Recombinant Gorilla gorilla gorilla NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 11, mitochondrial (NDUFB11)

What is NDUFB11 and what are its primary functions in cellular metabolism?

NDUFB11 (NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 11, mitochondrial) is a key subunit of complex I of the mitochondrial respiratory chain. It is primarily involved in the electron transfer process and plays crucial roles in complex I assembly and functional stability. Additionally, NDUFB11 participates in the regulation of respiratory function, apoptosis, and oxidative stress response in mitochondria . The protein is essential for maintaining energy metabolism within mitochondria, and deficiencies or mutations can lead to various pathological conditions including rare mitochondrial disorders characterized by symptoms such as muscle weakness, neurological disorders, and metabolic abnormalities .

How does recombinant Gorilla gorilla gorilla NDUFB11 differ from human NDUFB11?

Recombinant Gorilla gorilla gorilla NDUFB11 shares significant sequence homology with human NDUFB11 but has specific amino acid differences that may affect protein-protein interactions and functional properties. The Gorilla gorilla gorilla NDUFB11 protein has a specific amino acid sequence that includes regions involved in complex I assembly and electron transport . When designing experiments, researchers should consider these differences, particularly when using the gorilla protein as a model for human studies. Cross-species comparisons can provide valuable insights into evolutionarily conserved functions versus species-specific adaptations in mitochondrial respiratory complexes.

What are the optimal storage conditions for recombinant NDUFB11 to maintain its activity?

Recombinant NDUFB11 should be stored at -20°C for regular use, but for extended storage, conservation at -80°C is recommended. The protein is typically supplied in a Tris-based buffer with 50% glycerol, which has been optimized to maintain protein stability . Repeated freezing and thawing cycles should be avoided as they can significantly compromise protein activity. For working solutions, store aliquots at 4°C for up to one week to minimize degradation . When planning long-term research projects, consider preparing multiple small aliquots during initial reconstitution to avoid repeated freeze-thaw cycles of the stock solution.

How does NDUFB11 suppression affect mitochondrial complex I assembly and cellular respiration?

Suppression of NDUFB11 expression has been shown to profoundly impact mitochondrial complex I assembly, resulting in significant physiological consequences. Research has demonstrated that NDUFB11 suppression reduces cellular oxygen consumption linked to complex I by approximately two-thirds, an effect that can be bypassed by addition of the complex III substrate duroquinol . At the structural level, NDUFB11 suppression leads to a reduction in intact complex I and causes the accumulation of subcomplexes with molecular masses of approximately 815 kDa and 550 kDa .

The mitochondrial network, which typically appears as an interconnected structure in control cells, becomes fragmented following NDUFB11 suppression . This fragmentation suggests a broader impact on mitochondrial dynamics and quality control mechanisms. When designing NDUFB11 knockdown experiments, researchers should consider implementing multiple complementary approaches to evaluate mitochondrial function, including:

What methodological approaches can be used to study the interaction between NDUFB11 and other assembly factors in complex I formation?

Studying NDUFB11 interactions with other assembly factors requires a multi-faceted approach. When NDUFB11 expression is suppressed, various assembly factors associate with complex I subcomplexes, including C3orf1, which has been identified as a potential assembly factor through co-purification studies . To comprehensively investigate these interactions, researchers should employ the following methodological approaches:

• Co-immunoprecipitation (Co-IP) using anti-NDUFB11 antibodies can identify protein-protein interactions. The antibody 16720-1-AP has been validated for IP in mouse skeletal muscle tissue .

• Blue Native PAGE followed by western blotting can identify subcomplexes formed during assembly disruption.

• Proximity labeling techniques such as BioID or APEX2 can identify proteins in close spatial proximity to NDUFB11 in living cells.

• Crosslinking mass spectrometry (XL-MS) can provide detailed information about the architecture of NDUFB11-containing complexes.

• Cryo-electron microscopy can determine the structural organization of NDUFB11 within the fully assembled complex I.

When analyzing interaction data, it's important to distinguish between direct binding partners and proteins that associate with the same subcomplex but don't directly interact with NDUFB11.

How do mutations in NDUFB11 contribute to the pathophysiology of mitochondrial disorders?

