Recombinant Bovine Mitochondrial import inner membrane translocase subunit Tim23 (TIMM23)

What is the structural organization of bovine TIMM23 and how does it compare to human TIMM23?

Bovine TIMM23 shares high structural conservation with human TIMM23, functioning as a core component of the mitochondrial protein import machinery. The TIMM23 protein forms part of the TIM23 complex, which consists of multiple membrane proteins that create a channel through the inner mitochondrial membrane. The structure reveals a channel-like architecture that facilitates protein translocation, with the core complex showing remarkable evolutionary conservation between species .

Structural analysis indicates that TIMM23 interacts with other core components such as TIMM17A/B (human) or Tim17 (yeast), forming a stable complex that serves as the primary translocation channel for preproteins destined for the mitochondrial matrix . This high structural conservation suggests fundamental mechanistic similarities in protein import across mammalian species, including bovine models.

What is the primary function of TIMM23 in mitochondrial protein import?

TIMM23 functions as a central component of the TIM23 complex, which facilitates the translocation of nuclear-encoded proteins across the inner mitochondrial membrane into the matrix. After precursor proteins pass through the translocase of the outer membrane (TOM), they engage with the TIM23 complex, where TIMM23 forms part of the protein-conducting channel .

The import process mediated by TIMM23 requires two main driving forces: the membrane potential (Δψ) across the inner mitochondrial membrane, which acts as an electrophoretic force on the positively charged presequence, and ATP hydrolysis by matrix heat shock proteins (particularly mtHsp70), which pulls the rest of the precursor protein through to the matrix . This dual-drive mechanism ensures efficient and directional protein transport into the mitochondrial matrix.

How is TIMM23 expression regulated in normal bovine tissues compared to pathological conditions?

While the search results don't specifically address bovine TIMM23 regulation, research on human TIMM23 indicates significant expression differences between normal and pathological states. In non-small cell lung cancer (NSCLC), TIMM23 is consistently overexpressed compared to matched normal lung tissues, suggesting altered regulation in disease states .

In normal tissues, TIMM23 expression is tightly regulated to meet cellular energy demands. Comparative analysis would likely show similar patterns in bovine tissues, with expression levels corresponding to mitochondrial activity. The upregulation observed in pathological conditions like cancer suggests that TIMM23 expression responds to the altered metabolic demands of rapidly proliferating cells .

What experimental approaches are most effective for studying the interaction between bovine TIMM23 and other components of the TIM23 complex?

To effectively study TIMM23 interactions with other TIM23 complex components, researchers should employ a multi-faceted approach combining structural, biochemical, and functional methodologies:

• Structural analysis techniques:

  • Computational structure prediction tools similar to those used for human TIM23 complex analysis

  • Analysis of inter- and intramolecular interactions using programs like UCSF ChimeraX to identify contact points between TIMM23 and other complex components

  • Comparison of conformational states using root mean square deviation (RMSD) calculations to assess structural changes upon interaction

• Biochemical interaction studies:

  • Co-immunoprecipitation assays to identify direct protein-protein interactions

  • Crosslinking experiments followed by mass spectrometry for capturing transient interactions

  • Blue native PAGE to analyze intact complexes

• Functional translocation assays:

  • Real-time import assays using bioluminescence-based techniques that allow measurement of both rates and endpoints of protein import

  • Reconstitution experiments in liposomes to study minimal requirements for TIMM23-mediated translocation

These approaches collectively provide comprehensive insights into TIMM23's interactions within the import machinery.

How does mitochondrial membrane potential affect the functional efficiency of recombinant bovine TIMM23?

Mitochondrial membrane potential (Δψ) is critical for TIMM23-mediated protein import, with distinct effects on import kinetics and efficiency. Research indicates that Δψ serves two crucial functions in TIMM23-dependent import:

• Initial presequence insertion: Δψ (negative in the matrix) acts as an electrophoretic force on the positively charged mitochondrial targeting sequence (MTS), driving its initial insertion through the TIM23 channel .

