Recombinant Mouse UPF0466 protein C22orf32 homolog, mitochondrial

What is UPF0466 protein C22orf32 homolog and what is its function in mitochondrial biology?

UPF0466 protein C22orf32 homolog, also known as SMDT1 (Single-pass membrane protein with aspartate-rich tail 1, mitochondrial), functions as an Essential MCU (Mitochondrial Calcium Uniporter) regulator. This protein plays a critical role in mitochondrial calcium handling and homeostasis, which is fundamental to cellular energy production and signaling pathways. Research has established that this protein contains a distinctive aspartate-rich C-terminal domain that contributes to its functional properties in regulating calcium flux across the inner mitochondrial membrane . The protein's involvement in the MCU complex suggests it may serve as an important regulatory component in mitochondrial calcium uptake, which has implications for multiple cellular processes including metabolism, cell death, and signaling.

How is the mouse UPF0466 protein C22orf32 homolog structurally similar to homologs in other species?

Comparative analysis between mouse UPF0466 protein C22orf32 homolog and its counterparts in other species reveals notable structural conservation. For instance, the Xenopus laevis (African clawed frog) homolog contains 60 amino acids in its mature form (residues 38-97) with the sequence VIASSAGAILPKPEKVSFGLLRVFTVVIPFLYIGTLISKNFAAVLEEHDIFVPEDDDDDD . This sequence demonstrates the characteristic aspartate-rich tail that is conserved across species. Both mouse and Xenopus variants are classified as mitochondrial proteins, suggesting evolutionary conservation of subcellular localization and potentially similar functional roles in mitochondrial calcium regulation across vertebrate species. The high degree of conservation indicates the protein's fundamental importance in mitochondrial physiology throughout evolutionary history.

What expression patterns does UPF0466 protein C22orf32 homolog exhibit across different tissue types?

While comprehensive tissue-specific expression data for mouse UPF0466 protein C22orf32 homolog is still emerging, research indicates that this protein is expressed in multiple tissues with particular abundance in metabolically active cells. Given its role in mitochondrial function, expression levels tend to correlate with tissues that have high energy demands, such as cardiac and skeletal muscle, neuronal tissue, and liver. Expression patterns may vary during development and in response to metabolic demands or stress conditions. For experimental design considerations, researchers should note that baseline expression levels should be established for each tissue of interest when conducting comparative studies, as this provides essential context for interpreting experimental results related to altered expression under different physiological or pathological conditions.

What are the optimal storage and reconstitution protocols for recombinant UPF0466 protein C22orf32 homolog?

For optimal preservation of recombinant UPF0466 protein C22orf32 homolog activity, the protein should be stored according to the following protocol: Store lyophilized powder at -20°C/-80°C upon receipt, with aliquoting necessary for multiple use scenarios to avoid repeated freeze-thaw cycles which can compromise protein integrity . For reconstitution, it is recommended to briefly centrifuge the vial prior to opening to ensure all content is at the bottom. The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . The addition of glycerol to a final concentration of 25-50% is recommended for long-term storage at -20°C/-80°C, with 50% being the standard concentration for optimal stability . The reconstituted protein should be stored in working aliquots at 4°C for no more than one week to maintain functional integrity.

How can researchers design siRNA knockdown experiments to study UPF0466 protein C22orf32 homolog function in vitro?

When designing siRNA knockdown experiments for UPF0466 protein C22orf32 homolog, researchers should begin with multiple siRNA constructs targeting different regions of the transcript to identify the most effective construct. Based on methodologies applied to similar proteins, an effective approach involves transfecting cells with candidate siRNAs and measuring knockdown efficiency via RT-qPCR and western blot analysis. Studies with related proteins have demonstrated that knockdown efficiency of >50% can be achieved with optimized siRNA constructs . Researchers should include appropriate controls, such as non-targeting siRNA (si-NC) to account for non-specific effects of the transfection process. A time-course analysis (24h, 48h, and 72h post-transfection) is recommended to determine the optimal time point for functional assays, as protein knockdown effects may become more pronounced at later time points . The functional consequences of knockdown should be assessed through mitochondrial calcium flux assays, mitochondrial membrane potential measurements, ATP production, and cell viability assays.

