Recombinant Drosophila melanogaster UPF0466 protein CG17680, mitochondrial (CG17680)

What is UPF0466 protein CG17680 and what are its key identifiers?

UPF0466 protein CG17680 is a mitochondrial protein found in Drosophila melanogaster (fruit fly). It is identified through several database entries:

• UniProt ID: Q7JX57

• SwissProt ID: U466_DROME

• NCBI gene ID: 37071

• FlyBase ID: FBgn0062440

• Also known as EMRE (Essential MCU regulator, mitochondrial)

The protein is a member of the UPF (Uncharacterized Protein Family) 0466 classification, indicating it was initially identified without a clear function assigned. Current research suggests it functions as an essential regulator of the mitochondrial calcium uniporter (MCU) complex .

How is recombinant CG17680 typically produced for research purposes?

Recombinant CG17680 is typically produced in E. coli expression systems. Based on commercial protocols, the protein:

• Is expressed with an N-terminal His-tag for purification

• Encompasses the full-length mature protein (amino acids 36-97)

• Is usually purified to >90% homogeneity as determined by SDS-PAGE

• Is provided as a lyophilized powder for stability

• Should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Is typically stored in Tris/PBS-based buffer with 6% Trehalose (pH 8.0)

For long-term storage, the addition of 5-50% glycerol and aliquoting before freezing at -20°C/-80°C is recommended to prevent protein degradation from freeze-thaw cycles .

What is the relationship between CG17680 and mitochondrial calcium regulation?

CG17680, also known as EMRE (Essential MCU regulator, mitochondrial), functions as a critical component of the mitochondrial calcium uniporter (MCU) complex. The MCU complex is responsible for calcium uptake into the mitochondrial matrix, a process central to:

• Regulation of mitochondrial metabolism

• Cellular energy production

• Calcium homeostasis

• Cell death/apoptotic pathways

The protein contains a highly acidic C-terminal domain that faces the mitochondrial matrix and is thought to be involved in calcium sensing. Research indicates that EMRE/CG17680 may serve as both a regulator and scaffolding protein that facilitates the assembly of functional MCU complexes .

How does CG17680/EMRE compare with its orthologs in other species?

CG17680 has identified orthologs in several species, including:

While the sequence conservation may not be extremely high across all species, the functional conservation of EMRE-like proteins in the MCU complex appears to be significant. This makes Drosophila CG17680 a valuable model for studying fundamental aspects of mitochondrial calcium regulation relevant to human biology .

What phenotypes are associated with CG17680 mutations in Drosophila?

While the search results don't provide specific phenotypic data for CG17680 mutants, research on mitochondrial calcium uniporter components suggests potential phenotypes might include:

• Altered mitochondrial membrane potential

• Disrupted calcium homeostasis

• Metabolic abnormalities

• Developmental defects

• Potential neurological phenotypes (given the importance of calcium signaling in neurons)

• Changes in stress response and lifespan

These predictions are based on the known importance of mitochondrial calcium regulation across species rather than direct evidence for CG17680 specifically, highlighting a gap in the current literature .

What are the recommended protocols for studying CG17680 function in vitro?

For in vitro studies of CG17680 function, researchers typically employ the following methodologies:

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation with other MCU complex components

  • Yeast two-hybrid screening

  • Proximity labeling techniques (BioID, APEX)

  • Surface plasmon resonance for binding kinetics

• Structural Analysis:

  • Circular dichroism spectroscopy to assess secondary structure

  • Limited proteolysis to identify domains

  • NMR for solution structure (challenging for membrane proteins)

  • Reconstitution in liposomes for functional assays

• Calcium Transport Assays:

  • Reconstitution of MCU complex with purified components in liposomes

  • Calcium flux measurements using fluorescent indicators

  • Patch-clamp electrophysiology of reconstituted channels

How can CRISPR-Cas9 technologies be applied to study CG17680 in vivo?

CRISPR-Cas9 gene editing provides powerful approaches to study CG17680 in Drosophila:

• Knockout Generation:

  • Design gRNAs targeting coding regions of CG17680

  • Create precise deletions or insertions to generate null alleles

  • Validate knockout efficiency by qPCR and Western blot

• Knock-in Approaches:

  • Tag endogenous CG17680 with fluorescent proteins for localization studies

  • Introduce specific point mutations to assess structure-function relationships

  • Create humanized versions by replacing with human ortholog sequences

• Tissue-Specific Manipulation:

  • Combine with GAL4-UAS system to achieve spatial control

  • Use temperature-sensitive or drug-inducible Cas9 for temporal control

  • Generate conditional alleles with loxP sites for tissue-specific deletion

• Validation and Controls:

  • Include off-target analysis

  • Perform rescue experiments with wild-type constructs

  • Use multiple independent lines to confirm phenotypes

What methods are effective for studying the impact of CG17680 on mitochondrial function?

