ALDH6A1 Human

What is ALDH6A1 and what are its primary functions in human metabolism?

ALDH6A1 (Aldehyde Dehydrogenase 6 Family Member A1) encodes the mitochondrial enzyme methylmalonate semialdehyde dehydrogenase (MMSDH), predominantly expressed in the liver, kidney, and heart, with lower levels in muscle and brain . This enzyme belongs to the aldehyde dehydrogenase family and plays crucial roles in:

• Catalyzing the irreversible oxidative decarboxylation of malonate and methylmalonate semialdehydes to acetyl- and propionyl-CoA

• Serving as a key enzyme in the catabolic pathways of valine and pyrimidine

• Contributing to the metabolism of leucine, isoleucine, uracil, and thymine

• Functioning as the only known CoA-dependent aldehyde dehydrogenase

• Detoxifying harmful aldehydes to protect cells and mitochondria from oxidative stress

How is ALDH6A1 expression distributed across human tissues and what implications does this have for research design?

ALDH6A1 demonstrates a tissue-specific expression pattern that researchers should consider when designing experiments:

• High expression levels: liver, kidney, and heart tissues

• Moderate to low expression: skeletal muscle and brain tissues

• Expression patterns correlate with tissues having high oxidative metabolism demands

Methodological considerations for tissue-specific research:

• When investigating metabolic disorders, prioritize high-expression tissues

• For neurological studies, consider the lower but physiologically significant expression in brain tissue

• Employ tissue-specific cell lines that naturally express ALDH6A1 for in vitro studies

• Account for tissue-specific cofactors and regulatory mechanisms when measuring enzyme activity

What is known about the structural organization of ALDH6A1 and how does it relate to its function?

Recent structural analysis has revealed critical insights about ALDH6A1's architecture:

• ALDH6A1 in its apo form has a tetrameric structure with a resolution of 2.75 Å, featuring tightly interacting monomers

• This quaternary structure is essential for proper enzymatic function

• Compared to other ALDH family members, ALDH6A1 exhibits a unique binding model with NAD+

• The structural organization supports its dual function of converting methylmalonate semialdehyde to propionyl-CoA and malonate semialdehyde to acetyl-CoA

How should researchers approach experimental design when studying ALDH6A1 enzyme kinetics?

Effective experimental approaches for ALDH6A1 enzyme kinetics studies should include:

• Substrate preparation:

  • Synthesize or obtain purified methylmalonate semialdehyde and malonate semialdehyde

  • Prepare fresh substrates before each experiment due to potential instability

• Reaction conditions optimization:

  • Buffer composition: typically phosphate buffer (pH 7.4-8.0)

  • Temperature: 37°C for physiological relevance

  • Cofactors: ensure sufficient NAD+ and CoA availability

• Activity measurement methods:

  • Spectrophotometric assays tracking NAD+ to NADH conversion (340 nm)

  • HPLC analysis of acetyl-CoA and propionyl-CoA formation

  • Mass spectrometry for precise quantification of reaction products

• Control experiments:

  • Include enzyme-free and substrate-free controls

  • Use known inhibitors as positive controls for inhibition studies

  • Compare with other ALDH family enzymes to highlight ALDH6A1's unique properties

What approaches are recommended for comprehensive genetic analysis of ALDH6A1 variants?

When investigating ALDH6A1 genetic variants, researchers should implement a multi-faceted approach:

• Sequencing considerations:

  • Utilize clinical-grade NGS assays that can detect non-coding disease-causing variants

  • Be aware that certain test limitations exist, including difficulty detecting complex inversions, gene conversions, and balanced translocations

  • Consider test sensitivity limitations for low-level mosaicism (variants with minor allele fraction of 14.6% are detected with 90% probability)

• Variant classification strategy:

  • Apply rigorous variant classification schemes

  • Employ systematic clinical interpretation workflows using specialized software

  • Create comprehensive clinical statements documenting findings

• Analytical validation:

  • Confirm novel variants using orthogonal methods

  • Assess segregation patterns in families when possible

  • Evaluate evolutionary conservation and in silico prediction tools

How should researchers approach the functional characterization of novel ALDH6A1 variants?

