Recombinant Human UPF0723 protein C11orf83 (C11orf83)

What is UPF0723 protein C11orf83 and what are its alternative names?

C11orf83 was initially identified as an uncharacterized protein encoded by an open reading frame on chromosome 11. Further characterization has established that this protein, now also known as UQCC3 (ubiquinol-cytochrome c reductase complex assembly factor 3), is a functional component of the mitochondrial respiratory chain. The protein plays dual roles in cellular physiology: it functions in the assembly of the bc1 complex (complex III) of the electron transport chain and independently serves as an antiviral protein .

The protein has been referenced in scientific literature under several names:

• C11orf83 (Chromosome 11 Open Reading Frame 83)

• UQCC3 (Ubiquinol-Cytochrome c Reductase Complex Assembly Factor 3)

• UPF0723 protein C11orf83 homolog

For comprehensive literature searches, researchers should use all these designations to ensure complete retrieval of relevant information.

Where is C11orf83 localized in cells and what is its structure?

C11orf83 is specifically localized to the mitochondrial inner membrane, with its functional domains facing the intermembrane space . This precise localization is essential for its role in bc1 complex assembly and stabilization.

The protein structure includes:

• A transmembrane domain anchoring it to the inner mitochondrial membrane

• Key α-helices (specifically α-helices 2 and 3) that mediate binding to cardiolipin, a critical phospholipid for mitochondrial membrane structure and function

• Regions that interact with components of the bc1 complex during assembly

The protein shows structural and functional similarities to Cbp4p, a yeast bc1 complex assembly factor, suggesting evolutionary conservation of function. This homology has led researchers to propose that C11orf83/UQCC3 is the functional human equivalent of Cbp4p .

What are the optimal expression systems for recombinant C11orf83 production?

The selection of an expression system for recombinant C11orf83 depends on research objectives, particularly regarding protein yield, turnaround time, and the importance of posttranslational modifications.

How does C11orf83 contribute to mitochondrial function?

C11orf83/UQCC3 plays multiple essential roles in mitochondrial function:

• Assembly of bc1 complex (Complex III): C11orf83 is involved in the early stages of bc1 complex assembly by stabilizing the bc1 core complex . This function is essential for the proper formation of the electron transport chain, critical for cellular respiration and ATP production.

• Stabilization of respiratory supercomplexes: Beyond its role in bc1 complex assembly, C11orf83 contributes to the stabilization of bc1 complex-containing supercomplexes, especially the III2/IV supercomplex . These supercomplexes enhance the efficiency of electron transfer during oxidative phosphorylation.

• Cardiolipin binding: C11orf83 binds to cardiolipin through its α-helices 2 and 3 . This interaction may contribute to the proper organization of the inner mitochondrial membrane and positioning of respiratory complexes.

Experimental evidence for these functions comes from depletion studies: When C11orf83 is depleted in cells, researchers observe:

• Abnormal crista morphology in mitochondria

• Decreased ATP levels due to impaired respiration

• Subtle but significant changes in cardiolipin composition

• Higher sensitivity to apoptosis

What antiviral properties does C11orf83 possess?

C11orf83 exhibits significant antiviral activity independent of the classical interferon-mediated antiviral response. Key aspects include:

• Increased expression during viral infection: C11orf83 expression significantly increases in response to viral infection, suggesting it forms part of the cellular defense mechanism against viruses .

• Inhibition of viral replication: Cells with higher expression of C11orf83 demonstrate enhanced capability to inhibit viral replication, while deletion of C11orf83 makes cells more vulnerable to viral infection and killing .

• Activation of OAS3-RNase L system: C11orf83's antiviral effect is primarily mediated through triggering the OAS3-RNase L system, a known antiviral pathway that degrades viral RNA .

• Interferon independence: Remarkably, the signaling from C11orf83 to the OAS3-RNase L system operates independently of interferon production, representing an alternative antiviral pathway .

This dual functionality of C11orf83 as both a mitochondrial protein and an antiviral factor highlights an intriguing connection between cellular metabolism and viral defense mechanisms.

How does C11orf83 depletion affect cellular metabolism and mitochondrial structure?

C11orf83 depletion has profound effects on both mitochondrial structure and cellular metabolism, providing insights into the protein's importance.

Impact on mitochondrial structure:
When C11orf83 is depleted in cells, electron microscopy reveals significant abnormalities in mitochondrial crista morphology . These structural changes likely result from both:

• Altered assembly of the bc1 complex and respiratory supercomplexes

• Changes in cardiolipin composition and distribution in the inner mitochondrial membrane

Impact on cellular metabolism:
C11orf83 depletion leads to:

• Decreased ATP levels due to impaired respiration, reflecting dysfunction of the electron transport chain

• Potential metabolic reprogramming toward increased glycolysis to compensate for reduced oxidative phosphorylation

• Higher sensitivity to apoptosis, suggesting compromised mitochondrial integrity

Methodological approaches for comprehensive analysis:

To thoroughly investigate the impact of C11orf83 depletion, researchers should employ a multi-faceted approach:

• Structural analysis:

  • Transmission electron microscopy to evaluate crista morphology

  • Super-resolution microscopy with appropriate markers to assess mitochondrial network dynamics

  • Quantitative image analysis to measure mitochondrial morphological parameters

• Functional assessment:

  • Respirometry to measure oxygen consumption rate and extracellular acidification rate

  • ATP assays to quantify cellular energy production

  • Membrane potential measurements using potentiometric dyes

  • ROS measurements to assess oxidative stress as a consequence of respiratory chain dysfunction

• Metabolic profiling:

  • Targeted metabolomics focusing on TCA cycle intermediates

  • Isotope tracing to track metabolic flux through key pathways

  • Analysis of NAD+/NADH and ATP/AMP ratios as indicators of cellular energy status

What is the mechanism of C11orf83's interaction with cardiolipin and why is it significant?

