Fumarase: a mitochondrial metabolic enzyme and a cytosolic/nuclear component of the DNA damage response

Abstract

In eukaryotes, fumarase (FH in human) is a well-known tricarboxylic-acid-cycle enzyme in the mitochondrial matrix. However, conserved from yeast to humans is a cytosolic isoenzyme of fumarase whose function in this compartment remains obscure. A few years ago, FH was surprisingly shown to underlie a tumor susceptibility syndrome, Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC). A biallelic inactivation of FH has been detected in almost all HLRCC tumors, and therefore FH was suggested to function as a tumor suppressor. Recently it was suggested that FH inhibition leads to elevated intracellular fumarate, which in turn acts as a competitive inhibitor of HPH (HIF prolyl hydroxylase), thereby causing stabilization of HIF (Hypoxia-inducible factor) by preventing proteasomal degradation. The transcription factor HIF increases the expression of angiogenesis regulated genes, such as VEGF, which can lead to high microvessel density and tumorigenesis. Yet this mechanism does not fully explain the large cytosolic population of fumarase molecules. We constructed a yeast strain in which fumarase is localized exclusively to mitochondria. This led to the discovery that the yeast cytosolic fumarase plays a key role in the protection of cells from DNA damage, particularly from DNA double-strand breaks. We show that the cytosolic fumarase is a member of the DNA damage response that is recruited from the cytosol to the nucleus upon DNA damage induction. This function of fumarase depends on its enzymatic activity, and its absence in cells can be complemented by high concentrations of fumaric acid. Our findings suggest that fumarase and fumaric acid are critical elements of the DNA damage response, which underlies the tumor suppressor role of fumarase in human cells and which is most probably HIF independent. This study shows an exciting crosstalk between primary metabolism and the DNA damage response, thereby providing a scenario for metabolic control of tumor propagation.

Figure 1. Expression of fumarase from the mitochondrial genome.

(A) Mitochondrial encoded fumarase is enzymatically active. WT, Δfum1, and Δfum1/Fum1^m (Fum1^m) strains were serially diluted (10⁻¹, 10⁻², 10⁻³, 10⁻⁴) and grown on fermentable, glucose or non-fermentable, glycerol medium. (B) Mitochondrial encoded fumarase is exclusively localized to mitochondria. WT and Fum1^m strains were subjected to subcellular fractionation as previously described . Equivalent portions of the total (Tot), cytosolic (Cyt), and mitochondrial (Mit) fractions were analyzed by Western blotting using the indicated antibodies. Hsp60 and HxK1 were used as markers for the mitochondria and the cytosol, respectively.

Figure 2. The Δfum1/Fum1^m strain is sensitive to DNA damage.

(A) WT and FUM1^M were exposed to IR (150 or 200 Gy) and then serially diluted onto YPD plates. (B) Yeast, as above, were serially diluted onto YPD plates containing Hydroxyurea (HU) as indicated. (C) Fumarase cytosolic knockout suppresses the Δrev3 and Δrad10 phenotypes. Deletion (Δrev3, Δrev10, or Δrev51) and double deletion (Fum1^m and Δrev3, Δrev10, or Δrev51) strains were serially diluted and grown on YPD or YPD+MMS plates. Where indicated, plates were irradiated with 10 J/m² UV. (D) WT, Fum1^m, and Fum1^m+pΔMTS-Fum1 (the latter is a plasmid expressing cytosolic fumarase) were transformed with a plasmid expressing the galactose inducible HO double-stranded DNA endonuclease (pGal-HO). The strains were grown to logarithmic phase in glucose or galactose medium and then serially diluted onto glucose or galactose medium plates as indicated.

Figure 3. The enzymatic activity of fumarase is crucial for its extra-mitochondrial function.

(A) The Δfum1 strain, harboring the indicated fumarase derivatives, was serially diluted and grown on glucose or glycerol medium plates as indicated. (B) The Fum1^m strain harboring the indicated plasmids and expressing the inducible HO double-stranded DNA endonuclease was serially diluted and grown on glucose or galactose medium plates. (C) High levels of fumarate (but not malate) can suppress the sensitivity of the cytosolic knockout of fumarase to DSBs. WT and Fum1^M strains expressing the inducible HO double-stranded DNA endonuclease were diluted and grown on glucose or galactose medium plates containing 250 mM of phosphate buffer (pH = 8) and the indicated levels of each ester form of the indicated organic acids.

Figure 4. WT yeast fumarase and human FH are overexpressed in response to DNA damage.