• Mature domain translocation: Contrary to previous models, Δψ also drives the transport of the mature domain of precursor proteins, not exclusively the MTS. This is evident from the emergence of length-dependence on import kinetics under conditions of Δψ depletion .

In experimental systems, depletion of Δψ significantly impacts the import rates but not necessarily the total amount of protein imported (amplitude). The membrane potential appears to be particularly important for the second kinetic phase of import, which involves the engagement of the MTS with the TIM23 complex .

What are the structural differences between TIMM17A and TIMM17B variants of the TIM23 complex, and how do they affect interactions with bovine TIMM23?

Human cells possess two orthologs of yeast Tim17: TIMM17A and TIMM17B, which form two distinct populations of highly similar TIM23 complexes. Both variants interact with TIMM23 and ROMO1 (reactive oxygen species modulator 1, a homolog of yeast Mgr2) .

Structural analysis reveals:

The high conservation of these interactions across species suggests that findings from human complexes likely apply to bovine TIMM23 as well.

What are the optimal conditions for expressing and purifying recombinant bovine TIMM23 for structural studies?

For optimal expression and purification of recombinant bovine TIMM23, researchers should consider the following protocol based on approaches used for similar membrane proteins:

Expression System Selection:

• Bacterial systems (E. coli): Suitable for domains but challenging for full-length membrane proteins

• Yeast systems (P. pastoris): Better for full-length expression with proper folding

• Insect cell systems: Optimal for complex membrane proteins requiring eukaryotic processing

Expression Optimization:

• Use a construct with a cleavable affinity tag (His6 or GST)

• Consider fusion partners to enhance solubility

• Express at lower temperatures (16-18°C) to improve folding

• Include mild detergents in lysis buffers to solubilize membrane proteins

Purification Strategy:

• Membrane isolation followed by detergent solubilization

• Affinity chromatography using the engineered tag

• Size exclusion chromatography for final purification

• Consider amphipols or nanodiscs for stabilization

Quality Assessment:

• Circular dichroism to confirm secondary structure

• Limited proteolysis to verify folding

• Functional assays to confirm activity

While these recommendations are based on general membrane protein methodologies, they should be adapted for the specific properties of bovine TIMM23.

What techniques can be used to study the impact of TIMM23 mutations on mitochondrial protein import efficiency?

Several complementary techniques can be employed to study how TIMM23 mutations affect import efficiency:

Site-Directed Mutagenesis Approaches:

• Structure-guided mutagenesis targeting conserved residues identified through alignment between bovine and other species

• Systematic alanine scanning of transmembrane domains

• Charge reversal mutations in regions interacting with presequences

Functional Assessment Methods:

• In vitro import assays: Using isolated mitochondria containing mutant TIMM23 to measure import kinetics of radiolabeled or fluorescently labeled precursor proteins

• Real-time bioluminescence assays: For precise measurement of import kinetics, allowing differentiation between effects on rate versus total import capacity

• Membrane potential measurements: To determine if mutations affect Δψ maintenance, which is crucial for import

Cellular Phenotype Analysis:

• Mitochondrial function assays including:

  • Complex I activity measurements

  • ATP production quantification

  • Membrane potential assessment

  • Oxidative stress evaluation

• Cell viability and proliferation assays to connect molecular defects to cellular outcomes

Structural Impact Assessment:

• Computational modeling to predict how mutations affect protein-protein interactions within the TIM23 complex

• Distance calculations between interacting residues to quantify perturbations in complex stability

This multi-level approach provides comprehensive insights into structure-function relationships of TIMM23.

How does TIMM23 overexpression contribute to cancer cell growth and survival mechanisms?

TIMM23 overexpression significantly contributes to cancer cell growth and survival through enhanced mitochondrial function and bioenergetics. Research on NSCLC cells reveals several mechanisms:

• Enhanced mitochondrial bioenergetics: TIMM23 overexpression leads to increased mitochondrial complex I activity and ATP production, providing the energy required for rapid cell proliferation .

