CCBL1 Human

What is CCBL1 and what are its alternative nomenclatures in scientific literature?

CCBL1 (Cysteine conjugate-beta lyase 1) is known by several alternative names in scientific literature, including kynurenine aminotransferase I (KAT I), glutamine transaminase K (GTK), kynurenine-oxoglutarate transaminase I, and KYAT1. This enzyme is part of the aminotransferase family and plays crucial roles in multiple metabolic pathways . The diversity in nomenclature reflects the multifunctional nature of this enzyme, which participates in both aminotransferase reactions and beta-elimination processes. Researchers should be aware of these alternative designations when conducting literature searches to ensure comprehensive coverage of relevant studies.

What are the primary enzymatic functions of human CCBL1?

Human CCBL1 catalyzes several distinct biochemical reactions:

• Transamination of L-kynurenine to form kynurenic acid (KA), an intermediate in the tryptophan catabolic pathway and a broad-spectrum antagonist of ionotropic excitatory amino acid receptors .

• Beta-elimination of S-conjugates and Se-conjugates of L-(seleno)cysteine, resulting in cleavage of C-S or C-Se bonds .

• Metabolism of cysteine conjugates of halogenated alkenes and alkanes to form reactive metabolites .

• Participation in glutamine transaminase reactions with various amino acids .

The enzyme's reactivity can be summarized by the following reactions:

• L-kynurenine + 2-oxoglutarate → kynurenic acid + L-glutamate + H₂O + H⁺

• L-cysteine-S-conjugate + H₂O → thiol + pyruvate + ammonium

• L-glutamine + 4-(methylsulfanyl)-2-oxobutanoate ↔ 2-oxoglutaramate + L-methionine

• L-glutamine + 3-phenyl-2-oxopropanoate ↔ 2-oxoglutaramate + L-phenylalanine

What is the genomic organization and cellular localization of CCBL1?

The human CCBL1 gene is located on chromosome 9q34.11 and encodes a protein of approximately 422 amino acids (for the cytoplasmic isoform) . Current evidence suggests that CCBL1 exists in multiple isoforms with different subcellular localizations. The primary isoform (422 amino acids) is targeted to the cytoplasm, while an alternative isoform (516 amino acids) appears to be targeted to mitochondria . The genomic structure shows similarities to other KAT family members, particularly KAT III (CCBL2), with which it shares 51.7% sequence identity . The gene's expression pattern varies across tissues, with significant implications for its function in specific biological contexts.

How does CCBL1 contribute to cisplatin-induced nephrotoxicity?

CCBL1 plays a critical role in cisplatin-induced nephrotoxicity through its metabolic activity in renal tubular cells. When cisplatin infiltrates these cells, CCBL1 metabolizes it to form highly reactive thiol-cisplatin conjugates . This metabolic activation pathway appears to be a key mechanism underlying cisplatin's nephrotoxicity. Research has demonstrated that inhibition of CCBL1 may protect against cisplatin-induced nephrotoxicity without compromising the anticancer efficacy of cisplatin .

The mechanistic pathway involves:

• Infiltration of cisplatin into renal tubular cells

• Metabolism by CCBL1 to form reactive thiol-cisplatin

• Subsequent cellular damage leading to nephrotoxicity

Experimental evidence supports this mechanism, as inhibition of CCBL1 with compounds such as 2′,4′,6′-trihydroxyacetophenone (THA) significantly attenuates cisplatin-induced increases in blood urea nitrogen, creatinine, and renal tubular cell apoptosis in mouse models .

What is the relationship between CCBL1 genetic variation and disease susceptibility?

Genetic variations in CCBL1 have been associated with differential disease susceptibility, particularly in the context of environmental exposures. Research has identified several CCBL1-tagging single nucleotide polymorphisms (SNPs) that may modify risk in specific exposure scenarios .

In studies examining occupational trichloroethylene (TCE) exposure and renal carcinoma risk, evidence of heterogeneity was observed among TCE-exposed subjects with ≥1 minor allele of several CCBL1-tagging SNPs, including rs2293968, rs2280841, rs2259043, and rs941960 . This suggests that genetic variation in CCBL1 may influence individual susceptibility to TCE-related renal carcinogenesis, potentially through altered metabolism of TCE conjugates.

These findings highlight the importance of considering gene-environment interactions in studies of CCBL1 function and suggest that genetic screening may help identify individuals at higher risk for specific toxic exposures.

What methodologies are available for measuring CCBL1 enzymatic activity?

Several experimental approaches can be employed to measure CCBL1 enzymatic activity:

• Spectrophotometric Assays: Monitoring the formation of kynurenic acid from L-kynurenine by measuring absorbance changes or fluorescence.

