Recombinant Human NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 11 (NDUFA11)

What is NDUFA11 and what is its primary function in cellular metabolism?

NDUFA11 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 11) is a subunit of mitochondrial respiratory chain complex I. Its primary function involves the formation of respiratory chain protein complexes within mitochondria. NDUFA11 plays a crucial role in maintaining cellular respiration, and abnormal expression or structural changes in this protein can lead to defects in the cellular respiratory chain . The protein is part of the electron transport chain, which is essential for ATP production through oxidative phosphorylation.

How does NDUFA11 interact with other components of the mitochondrial respiratory chain?

NDUFA11 primarily interacts with NDUFS1 (NADH: ubiquinone oxidoreductase core subunit S1) to form functional protein complexes within the mitochondrial respiratory chain. Protein-protein interaction (PPI) analysis has revealed that NDUFS1 interacts with NDUFA11, NUBPL, and LRPPRC . These interactions are critical for the assembly and function of complex I. Specifically, the NDUFS1-NDUFA11 protein complex plays a significant role in maintaining mitochondrial respiratory chain function. Disruptions in these interactions can lead to respiratory chain dysfunction and potentially contribute to various pathological conditions.

What are the known mutations in NDUFA11 and their functional consequences?

While the search results don't specifically mention mutations in NDUFA11, research on related complex I subunits like NDUFA2 provides insight into how mutations might affect function. For instance, homozygous mutations in NDUFA2 (p.Lys45Glu, p.Glu57Ala) have been found to disrupt binding with NDUFS1, potentially destabilizing complex I . By analogy, mutations in NDUFA11 might similarly affect its interaction with NDUFS1 and other proteins. Patients with NDUFA2 mutations presented with symptoms including epilepsy, movement disorders, abnormal white matter in the brain, and microcephaly , suggesting that mutations in supernumerary subunits of complex I can have serious neurological consequences.

What are the most effective experimental models for studying NDUFA11 function?

Based on current research, two primary experimental models have proven effective for studying NDUFA11 function:

• Oxygen-Glucose Deprivation/Reoxygenation (OGD/R) models: This in vitro model simulates ischemic conditions and has been successfully used to detect changes in NDUFA11 expression. Research has shown significant reduction in NDUFA11 expression in OGD/R models .

• Middle Cerebral Artery Occlusion (MCAO) models: This in vivo model creates ischemic conditions in the brain and has been used to verify NDUFA11 expression changes. Western blot analyses confirmed that NDUFA11 expression decreased significantly in the MCAO group .

Additionally, immunofluorescence (IF) methods have been employed to demonstrate that NDUFA11 is expressed in neurons and cortical regions, making these cell types and tissues suitable for NDUFA11 research .

What techniques are recommended for detecting NDUFA11 protein-protein interactions?

For investigating NDUFA11 protein-protein interactions, the following techniques have proven effective:

• Co-Immunoprecipitation (Co-IP) assay: This technique has been successfully used to analyze the relationship between NDUFS1 and NDUFA11, confirming the formation of protein complexes. In ischemic conditions, Co-IP demonstrated that the NDUFS1-NDUFA11 protein complex decreased significantly .

• Protein-Protein Interaction (PPI) analysis: This computational approach can identify potential interaction partners. PPI analysis revealed that NDUFS1 interacts with NDUFA11, suggesting functional relationships that can be further investigated experimentally .

• Blue Native Gel Electrophoresis (BN-PAGE): Although not explicitly mentioned in the search results, this technique is commonly used to analyze intact protein complexes, including respiratory chain complexes, and would be valuable for studying NDUFA11's interactions within complex I.

How can researchers effectively measure changes in NDUFA11 expression levels?

To accurately measure changes in NDUFA11 expression levels, researchers should employ a combination of the following methods:

• Western Blot analysis: This technique has been effectively used to quantify NDUFA11 protein expression. Studies have demonstrated significant decreases in NDUFA11 expression in both OGD/R and MCAO models .

• RT-PCR (Reverse Transcription Polymerase Chain Reaction): This method has been used to measure NDUFA11 mRNA expression levels in experimental models and clinical samples .

• Immunofluorescence (IF): This technique allows visualization of NDUFA11 expression in specific cell types and tissues, confirming its presence in neurons and cortical regions .

For clinical applications, analyzing NDUFA11 expression in blood samples from patients has proven valuable, with studies showing that NDUFA11 expression in the blood of ischemic stroke patients decreases to approximately 20.9% compared to normal controls .

