Research

Thesis title: "FA-channeling via UCP3 in mitochondrial subtypes"

Thesis outline: Mitochondrial energy metabolism plays a central role in various diseases, including neurodegenerative disorders, obesity, type 2 diabetes, and cardiovascular conditions. Mitochondrial proteins coordinate key metabolic pathways such as fatty acid oxidation and glucose metabolism. These processes are orchestrated by mitochondrial proteins. Besides, studies suggest that there are several mitochondrial subpopulations including the peridroplet mitochondria and the cytoplasmic mitochondria.

Uncoupling protein 3 (UCP3), located in the inner mitochondrial membrane of non-proliferating cells including brown adipocytes, cardiomyocytes, and skeletal muscle cells, is upregulated during β-oxidation, characterized by elevated ATP production and maintained mitochondrial membrane potential (Φm). It has been proposed that proton transport via UCP3 is coupled to fatty acid anion export, contributing to Φm regulation. Despite its significance, the (patho-)physiological function of UCP3 and family members such as UCP2, involved in C4 metabolite transport, remains elusive, in part due to limitations in antibody specificity.

To address this, we employed transient chemical transfection of H9c2 cells with ALFA-tagged UCP3 (UCP3-ALFA), suitable as a positive control system for our endogenously tagged UCP2-ALFA THP-1 cell line. The ALFA tag, a stable 15-amino-acid α-helix, enables highly specific detection via anti-ALFA antibodies. While not establishing a stable cell line, our aim was to validate microscopy staining protocols and optimize affinity purification of native UCP3-ALFA under physiological conditions.

The aims are as followed:

• Isolation und purification of UCP2ALFA from tagged UCP2-ALFA THP-1 cells via column chromatography
• Visualizing the mitochondrial localization of UCP2ALFA in tagged UCP2-ALFA THP-1 cells via immunofluorescence microscopy
• To localize tagged UCP3 variant in PDM and CM mitochondria in H9c2/HL-1 cells.
• To determine the involvement of UCP3 in mitochondrial outward FA-channeling and FAO in H9c2/HL-1 (UCP3-ALFA).
• To quantify UCP3 in PDM and CM of H9c2/HL-1 (UCP3-ALFA) cells.

We hypothesize that UCP3 is enriched in β-oxidative cytoplasmic mitochondria (CM), where it transports FA out of the mitochondrial matrix, possibly via an FA-channeling interactome.

The novelty of this project lies in the approach of analyzing UCP3 function in a mitochondrial subtype-related manner.

Funding: University Assistant (predoctoral)

Supervisors: Jürgen König, Felix Sternberg

Back