Recombinant Mouse AFG3-like protein 1 (Afg3l1)

What is Afg3l1 and what is its role in mitochondria?

Afg3l1 (AFG3-like protein 1) is a nuclear-encoded subunit of the mitochondrial m-AAA protease complex. It belongs to the AAA+ (ATPases Associated with diverse cellular Activities) family of proteins that use ATP hydrolysis to drive protein translocation and degradation. As part of the m-AAA protease complex, Afg3l1 plays crucial roles in protein quality control within mitochondria, specifically on the matrix-facing surface of the inner mitochondrial membrane .

The m-AAA protease complex functions to:

• Degrade misfolded or damaged proteins

• Process specific mitochondrial proteins

• Maintain mitochondrial proteostasis

• Regulate mitochondrial dynamics and function

Afg3l1 contains both ATPase and protease domains that work in concert to unfold and degrade substrate proteins. The protein undergoes autocatalytic processing upon import into mitochondria, which is essential for its proper function .

How does Afg3l1 interact with other m-AAA protease subunits?

Afg3l1 forms heteromeric complexes with other m-AAA protease subunits, particularly paraplegin and Afg3l2. These interactions are critical for the assembly and function of the complete m-AAA protease complex. Experimental evidence from coimmunoprecipitation studies demonstrates that:

• Afg3l1 can interact directly with both paraplegin and Afg3l2

• The maturation of paraplegin depends on the presence of functional Afg3l1 and Afg3l2

• Afg3l1 can form both homomeric complexes and heteromeric complexes with other subunits

The interactions between these subunits can be studied using techniques such as coimmunoprecipitation and blue-native gel electrophoresis (BN-PAGE). When one subunit is depleted through RNA interference (RNAi), the formation and function of the m-AAA protease complex is affected, indicating the interdependence of these subunits for proper complex assembly and function .

What are the common methods to express and purify recombinant Afg3l1?

Expression and purification of recombinant Afg3l1 typically involves several key methodological approaches:

Expression Systems:

• Yeast expression systems: Afg3l1 can be expressed under the control of promoters such as the YTA10 promoter in yeast using multicopy vectors (e.g., YEplac181, YEplac195)

• Mammalian expression systems: For studies requiring mammalian post-translational modifications

• In vitro transcription/translation: For smaller-scale studies, Afg3l1 can be cloned into vectors like pGEM4 under the control of the SP6 promoter for in vitro transcription

Purification Approaches:

• Addition of affinity tags: C-terminal hexahistidine-tags can be incorporated to facilitate purification by affinity chromatography

• Expression optimization: Including 3′UTR elements (such as 250 base pairs of the 3′UTR of the YTA10 gene) can enhance expression levels

Validation Methods:

• Immunoblotting with Afg3l1-specific antisera

• Functional assays to assess ATPase and protease activities

• Size exclusion chromatography to verify complex assembly

When designing expression constructs, it is important to consider that Afg3l1 undergoes autocatalytic processing upon import into mitochondria, which may affect the choice of tags and their placement within the construct .

What experimental approaches are most effective for studying Afg3l1 function in vitro and in vivo?

Comprehensive investigation of Afg3l1 function requires multiple complementary experimental approaches:

In Vitro Systems:

• Reconstitution of purified components: Using recombinant proteins to reconstitute the m-AAA protease complex and assess its activity on model substrates

• ATP hydrolysis assays: Measuring the ATPase activity to evaluate the energy-dependent functions of Afg3l1

• Protease activity assays: Using fluorogenic peptides or defined protein substrates to assess proteolytic function

Cellular Models:

• RNAi-mediated knockdown: Specific Stealth RNAi (e.g., 5′-GCGAAACCAUGGUGGAGAAGCCAUA-3′ for AFG3L1) can be used to down-regulate Afg3l1 expression in cell culture models like MEFs

• CRISPR/Cas9 gene editing: For generating knockout or knock-in mutations

• Overexpression studies: Using vectors with strong promoters like ADH1 to express wildtype or mutant forms of Afg3l1

In Vivo Models:

• Transgenic mouse models: Including knockout models and models with tissue-specific expression

• Rescue experiments: Reintroducing wildtype or mutant Afg3l1 into knockout backgrounds

Analytical Techniques:

• Blue-native gel electrophoresis (BN-PAGE): For analyzing native protein complexes and their assembly

• Coimmunoprecipitation: Using subunit-specific antibodies to isolate protein complexes and identify interaction partners

• Cryo-EM structural analysis: Similar to approaches used for AFG3L2, structural studies can provide insights into the molecular mechanism of action

How does the autocatalytic processing of Afg3l1 occur and what is its significance?

