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A Novel Approach: Investigating the Intracellular Clearance Mechanism of Glyceraldehyde-Derived Advanced Glycation End-Products Using the Artificial Checkpoint Kinase 1 d270KD Mutant as a Substrate Model.

ABSTRACT: Advanced glycation end-products (AGEs), formed through glyceraldehyde (GA) as an intermediate in non-enzymatic reactions with intracellular proteins, are cytotoxic and have been implicated in the pathogenesis of various diseases. Despite their significance, the mechanisms underlying the degradation of GA-derived AGEs (GA-AGEs) remain unclear. In the present study, we found that N-terminal checkpoint kinase 1 cleavage products (CHK1-CPs) and their mimic protein, d270WT, were degraded intracellularly post-GA exposure. Notably, a kinase-dead d270WT variant (d270KD) underwent rapid GA-induced degradation, primarily via the ubiquitin-proteasome pathway. The high-molecular-weight complexes formed by the GA stimulation of d270KD were abundant in the RIPA-insoluble fraction, which also contained high levels of GA-AGEs. Immunoprecipitation experiments indicated that the high-molecular-weight complexes of d270KD were modified by GA-AGEs and that p62/SQSTM1 was one of its components. The knockdown of p62 or treatment with chloroquine reduced the amount of high-molecular-weight complexes in the RIPA-insoluble fraction, indicating its involvement in the formation of GA-AGE aggregates. The present results suggest that the ubiquitin-proteasome pathway and p62 play a role in the degradation and aggregation of intracellular GA-AGEs. This study provides novel insights into the mechanisms underlying GA-AGE metabolism and may lead to the development of novel therapeutic strategies for diseases associated with the accumulation of GA-AGEs.

SUBMITTER: Takeda K

PROVIDER: S-EPMC10741459 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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A Novel Approach: Investigating the Intracellular Clearance Mechanism of Glyceraldehyde-Derived Advanced Glycation End-Products Using the Artificial Checkpoint Kinase 1 d270KD Mutant as a Substrate Model.

Takeda Kenji K Sakai-Sakasai Akiko A Kajinami Kouji K Takeuchi Masayoshi M

Cells 20231214 24

Advanced glycation end-products (AGEs), formed through glyceraldehyde (GA) as an intermediate in non-enzymatic reactions with intracellular proteins, are cytotoxic and have been implicated in the pathogenesis of various diseases. Despite their significance, the mechanisms underlying the degradation of GA-derived AGEs (GA-AGEs) remain unclear. In the present study, we found that N-terminal checkpoint kinase 1 cleavage products (CHK1-CPs) and their mimic protein, d270WT, were degraded intracellula ...[more]

PMID: 38132156

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Project description:BackgroundSarcopenia is a progressive condition that is characterized by decreases in skeletal muscle mass and function. Although sarcopenia is associated with lifestyle-related diseases (LSRD), the mechanisms underlying cell death in myoblasts, which differentiate to myotubes, remain unclear. We previously designated glyceraldehyde (an intermediate of glucose/fructose metabolism)-derived advanced glycation end-products (AGEs) as toxic AGEs (TAGE) because of their cytotoxicity and involvement in LSRD, and hypothesized that TAGE contribute to cell death in myoblasts.MethodsC2C12 cells, which are murine myoblasts, were treated with 0, 0.5, 1, 1.5, and 2 mM glyceraldehyde for 24 h. Cell viability and intracellular TAGE were then assessed using 5-[2,4,-bis(sodioxysulfonyl)phenyl]-3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazole-3-ium (WST-8) and slot blot assays. Cells were pretreated with 8 mM aminoguanidine, an inhibitor of AGE production, for 2 h, followed by 0, 1.5, and 2 mM glyceraldehyde for 24 h. Cell viability and intracellular TAGE levels were then assessed. Serum TAGE levels in STAM mice, in which there were four stages (no steatosis, simple steatosis, steatohepatitis, and fibrosis), were measured using a competitive enzyme-linked immunosorbent assay. Results were expressed as TAGE units (U) per milliliter of serum, with 1 U corresponding to 1.0 μg of glyceraldehyde-derived AGE-bovine serum albumin (BSA) (TAGE-BSA). The viability of cells treated with 20, 50, and 100 μg/mL non-glycated BSA and TAGE-BSA for 24 h was assessed using the WST-8 assay.ResultsIn C2C12 cells treated with 1.5 and 2 mM glyceraldehyde, cell viability decreased to 47.7% (p = 0.0021) and 5.0% (p = 0.0001) and intracellular TAGE levels increased to 6.0 and 15.9 μg/mg protein, respectively. Changes in cell viability and TAGE production were completely inhibited by 8 mM aminoguanidine. Serum TAGE levels at the steatohepatitis and fibrosis stages were 10.51 ± 1.16 and 10.44 ± 0.95 U/mL, respectively, and were higher than those at the no steatosis stage (7.27 ± 0.18 U/mL). Cell death was not induced by 20 or 50 μg/mL TAGE-BSA. The viabilities of C2C12 cells treated with 100 μg/mL non-glycated BSA and TAGE-BSA were 105.0% (p = 0.2890) and 85.3% (p = 0.0217), respectively.ConclusionIntracellular TAGE strongly induced cell death in C2C12 cells and may also induce myoblast cell death in LSRD model mice.

Dataset Information

A Novel Approach: Investigating the Intracellular Clearance Mechanism of Glyceraldehyde-Derived Advanced Glycation End-Products Using the Artificial Checkpoint Kinase 1 d270KD Mutant as a Substrate Model.

Publications

A Novel Approach: Investigating the Intracellular Clearance Mechanism of Glyceraldehyde-Derived Advanced Glycation End-Products Using the Artificial Checkpoint Kinase 1 d270KD Mutant as a Substrate Model.

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