively characterize the impact of recombinant bovine UPF0466 protein C22orf32 on mitochondrial function, researchers should employ a multiparametric approach similar to that used in MTCH2 studies . Key measurements should include:

• Mitochondrial respiration: Using Seahorse XF analyzers to measure oxygen consumption rate (OCR) in both basal and maximal conditions

• Mitochondrial complex activities: Particularly complex-I activity, which showed significant reduction upon MTCH2 silencing in NSCLC cells

• ATP production: Quantitative luminescence-based assays to assess cellular energy status

• Mitochondrial membrane potential: Using JC-1 dye to detect shifts from red aggregates (polarized) to green monomers (depolarized)

• ROS production: Measuring reactive oxygen species using fluorescent probes like CellROX or DCF-DA

In studies of MTCH2, a mitochondrial protein with distinct function from SMDT1, researchers documented the following changes upon protein knockdown:

A similar experimental approach could be applied to studying UPF0466 protein C22orf32.

What molecular techniques are most effective for analyzing UPF0466 protein C22orf32 interactions with other mitochondrial proteins?

For analyzing protein-protein interactions involving recombinant bovine UPF0466 protein C22orf32, researchers should implement complementary approaches:

• Co-immunoprecipitation (Co-IP) with antibodies against UPF0466 protein C22orf32 or suspected interaction partners

• Proximity ligation assay (PLA) for in situ visualization of protein interactions within the mitochondrial environment

• FRET/BRET analysis for live-cell interaction studies

• Cross-linking mass spectrometry (XL-MS) to identify direct binding interfaces

• Blue native PAGE to preserve native protein complexes during electrophoretic separation

These techniques should be complemented with functional assays to correlate physical interactions with physiological outcomes, similar to the integrated approach used in studies of other mitochondrial proteins .

What are the most effective strategies for CRISPR/Cas9-mediated manipulation of bovine UPF0466 protein C22orf32 expression?

Based on approaches documented for other mitochondrial proteins like MTCH2 , effective CRISPR/Cas9-mediated manipulation of bovine UPF0466 protein C22orf32 requires careful consideration of:

• Guide RNA (gRNA) design: Target conserved exonic regions, avoiding regions with potential off-target effects

• Delivery method: Lentiviral vectors for stable Cas9 expression followed by puromycin selection, as demonstrated effective for MTCH2 knockout

• Single-cell selection: Isolate and screen single-cell-derived selections to ensure complete knockout

• Validation: Confirm knockout via Western blotting, with particular attention to specific elimination of UPF0466 protein C22orf32 without affecting related proteins (similar to MTCH2 knockout validation, which showed no effect on MTCH1)

• Functional assessment: Evaluate phenotypic consequences including mitochondrial parameters

For comparative purposes, the approach used with MTCH2 resulted in complete protein elimination while MTCH1 expression remained unaffected , demonstrating the specificity achievable with properly designed CRISPR-based approaches.

How can researchers develop stable cell lines for studying long-term effects of UPF0466 protein C22orf32 overexpression?

To develop stable cell lines overexpressing bovine UPF0466 protein C22orf32, researchers should:

• Clone the full-length cDNA into a mammalian expression vector containing a strong promoter (CMV/EF1α)

• Include a selection marker (puromycin/neomycin resistance) for stable integration

• Perform lentiviral transduction for efficient delivery, as successfully employed in MTCH2 overexpression studies

• Select stable cells through antibiotic pressure

• Validate expression through qRT-PCR and Western blotting

• Characterize functional consequences through mitochondrial assays

In studies of MTCH2, researchers demonstrated substantial elevation of mRNA levels in transduced cells without affecting related genes (e.g., MTCH1 mRNA remained unaffected) . Similar approaches would be applicable to UPF0466 protein C22orf32 studies.

How does recombinant bovine UPF0466 protein C22orf32 compare with human SMDT1 in functional assays?

Cross-species comparisons between bovine UPF0466 protein C22orf32 and human SMDT1 should examine both structural and functional conservation. Methodologically, researchers should:

• Perform sequence alignment and structural prediction to identify conserved domains

• Express both proteins recombinantly under identical conditions

• Compare functional parameters including calcium handling capacity, interaction with MCU complex components, and effects on mitochondrial bioenergetics

• Assess species-specific differences in post-translational modifications

• Evaluate cross-complementation capacity in knockout cellular models

Such comparative analyses would provide insights into evolutionary conservation of mitochondrial calcium regulation mechanisms and identify species-specific adaptations.

What are the methodological considerations for studying UPF0466 protein C22orf32 in the context of metabolic reprogramming?

When investigating UPF0466 protein C22orf32's role in metabolic reprogramming, researchers should implement integrated approaches that connect mitochondrial calcium regulation with broader cellular metabolism. Key methodological considerations include:

• Metabolic flux analysis using isotope-labeled substrates to track carbon routing

• Comprehensive metabolomic profiling before and after manipulation of UPF0466 protein C22orf32 expression

• Assessment of glycolysis versus oxidative phosphorylation balance using Seahorse technology

• Analysis of protein synthesis capacity using RNA/protein ratios as performed in metabolic studies

• Evaluation of substrate preference (lipids, carbohydrates, proteins) under various conditions

In metabolic studies involving other recombinant proteins, researchers documented notable changes in white muscle composition:

Similar methodologies could reveal how UPF0466 protein C22orf32 influences cellular energy metabolism.