CTH Human

What is CTH Human and what is its biochemical function?

CTH (Cystathionine Gamma-Lyase) is a cytoplasmic enzyme that plays a crucial role in the trans-sulfuration pathway. It catalyzes the final step in converting methionine to cysteine by cleaving L,L-cystathionine into L-cysteine, ammonia, and 2-oxobutanoate in a pyridoxal-5'-phosphate (PLP)-dependent manner . This process is essential for the biosynthesis of glutathione, a ubiquitous antioxidant . Beyond its primary function, CTH also contributes to the production of hydrogen sulfide (H2S), an important gaseous signaling molecule, by utilizing L-cysteine and L-homocysteine as substrates (though at lower rates than with L,L-cystathionine) .

The enzyme's multifunctional nature extends to its ability to act as a cysteine-protein sulfhydrase, mediating sulfhydration of target proteins by converting -SH groups into -SSH on specific cysteine residues. This post-translational modification affects the function of various proteins, including GAPDH, PTPN1, and NF-kappa-B subunit RELA .

What are the structural characteristics of CTH Human protein?

Human CTH has distinct structural and biochemical properties that researchers should consider when designing experiments:

Understanding these structural characteristics is essential for experimental design, particularly when working with recombinant versions of the protein or developing assays to measure enzymatic activity.

What diseases are associated with CTH dysfunction?

CTH dysfunction has been linked to several pathological conditions that provide important research contexts:

• Cystathioninuria - A metabolic disorder characterized by the accumulation of cystathionine in urine due to mutations in the CTH gene .

• Hyperhomocysteinemia - A condition featuring elevated blood homocysteine levels, which is a risk factor for cardiovascular disease, diabetes, and Alzheimer's disease .

These disease associations highlight the importance of CTH in maintaining sulfur amino acid homeostasis. Research into these conditions often requires specialized experimental models that accurately recapitulate the metabolic disturbances observed in human patients.

How should researchers optimally store and handle CTH Human samples?

Proper storage and handling of CTH Human samples is critical for maintaining enzymatic activity and experimental reproducibility:

These parameters are particularly important when working with recombinant protein preparations. Researchers should validate protein activity after extended storage periods and minimize exposure to conditions that might promote denaturation or aggregation.

What experimental controls are essential when studying CTH enzymatic activity?

When designing experiments to measure CTH activity, several controls are necessary:

• Enzyme-free control reactions to account for non-enzymatic background reactions.

• Heat-inactivated enzyme controls to distinguish enzymatic from non-specific chemical reactions.

• PLP cofactor controls to ensure optimal enzymatic function and account for potential limitations.

• Substrate specificity controls using structural analogs of cystathionine to confirm reaction specificity.

• Time course measurements to ensure linearity of the enzymatic reaction within the experimental timeframe.

These controls help ensure experimental rigor and facilitate accurate interpretation of results when studying CTH's complex catalytic functions.

What are the most effective experimental design approaches for studying CTH across different tissues?

Studying CTH across different tissues requires careful experimental design considerations:

By incorporating these design elements, researchers can generate more reliable cross-tissue comparisons and identify tissue-specific regulation of CTH activity.

How can researchers effectively investigate CTH protein interactions and post-translational modifications?

Investigating CTH protein interactions and post-translational modifications requires specialized methodological approaches:

• Protein Interaction Screening

  Employ complementary techniques including co-immunoprecipitation followed by mass spectrometry, proximity labeling methods (BioID, APEX), and yeast two-hybrid screening to identify interaction partners. Validation should include orthogonal methods to confirm primary findings.

• Sulfhydration Detection

  Utilize modified biotin-switch techniques specifically adapted for detecting protein S-sulfhydration. This involves selective labeling of -SSH groups followed by biotin tagging and detection via Western blotting or mass spectrometry.

• Site-Specific Modification Analysis

  Implement site-directed mutagenesis of specific cysteine residues followed by functional assays to determine the impact of sulfhydration on protein function. Mass spectrometry-based approaches can provide direct evidence for modification sites.