C11orf83 binds specifically to cardiolipin, a unique phospholipid found predominantly in the inner mitochondrial membrane, through its α-helices 2 and 3 . This interaction is crucial for both C11orf83 function and mitochondrial membrane organization.

Mechanism of interaction:
The binding between C11orf83 and cardiolipin likely involves:

• Electrostatic interactions between positively charged residues in the α-helices and the negatively charged phosphate groups of cardiolipin

• Potential hydrophobic interactions with the acyl chains of cardiolipin

• Specific recognition of the unique dimeric structure of cardiolipin

Functional significance:
This interaction is significant for several reasons:

• Supercomplex stabilization: C11orf83's binding to cardiolipin contributes to the stabilization of bc1 complex-containing supercomplexes, especially the III2/IV supercomplex . Cardiolipin is required for respiratory supercomplex formation, and C11orf83 may serve as a bridge between cardiolipin and protein components.

• Membrane organization: By interacting with cardiolipin, C11orf83 likely participates in organizing the inner mitochondrial membrane, particularly in regions where respiratory complexes assemble.

• Reciprocal regulation: While C11orf83 binds to cardiolipin, the depletion of C11orf83 leads to changes in cardiolipin composition, suggesting a complex regulatory relationship .

• Mitochondrial quality control: The OMA1-mediated cleavage of C11orf83 in response to mitochondrial depolarization may be linked to changes in cardiolipin distribution, potentially serving as a mechanism for detecting mitochondrial damage .

How does the OMA1-mediated cleavage of C11orf83 regulate mitochondrial quality control?

The OMA1 metalloprotease cleaves C11orf83 in response to mitochondrial depolarization, suggesting a role in mitochondrial quality control and the selection of damaged mitochondria for elimination through apoptosis .

Mechanism of OMA1-mediated cleavage:

• OMA1 is a zinc metalloprotease located in the inner mitochondrial membrane

• Under normal conditions, OMA1 exists in an inactive form

• Mitochondrial stress, particularly membrane depolarization, activates OMA1

• Activated OMA1 cleaves C11orf83, likely altering its function

• This process parallels OMA1's role in cleaving OPA1, a protein involved in mitochondrial fusion and cristae remodeling

Functional significance for mitochondrial quality control:

• Disruption of bc1 complex assembly: Cleavage of C11orf83 likely impairs its ability to participate in bc1 complex assembly, potentially preventing further assembly of respiratory complexes in damaged mitochondria

• Altered supercomplex stability: The cleavage may destabilize existing supercomplexes, contributing to cristae remodeling during apoptosis

• Selection of damaged mitochondria: This mechanism may help mark severely damaged mitochondria for subsequent elimination by mitophagy or trigger cellular elimination by apoptosis

Methodological approaches to study this process:

• Cleavage site identification:

  • Mass spectrometry analysis of C11orf83 fragments after OMA1 activation

  • Generation of cleavage site mutants resistant to OMA1 processing

  • Time-course analysis of C11orf83 cleavage after mitochondrial depolarization

• Integration with mitochondrial quality control:

  • Analysis of the relationship between C11orf83 cleavage and mitophagy induction

  • Investigation of the temporal relationship between C11orf83 cleavage and apoptotic events

  • Evaluation of the impact of C11orf83 cleavage on mitochondrial recovery mechanisms

What are the methodological approaches for studying C11orf83-mediated antiviral effects?

C11orf83 functions as a novel antiviral protein through the OAS3-RNase L pathway, independent of interferon production . Studying this unique mechanism requires specialized approaches.

Comprehensive experimental workflow:

• Modulation of C11orf83 expression:

  • Overexpression systems using plasmid transfection or viral vectors

  • Knockdown approaches using siRNA, shRNA, or CRISPR/Cas9

  • Generation of stable cell lines with controlled expression levels

  • Inducible expression systems to study temporal effects

• Viral challenge models:

  • Selection of appropriate virus models (RNA viruses are particularly relevant for OAS3-RNase L studies)

  • Optimization of viral infection protocols

  • Measurement of viral replication using plaque assays, qPCR, immunofluorescence, or reporter systems

• Analysis of OAS3-RNase L pathway activation:

  • Quantification of OAS3 expression levels

  • Assessment of OAS3 enzymatic activity

  • Measurement of RNase L activation through RNA degradation analysis

  • OAS3 or RNase L knockdown to confirm pathway dependency

• Interferon-independence verification:

  • Use of interferon receptor knockout cells or blocking antibodies

  • JAK inhibitor treatment to block interferon signaling

  • Measurement of interferon production

  • Analysis of interferon-stimulated gene induction

Key experimental controls and considerations:

How do posttranslational modifications affect C11orf83 function and preservation in recombinant expression systems?

Posttranslational modifications (PTMs) are likely critical for C11orf83's proper function. Understanding and preserving these modifications in recombinant expression systems is essential for studying the protein's authentic activities.

Potential posttranslational modifications of C11orf83:

• N-terminal processing: As a mitochondrial protein, C11orf83 likely undergoes cleavage of a mitochondrial targeting sequence upon import

• Phosphorylation: Potential regulation in response to changes in cellular energy status

• Other modifications: May include acetylation, ubiquitination, or oxidative modifications given its mitochondrial localization

Strategies for preserving PTMs in recombinant expression systems:

While E. coli and yeast offer the best yields and shorter turnaround times , expression in insect cells with baculovirus or mammalian cells can provide many of the posttranslational modifications necessary for correct protein folding and activity . When conducting studies that depend on C11orf83's native function, researchers should carefully consider the expression system to ensure the preservation of critical modifications.