Recombinant Mouse NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 5, mitochondrial (Ndufb5)

What is the structure and function of NDUFB5 in mitochondrial metabolism?

NDUFB5 (NADH:ubiquinone oxidoreductase subunit B5) is a key component of Complex I of the electron transport chain, playing an essential role in maintaining mitochondrial respiration. The protein is located in the mitochondrial inner membrane and functions as part of the respiratory chain complex I .

Structurally, NDUFB5 belongs to the NDUFB5/SGDH subunit family with conserved domains across species. In zebrafish, the protein contains 186 amino acids . The primary function of NDUFB5 involves facilitating the oxidation of NADH through the respiratory chain complexes, which is essential for the oxidative phosphorylation process that provides cellular energy.

Methodological considerations for structural studies:

• Purification of mitochondrial fractions using density gradient centrifugation

• Blue native PAGE for analyzing intact Complex I

• Cryo-electron microscopy for high-resolution structural analysis

• Comparative bioinformatics analysis with homologs from other species

NDUFB5 contributes significantly to energy metabolism by maintaining the electron flow through Complex I, which is critical for producing ATP via oxidative phosphorylation. Disruption of NDUFB5 function can lead to impaired mitochondrial respiration and energy deficits in affected tissues.

How does NDUFB5 expression correlate with wound healing mechanisms in diabetic models?

NDUFB5 plays a crucial role in diabetic wound healing processes. Research has demonstrated that upregulation of NDUFB5 can accelerate diabetic wound healing, specifically in the context of diabetic foot ulcers (DFU) .

Skin wound healing requires substantial energy, primarily provided by mitochondrial respiration through oxidative phosphorylation. As a key component of Complex I, NDUFB5 is essential for maintaining mitochondrial respiratory chain function, thereby supporting the energy-intensive process of wound healing .

Experimental findings:

• NDUFB5 promotes cell viability, migration, and mitochondrial respiration in advanced glycation end products (AGEs)-treated human umbilical vein endothelial cells (HUVECs)

• NDUFB5 enhances wound healing in diabetic mice models

• NDUFB5 expression is reduced in diabetic skeletal muscle, correlating with altered carbohydrate, energy, and amino acid metabolism

Research methodologies:

• In vivo wound healing assays in diabetic mouse models

• Cell migration and proliferation assays in AGEs-treated cells

• Measurements of mitochondrial respiratory parameters using Seahorse technology

• Immunohistochemistry of wound tissues to assess vascularization and healing markers

What knockout/knockdown models exist for studying NDUFB5 function?

While the search results don't mention specific NDUFB5 knockout models, analogous approaches used for other Complex I subunits provide methodological insights:

Cell culture models:

• CRISPR/Cas9-mediated gene editing to create complete or conditional knockouts

• RNA interference (siRNA/shRNA) for transient knockdown studies

• Lentiviral expression systems for rescue experiments using recombinant NDUFB5

Animal models considerations:

• Complete knockout of essential mitochondrial proteins is often embryonically lethal, as seen with the related subunit NDUFA5

• Conditional tissue-specific knockout strategies using Cre-lox systems

• Temporal control using inducible knockout systems

• Hypomorphic alleles for partial loss of function studies

Phenotypic assessments:

• Mitochondrial respiratory function measurement

• Analysis of complex I assembly intermediates

• Metabolomic profiling of energy metabolism pathways

• Tissue-specific phenotypes in conditional knockout models

For effective NDUFB5 functional studies, the commercial availability of expression-ready lentiviral ORF clones with Myc-DDK tags provides tools for rescue experiments and overexpression studies .

How does Complex I assembly depend on NDUFB5 and its interactions?

While specific details about NDUFB5's role in Complex I assembly aren't explicit in the search results, insights can be drawn from studies of other accessory subunits:

Complex I assembly follows a modular pattern, with different subassemblies forming and then integrating. Accessory subunits like NDUFB5 are generally thought to play roles in the assembly, stability, and regulation of Complex I function.

Comparative insights from NDUFA5 (another accessory subunit):

• Knockout of NDUFA5 in HEK293T cells results in incomplete assembly of Complex I with accumulation of a 460 kDa subcomplex composed of membrane arm subunits but lacking Q- or N-module subunits

• NDUFA5 depletion by RNA interference leads to significant decrease in Complex I activity

• NDUFA5 is required for the stability of the Q-module and formation of functional Complex I

Assembly dynamics investigation methods:

• Blue native PAGE to separate assembly intermediates

• Pulse-chase labeling to track assembly kinetics

• Proximity labeling to identify interaction partners during assembly

• Time-course analysis following gene induction or repression

By analogy, NDUFB5 likely has specific interaction interfaces with neighboring subunits in the assembled Complex I that contribute to its stability and function.

How does METTL3-mediated m6A modification regulate NDUFB5 expression and function in cellular stress responses?

METTL3-mediated m6A modification plays a crucial role in regulating NDUFB5 expression, particularly in the context of cellular responses to stress conditions such as exposure to advanced glycation end products (AGEs).

Key findings:

• METTL3-mediated m6A modification enhances NDUFB5 expression in human umbilical vein endothelial cells (HUVECs)

• Insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) recognizes the m6A modification on NDUFB5 mRNA, stabilizing its expression

• METTL3 promotes cell viability, migration, and mitochondrial respiration in AGEs-treated HUVECs by increasing NDUFB5 expression

Experimental pathway:

• AGEs induce cellular stress in HUVECs

• METTL3 mediates m6A modification of NDUFB5 mRNA

• IGF2BP2 recognizes and binds to the modified mRNA

• This interaction enhances NDUFB5 expression

• Increased NDUFB5 improves mitochondrial respiration

• Enhanced mitochondrial function promotes cell viability and migration

Methodology for studying this regulatory mechanism:

• Methylated RNA immunoprecipitation sequencing (MeRIP-seq) to identify m6A modification sites

• RNA immunoprecipitation to detect protein-RNA interactions

• Site-directed mutagenesis of m6A sites to confirm functional importance

• METTL3 knockout/knockdown and overexpression studies

• Functional assays for cell viability, migration, and mitochondrial respiration

This regulatory mechanism represents a potential therapeutic target for conditions involving mitochondrial dysfunction, particularly in diabetic complications like DFU.