What is the role of NDUFA11 in ischemic stroke pathophysiology?

NDUFA11 has been identified as a differentially expressed disulfidptosis-related biomarker (DE-DRB) in ischemic stroke (IS). Its expression is significantly downregulated in patients with IS, with levels at approximately 20.9% compared to normal controls . This reduction has been confirmed in both in vitro (OGD/R) and in vivo (MCAO) models of ischemic stroke.

The pathophysiological significance lies in the decrease of the NDUFS1-NDUFA11 protein complex during ischemic conditions, suggesting that IS may damage respiratory chain protein complex I in neuronal mitochondria . This damage can impair cellular oxygen consumption and increase reactive oxygen species (ROS) levels, leading to mitochondrial dysfunction and neuronal death.

Mitochondrial respiratory chain protein complex I is particularly sensitive to ischemic stroke and vulnerable to disruption . The damage to this complex can promote reverse electron transport-derived ROS production, further exacerbating mitochondrial damage and contributing to neuronal injury.

How does NDUFA11 contribute to the process of disulfidptosis?

Disulfidptosis is a novel programmed cell death mechanism resulting from abnormal accumulation of cytotoxic disulfides, which induces disulfide stress, causes breakdown of the filamentous actin network, and leads to cell death . Conventional inhibitors of ferroptosis, apoptosis, necroptosis, and autophagy cannot inhibit this mechanism.

NDUFA11 has been identified as a disulfidptosis-related biomarker (DRB) in ischemic stroke. The connection between NDUFA11 and disulfidptosis likely involves its role in maintaining mitochondrial respiratory chain function:

• Ischemic conditions lead to glucose deficiency, which can cause abnormal accumulation of cytotoxic disulfides.

• Decreased NDUFA11 expression disrupts the formation of the NDUFS1-NDUFA11 protein complex.

• This disruption damages respiratory chain protein complex I in neuronal mitochondria.

• Mitochondrial dysfunction increases ROS production and promotes disulfide stress.

• The resulting disulfide stress triggers disulfidptosis, contributing to neuronal death in ischemic stroke.

The identification of NDUFA11 as a specific DRB for ischemic stroke suggests it plays a critical role in the disulfidptosis pathway during ischemic conditions .

What is the relationship between NDUFA11 and NDUFS1 in normal versus pathological conditions?

In normal conditions, NDUFA11 forms a stable protein complex with NDUFS1, which is essential for the proper functioning of mitochondrial respiratory chain complex I. This interaction is part of the normal assembly and maintenance of the electron transport chain.

In pathological conditions such as ischemic stroke:

• The expression of NDUFA11 is significantly downregulated (to approximately 20.9% of normal levels) .

• The number of formed complexes between NDUFS1 and NDUFA11 decreases significantly, as demonstrated by Co-IP assays in both OGD/R and MCAO models .

• This reduction in NDUFS1-NDUFA11 protein complexes suggests damage to respiratory chain protein complex I in neuronal mitochondria.

• The disruption of this interaction likely contributes to mitochondrial dysfunction, increased ROS production, and neuronal injury.

Interestingly, while NDUFA11 expression and NDUFS1-NDUFA11 complex formation decrease in ischemic conditions, another related interaction, the NDUFS1-LRPPRC protein complex, does not change significantly . This differential effect suggests specific pathways and mechanisms in the pathological response.

How can NDUFA11 be utilized as a biomarker for ischemic stroke?

NDUFA11 has significant potential as a biomarker for ischemic stroke based on the following research findings:

• Blood-based detection: NDUFA11 expression in the blood of patients with ischemic stroke decreases to approximately 20.9% compared to normal controls, making it a potentially accessible biomarker .

• Machine learning applications: Research has identified the Support Vector Machine (SVM) model as the optimal machine-learning model for predicting ischemic stroke using NDUFA11 and other biomarkers. This model demonstrated high accuracy in distinguishing between ischemic stroke patients and healthy controls .

• Specific association with disulfidptosis: NDUFA11 has been identified as a specific disulfidptosis-related biomarker (DRB) for ischemic stroke, distinguishing it from other stroke biomarkers that may be associated with different cell death mechanisms .

To implement NDUFA11 as a clinical biomarker, researchers should consider:

• Using RT-PCR or Western blot analysis for quantitative measurements in blood samples

• Combining NDUFA11 with other biomarkers for improved diagnostic accuracy

• Developing standardized assays suitable for clinical laboratory use

What are the challenges in developing therapeutic strategies targeting NDUFA11?