• High-Performance Liquid Chromatography (HPLC): Quantifying reaction products such as kynurenic acid or pyruvate.

• Beta-Elimination Activity Assays: Using specific cysteine S-conjugate substrates and measuring the release of thiols or pyruvate.

• High-Throughput Screening Assays: As described in research identifying THA as a CCBL1 inhibitor, these assays can be adapted to measure enzyme activity in the presence of potential inhibitors .

When designing activity assays, researchers should consider factors such as pH optima, cofactor requirements (e.g., pyridoxal 5'-phosphate), and potential competing reactions. Control experiments should include specific inhibitors or competitive substrates to confirm the specificity of the measured activity.

How can researchers effectively express and purify recombinant CCBL1 for in vitro studies?

Expression and purification of recombinant CCBL1 can be achieved using several approaches:

• Bacterial Expression Systems: Recombinant human CCBL1 has been successfully expressed in Escherichia coli with high purity (>90%) . The full-length protein (amino acids 1-442) can be expressed with N-terminal tags (such as His-tag) to facilitate purification.

• Purification Strategies: Standard purification methods include:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged proteins

  • Ion exchange chromatography

  • Size exclusion chromatography for final polishing

• Quality Control: Verify protein identity and activity through:

  • SDS-PAGE analysis

  • Western blotting

  • Mass spectrometry

  • Enzymatic activity assays with known substrates

The purified recombinant protein can be used for various applications including structural studies, inhibitor screening, and biochemical characterization. Researchers should ensure that the recombinant protein retains native enzymatic activities by comparing its kinetic parameters with those reported for the native enzyme.

What are effective approaches to inhibit CCBL1 activity in experimental models?

Several approaches can be used to inhibit CCBL1 activity in experimental models:

• Small Molecule Inhibitors:

  • 2′,4′,6′-trihydroxyacetophenone (THA) has been identified as an effective inhibitor of CCBL1 β-elimination activity . In high-throughput screening assays, THA inhibited human CCBL1 in a concentration-dependent manner.

  • Other potential inhibitors may be identified through structure-based drug design approaches based on the enzyme's active site.

• Genetic Approaches:

  • siRNA or shRNA knockdown of CCBL1 expression

  • CRISPR-Cas9 gene editing to create CCBL1 knockout models

  • Antisense oligonucleotides targeting CCBL1 mRNA

• Competitive Substrates:

  • Non-metabolizable analogs of natural substrates can be used to competitively inhibit CCBL1 activity.

When evaluating inhibitor efficacy, researchers should assess both biochemical inhibition (using purified enzyme) and cellular/tissue inhibition (in relevant model systems). The specificity of inhibition should be validated against related enzymes, particularly other KAT family members.

How does CCBL1 interact with other enzymes in tryptophan metabolism?

CCBL1 (KAT I) is one of several kynurenine aminotransferases that catalyze the irreversible transamination of L-kynurenine to form kynurenic acid in the tryptophan degradation pathway . This places CCBL1 within a complex network of enzymes involved in tryptophan metabolism:

• CCBL1 acts downstream of enzymes that convert tryptophan to kynurenine (tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase).

• It competes with kynurenine 3-monooxygenase, which converts kynurenine to 3-hydroxykynurenine, and kynureninase, which produces anthranilic acid from kynurenine.

• The product of CCBL1 activity, kynurenic acid, is a neuroactive metabolite that acts as an antagonist of ionotropic excitatory amino acid receptors .

The balance between these enzymatic activities influences the levels of neuroactive metabolites in the kynurenine pathway, with potential implications for neurological function and disease. Research has shown that alterations in CCBL1 activity can affect extracellular glutamate and dopamine levels in various brain regions, possibly by antagonizing presynaptic α7 nAChRs or by increasing acetylcholine release .

What is the role of CCBL1 in glutathione-mediated detoxification?

CCBL1 plays a significant role in glutathione-mediated detoxification pathways through its cysteine conjugate β-lyase activity . Key aspects of this function include:

• Processing of Xenobiotic Conjugates: CCBL1 metabolizes cysteine conjugates of halogenated alkenes and alkanes, which are formed as part of the mercapturic acid pathway of glutathione-dependent detoxification .

• Bioactivation of Nephrotoxins: Paradoxically, the β-lyase activity of CCBL1 can convert certain cysteine conjugates to reactive thiols that may contribute to nephrotoxicity. This is particularly relevant for halogenated compounds like trichloroethylene (TCE) .

• Integration with Glutathione Pathways: The enzyme functions downstream of glutathione S-transferases and peptidases that process glutathione conjugates to cysteine conjugates.