• Real-time Interaction Dynamics

  Apply FRET-based biosensors or bioluminescence resonance energy transfer (BRET) to monitor CTH interactions in living cells, providing insights into the temporal and spatial regulation of these interactions.

• Integrative Network Analysis

  Develop protein interaction networks incorporating CTH and its partners to contextualize findings within broader cellular pathways. This approach can reveal unexpected connections and functional implications.

These methodological approaches enable researchers to move beyond simple identification of CTH interactions to understanding their functional significance in different cellular contexts.

What methodological approaches can help resolve contradictions in published CTH research?

Researchers often encounter contradictory findings in the CTH literature, particularly regarding its role in H2S production and protein sulfhydration. Several methodological approaches can help address these contradictions:

These approaches can help reconcile contradictory findings and establish a more coherent understanding of CTH function across different experimental contexts.

How can advanced techniques be applied to study CTH's role in redox signaling and hydrogen sulfide production?

Investigating CTH's contributions to redox signaling and H2S production requires sophisticated methodological approaches:

• Real-time H2S Detection Systems

  Implement fluorescent H2S probes (e.g., SF7-AM, HSip-1) for spatiotemporal monitoring of H2S production in living cells. These probes can be combined with genetic manipulation of CTH to establish direct causality.

• Redox Proteomics Workflow

  Apply cysteine-targeted redox proteomics to identify proteins modified by CTH-generated H2S. This involves selective labeling of modified cysteines followed by mass spectrometry analysis to create comprehensive maps of sulfhydration targets.

• Genetic Manipulation Strategies

  Utilize CRISPR-Cas9 gene editing to create precise CTH mutations that separate its trans-sulfuration function from H2S production. This allows dissection of specific CTH functions in redox signaling pathways.

• Microfluidic Systems

  Develop microfluidic platforms that enable precise control over the cellular microenvironment while simultaneously monitoring redox status and H2S levels. These systems facilitate studies of CTH regulation under dynamically changing conditions.

• Mathematical Modeling

  Create computational models of CTH-mediated H2S production and consumption that incorporate enzyme kinetics, diffusion, and interaction with cellular targets. These models can predict system behavior under conditions difficult to test experimentally.

By integrating these advanced techniques, researchers can develop more comprehensive models of how CTH contributes to redox homeostasis through both its metabolic functions and secondary signaling roles.

What experimental design considerations are crucial when investigating CTH in disease models?

When studying CTH in disease contexts, researchers should address several critical methodological considerations:

• Model Selection and Validation

  Choose disease models that accurately recapitulate key aspects of human pathophysiology related to CTH dysfunction. For genetic diseases like cystathioninuria, consider both genetic models with CTH mutations and biochemical models that mimic metabolic disturbances.

• Experimental Design Requirements

  Implement factorial designs that can simultaneously assess multiple variables affecting CTH function in disease contexts . This approach is particularly valuable when investigating complex conditions like cardiovascular disease where multiple factors may influence outcomes.

• Translational Biomarker Development

  Establish reliable biomarkers that reflect CTH activity in both experimental models and clinical samples. This might include:

  Biomarker Type	Measurement Approach	Clinical Relevance
  Metabolic	LC-MS/MS quantification of cystathionine/cysteine ratio	Reflects trans-sulfuration pathway efficiency
  Functional	H2S production capacity in tissue samples	Indicates CTH contribution to H2S signaling
  Genetic	CTH polymorphism analysis	Identifies risk factors for disease susceptibility

• Longitudinal Study Design

  Implement time-course studies that capture disease progression and corresponding changes in CTH function . This approach is particularly important for chronic conditions where compensatory mechanisms may develop over time.

• Data Integration Framework

  Develop comprehensive databases that integrate phenotypic outcomes with molecular measurements to facilitate the identification of correlations between CTH function and disease manifestations .

By addressing these methodological considerations, researchers can develop more robust and clinically relevant insights into CTH's role in disease pathogenesis and potential therapeutic interventions.