What is the relationship between NDUFB5 function and cancer cell resistance to chemotherapeutic agents?

While the search results don't directly address NDUFB5's role in chemotherapy resistance, they provide insights into the broader relationship between mitochondrial metabolism and drug resistance, particularly to 5-fluorouracil (5FU).

Research findings on mitochondrial metabolism and chemoresistance:

• Increased mitochondrial metabolism promotes 5FU persistence and resistance in cancer cells

• CRISPR screen analysis revealed that approximately 7.3% of genes significant for 5FU resistance were metabolic, with significant enrichment for oxidative phosphorylation (OxPhos) genes

• Oxidative metabolism signatures predict response to 5FU-based chemotherapy treatment

Research approaches to investigate NDUFB5-specific effects:

• Expression analysis of NDUFB5 in sensitive versus resistant cancer cell lines

• CRISPR-mediated knockout or overexpression of NDUFB5 followed by drug sensitivity testing

• Metabolic flux analysis to measure changes in oxidative phosphorylation upon NDUFB5 modulation

• Correlation analysis of NDUFB5 expression with treatment outcomes in patient samples

As NDUFB5 is a component of Complex I involved in oxidative phosphorylation, its potential contribution to chemotherapy resistance warrants further investigation.

How can recombinant NDUFB5 be optimized for structure-function studies in mitochondrial research?

Optimization of recombinant NDUFB5 for functional studies requires careful consideration of expression systems, tagging strategies, and functional assays.

Available tools:

• Expression-ready ORF plasmid in lenti backbone (pLenti-C-Myc-DDK-P2A-Puro)

• Myc-DDK-tagged mouse NDUFB5 for detection and purification

Optimization strategies:

Methodological approaches:

• Verify mitochondrial localization using fluorescence microscopy

• Assess incorporation into Complex I using blue native PAGE

• Measure effects on mitochondrial respiration using oxygen consumption assays

• Evaluate protein-protein interactions using proximity labeling techniques

• Perform site-directed mutagenesis to create disease-associated variants

For structure determination studies, recombinant NDUFB5 can be incorporated into reconstituted Complex I for cryo-electron microscopy analysis, though this requires co-expression with other Complex I subunits.

What are the experimental challenges in studying NDUFB5's role in the "checkpoint hypothesis" of Complex I assembly?

The "checkpoint hypothesis" of Complex I assembly involves a coordinated sequence of subunit additions and assembly factor interactions. While NDUFB5's specific role isn't detailed in the search results, understanding the experimental challenges in studying this process is valuable for researchers.

The checkpoint hypothesis concept:
Based on search result , some Complex I accessory subunits act in concert with assembly factors as checkpoints, blocking full assembly until specific conditions are met. For example, NDUFAF2, NDUFS4, NDUFS6, and NDUFA12 form a checkpoint in the assembly pathway .

Experimental challenges and solutions:

Advanced techniques for studying assembly:

• Proximity-dependent biotin identification (BioID) to map dynamic protein interactions

• Time-resolved cryo-electron microscopy to capture assembly intermediates

• Multiplexed CRISPR screens to identify genetic interactions

• Mass spectrometry of crosslinked complexes to define interaction interfaces

• Single-particle tracking to monitor assembly kinetics in living cells

Understanding NDUFB5's position in the assembly pathway would require systematic analysis of assembly intermediates that accumulate in its absence, combined with interaction studies to identify its binding partners during the assembly process.

How does NDUFB5 modulation affect mitochondrial dynamics and quality control pathways?

Mitochondrial dynamics (fusion/fission) and quality control pathways are essential for maintaining mitochondrial function. While direct evidence for NDUFB5's role in these processes is limited in the search results, inferences can be made from available data.

Research insights:

• Mitochondrial fusion promoter M1 facilitates cell viability, migration, and mitochondrial oxidative respiration in NDUFB5 knockdown HUVECs

• This suggests that NDUFB5 may have functional connections to mitochondrial fusion processes

• As a Complex I component, NDUFB5 likely influences mitochondrial membrane potential, which is a key regulator of mitochondrial dynamics

Proposed mechanisms for investigation:

• NDUFB5 and mitochondrial membrane potential:

  • Complex I activity generates proton gradient for membrane potential

  • Membrane potential influences fusion/fission balance

  • NDUFB5 dysfunction could alter this balance

• NDUFB5 and mitochondrial quality control:

  • Dysfunctional Complex I can trigger mitophagy

  • NDUFB5 deficiency might activate PINK1/Parkin pathway

  • Potential interaction with mitochondrial proteostasis machinery

Experimental approaches:

• Live-cell imaging of mitochondrial networks in NDUFB5-modulated cells

• Measurement of mitochondrial membrane potential using potentiometric dyes

• Analysis of mitophagy flux using mt-Keima or similar reporters

• Assessment of mitochondrial proteome turnover rates

• Identification of potential NDUFB5 interactors outside of Complex I

Understanding how NDUFB5 influences these processes could provide insights into its role in pathological conditions like diabetic complications and potentially reveal new therapeutic approaches targeting mitochondrial function.