• Promotion of cell proliferation: Ectopic TIMM23 overexpression significantly enhances:

  • Cell viability (as measured by CCK-8 assay)

  • Colony formation capacity

  • Nuclear EdU incorporation (marker of DNA synthesis)

• Enhanced metastatic potential: TIMM23 overexpression promotes:

  • Increased cell migration

  • Enhanced invasive capabilities in Matrigel invasion assays

Conversely, silencing TIMM23 demonstrates its critical role in cancer cell survival:

• Impaired mitochondrial function characterized by reduced complex I activity

• ATP depletion

• Mitochondrial membrane potential dissipation

• Oxidative stress and lipid peroxidation

• Growth arrest and apoptosis induction

This data establishes TIMM23 as a critical pro-tumorigenic factor that enhances cancer cell growth and survival by optimizing mitochondrial function to meet the elevated bioenergetic demands of malignant cells.

What experimental approaches are most effective for studying TIMM23 as a potential therapeutic target in cancer treatment?

To evaluate TIMM23 as a therapeutic target in cancer, researchers should employ a multi-level approach that addresses both mechanistic understanding and therapeutic potential:

1. Target Validation Strategies:

• RNA interference (RNAi): Using multiple shRNAs targeting TIMM23 to establish dose-dependent effects on cancer phenotypes

• CRISPR/Cas9-mediated knockout: For complete ablation of TIMM23 expression to assess maximal therapeutic effects

• Rescue experiments: To confirm specificity by determining if exogenous ATP or antioxidants (e.g., N-acetylcysteine) can reverse growth inhibition

2. In Vitro Efficacy Assessment:

• Cell viability assays: CCK-8 or MTT assays to measure anti-proliferative effects

• Colony formation assays: To assess long-term growth inhibition

• EdU incorporation: To measure DNA synthesis inhibition

• Migration/invasion assays: To evaluate effects on metastatic potential

3. Mechanism Exploration:

• Mitochondrial function assessment:

  • Complex I activity measurements

  • ATP quantification

  • Membrane potential analysis

  • ROS detection

  • Lipid peroxidation analysis

4. In Vivo Efficacy Studies:

• Xenograft models: Using intratumoral delivery of TIMM23 shRNA-expressing adeno-associated virus to suppress tumor growth

• Analysis of tumor tissues for:

  • TIMM23 expression

  • ATP levels

  • Oxidative damage markers

  • Proliferation markers

  • Apoptotic indices

This comprehensive approach provides strong evidence for TIMM23 as a potential therapeutic target and identifies key mechanisms that could be exploited for drug development.

How can researchers differentiate between the roles of TIMM17A and TIMM17B variants in cancer-related mitochondrial dysfunction?

To differentiate between TIMM17A and TIMM17B roles in cancer-related mitochondrial dysfunction, researchers should employ the following strategies:

1. Expression Analysis in Cancer Tissues:

• Comparative qPCR and immunoblotting to quantify TIMM17A versus TIMM17B expression levels across cancer types

• Single-cell RNA sequencing to identify cell-type-specific expression patterns within heterogeneous tumors

• Correlation of expression ratios with clinical outcomes to determine prognostic significance

2. Variant-Specific Knockdown/Knockout:

• Selective silencing of TIMM17A or TIMM17B using targeted siRNAs or CRISPR/Cas9

• Comparative analysis of:

  • Mitochondrial import efficiency for different substrate classes

  • Bioenergetic consequences (ATP production, membrane potential)

  • Cell proliferation and survival outcomes

  • Impact on oxidative stress responses

3. Structural and Interaction Analysis:

• Comparison of TIMM17A and TIMM17B interaction interfaces with TIMM23 and ROMO1

• Analysis of distances between atom pairs at interaction surfaces

• Identification of variant-specific protein partners through proximity labeling approaches

4. Substrate Specificity Assessment:

• Import assays using model precursor proteins to determine if TIMM17A and TIMM17B variants preferentially transport different subsets of mitochondrial proteins

• Analysis of how variant-specific import affects the mitochondrial proteome in cancer cells

5. Therapeutic Targeting Assessment:

• Evaluation of whether selective targeting of TIMM17A versus TIMM17B produces differential anti-cancer effects

• Determination of whether cancer cells exhibit preferential dependence on one variant over the other

These approaches would help determine if the two variants play redundant or specialized roles in cancer mitochondrial function, potentially revealing more precise therapeutic opportunities.