This dual role in both detoxification and bioactivation highlights the complex nature of CCBL1 in xenobiotic metabolism and provides a mechanistic basis for understanding chemical-induced nephrotoxicity and potential cancer risk associated with certain environmental exposures .

How can researchers design experiments to investigate the role of CCBL1 in cisplatin-induced nephrotoxicity?

Designing experiments to investigate CCBL1's role in cisplatin-induced nephrotoxicity requires a multifaceted approach:

• In Vitro Models:

  • Renal tubular cell lines (e.g., LLC-PK1 cells) can be used to study the effects of cisplatin with and without CCBL1 inhibition .

  • Cell viability assays, apoptosis assays, and measurements of cellular damage markers can assess the protective effects of CCBL1 inhibition.

  • Mechanistic studies can be conducted by measuring the formation of thiol-cisplatin conjugates.

• In Vivo Models:

  • Mouse models of cisplatin-induced nephrotoxicity, with pretreatment using CCBL1 inhibitors like THA .

  • Assessment of kidney function through blood urea nitrogen and creatinine measurements.

  • Histopathological examination of renal tissues for cell damage and apoptosis.

  • Concurrent tumor models (e.g., subcutaneous syngeneic LLC tumors) to evaluate whether CCBL1 inhibition affects cisplatin's anticancer efficacy .

• Translational Approaches:

  • Analysis of CCBL1 expression and activity in patient-derived samples.

  • Correlation of CCBL1 genetic variants with clinical outcomes in cisplatin-treated patients.

  • Development of biomarkers for CCBL1 activity that could predict nephrotoxicity risk.

Key methodological considerations should include appropriate controls, dose-response relationships, timing of interventions, and comprehensive assessment of both renal function and tumor response (if applicable).

What are the implications of CCBL1 genetic polymorphisms for personalized medicine approaches?

CCBL1 genetic polymorphisms have significant implications for personalized medicine approaches:

• Risk Stratification: Several CCBL1-tagging SNPs (rs2293968, rs2280841, rs2259043, and rs941960) have been associated with differential susceptibility to TCE-induced renal carcinoma . Similar genetic variations might influence individual responses to cisplatin and other nephrotoxic agents.

• Pharmacogenetic Considerations:

  • Patients with specific CCBL1 variants might require adjusted dosing of cisplatin to minimize nephrotoxicity.

  • Genetic screening could identify individuals who would particularly benefit from CCBL1 inhibitors as nephroprotective agents.

• Research Design Implications:

  • Clinical studies should consider CCBL1 genotyping as part of patient stratification.

  • Analysis of treatment outcomes should include genetic subgroup analyses.

  • Development of CCBL1 inhibitors should consider potential genotype-specific effects.

These considerations highlight the potential for developing genotype-guided approaches to managing nephrotoxicity risk in cisplatin therapy and other contexts where CCBL1 plays a role in xenobiotic metabolism.

What are the most promising therapeutic applications targeting CCBL1?

The most promising therapeutic applications targeting CCBL1 include:

• Nephroprotection in Cisplatin Therapy: CCBL1 inhibitors like THA have shown potential to attenuate cisplatin-induced nephrotoxicity without compromising anticancer efficacy . This represents a significant opportunity to improve the therapeutic window of cisplatin-based chemotherapy regimens.

• Modulation of Kynurenine Pathway in Neurological Disorders: Given CCBL1's role in producing kynurenic acid, which affects glutamate and dopamine levels , targeted modulation of this enzyme might have applications in neurological and psychiatric disorders characterized by imbalances in these neurotransmitter systems.

• Protection Against Environmental Toxicants: For individuals with high-risk occupational exposures to compounds like TCE, CCBL1 inhibition might provide protection against long-term adverse health effects, particularly in genetically susceptible populations .

Future research should focus on developing more selective CCBL1 inhibitors, understanding tissue-specific roles of different CCBL1 isoforms, and conducting clinical trials to evaluate the safety and efficacy of CCBL1-targeted interventions in relevant patient populations.

What key methodological challenges remain in CCBL1 research?

Despite significant advances, several methodological challenges remain in CCBL1 research:

• Isoform-Specific Analysis: Developing tools and approaches to distinguish between cytoplasmic and mitochondrial CCBL1 isoforms and their respective contributions to different biological processes.

• Selective Inhibition: Creating inhibitors that specifically target CCBL1 without affecting other KAT family members or related enzymes, which is challenging due to structural similarities.

• In Vivo Activity Assessment: Developing non-invasive methods to measure CCBL1 activity in vivo, which would facilitate both research and potential clinical applications.

• Integration of Multi-Omics Data: Combining genomic, transcriptomic, proteomic, and metabolomic data to fully understand CCBL1's role in complex biological networks and disease processes.