Autocatalytic processing is a critical step in the maturation of Afg3l1 upon import into mitochondria. This process involves several distinct steps and mechanisms:

Processing Mechanism:

• Initial import: Nuclear-encoded Afg3l1 is translated in the cytosol and contains a mitochondrial targeting sequence

• Membrane insertion: After import into mitochondria, Afg3l1 is inserted into the inner mitochondrial membrane

• Autocatalytic cleavage: The protease domain of Afg3l1 catalyzes its own processing, removing the N-terminal targeting sequence

• Complex assembly: Processed Afg3l1 assembles with other m-AAA protease subunits to form functional complexes

Functional Significance:

• Activation: Autocatalytic processing converts Afg3l1 from an inactive precursor to an active protease

• Regulation: The processing step provides a regulatory checkpoint for m-AAA protease assembly

• Integration: Ensures that only properly imported and membrane-inserted Afg3l1 becomes activated

Experimental Evidence:
Research has demonstrated that protease-inactive mutants of Afg3l1 (such as those with mutations in the catalytic site) fail to undergo proper processing. Similarly, experiments in which the ATPase function is compromised through Walker B mutations (e.g., E408Q) reveal the importance of ATP hydrolysis in the conformational changes needed for proper processing and substrate engagement .

The autocatalytic processing of Afg3l1 represents an elegant mechanism by which the protein regulates its own activation, ensuring that protease activity is only engaged after proper mitochondrial import and membrane insertion, thereby preventing inappropriate proteolysis in cellular compartments outside the mitochondria .

What are the structural features of Afg3l1 that dictate its substrate specificity?

While the search results don't provide direct structural information specifically for Afg3l1, insights can be drawn from structural studies of the related AFG3L2 protein, which shares significant homology with Afg3l1:

Key Structural Elements:

• Pore loops: Specialized loops (pore-loop 1 and pore-loop 2) within the ATPase domain that engage substrates and help translocate them through the central pore of the hexameric complex

• Central protrusion: Formed by residues within the protease domain that project upward toward the incoming substrate, potentially playing a role in substrate recognition and processing

• N-terminal domain: May form an additional spiral staircase above the ATPase domains that surrounds and contacts translocating substrates

Substrate Recognition Mechanisms:

• Hydrophobic interactions: Residues like phenylalanine (comparable to F421 in AFG3L2) in pore-loop 2 may form hydrophobic interactions with substrates

• Sequential ATP hydrolysis: The asymmetric ATPase spiral structure, with different nucleotide states (ATP, ADP, apo) in different subunits, creates a coordinated power stroke that drives substrate translocation

• Transfer mechanism: The extended pore-loop 2 and central protease loops likely facilitate substrate transfer from the ATPase spiral to the proteolytic ring

Experimental Approaches to Study Specificity:

• Site-directed mutagenesis: Mutating key residues in the pore loops or central protrusion to assess their impact on substrate processing

• Substrate competition assays: Using defined substrates to assess preferential processing

• Structural studies: Cryo-EM analysis of substrate-bound complexes, similar to those performed for AFG3L2

It's worth noting that the creation of chimeric proteins, where domains are swapped between Afg3l1 and other m-AAA protease subunits, can help identify regions responsible for substrate specificity differences between these closely related proteins.

What single-case experimental designs are most appropriate for studying Afg3l1 function in rare mitochondrial disorders?

