GRP78 protects CHO cells from ribosylation

Area of Science:

• Biochemistry
• Cell Biology
• Metabolic Disorders

Background:

• d-Ribose (Rib) is a reactive glycation compound found in organisms.
• Elevated Rib levels in diabetic patients' urine contribute to advanced glycation end products (AGEs) and cell dysfunction.
• The sensitivity of cellular proteins to Rib-induced glycation remains largely unknown.

Purpose of the Study:

• To investigate the accumulation of cellular AGEs in Chinese hamster ovary (CHO) cells treated with d-Ribose.
• To identify cellular proteins susceptible to Rib-induced glycation.
• To elucidate the role of glucose-regulated protein 78 (GRP78) in d-Ribose-induced cytotoxicity.

Main Methods:

• Cell culture (CHO cells) with varying d-Ribose concentrations and incubation times.
• Mass spectrometry to identify AGE-modified proteins.
• Co-immunoprecipitation and Western blotting to confirm protein interactions and expression levels.
• Kinetic studies measuring lactate dehydrogenase (LDH) release.
• RNA interference (siRNA) and pharmacological inhibition (EGCG) to modulate GRP78 expression.
• GRP78 overexpression studies.

Main Results:

• Cellular AGEs accumulated in CHO cells with increased Rib concentration and duration.
• Glucose-regulated protein 78 (GRP78) was identified as a major ribosylated protein.
• d-Ribose induced significantly higher AGEs and cytotoxicity compared to d-glucose.
• GRP78 expression was initially upregulated then downregulated during Rib treatment.
• Suppression of GRP78 enhanced AGE levels and reduced cell viability, while GRP78 overexpression conferred protection.

Conclusions:

• GRP78 is a key cellular target of d-Ribose-induced glycation.
• GRP78 plays a protective role against d-Ribose-induced cytotoxicity in CHO cells.
• These findings suggest GRP78 is a potential therapeutic target for managing Rib-induced cellular damage.