NDUFB11 mutations are associated with a rare mitochondrial disorder known as NDUFB11-deficient mitochondriopathy . The pathophysiological mechanisms involve disruption of complex I assembly and function, leading to impaired electron transport and ATP production. This energy deficit particularly affects tissues with high energy demands, such as muscle and nervous system tissues.

The disease manifestations include a spectrum of symptoms:

• Muscle weakness and exercise intolerance

• Neurological disorders

• Metabolic abnormalities

• Cardiomyopathy in some cases

When investigating the pathophysiological consequences of NDUFB11 mutations, researchers should consider:

• Conducting functional studies comparing wild-type and mutant NDUFB11 proteins

• Analyzing the impact on complex I assembly using blue native PAGE

• Measuring respiratory chain function in patient-derived cells or model systems

• Assessing ROS production and oxidative stress markers

• Evaluating cellular adaptations, including metabolic reprogramming and mitochondrial quality control

Patient-derived fibroblasts, induced pluripotent stem cells (iPSCs), or CRISPR-engineered cell lines carrying specific NDUFB11 mutations can serve as valuable models for such studies.

What are the recommended protocols for detecting NDUFB11 in different experimental systems?

Detection of NDUFB11 can be accomplished through various techniques, with western blotting being the most common. Based on validated antibody data, the following methodological approaches are recommended :

For optimal results in western blotting, the following protocol is recommended:

• Prepare samples in RIPA buffer with protease inhibitors

• Load 20-30 μg protein per lane

• Separate proteins on 12-15% SDS-PAGE gels

• Transfer to PVDF membrane at 100V for 1 hour

• Block with 5% non-fat milk in TBST for 1 hour

• Incubate with primary antibody (anti-NDUFB11) at 4°C overnight

• Wash 3× with TBST

• Incubate with HRP-conjugated secondary antibody for 1 hour

• Develop using ECL substrate

Always include appropriate positive controls (e.g., HepG2 cells) and negative controls (e.g., NDUFB11 knockdown samples) to validate specificity .

What approaches can be used to study the functional impact of NDUFB11 in cellular models?

To study NDUFB11 function, several complementary approaches can be implemented:

• Gene Knockdown/Knockout: siRNA, shRNA, or CRISPR-Cas9 can be used to reduce or eliminate NDUFB11 expression. The effectiveness of knockdown should be verified by western blotting, with expected phenotypes including reduced complex I activity, accumulation of subcomplexes, and mitochondrial network fragmentation .

• Overexpression Studies: Wild-type or mutant NDUFB11 can be overexpressed to study gain-of-function effects or perform rescue experiments. The recombinant protein can serve as a positive control .

• Mitochondrial Function Assays:

  • Oxygen consumption rate measurement using respirometry

  • ATP production assays

  • Mitochondrial membrane potential assessment

  • ROS production measurement

• Complex I Assembly Analysis: Blue Native PAGE followed by western blotting or in-gel activity assays can reveal assembly defects.

• Protein-Protein Interaction Studies: Co-immunoprecipitation can identify binding partners, while proximity labeling can map the protein's interaction network in living cells.

When designing functional studies, it's crucial to consider cell type-specific effects, as NDUFB11 dependency may vary across tissues. Skeletal muscle cells, neurons, and cardiomyocytes are particularly relevant models due to their high energy demands.

How should researchers analyze and interpret changes in NDUFB11 expression in disease models?

When analyzing NDUFB11 expression changes in disease models, researchers should employ a comprehensive approach that integrates multiple analytical methods:

• Expression Level Analysis:

  • Quantify NDUFB11 mRNA using RT-qPCR

  • Measure protein levels via western blotting with appropriate normalization

  • Consider single-cell analysis to detect cell-specific alterations

• Contextual Analysis:

  • Evaluate co-expression patterns with other complex I subunits

  • Analyze expression in relation to mitochondrial content markers

  • Consider post-translational modifications that may affect function without changing expression levels

• Functional Correlation:

  • Correlate expression changes with complex I activity measurements

  • Assess the relationship between NDUFB11 levels and cellular respiration rates

  • Evaluate disease-specific phenotypes in relation to NDUFB11 expression

For instance, in atherosclerosis and chronic stress models, NDUFB11 has been implicated in disease pathophysiology through differential expression analysis and weighted gene co-expression network analysis (WGCNA) . When interpreting such findings, consider whether NDUFB11 changes are primary drivers or secondary adaptations in the disease process.