How does UPF0466 protein C22orf32 homolog contribute to mitochondrial dysfunction in disease models?

UPF0466 protein C22orf32 homolog (SMDT1) plays a critical role in regulating mitochondrial calcium homeostasis, making its dysfunction potentially significant in multiple disease pathologies. Altered expression or function of this protein may contribute to pathological calcium overload or deficient calcium signaling in mitochondria. While specific disease associations for UPF0466 protein C22orf32 homolog are still being elucidated, research on related mitochondrial proteins suggests potential involvement in neurodegenerative conditions, cardiovascular diseases, and metabolic disorders. Investigation of this protein in disease contexts should employ both in vitro cellular models and in vivo animal models with genetic modifications or disease-relevant stressors. Researchers should consider examining not only expression levels but also post-translational modifications and protein-protein interactions that may be altered in disease states. The contribution of UPF0466 protein C22orf32 homolog to mitochondrial permeability transition pore opening, a key event in cell death pathways, represents an important area for investigation in disease models.

What are the current challenges in characterizing post-translational modifications of UPF0466 protein C22orf32 homolog?

Characterizing post-translational modifications (PTMs) of UPF0466 protein C22orf32 homolog presents several significant challenges. The protein's small size and mitochondrial localization make isolation and enrichment technically demanding. Current methodologies for studying PTMs of this protein should incorporate mass spectrometry-based approaches, which require careful sample preparation to maintain modification integrity. Potential phosphorylation sites, particularly on serine and threonine residues, may regulate the protein's interaction with other MCU complex components. Researchers should consider employing phosphorylation-specific antibodies or phospho-proteomic approaches to identify modification sites. Additionally, the aspartate-rich tail may undergo modifications that affect calcium binding properties. Site-directed mutagenesis of potential modification sites combined with functional assays can help determine the physiological relevance of identified PTMs. The development of modification-specific antibodies remains an important goal to facilitate more routine analysis of these modifications in experimental and clinical samples.

How can high-throughput screening approaches be optimized to identify small molecule modulators of UPF0466 protein C22orf32 homolog function?

Optimizing high-throughput screening (HTS) for small molecule modulators of UPF0466 protein C22orf32 homolog function requires careful consideration of assay design and screening parameters. Researchers should develop cell-based assays that monitor mitochondrial calcium dynamics as a primary readout, using fluorescent calcium indicators with mitochondrial targeting sequences. The assay should be validated using known modulators of mitochondrial calcium transport before proceeding to large-scale screening. Compound libraries should include molecules with diverse chemical scaffolds and known ability to penetrate mitochondrial membranes. Secondary assays should evaluate effects on mitochondrial membrane potential, ATP production, and cell viability to eliminate compounds with non-specific toxicity. Tertiary validation should include direct binding assays with purified recombinant UPF0466 protein C22orf32 homolog to confirm target engagement. Structure-activity relationship studies of hit compounds can guide optimization for potency and selectivity. This systematic approach will help identify specific modulators of UPF0466 protein C22orf32 homolog function that can serve as valuable research tools and potential therapeutic leads.

How does the function of UPF0466 protein C22orf32 homolog compare with related C22orf32 gene products?

Comparing UPF0466 protein C22orf32 homolog with other C22orf32 gene products reveals important functional distinctions. While UPF0466 protein C22orf32 homolog functions as a mitochondrial regulator protein, the lncRNA C22orf32-1 has been identified as having oncogenic properties, particularly in nasopharyngeal carcinoma (NPC) . Research has demonstrated that lncRNA C22orf32-1 is significantly upregulated in NPC tissues compared to normal nasopharyngeal epithelial tissues . Functional studies using siRNA knockdown of lncRNA C22orf32-1 have shown that it promotes cell proliferation, migration, and invasion while inhibiting apoptosis in NPC cells . These contrasting functions highlight the diversity of gene products from the C22orf32 locus on chromosome 22, with the protein product functioning in mitochondrial regulation and the lncRNA functioning in cancer progression. Researchers should be aware of these distinctions when designing experiments and interpreting results related to C22orf32 gene products.

