Recombinant Human AFG3-like protein 2 (AFG3L2)

Basic Research Questions

• What is the molecular structure and cellular localization of AFG3L2?

AFG3L2 is a zinc metalloprotease and ATPase localized in the inner mitochondrial membrane. The full-length human AFG3L2 protein consists of 797 amino acids with a molecular weight of approximately 89.5 kDa . Structurally, AFG3L2 contains several functional domains critical for its proteolytic and ATPase activities. Recombinant expression systems typically use full-length proteins (AA 1-802 or AA 1-797) or specific fragments for antibody production and functional studies .

For proper localization studies, fluorescence microscopy of AFG3L2-GFP fusion proteins demonstrates clear mitochondrial localization, with expression patterns throughout the mitochondrial network . When conducting subcellular fractionation, researchers should expect AFG3L2 to exclusively partition with the mitochondrial fraction, specifically in the inner mitochondrial membrane.

• What are the primary functions of AFG3L2 in mitochondrial homeostasis?

AFG3L2 functions as an ATP-dependent protease essential for mitochondrial quality control through several mechanisms:

• Protein quality control: AFG3L2 mediates degradation of misfolded or damaged proteins in the inner mitochondrial membrane

• Processing of mitochondrial proteins: Required for the maturation of paraplegin (SPG7) after its cleavage by mitochondrial-processing peptidase (MPP), converting it into a proteolytically active form

• PINK1 processing: Essential for the maturation of PINK1 into its 52 kDa mature form after MPP cleavage

• Calcium homeostasis regulation: In neurons, mediates degradation of SMDT1/EMRE before its assembly with the uniporter complex, limiting SMDT1/EMRE availability for MCU assembly

• OPA1 processing regulation: Involved in the regulation of OMA1-dependent processing of OPA1, affecting mitochondrial dynamics

Research methodologies examining these functions should incorporate multiple approaches including immunoblotting for processed protein forms, protease activity assays, and functional measurements of mitochondrial processes affected by AFG3L2 activity.

• How do mutations in AFG3L2 contribute to neurological disorders?

AFG3L2 mutations are causally linked to several neurological conditions through distinct pathophysiological mechanisms:

The pathophysiological consequences include :

• Mitochondrial network fragmentation

• Disruption of Bergmann glial cells affecting glutamate clearance at Purkinje cell synapses

• Upregulation of necroptotic factor ZBP1 leading to neuroinflammation

• Altered Ca²⁺ homeostasis causing cellular stress and neuronal death

• Impaired respiratory complex I and III activity due to inadequate assembly

• Formation of swollen mitochondria with damaged cristae

When investigating these disorders, researchers should consider combining genetic screening, patient-derived cell models, and functional mitochondrial assays to establish pathogenicity of novel mutations .

• What expression systems and purification strategies are optimal for recombinant AFG3L2?

Several expression systems have been successfully employed for recombinant AFG3L2 production:

For optimal purification:

• Use affinity tags (His-tag, Strep-tag) for one-step purification

• Verify purity by SDS-PAGE, Western blot, and analytical SEC (HPLC)

• Store purified protein at -80°C to prevent freeze-thaw degradation

• Include appropriate detergents for solubilization of this membrane protein

Researchers should select expression systems based on their specific experimental requirements, with mammalian systems preferable for functional studies .

Intermediate Research Questions

• How does AFG3L2 regulate mitochondrial calcium homeostasis?

AFG3L2 plays a critical role in mitochondrial calcium homeostasis through regulation of the mitochondrial calcium uniporter (MCU) complex:

• AFG3L2 mediates degradation of SMDT1/EMRE before its assembly with the uniporter complex

• This limits SMDT1/EMRE availability for MCU assembly

• Promotes efficient assembly of gatekeeper subunits with MCU

• AFG3L2 deficiency leads to altered calcium homeostasis

Research shows that AFG3L2 dysfunction significantly impacts calcium handling :

• Patient-derived cells with AFG3L2 mutations show approximately 5-fold elevation in mitochondrial calcium concentration

• This calcium overload correlates with reduced phosphorylation levels of PDHA1 (44.2%±7.2% reduction)

• Altered MICU1 protein levels are observed while MCU and MICU2 remain unaffected

Methodologically, researchers can assess mitochondrial calcium using:

• Fluorescent calcium indicators like Fura-2 AM for intracellular calcium

• Mitochondria-specific calcium probes

• Measurement of PDHA1 phosphorylation as an indirect marker of mitochondrial calcium levels

• Western blot analysis of MCU complex components

• What techniques are most effective for analyzing AFG3L2's role in mitochondrial dynamics?