What bioinformatic approaches are recommended for analyzing NDUFB11 in multi-omics datasets?

For comprehensive multi-omics analysis involving NDUFB11, the following bioinformatic approaches are recommended:

• Differential Expression Analysis: Identify significant changes in NDUFB11 expression across conditions using tools like DESeq2 or limma.

• Network Analysis: Weighted gene co-expression network analysis (WGCNA) can identify modules of co-expressed genes that include NDUFB11, revealing functional relationships. This approach has been successfully used to identify NDUFB11's role in complex diseases .

• Pathway Enrichment: Tools like GO-stats and GOrilla can identify enriched biological processes, cellular components, and molecular functions associated with NDUFB11 and its network partners .

• Protein-Protein Interaction Networks: Construct PPI networks to understand NDUFB11's functional context.

• Integration of Multiple Data Types:

  • Correlate transcriptomic changes with proteomic data

  • Integrate metabolomics to assess functional consequences

  • Incorporate epigenomic data to understand regulatory mechanisms

When applying these approaches, use appropriate statistical methods to account for multiple comparisons, and validate key findings experimentally. For instance, the GOrilla algorithm has been successfully used for illustrating GO annotation enrichment and probing for annotation enrichment of selected groups of genes against a background set of all network genes .

What are common challenges in working with recombinant NDUFB11 and how can they be addressed?

Researchers working with recombinant NDUFB11 may encounter several challenges:

• Protein Stability Issues:

  • Challenge: Recombinant NDUFB11 may degrade during storage or experimental handling.

  • Solution: Store in recommended buffer (Tris-based buffer with 50% glycerol) at -20°C for short-term or -80°C for long-term storage. Avoid repeated freeze-thaw cycles by preparing small working aliquots .

• Specificity in Detection:

  • Challenge: Cross-reactivity with other complex I subunits.

  • Solution: Use validated antibodies with confirmed specificity (e.g., 16720-1-AP). Include appropriate controls in each experiment, such as NDUFB11 knockdown samples .

• Functional Assays:

  • Challenge: Distinguishing NDUFB11-specific effects from general mitochondrial dysfunction.

  • Solution: Include complementary assays and appropriate controls. For example, when measuring respiration, test complex I-specific substrates alongside substrates that bypass complex I.

• Species Differences:

  • Challenge: Functional differences between gorilla and human NDUFB11.

  • Solution: When using gorilla NDUFB11 as a model for human studies, validate key findings using human systems. Compare sequence alignment to identify conserved and divergent regions.

• Expression Systems:

  • Challenge: Achieving proper folding and post-translational modifications.

  • Solution: Consider using mammalian expression systems for functional studies rather than bacterial systems, especially when studying protein-protein interactions.

How can researchers design experiments to study the role of NDUFB11 in atherosclerosis and chronic stress conditions?

Recent research has implicated NDUFB11 in atherosclerosis and chronic stress . To investigate this relationship, researchers should design experiments that address the following aspects:

• Expression Profiling:

  • Analyze NDUFB11 expression in relevant tissues from atherosclerosis models and chronic stress conditions

  • Compare expression patterns across different disease stages

  • Use both transcriptomic and proteomic approaches for comprehensive assessment

• Functional Studies:

  • Develop cell culture models that recapitulate key aspects of atherosclerosis (e.g., foam cell formation, endothelial dysfunction)

  • Manipulate NDUFB11 expression in these models to assess functional consequences

  • Measure parameters such as mitochondrial function, ROS production, inflammatory signaling, and lipid metabolism

• Animal Models:

  • Generate tissue-specific NDUFB11 knockout or overexpression models

  • Subject these models to chronic stress protocols or atherosclerotic stimuli

  • Evaluate disease progression and severity compared to controls

• Mechanistic Investigations:

  • Identify signaling pathways connecting mitochondrial dysfunction and disease phenotypes

  • Assess whether targeting NDUFB11 can modulate disease outcomes

  • Investigate potential therapeutic approaches based on findings

• Clinical Correlations:

  • Analyze NDUFB11 expression in human atherosclerotic plaques

  • Correlate expression with clinical parameters and disease severity

  • Investigate genetic variants affecting NDUFB11 function in relation to disease risk

When designing these experiments, controls should include other mitochondrial proteins to determine whether observed effects are NDUFB11-specific or represent general mitochondrial dysfunction.