What experimental models are most appropriate for investigating the evolutionary conservation of UPF0466 protein C22orf32 homolog function?

To investigate evolutionary conservation of UPF0466 protein C22orf32 homolog function, researchers should employ a diverse array of model organisms spanning evolutionary distance. Comparative studies using recombinant proteins from mouse, Xenopus laevis (African clawed frog), and human sources can provide insights into structural and functional conservation . Cell-based systems should include both mammalian cell lines (mouse and human) and amphibian cell lines (such as Xenopus) to assess functional conservation in cellular contexts. Heterologous expression systems, where the protein from one species is expressed in cells from another species, can determine the degree of functional interchangeability. Genetic models including knockout and knockin mice, zebrafish, and other model organisms allow for in vivo assessment of conserved functions. Sequence analysis and structural modeling should complement experimental approaches by identifying conserved domains and motifs across species. Together, these approaches provide a comprehensive understanding of evolutionary conservation and divergence in UPF0466 protein C22orf32 homolog function.

What are the implications of UPF0466 protein C22orf32 homolog research for understanding broader mitochondrial calcium signaling networks?

Research on UPF0466 protein C22orf32 homolog has significant implications for our understanding of mitochondrial calcium signaling networks and their role in cellular homeostasis. As an essential regulator of the Mitochondrial Calcium Uniporter (MCU), this protein represents a key control point in the complex network regulating mitochondrial calcium uptake. Understanding its molecular interactions and regulatory mechanisms provides insights into how cells fine-tune mitochondrial calcium levels in response to various physiological signals. The protein's aspartate-rich tail suggests potential calcium-binding properties that may serve as a calcium-sensing mechanism within the MCU complex. Research on this protein contributes to our understanding of how mitochondrial calcium handling is integrated with other cellular signaling pathways, including those involving reactive oxygen species, ATP production, and apoptotic signaling. Furthermore, elucidating the function of UPF0466 protein C22orf32 homolog may reveal new therapeutic targets for diseases characterized by dysregulated mitochondrial calcium handling, such as neurodegenerative disorders, ischemia-reperfusion injury, and certain cancers.

What are the key specifications for commercially available recombinant UPF0466 protein C22orf32 homolog preparations?

The following table summarizes key specifications for commercially available recombinant UPF0466 protein C22orf32 homolog preparations:

This data compilation is based on available information from commercial suppliers . Researchers should verify current specifications with suppliers before designing experiments, as formulations may be updated over time.

What analytical methods are most reliable for verifying the identity and purity of recombinant UPF0466 protein C22orf32 homolog preparations?

For reliable verification of recombinant UPF0466 protein C22orf32 homolog identity and purity, researchers should employ a combination of complementary analytical methods. SDS-PAGE analysis provides basic size verification and purity assessment, with commercial preparations typically showing >90% purity . Western blotting using antibodies against the protein itself or tag epitopes (such as His-tag) confirms identity and can detect potential degradation products. Mass spectrometry offers the most definitive confirmation of protein identity and can verify the amino acid sequence, with MALDI-TOF MS appropriate for molecular weight confirmation and LC-MS/MS providing sequence coverage. For higher-order structure assessment, circular dichroism spectroscopy can verify proper protein folding. Functional assays, such as calcium-binding assays for the aspartate-rich tail region, provide validation of biological activity. Size-exclusion chromatography can detect aggregation states and higher-order structures. For tagged proteins, tag-specific assays (such as nickel-affinity binding for His-tagged proteins) provide additional confirmation of proper expression. These methods should be used in combination for comprehensive characterization of recombinant protein preparations.