To assess the impact of CG17680 on mitochondrial function, researchers can employ:

• Mitochondrial Calcium Measurements:

  • Genetically-encoded calcium indicators targeted to mitochondria (mito-GCaMP)

  • Calcium-sensitive dyes (Rhod-2, Rhod-FF) with mitochondrial localization

  • Dual-wavelength ratiometric imaging for quantitative measurements

• Mitochondrial Physiology Assays:

  • Oxygen consumption rate measurements (respirometry)

  • Membrane potential assessment using potentiometric dyes (TMRM, JC-1)

  • Reactive oxygen species (ROS) production using fluorescent indicators

  • ATP production assays

• Morphological Analysis:

  • Electron microscopy for ultrastructural changes

  • Super-resolution microscopy for dynamic assessment

  • Mitochondrial network analysis using automated image processing

• Integration with Omics Approaches:

  • Proteomics to identify changes in mitochondrial protein composition

  • Metabolomics to assess impact on TCA cycle intermediates

  • Transcriptomics to identify compensatory responses

How should contradictory findings about CG17680 function be approached?

When encountering contradictory findings regarding CG17680 function, researchers should systematically:

• Evaluate Experimental Conditions:

  • Compare protein expression levels across studies (overexpression vs. endogenous)

  • Assess differences in model systems (cell lines, tissue types, developmental stages)

  • Examine environmental conditions (temperature, nutrient availability, stress)

• Consider Technical Variations:

  • Evaluate antibody specificity and validation

  • Compare detection methods and their sensitivity

  • Assess the impact of tags on protein function

• Integrate Multiple Approaches:

  • Combine genetic, biochemical, and physiological evidence

  • Perform epistasis experiments to place CG17680 in a pathway context

  • Use structure-function analysis to reconcile different observations

• Statistical Considerations:

  • Reassess statistical power across studies

  • Consider different statistical models for data interpretation

  • Perform meta-analysis when multiple datasets are available

What are the key considerations when designing controls for CG17680 functional studies?

Robust control design is essential for CG17680 functional studies:

• Genetic Controls:

  • Use multiple independent CRISPR knockout lines

  • Include precise revertants that restore the wild-type sequence

  • For transgenic rescue, include both active and inactive versions of the protein

• Expression Controls:

  • Match expression levels between experimental and control conditions

  • Monitor subcellular localization to ensure proper targeting

  • Assess stability of mutant proteins compared to wild-type

• Functional Controls:

  • Include positive controls that demonstrate assay sensitivity

  • Use both gain-of-function and loss-of-function approaches

  • Perform parallel analysis of known MCU complex components

• Environmental Controls:

  • Standardize temperature, media composition, and culture conditions

  • Control for circadian effects (particularly relevant in Drosophila)

  • Account for genetic background effects through backcrossing

How can machine learning approaches enhance the analysis of CG17680 structure-function relationships?

Machine learning techniques offer powerful tools for CG17680 research:

• Structural Prediction:

  • Deep learning models can predict protein structure from sequence

  • Identify potential binding interfaces and regulatory motifs

  • Model conformational changes upon calcium binding or protein interaction

• Functional Classification:

  • Train models to identify patterns in calcium flux data

  • Classify mitochondrial morphological changes from microscopy images

  • Predict functional impact of mutations based on evolutionary conservation

• Systems-Level Integration:

  • Network analysis to place CG17680 in the broader context of mitochondrial function

  • Identify potential compensatory mechanisms in genetic perturbation experiments

  • Predict off-target effects of experimental manipulations

• Implementation Considerations:

  • Requires sufficient training data specific to mitochondrial proteins

  • Demands rigorous validation of model predictions

  • Benefits from cross-disciplinary collaboration between wet-lab and computational researchers

How can CG17680 research inform our understanding of mitochondrial diseases?

Research on CG17680/EMRE has significant implications for understanding mitochondrial diseases:

• Translational Relevance:

  • Drosophila CG17680 studies can provide insights into the role of human EMRE in disease

  • Identify conserved mechanisms of calcium dysregulation in pathological states

  • Develop potential therapeutic strategies targeting MCU complex assembly

• Disease Modeling:

  • Engineer Drosophila CG17680 to mimic human disease-associated mutations

  • Assess how altered calcium homeostasis contributes to neurodegeneration

  • Investigate connections to metabolic disorders given mitochondrial calcium's role in metabolism

• Therapeutic Exploration:

  • Screen for compounds that modulate CG17680/MCU function

  • Assess whether manipulating mitochondrial calcium can ameliorate disease phenotypes

  • Develop gene therapy approaches for EMRE-related disorders

What are the emerging technologies that could advance CG17680 research?

Several cutting-edge technologies hold promise for furthering CG17680 research:

• Advanced Imaging:

  • Cryo-electron microscopy for high-resolution structural analysis

  • Live-cell super-resolution microscopy to track protein dynamics

  • Correlative light and electron microscopy to connect function and structure

• Genetic Technologies:

  • Base editing for precise mutation introduction

  • Optogenetic control of CG17680 function

  • Single-cell transcriptomics to assess cell-specific responses

• Biochemical Innovations:

  • Nanobody development for improved detection and manipulation

  • Advanced mass spectrometry for post-translational modification analysis

  • Microfluidic platforms for high-throughput functional screening

• Computational Approaches:

  • Molecular dynamics simulations of the MCU complex

  • Integrative modeling combining diverse experimental data

  • Systems biology approaches to model calcium dynamics