Single-case experimental designs (SCEDs) offer valuable approaches for studying Afg3l1 function, particularly in the context of rare mitochondrial disorders where large sample sizes may be unavailable:

Applicable SCED Approaches:

• Reversal designs: Can be used to study the effects of Afg3l1 interventions by implementing an A1B1A2B2 design, where:

  • A phases represent baseline or no treatment

  • B phases represent Afg3l1-targeted interventions

  • Multiple replications demonstrate experimental control

• Multiple baseline designs: Particularly useful for testing Afg3l1-targeted treatments across:

  • Different patients with similar mitochondrial disorders

  • Different cell types within the same patient

  • Different biochemical outcomes in the same experimental system

• Combined designs: Integrating multiple baseline and reversal approaches to provide robust evidence of treatment effects

Implementation Considerations:

• Stability criteria: Data points should fall within a 15% range of the median for a condition to establish stability

• Phase length: A minimum of 5 data points per phase is recommended, with flexibility to determine stability and trend

• Replications: At least three replications of treatment effects are needed to demonstrate experimental control

Applications to Afg3l1 Research:

• Treatment development: Testing different Afg3l1-modulating compounds on cellular models derived from patients

• Dose-response relationships: Using designs like A1B1C1B2C2, where B and C represent different doses of an Afg3l1-targeted treatment

• Personalized interventions: Identifying optimal treatment approaches for individual patients based on their specific Afg3l1 mutations

These experimental designs are particularly valuable for translational research on Afg3l1, bridging the gap between basic scientific understanding and clinical applications in the treatment of mitochondrial disorders .

How can RNAi approaches be optimized for studying Afg3l1 function in cellular models?

RNA interference (RNAi) is a powerful technique for studying Afg3l1 function through targeted knockdown of gene expression. Optimizing RNAi approaches for Afg3l1 studies requires careful consideration of several factors:

siRNA Design and Selection:

• Sequence specificity: Use carefully validated sequences such as 5′-GCGAAACCAUGGUGGAGAAGCCAUA-3′ for AFG3L1 to ensure target specificity

• Control selection: Include non-targeting Stealth RNAi Negative Controls to distinguish specific from non-specific effects

• Multiple siRNAs: Test at least three different oligonucleotides specific for Afg3l1 to confirm consistent phenotypes and rule out off-target effects

Transfection Protocol Optimization:

• Transfection reagent: Lipofectamine RNAiMAX has been successfully used for Afg3l1 knockdown in MEFs

• siRNA concentration: Concentrations around 10 nM have shown effective knockdown

• Multiple transfections: Sequential transfections (e.g., two separate transfections) can enhance knockdown efficiency

Validation and Analysis:

• Timing: Optimal protein depletion is typically observed ~2.5 days after transfection for Afg3l1

• Verification methods: Use immunoblot analysis with Afg3l1-specific antisera to confirm knockdown efficiency

• Functional assays: Combine knockdown with functional assays to assess the impact on mitochondrial function

Combined Approaches:

• Sequential knockdown: When studying interactions between Afg3l1 and other m-AAA protease subunits, sequential or simultaneous knockdown of multiple targets can be informative

• Rescue experiments: Introducing siRNA-resistant Afg3l1 constructs can confirm specificity of observed phenotypes

• Complementary methods: Combine RNAi with pharmacological approaches or genetic models for comprehensive analysis

Experimental Controls and Considerations:

• Cell type selection: Mouse embryonic fibroblasts (MEFs) have been successfully used for Afg3l1 studies, but optimization may be needed for other cell types

• Background selection: Consider using cells from relevant genetic backgrounds (e.g., Spg7+/+ or Spg7−/− mice) when studying interactions with paraplegin

• Transfection efficiency: Monitor and optimize transfection efficiency for each cell type

When properly optimized, RNAi approaches provide a valuable tool for dissecting Afg3l1 function in cellular contexts, particularly for studying its interactions with other m-AAA protease subunits and its role in mitochondrial proteostasis .

What are the most common challenges in purifying active recombinant Afg3l1?

Purification of active recombinant Afg3l1 presents several challenges due to its complex structure, membrane association, and functional requirements:

Common Challenges and Solutions:

• Maintaining Native Conformation:

  • Challenge: Afg3l1 contains both soluble and membrane-embedded domains

  • Solution: Use mild detergents (e.g., n-dodecyl β-D-maltoside) for extraction; consider nanodiscs or amphipols for mimicking membrane environment

• Preserving ATPase Activity:

  • Challenge: ATPase activity can be compromised during purification

  • Solution: Include ATP or non-hydrolyzable analogs (e.g., AMP-PNP) during purification; avoid oxidizing conditions that could affect nucleotide binding sites

• Maintaining Oligomeric State:

  • Challenge: Afg3l1 functions as part of hexameric complexes

  • Solution: Use techniques that preserve native complexes, such as blue-native gel electrophoresis for analysis; consider crosslinking approaches