Developing therapeutic strategies targeting NDUFA11 presents several significant challenges:

• Mitochondrial targeting: As a mitochondrial protein, NDUFA11 is located within the mitochondrial inner membrane, making drug delivery challenging due to the need to cross both the cell membrane and mitochondrial membranes.

• Specificity concerns: Given that NDUFA11 is part of the essential respiratory chain complex I, interventions must be highly specific to avoid disrupting normal mitochondrial function in unaffected tissues.

• Timing of intervention: Since NDUFA11 expression decreases rapidly during ischemic conditions, therapeutic interventions would need to be administered very early in the course of ischemic stroke to be effective.

• Complex interactions: NDUFA11 forms protein complexes with multiple partners, including NDUFS1. Therapeutic strategies would need to account for these interactions and their dynamics during pathological conditions.

Despite these challenges, network pharmacological analysis has identified metformin hydrochloride as a potential target drug for NDUFA11 . This suggests that existing drugs might be repurposed to target NDUFA11-related pathways, potentially offering a more expedient path to therapeutic development.

How might recombinant NDUFA11 be utilized in experimental designs to investigate mitochondrial complex I assembly?

Recombinant NDUFA11 could be utilized in several innovative experimental approaches to investigate mitochondrial complex I assembly:

• Reconstitution experiments: Purified recombinant NDUFA11 could be used to reconstitute complex I assembly in cell-free systems or in cells with NDUFA11 deficiency. This would allow researchers to study the specific role of NDUFA11 in complex I assembly and function.

• Structure-function studies: By creating modified versions of recombinant NDUFA11 with specific mutations or deletions, researchers could identify critical regions or residues involved in protein-protein interactions, particularly with NDUFS1.

• Interaction kinetics analysis: Surface plasmon resonance or isothermal titration calorimetry using recombinant NDUFA11 and its binding partners could reveal the kinetics and thermodynamics of these interactions, providing insights into complex I assembly mechanisms.

• Rescue experiments: Introducing recombinant NDUFA11 into cellular or animal models of ischemic stroke could determine if restoring NDUFA11 levels can prevent or reduce mitochondrial dysfunction and disulfidptosis.

• Protein complex stability studies: Recombinant NDUFA11 could be used to investigate factors affecting the stability of the NDUFS1-NDUFA11 protein complex under various conditions, including oxidative stress, pH changes, or ionic environment alterations that might occur during ischemia.

What omics approaches can be integrated to better understand NDUFA11 function?

An integrated multi-omics approach would provide comprehensive insights into NDUFA11 function:

• Genomics: Whole-genome or targeted sequencing to identify genetic variants in NDUFA11 that may predispose individuals to ischemic stroke or affect mitochondrial function.

• Transcriptomics: RNA-seq analysis to examine changes in gene expression profiles associated with altered NDUFA11 levels, as demonstrated in the GSE16561 and GSE58294 datasets used to identify NDUFA11 as a differentially expressed gene in ischemic stroke .

• Proteomics: Mass spectrometry-based approaches to identify post-translational modifications of NDUFA11 and changes in the mitochondrial proteome in response to NDUFA11 dysregulation.

• Metabolomics: Analysis of metabolic changes associated with NDUFA11 dysfunction, particularly focusing on energy metabolism and oxidative stress markers.

• Interactomics: Comprehensive protein-protein interaction studies to map the entire interactome of NDUFA11, expanding beyond the known interactions with NDUFS1.

Integration of these omics data using systems biology approaches could reveal novel aspects of NDUFA11 function and identify potential therapeutic targets. Current research has already demonstrated the value of integrating bioinformatic analyses with experimental validation to understand NDUFA11's role in disulfidptosis and ischemic stroke .

How do changes in NDUFA11 expression correlate with clinical outcomes in ischemic stroke patients?

• Measuring NDUFA11 expression levels in blood samples from a cohort of ischemic stroke patients at different time points.

• Correlating these measurements with:

  • Stroke severity (using standardized scales like NIHSS)

  • Infarct volume (measured by MRI)

  • Functional outcomes (using scales like mRS)

  • Recovery trajectories

  • Response to treatment interventions

The development of a nomogram model for predicting ischemic stroke risk based on disulfidptosis-related biomarkers, including NDUFA11, suggests that quantitative measurements of NDUFA11 could have prognostic value . The machine learning models developed using these biomarkers demonstrated relatively high accuracy, suggesting potential clinical utility.