For functional characterization of novel ALDH6A1 variants, implement this methodological framework:

• Expression system selection:

  • Prokaryotic systems for high protein yield

  • Mammalian cell lines for proper post-translational modifications

  • Consider using tissue-specific cell lines (hepatic, renal) for physiological relevance

• Functional assays:

  • Enzyme activity measurements comparing wild-type vs. variant proteins

  • Protein stability analysis using thermal shift assays

  • Structural impact assessment using circular dichroism or crystallography

  • Subcellular localization studies using fluorescent tagging

• Metabolic profiling:

  • Measure levels of substrate accumulation and product formation

  • Assess impact on connected metabolic pathways

  • Quantify cellular markers of oxidative stress

• Data interpretation framework:

  • Correlate functional deficits with clinical phenotypes

  • Compare with known pathogenic variants

  • Consider the impact on tissue-specific functions

What is the current understanding of ALDH6A1 deficiency syndrome and how should it be investigated?

ALDH6A1 deficiency (also known as methylmalonate semialdehyde dehydrogenase deficiency) is an autosomal recessive disorder characterized by:

• Elevated levels of beta-alanine, 3-hydroxypropionic acid, and both isomers of 3-amino and 3-hydroxyisobutyric acids in urine organic acids

• Psychomotor developmental delay in affected individuals

• Disruption of valine and pyrimidine catabolic pathways

Research approach for investigating ALDH6A1 deficiency:

• Metabolic profiling:

  • Comprehensive urine organic acid analysis using GC-MS

  • Plasma amino acid profiling focusing on valine metabolites

  • Measurement of beta-alanine and related metabolites

• Functional studies:

  • Enzyme activity assays in patient-derived cells

  • Metabolic flux analysis using isotope-labeled precursors

  • Expression studies of compensatory enzymes

• Animal and cellular models:

  • Generate ALDH6A1 knockout or knock-in models with patient-specific mutations

  • Develop induced pluripotent stem cell (iPSC) models from patient cells

  • Create tissue-specific conditional knockout models to study organ-specific effects

How is ALDH6A1 implicated in insulin resistance and metabolic disorders?

Recent research has identified important connections between ALDH6A1 and metabolic health:

• Downregulation of ALDH6A1 has been identified as a new marker of muscle insulin resistance, a key factor in type 2 diabetes mellitus pathophysiology

• ALDH6A1 is upregulated during lipogenesis and is involved in the maturation of fibroblasts into adipocytes

• The enzyme's role in detoxifying harmful aldehydes may protect against oxidative stress-related metabolic damage

Methodological considerations for metabolic research:

• Tissue-specific analysis:

  • Compare ALDH6A1 expression between insulin-sensitive and insulin-resistant tissues

  • Investigate regulatory mechanisms in different metabolic states (fed vs. fasted)

  • Assess the impact of ALDH6A1 manipulation on glucose uptake and insulin signaling

• Integration with metabolic pathways:

  • Analyze connections between ALDH6A1 activity and mitochondrial function

  • Investigate relationships with BCAA metabolism and insulin sensitivity

  • Study the impact on energy substrate utilization and flexibility

What are the cutting-edge approaches for targeting ALDH6A1 in therapeutic development?

Several promising therapeutic approaches targeting ALDH6A1 are emerging:

• Small molecule modulators:

  • Alda-1, originally developed as an ALDH2 agonist, has been found to enhance ALDH6A1 activity as well

  • Structure-based drug design utilizing the recently determined ALDH6A1 structure could lead to the development of specific modulators

• Advanced therapeutic strategies:

  • Gene therapy approaches to restore functional ALDH6A1 in deficiency states

  • RNA-based therapeutics to modulate ALDH6A1 expression

  • Metabolic bypass strategies targeting downstream pathways

• Experimental design considerations:

  • Employ the molecular docking approaches that identified the ALDH6A1-NAD+ interaction mechanism

  • Develop assays to screen compound libraries for ALDH6A1 modulators

  • Establish relevant cellular and animal models for preclinical testing

How should researchers approach the investigation of ALDH6A1's role in cancer biology?

ALDH6A1 has emerged as a biomarker for several types of severe cancer , necessitating specialized research approaches:

• Expression analysis in cancer tissues:

  • Compare ALDH6A1 expression between tumor and matched normal tissues

  • Correlate expression levels with clinical outcomes and treatment responses

  • Investigate epigenetic regulation of ALDH6A1 in cancer contexts

• Functional studies in cancer models:

  • Manipulate ALDH6A1 expression in cancer cell lines using CRISPR/Cas9 or RNAi

  • Assess impacts on proliferation, migration, invasion, and resistance to therapy

  • Investigate interactions with known cancer-associated pathways

• Metabolic reprogramming investigation:

  • Analyze how ALDH6A1 alterations affect cancer cell metabolism

  • Study the connection to mitochondrial function in cancer cells

  • Evaluate potential synthetic lethal interactions with other metabolic enzymes

• Translational applications:

  • Develop ALDH6A1-based diagnostic or prognostic biomarkers

  • Explore ALDH6A1 as a potential therapeutic target in specific cancer types

  • Investigate combination approaches targeting ALDH6A1 alongside established therapies

What are the optimal experimental conditions for measuring ALDH6A1 activity in different sample types?