• Expression Yield:

  • Challenge: Low expression levels in heterologous systems

  • Solution: Optimize codon usage; include relevant 3′UTR elements (e.g., 250 bp of YTA10 3′UTR); use strong promoters like ADH1

• Autoproteolysis:

  • Challenge: Active Afg3l1 may undergo self-cleavage during purification

  • Solution: Consider using protease-inactive mutants (e.g., E575Q equivalent) for structural studies; perform purification at lower temperatures

Validation of Purified Protein:

• ATPase activity assays to confirm functionality

• Limited proteolysis to assess proper folding

• Size exclusion chromatography to verify oligomeric state

• Negative stain EM to confirm complex formation

Researchers should consider that modifications introduced for purification purposes, such as C-terminal hexahistidine tags, may affect the activity or assembly of Afg3l1. Validation of the purified protein's functional characteristics against native Afg3l1 is essential to ensure that the recombinant protein accurately represents the in vivo state .

How can one differentiate between the effects of Afg3l1 and Afg3l2 in experimental systems?

Differentiating between Afg3l1 and Afg3l2 functions is crucial for understanding their specific roles, as these proteins share significant sequence homology and functional overlap. Several experimental approaches can help distinguish their individual contributions:

Selective Knockdown Approaches:

• siRNA specificity: Use validated siRNA sequences specific to each protein:

  • For Afg3l1: 5′-GCGAAACCAUGGUGGAGAAGCCAUA-3′

  • For Afg3l2: 5′-CCUGCCUCCGUACGCUCUAUCAAUA-3′

• Sequential knockdown: Deplete one protein followed by the other to assess stepwise effects

• Rescue experiments: Selectively reintroduce either Afg3l1 or Afg3l2 into double-knockdown cells

Biochemical Discrimination:

• Subunit-specific antibodies: Use validated antibodies that specifically recognize either Afg3l1 or Afg3l2

• Tagged versions: Express differentially tagged versions (e.g., HA-Afg3l1 and FLAG-Afg3l2) to track individual proteins

• Immunoprecipitation: Use subunit-specific antibodies for coimmunoprecipitation to isolate complexes containing specific subunits

Genetic Approaches:

• Knockout models: Compare phenotypes of Afg3l1-/- vs. Afg3l2-/- cells or organisms

• Domain swapping: Create chimeric proteins to identify functionally distinct domains

• Species-specific differences: Exploit the fact that humans only express AFG3L2 (AFG3L1 is a pseudogene in humans)

Substrate Specificity Analysis:

• Comparative proteomics: Identify differentially processed substrates following selective depletion

• In vitro processing: Test substrate processing using purified Afg3l1 or Afg3l2 complexes

• Binding assays: Compare substrate binding affinities between Afg3l1 and Afg3l2

By employing these complementary approaches, researchers can dissect the specific functions of Afg3l1 and Afg3l2, identifying both their unique and overlapping roles in mitochondrial protein quality control and processing .

What are the best practices for designing experiments to study Afg3l1 and paraplegin interactions?

The interaction between Afg3l1 and paraplegin is critical for the formation and function of heteromeric m-AAA protease complexes. Designing robust experiments to study these interactions requires careful consideration of multiple factors:

Experimental Design Principles:

• Genetic Manipulation Strategies:

  • Use cells from relevant genetic backgrounds (e.g., Spg7+/+ or Spg7−/− MEFs) to provide clean backgrounds for interaction studies

  • Implement selective knockdown of individual subunits using validated siRNAs

  • Consider the use of inducible expression systems for controlled expression

• Biochemical Interaction Analysis:

  • Coimmunoprecipitation using subunit-specific antibodies to isolate native complexes

  • Blue-native gel electrophoresis (BN-PAGE) to analyze intact complexes

  • Chemical crosslinking followed by mass spectrometry to identify interaction interfaces

• Functional Interdependence Assessment:

  • Study the maturation of paraplegin in Afg3l1-depleted cells to assess processing dependencies

  • Evaluate the assembly of m-AAA protease complexes in the absence of individual subunits

  • Conduct rescue experiments with wildtype or mutant versions of either protein

Controls and Validations:

• Include both positive controls (known interacting proteins) and negative controls

• Verify knockdown or knockout efficiency using immunoblotting

• Use multiple, complementary approaches to confirm interactions

• Consider the potential impact of tags on protein interactions

Advanced Approaches:

• Proximity labeling methods (BioID, APEX) to identify in situ interaction partners

• Single-molecule techniques to study dynamics of complex assembly

• Structural studies (similar to those conducted for AFG3L2) to identify interaction interfaces

By combining these approaches, researchers can comprehensively characterize the interactions between Afg3l1 and paraplegin, providing insights into the assembly, regulation, and function of m-AAA protease complexes in mitochondrial protein quality control .

