An Ultrasensitive Biosensor for Probing Subcellular Distribution and Mitochondrial Transport of l-2-Hydroxyglutarate

Area of Science:

• Biochemistry
• Cell Biology
• Metabolomics

Background:

• l-2-Hydroxyglutarate (l-2-HG) is a key metabolite with unclear subcellular localization and transport mechanisms.
• Technical limitations have hindered the detailed analysis of l-2-HG's role in physiological processes.

Purpose of the Study:

• To develop and validate an ultrasensitive biosensor for monitoring l-2-HG metabolism and distribution.
• To investigate the subcellular localization of l-2-HG and its mitochondrial transport.
• To identify the role of SLC1A1 in mitochondrial l-2-HG uptake and develop a diagnostic tool for l-2-HG-related diseases.

Main Methods:

• Development of an ultrasensitive l-2-HG biosensor (sfLHGFRH) using fluorescent protein and a transcriptional regulator.
• Application of the biosensor in human embryonic kidney cells (HEK293FT) and macrophages for spatiotemporal monitoring.
• Utilizing the biosensor to elucidate the function of the l-glutamate transporter SLC1A1 in mitochondrial l-2-HG uptake.
• Development of a second biosensor (sfLHGFR L) for point-of-care diagnostics.

Main Results:

• The sfLHGFRH biosensor successfully enabled analysis of l-2-HG metabolism in cellular models.
• Spatiotemporal monitoring revealed the subcellular distribution of l-2-HG, indicating lower abundance in mitochondria.
• The study identified SLC1A1 as a transporter involved in mitochondrial l-2-HG uptake.
• The sfLHGFR L biosensor detected elevated l-2-HG in the urine of kidney cancer patients.

Conclusions:

• The developed biosensors provide novel tools for studying l-2-HG metabolism and subcellular distribution.
• The findings clarify the mitochondrial transport of l-2-HG and its dependence on SLC1A1.
• The sfLHGFR L biosensor shows potential for early diagnosis of l-2-HG-associated pathologies like kidney cancer.