How can structural analysis methods be applied to understand species-specific differences in TIMM23 function?

To understand species-specific differences in TIMM23 function, researchers can apply several complementary structural analysis methods:

Computational Structure Prediction:

• Use of AlphaFold or similar tools to generate structural models for TIMM23 from different species

• Comparative analysis of predicted structures to identify conserved and divergent regions

Structural Alignment and Conservation Analysis:

• Superimposition of bovine, human, and yeast TIMM23 structures to identify structural differences

• Calculation of root mean square deviation (RMSD) between aligned structures

• Coloring protein structures according to RMSD values to visualize regions of structural divergence

Interaction Surface Analysis:

• Identification of contacts between TIMM23 and partner proteins (TIMM17A/B, ROMO1/Mgr2)

• Contacts defined where distances between atom pairs are ≤ sum of van der Waals radii plus 0.4 Å

• Comparison of interaction interfaces across species to identify conserved binding motifs

Electrostatic and Hydrophobicity Analysis:

• Calculation of surface electrostatic potential to identify species-specific charge distributions

• Analysis of surface hydrophobicity patterns that might affect protein-protein interactions

Functional Domain Comparison:

• Identification of species-specific insertions/deletions that might confer functional specialization

• Analysis of channel-forming regions and substrate-binding sites

Molecular Dynamics Simulations:

• Simulation of TIMM23 dynamics in membrane environments

• Comparison of conformational flexibility and gating mechanisms across species

This multi-faceted approach would reveal both structural conservation and species-specific adaptations in TIMM23 function, providing insights into evolutionary adaptations of the mitochondrial import machinery.

What are the most effective approaches for reconstituting functional bovine TIMM23 complexes in vitro?

Reconstituting functional bovine TIMM23 complexes in vitro requires careful consideration of membrane protein biochemistry and complex assembly. The following approaches represent current best practices:

Protein Component Preparation:

• Expression systems:

  • Insect cell expression for complex membrane proteins

  • Cell-free systems supplemented with lipids for direct integration into membranes

  • Careful optimization of detergent solubilization conditions

• Purification strategy:

  • Tandem affinity purification to isolate intact complexes

  • Gradient centrifugation to separate different assembly states

  • Mild detergents to maintain native-like interactions

Membrane Mimetic Systems:

• Liposome reconstitution:

  • Use of defined lipid compositions mimicking mitochondrial inner membrane

  • Controlled protein:lipid ratios to achieve physiological densities

  • Size control through extrusion techniques

• Nanodiscs:

  • MSP (membrane scaffold protein) nanodiscs for single-complex studies

  • Larger SMALPs (styrene-maleic acid lipid particles) for multi-protein assemblies

  • Direct extraction from membranes to maintain native lipid environment

Functional Verification Methods:

• Electrical measurements:

  • Planar lipid bilayer recordings to measure channel activity

  • Patch-clamp of proteoliposomes to assess gating properties

• Import assays:

  • Fluorescence-based substrate translocation monitoring

  • FRET assays to measure substrate-channel interactions

  • Real-time protein import with bioluminescent reporters

Complex Assembly Assessment:

• Analytical ultracentrifugation to determine complex stoichiometry

• Native gel electrophoresis to verify assembly state

• Chemical crosslinking followed by mass spectrometry to map interaction interfaces

These approaches collectively provide powerful tools for reconstituting and studying functional bovine TIMM23 complexes, offering insights impossible to obtain from cellular systems alone.