How should researchers interpret conflicting data regarding Afg3l1 function across different experimental systems?

Sources of Experimental Variability:

• Model System Differences:

  • Species variations: Mouse studies may not directly translate to human systems, especially since AFG3L1 is a pseudogene in humans

  • Cell type specificity: Afg3l1 function may vary between different cell types (e.g., fibroblasts vs. neurons)

  • Compensatory mechanisms: Different model systems may have varying abilities to compensate for Afg3l1 deficiency

• Methodological Factors:

  • Knockdown efficiency: Incomplete vs. complete depletion of Afg3l1

  • Timing: Acute vs. chronic depletion may yield different phenotypes

  • Experimental conditions: Growth conditions, stress levels, and media composition

Reconciliation Strategies:

• Systematic Comparison:

  • Create standardized experimental conditions across systems

  • Perform side-by-side comparisons using identical readouts

  • Quantify the degree of discrepancy to determine if differences are significant

• Multi-level Analysis:

  • Examine effects at different biological levels (molecular, cellular, organismal)

  • Consider temporal dynamics of Afg3l1 function

  • Assess both direct effects and compensatory responses

• Validation Approaches:

  • Confirm knockdown/knockout efficiency with multiple methods

  • Use multiple, independent siRNAs or genetic approaches

  • Perform rescue experiments with wildtype Afg3l1

Interpretation Framework:

When publishing research on Afg3l1, transparency about experimental conditions, limitations, and potential sources of variability is essential. By systematically addressing discrepancies and exploring their biological basis, researchers can transform conflicting data into deeper insights about the context-dependent functions of Afg3l1 .

What statistical approaches are most appropriate for analyzing Afg3l1 knockdown or knockout experiments?

Selecting appropriate statistical approaches for Afg3l1 functional studies requires consideration of experimental design, data types, and research questions. Here are recommended statistical methods for different experimental scenarios:

For Single-Case Experimental Designs (SCEDs):

• Visual analysis: Systematic examination of level, trend, and variability within and between phases

• Effect size calculations: Measuring the magnitude of treatment effects using metrics like percentage of non-overlapping data (PND) or Tau-U

• Randomization tests: When treatment assignment can be randomized across phases

For Group Comparison Studies:

• Parametric tests: t-tests (paired or unpaired) for comparing two conditions; ANOVA for multiple conditions

• Non-parametric alternatives: Mann-Whitney U or Wilcoxon signed-rank tests when normality assumptions are violated

• Mixed-effects models: For longitudinal data with repeated measurements

For Dose-Response or Time-Course Experiments:

• Regression analysis: Linear or non-linear regression to model relationships between Afg3l1 levels and outcomes

• Time-series analysis: For temporal patterns in response to Afg3l1 manipulation

• Area under the curve (AUC) analysis: To quantify cumulative effects over time

Statistical Considerations for Afg3l1 Studies:

• Sample Size Determination:

  • Power analysis to determine adequate sample size

  • Consider biological variability in Afg3l1 expression and function

  • For rare conditions, consider adaptive designs or sequential analysis

• Controlling for Confounders:

  • Account for variability in knockdown efficiency

  • Control for off-target effects of siRNAs

  • Consider cell cycle effects in proliferating cells

• Multiple Testing Correction:

  • Use appropriate corrections (e.g., Bonferroni, Benjamini-Hochberg) when testing multiple hypotheses

  • Consider false discovery rate (FDR) control for high-dimensional data

Data Visualization Recommendations:

• Box plots with individual data points to show distribution

• Time-course graphs with appropriate error bars

• Correlation plots for relationships between Afg3l1 levels and functional outcomes

For all statistical approaches, researchers should report effect sizes alongside p-values, provide clear descriptions of statistical methods, and ensure transparency about data exclusions and transformations .

