SIRT3 and GCN5L regulation of NADP+- and NADPH-driven reactions of mitochondrial isocitrate dehydrogenase IDH2

Wild type mitochondrial isocitrate dehydrogenase (IDH2) was previously reported to produce oncometabolite 2-hydroxyglutarate (2HG). Besides, mitochondrial deacetylase SIRT3 has been shown to regulate the oxidative function of IDH2. However, regulation of 2HG formation by SIRT3-mediated deacetylation was not investigated yet. We aimed to study mitochondrial IDH2 function in response to acetylation and deacetylation, and focus specifically on 2HG production by IDH2. We used acetylation surrogate mutant of IDH2 K413Q and assayed enzyme kinetics of oxidative decarboxylation of isocitrate, 2HG production by the enzyme, and 2HG production in cells. The purified IDH2 K413Q exhibited lower oxidative reaction rates than IDH2 WT. 2HG production by IDH2 K413Q was largely diminished at the enzymatic and cellular level, and knockdown of SIRT3 also inhibited 2HG production by IDH2. Contrary, the expression of putative mitochondrial acetylase GCN5L likely does not target IDH2. Using mass spectroscopy, we further identified lysine residues within IDH2, which are the substrates of SIRT3. In summary, we demonstrate that 2HG levels arise from non-mutant IDH2 reductive function and decrease with increasing acetylation level. The newly identified lysine residues might apply in regulation of IDH2 function in response to metabolic perturbations occurring in cancer cells, such as glucose-free conditions.


Cell fractionation
Cells were harvested from three 150 cm 2 culture dishes by treatment with 0.5% trypsin and washed with PBS. A small part of the cells was lysed in RIPA buffer (lysate) and protein concentration determined. The larger part of cells was resuspended in 2 ml of cold isolation buffer (20 mM HEPES pH 7.2, 1 mM EGTA, 210 mM mannitol, 70 mM sucrose, with 0.1 % BSA and 1 mM PMSF), and homogenized on ice using glass homogenizer. Homogenate was centrifuged 10 min at 1000 × g, 4°C, the supernatant was carefully removed and collected into a new test tube (pellet was discarded) and centrifuged 15 min at 14000 × g, 4°C. The upper part of supernatant (1 ml) was collected again, and the concentration of protein was determined (cytosol). The rest of the supernatant was discarded. Then the pellet (mitochondrial fraction) was washed 4-times with 1.5 ml of cold isolation buffer with BSA and once with 1.5 ml of cold isolation buffer without BSA.
Finally, the pellet was resuspended in 1.5 ml of KCl buffer (5 mM EGTA, 180 mM KCl, 1 mM HEPES, pH 7.2), centrifuged and resuspended in 200 µl of isolation buffer without BSA (mitochondria); protein concentration was determined. Western blot was loaded with 50 µg of whole cell lysate, cytosolic, and mitochondrial protein, respectively.

Biophysical techniques
IDH2 WT and K413Q were purified as described above and diluted in the PBS, pH 7.4. Protein concentration was determined and diluted to 0.1 mg/ml for subsequent analyses.
CD spectra were recorded using Chirascan™-plus (Applied Photophysics) spectrometer in steps of 1 nm over the wavelength range of 195-260 nm. Samples at a concentration of 0.1 mg/ml were placed into 0.1 cm path-length quartz cell to the holder and individual spectra were recorded at room temperature. The CD signal was expressed as the ellipticity and the resulting spectra were buffer-subtracted. We used the CDNN software provided with Chirascan CD spectrometer to analyze the ratio of the secondary structures 23 .
Thermal stability measurements were performed by NanoDSF (differential scanning fluorimetry) on Prometheus NT.48 instrument (NanoTemper Technologies), label-free technique based on changes of tryptophan and tyrosine fluorescence. 10 μl of IDH2 WT and K413Q proteins were placed into the capillaries and fluorescence was screened over a temperature range from 25°C to 95°C using a heating gradient of 1.5°C×min −1 . Unfolding transition midpoints were determined automatically from the first derivative of the ratio of the fluorescence intensities (F350/F330).