Future research could focus on constructing a longitudinal database correlating NDUFA11 expression changes with clinical outcomes to determine if this biomarker can predict recovery potential or guide personalized treatment approaches.

What is the comparative analysis of NDUFA11 and other complex I subunits in neurodegenerative disorders?

A comparative analysis of NDUFA11 and other complex I subunits in neurodegenerative disorders would reveal important insights into mitochondrial dysfunction mechanisms:

The mechanistic differences between these subunits provide important insights:

• Supernumerary subunits like NDUFA11 and NDUFA2 appear particularly important for stable complex I assembly and function.

• Disruption of protein-protein interactions within complex I, whether through mutations (as with NDUFA2) or altered expression (as with NDUFA11), can lead to mitochondrial dysfunction and neurological conditions.

• Different subunits may be associated with specific pathological mechanisms - NDUFA11 with disulfidptosis in ischemic stroke, and NDUFA2 with complex I deficiency leading to epilepsy and movement disorders.

Further comparative studies could reveal whether targeting common pathways affected by these subunits might provide therapeutic benefits across multiple neurodegenerative conditions.

What are the optimal expression systems for producing functional recombinant human NDUFA11?

While the search results don't specifically address expression systems for recombinant NDUFA11, based on general principles for producing mitochondrial proteins, the following systems would be optimal:

• Mammalian expression systems: HEK293 or CHO cells would likely provide the appropriate post-translational modifications and folding environment for human NDUFA11. These systems would be particularly valuable for producing protein intended for functional studies or structural analysis.

• Insect cell expression systems: Baculovirus-infected insect cells (Sf9 or Hi5) offer advantages for membrane-associated proteins like NDUFA11, potentially yielding higher quantities of properly folded protein compared to bacterial systems.

• Cell-free protein synthesis: This approach might be suitable for producing NDUFA11 for structural studies, especially when supplemented with appropriate chaperones and membrane mimetics.

For experimental considerations:

• Include appropriate mitochondrial targeting sequences if necessary for localization studies

• Consider fusion tags that can be removed without affecting protein function

• Verify protein functionality by assessing its ability to form complexes with NDUFS1

• Validate proper folding using circular dichroism or limited proteolysis

How can researchers verify the functional integrity of recombinant NDUFA11?

To verify the functional integrity of recombinant NDUFA11, researchers should implement a multi-faceted approach:

• Binding assays with NDUFS1: Co-immunoprecipitation or surface plasmon resonance to confirm that recombinant NDUFA11 can bind to its primary interaction partner, NDUFS1. This interaction is critical for NDUFA11's function .

• Complex I assembly complementation: Introduction of recombinant NDUFA11 into cells with NDUFA11 deficiency should restore complex I assembly and function if the recombinant protein is functional.

• Mitochondrial respiratory chain activity: Oxygen consumption rate measurements using platforms like Seahorse XF Analyzer could assess whether recombinant NDUFA11 restores proper electron transport chain function in deficient cells.

• Protection against disulfidptosis: Since NDUFA11 is associated with disulfidptosis in ischemic conditions , functional recombinant NDUFA11 should provide protection against disulfide stress-induced cell death in appropriate experimental models.

• Structural integrity assessment: Circular dichroism spectroscopy and thermal shift assays could confirm proper folding and stability of the recombinant protein.

What are the critical quality control parameters for recombinant NDUFA11 in research applications?

For research applications using recombinant NDUFA11, the following quality control parameters are critical:

• Purity assessment:

  • SDS-PAGE with Coomassie or silver staining (>95% purity recommended)

  • Western blot using specific antibodies to confirm identity

  • Mass spectrometry to verify molecular weight and potential modifications

• Functional verification:

  • Binding kinetics with NDUFS1 and other interaction partners

  • Complex I assembly rescue capability in appropriate cellular models

  • Mitochondrial localization if the protein includes targeting sequences

• Stability parameters:

  • Thermal stability profile

  • pH stability range

  • Storage condition optimization

  • Freeze-thaw stability assessment

• Endotoxin testing: For applications involving cell culture or in vivo studies, endotoxin levels should be below 0.1 EU/μg protein.

• Batch-to-batch consistency: Establishing reproducible production protocols with defined acceptance criteria for critical parameters to ensure consistent experimental outcomes.

Documenting these quality control parameters in research publications would enhance reproducibility and enable proper interpretation of experimental results using recombinant NDUFA11.