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Role of Chaperones and Tryptophan Metabolites in the Glyoxal Stress Response of Escherichia coli

  • 유정화 (아주대학교 일반대학원)

초록/요약

Reactive Carbonyl Species (RCS), particularly glyoxal (GO) and methylglyoxal (MGO), are toxic metabolic byproducts that threaten cellular homeostasis. This thesis investigates the cytotoxic mechanisms of RCS and the corresponding adaptive responses across biological systems, bridging molecular findings in Escherichia coli to clinical pathology in human neurodegenerative disease. First, using an E. coli chaperone-deletion library, we elucidated the molecular defense mechanisms against RCS. We discovered that cells rely on a highly selective subset of chaperones rather than a general stress response. DnaK emerged as the primary defender, essential for maintaining proteostasis by stabilizing pre-existing proteins against RCS-induced denaturation. Additionally, the redox-regulated chaperone HslO was identified as critical for countering the secondary oxidative stress generated by RCS. Intriguingly, deletion of YajL (DJ-1 homolog) conferred resistance to GO, suggesting that its loss triggers a compensatory upregulation of alternative detoxification networks. Second, we uncovered a previously unrecognized metabolic vulnerability and its adaptive resolution. Tryptophanase (TnaA), an enzyme typically beneficial for amino acid catabolism, becomes a liability under RCS stress. Active TnaA imposes a "double penalty": it simultaneously depletes protective tryptophan reserves while generating toxic indole, thereby exacerbating GO-induced damage. Critically, we discovered that GO functionally inactivates TnaA by disrupting its PLP cofactor binding—an elegant example of substrate-directed enzyme inhibition. This inactivation represents an adaptive metabolic switch: by halting catabolism, cells redirect resources toward tryptophan conservation, a survival mechanism that operates independently of canonical GloA/YqhD detoxification pathways. Comparative analysis with non-substrate amino acids (phenylalanine, histidine) confirmed that protection is specific to TnaA-mediated tryptophan metabolism, not chemical scavenging. Finally, extending to human pathology, we investigated the impact of these stresses in patients with Parkinson's disease (PD) and Type 2 Diabetes (T2DM). We identified a divergence in RCS dynamics within "Dual Pathology" (DP) patients. GO levels specifically correlated with the severity of dopaminergic neurodegeneration (DAT loss), supporting a "synergistic toxicity" model. In contrast, MGO levels were associated with cerebral perfusion deficits, reflecting cerebrovascular burden. Notably, DP patients exhibited a paradoxical "uncoupling" of the glucose-glyoxal axis, suggesting non-glucose sources like lipid peroxidation drive neurotoxicity in this high-risk group. In conclusion, this study establishes a comprehensive model of RCS stress responses: bacteria employ dual-layered defenses—selective chaperone networks (DnaK/HslO) to protect the proteome, and metabolic reprogramming via TnaA inactivation to preserve critical amino acid pools—whereas in human metabolic disease, the chronic failure of analogous protective mechanisms contributes to specific neurodegenerative and neurovascular pathologies. Keywords: Reactive Carbonyl Species (RCS), Tryptophanase (TnaA), Amino acid metabolism, Chaperone, Parkinson's disease

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목차

PART 1. Molecular mechanisms of Glyoxal/Methylglyoxal stress sensing and adaptation in Escherichia coli 1
Ⅰ. Introduction 1
Ⅱ. Results 3
2.1 Chaperone deletion strains show differential sensitivity to GO and MGO stress 3
2.2 DnaK is required for maintaining protein stability under GO stress 5
Ⅲ. Discussion 6
Ⅳ. Methods 19
4.1 P1 phage transduction 19
4.2 GO and MGO stress spot assay 20
4.3 Protein Expression Monitoring by Luminescence 21
PART 2. Tryptophanase Inactivation as an Adaptive Metabolic Strategy Against Glyoxal Stress in Escherichia coli 23
Ⅰ. Introduction 23
Ⅱ. Results 25
2.1 GO stress alters protein expression patterns including tryptophanase 25
2.2 TnaA deficiency enhances resistance to GO stress 26
2.3 TnaA-mediated GO sensitivity operates independently of major detoxification pathways 27
2.4 GO treatment reduces indole production even with constitutive TnaA overexpression 29
2.5 Exogenous indole increases GO sensitivity while tryptophan provides protection 31
2.6 Cysteine, an alternative TnaA substrate, provides protection against GO stress 33
2.7 Non-TnaA substrate amino acids fail to provide substantial protection against GO stress, suggesting that TnaA-mediated metabolism is essential 35
2.8 GO directly inhibits TnaA enzymatic activity in vitro 38
2.9 GO treatment preserves extracellular tryptophan levels by inhibiting TnaA activity 39
Ⅲ. Discussion 42
Ⅳ. Methods 64
4.1 GO-Induced Protein Analysis 64
4.2 GO and MGO stress spot assay 65
4.3 Indole production assay 65
4.4 Growth Curve Analysis under GO Stress 66
4.5 TnaA protein purification 67
4.6 In vitro TnaA Enzymatic Activity Assay 68
4.7 Extracellular Tryptophan Measurement 69
PART 3. Metabolic Characteristics and Reactive Glyoxal/Methylglyoxal Species Levels in Type 2 Diabetes Patients Developing Parkinson's Disease 71
Ⅰ. Introduction 71
Ⅱ. Results 74
2.1 Measurement of Whole Blood RCS and Correction 74
2.2 Correlations Between Whole Blood RCS Levels and Clinical Groups 77
2.3 Whole Blood RCS Levels in Relation to PD Status and Neuroimaging Biomarkers 79
Ⅲ. Discussion 81
Ⅳ. Method 94
4.1 Measurement of Whole Blood RCS 94
4.2 Data Preprocessing and Batch Effect Correction using the ComBat algorithm 95
4.3 Interaction Screening and Post-hoc Assessment 96
4.4 Western Blot Analysis 97
Data and Code Availability Statement 98
Reference 99

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