(A) WT strain was grown to logarithmic phase and HU was added to a final concentration of 300 mM. The levels of fumarase in the cells were determined at the indicated times by Western blot analysis. Anti-HxK1 was used as a loading control. (B) The chart presents the amount of fumarase normalized to the loading control HxK1 and relative to time 0 set at 1. The calculation is based on the optical density of the bands at each time point (n = 3; error bars indicate s.d.). (C) HeLa cells were grown in the presence of 1 mM HU for the indicated times. The levels of FH in the cells were determined at the indicated times by Western blot analysis. Anti-Actin was used as a loading control. (D) The chart presents the relative amount of FH, calculated (as above) by measuring the optical density of the FH versus actin bands at each time point (n = 3; error bars indicate s.d.).

Figure 5. FH is localized in the nucleus after DNA damage.

(A) HeLa cells were grown in the presence of HU for 3, 6, and 24 h or irradiated with 5 Gy of IR and left to recover for 3, 6, and 24 h. Cells were fixed, immunostained with anti-FH antibodies and DAPI, and visualized on a confocal microscope. (B) HeLa cells were irradiated with 5 Gy of IR. Cells were fixed and immunostained with anti-FH antibodies and then visualized on a confocal microscope. (C) The charts present the percent of cells of the total cell population containing nuclear FH (n = 3; error bars indicate s.d.). (D) HCT cells were grown for 3 h in the presence of the indicated concentration of HU. Nuclear extractions were preformed and analyzed by Western blotting using the indicated antibodies. H3 and Tubulin were used as markers for the nucleus and the cytosol, respectively. (E) WT yeast cells were grown in the presence of HU for 16 h, fixed, and immunostained with anti-fumarase and DAPI. (F) The charts present the percent of cells of the total cell population containing nuclear fumarase staining (n = 3; error bars indicate s.d.).

Figure 6. Specific FH shRNA efficiently knockdown the protein expression.

(A) HeLa and HEK293 cells were stably transfected with shRNA lentiviral plasmid. FH levels were determined by Western blot analysis. Anti-GAPDH was used as a loading control. (B) HCT116 cells were stably transfected with shRNA lentiviral plasmid and grown in the presence of HU. The levels of FH in the cells at the indicated HU concentrations were determined by Western blot analysis. Anti-GAPDH was used as a loading control. (C–D) FH knockdown cells are sensitive to IR. 1,500 cells were plated per 6 cm plates and incubated for 24 h. Cells were then irradiated with 4 Gy of IR and incubated for 6–8 d. Cells were fixed on the plates using 3.7% formaldehyde, stained with 0.1% crystal violet, and number of colonies was counted (n = 3; error bars indicate s.d.). (E) FH knockdown cells are sensitive to IR. HCT116 cells were irradiated with 10 Gy of IR and then incubated for the indicated times. Mortality was determined by Trypan Blue dye exclusion assay (n = 3; error bars indicate s.d.). (F) FH knockdown cells are sensitive to HU. HCT116 cells were grown in the presence of indicated concentrations of HU for 48 h. Mortality was determined by Trypan Blue dye exclusion assay (n = 3; error bars indicate s.d.).

Figure 7. In the absence of FH, timing and levels of histone H2AX phosphorylation are affected.

(A) HeLa cells were irradiated with 5 Gy of IR. Cells were fixed and double immunostained with anti-phospho-Ser¹³⁹-H2AX and -FH antibodies. (B) The charts present the percent of cells of the total cell population, presenting foci of phospho-H2AX (n = 3; error bars indicate s.d.). (C) Histone H2AX phosphorylation is impaired in knockdown for FH. HCT116 cells were irradiated with 20 Gy of IR and left to recover for 24 h. γH2AX levels were determined by Western blot using anti-phospho-Ser¹³⁹-H2AX antibodies. (D) Histone H2A phosphorylation is impaired in the Fum1^m strain. WT and Fum1^m yeast strains were grown in the presence or absence of HU (300 mM). Phospho H2A levels were determined, after the indicated times, by Western blot using anti-phospho-Ser¹²⁹-H2A antibodies.

Figure 8. In the absence of FH, the DNA damage checkpoint activation is impaired.

HCT cells and FH-shRNA cells were irradiated with 5 Gy (A) or grown in the presence of HU (B) for the indicated times. Phospho-CHK2 levels were determined by Western blot using anti-phospho-Chk2 (Thr⁶⁸) antibodies. (C) HCT WT and FH-shRNA cells were irradiated with 5 Gy of IR and left to recover for the indicated times. Cells were stained with propidium iodide and were analyzed for cell cycle distribution with a flow cytometer (FACS). WT HCT cell cycle histograms are black lined and unfilled, while those of FH-shRNA are filled gray. (D) Relative amounts of cell cycle stages G1 (black), S (gray), and G2 (white) were calculated from the propidium iodide staining shown in (C).