To investigate AFG3L2's impact on mitochondrial dynamics, researchers should employ multiple complementary approaches:

Morphological Analysis:

• Transmission electron microscopy (TEM) provides high-resolution visualization of mitochondrial ultrastructure, revealing changes in cristae morphology and swelling in AFG3L2-deficient cells

• Fluorescence microscopy with mitochondrial markers to assess network fragmentation

Molecular Analysis:

• Western blot analysis of key proteins involved in mitochondrial dynamics:

  • OPA1 processing (monitoring long and short forms)

  • OMA1 activation (precursor protein levels)

  • Detection of mitochondrial fission/fusion proteins

Quantitative Assessment:

• Image analysis software to quantify mitochondrial network parameters

• Mitochondrial aspect ratio and form factor measurement

• Live-cell imaging to track dynamic changes

Rescue Experiments:

• Transfection with wild-type AFG3L2 in knockout models can restore normal mitochondrial morphology, providing evidence for direct causality

Research demonstrates that AFG3L2 deficiency leads to:

• Increased OMA1 activation with reduced precursor OMA1 protein levels

• Enhanced processing of OPA1 from long to short forms

• Mitochondrial fragmentation and swelling

• Reduction in cristae number and abnormal cristae arrangement

• How can researchers assess the impact of AFG3L2 mutations on respiratory chain complexes?

AFG3L2 plays a crucial role in respiratory chain complex assembly and function. To comprehensively evaluate how AFG3L2 mutations affect these complexes, researchers should implement:

Functional Assays:

• Oxygen consumption rate (OCR) measurements using Seahorse XF analyzers

• Complex-specific activity assays for CI-CV

• ATP production assessment

• Membrane potential measurements using fluorescent probes

Assembly Analysis:

• Blue Native PAGE to visualize intact respiratory complexes and supercomplexes

• Assembly kinetics using pulse-chase experiments

• Immunoprecipitation of complex components to assess interaction partners

Protein Stability Studies:

• Cycloheximide chase assays to determine protein half-life

• Proteomic analysis to identify destabilized subunits

Research has shown that Afg3l2 mutant mice exhibit impaired respiratory complex I and III activity due to inadequate assembly, despite having no effect on mitochondrial protein synthesis . NADH dehydrogenase 1 (ND1), critical for initiating complex I formation, is degraded in an AFG3L2-dependent manner .

For accurate assessment, researchers should compare results from patient-derived cells with appropriate controls and validate findings using multiple methodological approaches.

Advanced Research Questions

• How can multi-omics approaches be utilized to characterize the cellular impact of AFG3L2 mutations?

A comprehensive multi-omics strategy provides deep insights into the cellular consequences of AFG3L2 mutations:

Integrated Multi-omics Workflow:

• Proteomics:

  • Quantitative proteomics using LC-MS/MS for global protein profiling

  • Phosphoproteomics to assess signaling changes

  • Protein-protein interaction studies via BioID or proximity labeling

• Lipidomics:

  • Targeted and untargeted lipidomic profiling

  • Membrane composition analysis

  • Sphingolipid and phospholipid quantification

• Metabolomics:

  • Central carbon metabolism assessment

  • Mitochondrial metabolite profiling

  • Stable isotope tracing for flux analysis

• Transcriptomics:

  • RNA-seq for global gene expression changes

  • miRNA analysis for post-transcriptional regulation

  • Ribosome profiling for translation efficiency

Key Findings from Multi-omics Studies of AFG3L2 Mutations:

Recent multi-omics analysis of patient-derived lymphoblastoid cells with biallelic AFG3L2 variants revealed :

• Proteomic dysregulation: 63 significantly altered proteins affecting:

  • Mitochondrial function (COX11, NFU1)

  • Cytoskeletal integrity

  • Cellular metabolism

  • Calcium homeostasis

• Lipidomic alterations:

  • Significant decreases in sphingomyelins

  • Reduced phosphatidylethanolamine and phosphatidylcholine

  • Disrupted membrane integrity

• Pathway analysis identified dysregulation in:

  • Lipid and steroid metabolism (upregulated)

  • Immune-related pathways

  • MAPK and PI3K-Akt signaling (downregulated)

  • Purine metabolism (downregulated)

This multi-dimensional approach enables researchers to construct comprehensive models of AFG3L2 dysfunction, identifying both primary effects and compensatory mechanisms .

• What mechanisms explain the neuronal specificity of AFG3L2-related disorders despite ubiquitous expression?