What are the most promising therapeutic targets related to Afg3l1 for mitochondrial disorders?

Research on Afg3l1 suggests several promising therapeutic approaches for mitochondrial disorders associated with m-AAA protease dysfunction:

Potential Therapeutic Strategies:

• Direct Modulation of Afg3l1 Activity:

  • Small molecule activators: Compounds that enhance remaining Afg3l1 activity in partial deficiency cases

  • Protease modulators: Drugs that modify proteolytic activity without affecting ATPase function

  • Allosteric regulators: Molecules that bind to regulatory sites to enhance function

• Enhancement of Complementary Pathways:

  • Upregulation of alternative mitochondrial proteases (e.g., i-AAA proteases like YME1)

  • Boosting mitochondrial chaperone systems to handle misfolded proteins

  • Activation of mitophagy to eliminate damaged mitochondria

• Gene Therapy Approaches:

  • Adeno-associated virus (AAV) delivery of functional Afg3l1

  • CRISPR-based correction of Afg3l1 mutations

  • RNA-based therapies to enhance expression or correct splicing defects

• Metabolic Bypass Strategies:

  • Supplementation with metabolites downstream of affected pathways

  • Ketogenic diets to provide alternative energy sources

  • Mitochondrial cofactor supplementation (CoQ10, riboflavin, lipoic acid)

Target Prioritization Matrix:

Personalized Medicine Approaches:
Given the complexity of m-AAA protease function and the specificity of different mutations, single-case experimental designs (SCEDs) could be particularly valuable for identifying optimal treatments for individual patients. Approaches like reversal designs (A1B1C1B2C2) could help determine the most effective interventions or combinations for specific genetic backgrounds .

Future therapeutic development should integrate insights from structural studies of m-AAA proteases (such as those available for AFG3L2) to design highly specific modulators that can restore function in disease states while minimizing off-target effects .

What emerging technologies will advance our understanding of Afg3l1 function?

Several cutting-edge technologies are poised to significantly advance our understanding of Afg3l1 function in the coming years:

Structural Biology Approaches:

• Cryo-electron microscopy (Cryo-EM): Building upon the techniques used for AFG3L2, high-resolution cryo-EM can reveal the dynamic structural changes in Afg3l1 during substrate processing

• Integrative structural biology: Combining cryo-EM with crosslinking mass spectrometry, molecular dynamics simulations, and other techniques to build comprehensive structural models

• Time-resolved structural methods: Capturing transient intermediates during substrate processing

Advanced Genetic Technologies:

• CRISPR base editing and prime editing: For precise introduction of specific mutations without double-strand breaks

• CRISPR screens: Genome-wide or targeted screens to identify genetic interactors of Afg3l1

• Single-cell genomics and transcriptomics: To understand cell-to-cell variability in Afg3l1 function and compensatory responses

Proteomic Innovations:

• Proximity labeling proteomics: Techniques like BioID or APEX to identify the dynamic interactome of Afg3l1 in living cells

• Targeted proteomics: Selective reaction monitoring (SRM) or parallel reaction monitoring (PRM) for precise quantification of Afg3l1 and its substrates

• Degradomics: Methods to systematically identify Afg3l1 substrates and cleavage sites

Advanced Imaging:

• Super-resolution microscopy: Techniques like STORM or PALM to visualize Afg3l1 distribution and dynamics at nanoscale resolution

• Live-cell imaging: Using split fluorescent proteins or FRET sensors to monitor Afg3l1 interactions in real time

• Correlative light and electron microscopy (CLEM): To connect functional observations with ultrastructural context

Computational and Systems Biology:

• Machine learning approaches: For prediction of Afg3l1 substrates and regulatory networks

• Molecular dynamics simulations: To model substrate translocation and processing mechanisms

• Systems biology modeling: To integrate Afg3l1 function into broader mitochondrial quality control networks

Translational Technologies:

• Organoid and microphysiological systems: For studying Afg3l1 function in tissue-specific contexts

• Patient-derived models: iPSC-derived cells from patients with m-AAA protease-related disorders

• In situ sequencing and spatial transcriptomics: To understand the spatial context of Afg3l1 function in tissues

The integration of these technologies, particularly when applied within well-designed experimental frameworks such as single-case experimental designs, will provide unprecedented insights into the molecular mechanisms and physiological roles of Afg3l1 in health and disease .