Researchers should consider these technical parameters when measuring ALDH6A1 activity:

• Sample preparation by tissue type:

  Tissue Type	Homogenization Buffer	Special Considerations
  Liver	50mM phosphate buffer (pH 7.4) with protease inhibitors	Highest ALDH6A1 content; use lower protein concentration
  Kidney	50mM phosphate buffer (pH 7.4) with protease inhibitors	Prone to proteolysis; add extra protease inhibitors
  Heart	100mM HEPES buffer (pH 7.4) with 1mM DTT	Requires membrane disruption for mitochondrial access
  Muscle	50mM Tris-HCl (pH 7.5) with 150mM NaCl	Lower abundance; may need concentration steps
  Cell cultures	Gentle lysis buffer with digitonin	Maintain mitochondrial integrity during extraction

• Activity assay optimization:

  • Temperature: 37°C for physiological relevance

  • pH optimum: 7.8-8.2 for maximum activity

  • Cofactor concentrations: 1-2mM NAD+, 0.1-0.5mM CoA

  • Substrate concentration: typically 50-200μM of methylmalonate semialdehyde

• Detection methods comparison:

  Method	Sensitivity	Advantages	Limitations
  Spectrophotometric	Medium	Simple, real-time	Potential interference
  HPLC	High	Direct product measurement	Time-consuming
  Mass spectrometry	Very high	Most specific and sensitive	Expensive, specialized equipment
  Radioisotope-based	Very high	Excellent for kinetic studies	Safety concerns, special facilities

How can researchers effectively differentiate ALDH6A1 activity from other ALDH family members?

Distinguishing ALDH6A1 activity from other ALDH family members requires careful methodological approaches:

• Specific substrate selection:

  • Use methylmalonate semialdehyde, which is preferentially metabolized by ALDH6A1

  • Include CoA as a cofactor, as ALDH6A1 is the only CoA-dependent ALDH

• Inhibitor profiles:

  • Employ differential inhibitor sensitivity patterns

  • Utilize Alda-1, which has recently been identified to enhance ALDH6A1 activity

• Genetic approaches:

  • Use siRNA/shRNA knockdown specific to ALDH6A1

  • Express recombinant ALDH6A1 in systems lacking endogenous enzyme

• Product analysis:

  • Measure CoA-activated products (acetyl-CoA and propionyl-CoA)

  • Track both dehydrogenation and CoA-activation steps

What emerging technologies should researchers consider for advancing ALDH6A1 research?

The field of ALDH6A1 research will benefit from adopting these cutting-edge technologies:

• Advanced structural biology techniques:

  • Cryo-EM for dynamic structural studies of ALDH6A1 in different conformational states

  • Hydrogen-deuterium exchange mass spectrometry for protein dynamics

  • Single-molecule FRET to study conformational changes during catalysis

• Systems biology approaches:

  • Multi-omics integration (proteomics, metabolomics, transcriptomics)

  • Computational modeling of ALDH6A1-dependent metabolic networks

  • Machine learning for predicting impacts of genetic variants

• Advanced genetic engineering:

  • Base editing and prime editing for precise genetic modifications

  • Tissue-specific and inducible CRISPR systems

  • Organoid models derived from patient cells

• Translational technologies:

  • High-throughput screening platforms for ALDH6A1 modulators

  • In vivo imaging of ALDH6A1 activity using specific probes

  • Nanobody-based approaches for targeting ALDH6A1 in specific cellular compartments

How should researchers approach the investigation of ALDH6A1's role in previously unexplored physiological processes?

When expanding ALDH6A1 research into novel areas, consider this methodological framework:

• Hypothesis generation:

  • Mining publicly available datasets for ALDH6A1 correlations with unexplored pathways

  • Leveraging interactome data to identify novel protein-protein interactions

  • Using comparative biology to identify evolutionarily conserved functions

• Validation strategy:

  • Initial screening in cell culture models with ALDH6A1 modulation

  • Confirmation in tissue-specific contexts

  • Mechanistic studies linking ALDH6A1 to the newly identified process

• Physiological relevance assessment:

  • Determine if the process is affected in ALDH6A1 deficiency

  • Evaluate tissue-specific implications

  • Connect to broader metabolic or signaling networks

• Translational potential evaluation:

  • Identify potential biomarker applications

  • Assess therapeutic targeting opportunities

  • Determine relevance to human diseases or conditions