Despite ubiquitous expression of AFG3L2, mutations predominantly affect specific neuronal populations. Several mechanisms contribute to this tissue specificity:

Cellular Energy Demands:

• Neurons have exceptionally high energy requirements

• Limited glycolytic capacity makes neurons heavily dependent on mitochondrial function

• Long axons require efficient mitochondrial transport and distribution

Cell-Type Specific Mitochondrial Dependencies:

• Purkinje cells show enhanced expression of AFG3L2 and particular vulnerability

• Bergmann glial cells rely on AFG3L2 for supporting Purkinje cell function through glutamate clearance

• Research shows that AFG3L2 deficiency in Bergmann glial cells causes mitochondrial fragmentation without OXPHOS dysfunction

Secondary Cellular Effects:

• AFG3L2 deficiency upregulates necroptotic factor ZBP1, triggering neuroinflammation

• Altered calcium homeostasis particularly affects excitatory neurons

• Purkinje cells show reduced dendrite formation and abnormal firing patterns

Experimental Approaches to Study Neuronal Specificity:

• Cell-type specific conditional knockout models

• Single-cell transcriptomics of affected tissues

• Comparison of mitochondrial proteomes across tissues

• iPSC-derived neuronal models of different subtypes

• In vivo calcium imaging of neuronal activity

Research shows that AFG3L2 expression patterns are not strictly related to the occurrence of SCA28 or SPAX5 development, suggesting unidentified proteins regulated by AFG3L2 may participate in disease pathophysiology .

• How does AFG3L2 function adapt under hypoxic conditions?

Under hypoxic conditions, AFG3L2 undergoes significant functional adaptations that contribute to mitochondrial remodeling:

Activation Mechanisms:

• AFG3L2 is activated in hypoxia along an HIF1α-mTORC1 signaling axis

• This activation enhances proteolytic activity toward specific substrate proteins

• Post-translational modifications may regulate AFG3L2 activity during hypoxia

Substrate Specificity Changes:

• Under hypoxia, AFG3L2-mediated proteolysis targets proteins involved in:

  • Mitochondrial protein import

  • Mitochondrial transcription

  • mRNA processing, modification, and stability

  • RNA granule formation

Functional Consequences:

• Restricts mitochondrial biogenesis during hypoxia

• Contributes to metabolic rewiring from oxidative phosphorylation to glycolysis

• Participates in rapid adaptation of the mitochondrial proteome to low oxygen

Experimental Approaches for Studying Hypoxic Regulation:

• Hypoxic chamber cultivation of cells with controlled oxygen levels

• Time-course proteomics to identify early vs. late AFG3L2 substrates

• SILAC-based approaches to measure protein degradation rates

• Proximity labeling to identify hypoxia-specific interaction partners

• Live-cell imaging of AFG3L2 activity using reporter substrates

This hypoxia-induced adaptation represents a critical aspect of AFG3L2 function, highlighting its importance beyond basal mitochondrial quality control to include stress response mechanisms .

• What methodological considerations are essential for studying AFG3L2-substrate interactions?

Investigating AFG3L2-substrate interactions requires specialized approaches due to the transient nature of protease-substrate relationships:

Substrate Trapping Approaches:

• Expression of catalytically inactive AFG3L2 (E408Q mutation in the proteolytic site)

• ATP-binding mutants (K354A) that can bind but not process substrates

• Pulse-chase analyses with proteasome inhibitors to stabilize degradation intermediates

Proximity-Based Methods:

• BioID or TurboID fusion proteins to identify proteins in proximity to AFG3L2

• APEX2-based proximity labeling for temporal resolution of interactions

• Split-GFP complementation for visualization of interactions in living cells

Structural and Biochemical Approaches:

• Cryo-EM analysis of AFG3L2 complexes with substrate peptides

• Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

• In vitro reconstitution of purified components for direct interaction assessment

Substrate Validation Strategy:

• Initial identification through proteomics or proximity labeling

• Confirmation of direct interaction (co-IP, in vitro binding)

• Demonstration of processing/degradation dependence on AFG3L2 activity

• Mapping of recognition sequences or degrons

• Functional consequences of preventing substrate processing

When designing these experiments, researchers should consider the membrane-embedded nature of AFG3L2, the potential for indirect effects, and the dynamic regulation of substrate recognition under different cellular conditions .

For recombinant AFG3L2 expression in substrate interaction studies, HEK-293 cells provide superior results with >90% purity, while maintaining proper folding and post-translational modifications essential for authentic substrate recognition .