Cryptic proteolytic activity of dihydrolipoamide dehydrogenase

Abstract

The mitochondrial enzyme, dihydrolipoamide dehydrogenase (DLD), is essential for energy metabolism across eukaryotes. Here, conditions known to destabilize the DLD homodimer enabled the mouse, pig, or human enzyme to function as a protease. A catalytic dyad (S456-E431) buried at the homodimer interface was identified. Serine protease inhibitors and an S456A or an E431A point mutation abolished the proteolytic activity, whereas other point mutations at the homodimer interface domain enhanced the proteolytic activity, causing partial or complete loss of DLD activity. In humans, mutations in the DLD homodimer interface have been linked to an atypical form of DLD deficiency. These findings reveal a previously unrecognized mechanism by which certain DLD mutations can simultaneously induce the loss of a primary metabolic activity and the gain of a moonlighting proteolytic activity. The latter could contribute to the metabolic derangement associated with DLD deficiency and represent a target for therapies of this condition.

Fig. 1.

Identification of mouse DLD as m-fxn degrading activity. (A) Time course of cleavage of m-fxn (1.5 μg per lane) in the presence (+) or absence (−) of 1.5 μg (total protein) of mouse liver Mono S fraction. Each reaction was incubated at 37°C in 10 mM Tris·HCl, pH 8.0, for the indicated time and analyzed by SDS/12% PAGE and SYPRO orange staining. (B) Cleavage sites in m-fxn substrate were determined by subjecting the d-fxn b, c, and d products to N-terminal sequencing. (C) Mouse Mono S fraction (3 μg of total protein) analyzed by SDS/12% PAGE and silver staining. (D) All visible protein bands (numbered 1 to 15) were excised from the gel in C, and each gel slice was extracted in 20 μl of 10 mM Tris·HCl, pH 8.0, and 1% Triton X-100 and assayed for proteolytic activity for 24 h. Activity was expressed as percent of the total activity recovered in band 6, which was identified as mouse DLD by MS. The DLD protein was also detected by MS in bands 2 and 3 (see Results). Results presented below suggest that a proteolytically active ≈14 kDa fragment of DLD was present in band 14. (E) An aliquot (≈25 μg of total protein) of mouse Mono S fraction was loaded on a Superdex 200 column (10 mm × 30 cm). Aliquots of fractions 1–11, comprising the void volume and the entire molecular mass range of the column (10–600 kDa), were analyzed for m-fxn-degrading activity as in Fig. 1A. S, reaction containing m-fxn substrate only. (F) Because of the limited amount of starting material, DLD or other proteins could not be detected by SDS/PAGE in aliquots from the Superdex 200 fractions described above. Therefore, active fractions 7 and 8 were pooled and concentrated, and total protein was precipitated with 10% trichloroacetic acid and analyzed by SDS/PAGE and silver staining. The arrow points at the ≈50-kDa DLD band, as determined in Fig. 1C. (G) A commercial preparation of pig heart DLD (L2002; Sigma, St. Louis, MO) was purified through Mono S, hydroxyapatite, and Superdex 200 chromatography, as described in SI Fig. 7 C–E. The purest fraction obtained (fraction 7 + 8 in SI Fig. 7E) was preincubated for 1 h with (+) or without (−) 1 mM DFP, an irreversible serine protease inhibitor, in 10 mM Hepes-KOH, pH 6.8, 100 mM NaCl, and 0.1% Triton X-100 [proteolytic reaction buffer (PRB)] at 37°C; m-fxn substrate was added, and activity was assayed for 2 h.

Fig. 2.

D444V mutation in the human DLD homodimer interface increases DLD proteolytic activity. (A and B) The two subunits of the human DLD homodimer (1ZMC) (36) are shown in ribbon representation, with the D444 and Y438 residues shown as sticks. Figures generated with PyMOL. (C) Time courses of proteolytic activity were performed with purified recombinant human WT or D444V DLD (5 μg per reaction) in proteolytic reaction buffer (PRB) with 1 μg per reaction of m-fxn substrate and were analyzed by SDS/PAGE and SYPRO orange staining. The percent residual m-fxn was determined by densitometry; values at the bottom of each blot represent the average of two independent experiments, one of which is shown. SDS/PAGE analysis of WT and D444V DLD is shown in SI Fig. 8A. (D) Purified D444V DLD (1.5 mg) was fractionated on a Superdex 75 gel filtration column. (Top and Middle) Ten microliters from each fraction were used to detect DLD protein (Top) or measure proteolytic activity (Middle). (Bottom) The graph shows the protein elution profile (blue) superimposed to that of the proteolytic activity (red), which was quantified by densitometry. A.U., arbitrary units.

Fig. 3.

Identification of serine protease active-site residues at the DLD homodimer interface. (A) One subunit of human DLD homodimer (1ZMC) is shown in ribbon representation with the dimer interface domain (residues 350–474) in green, and residues S456, H450, and E431 are shown as sticks. The figure created with PyMOL. (B) The C-term, corresponding to the dimer interface domain, was expressed in E. coli and purified (SI Methods and SI Table 4). C-term (5 μg) was analyzed by SDS/12% PAGE and SYPRO orange staining alongside the full-length human DLD. (C) Aliquots (5 or 10 μg) of C-term DLD were incubated with or without 1 mM diisopropyl fluorophosphate or phosphorofluoridate (DFP) in duplicate for 24 h at 37°C. All protein samples were resolved on SDS/12% PAGE, visualized by silver staining, eluted from gel slices by passive diffusion, and tested for proteolytic activity with m-fxn substrate as described in Fig. 1 C and D. Shown are the results obtained with eight independent gel slices. (D) WT and mutant DLD proteins were purified at the same time and assayed simultaneously in a 96-well plate by using a fluorogenic peptide substrate consisting of 13 aa from the m-fxn N-terminal region, with a fluorescent donor and a quenching acceptor (see Experimental Methods). Each reaction contained 2 μM DLD and 50 μM peptide in 100 μl of 10 mM Tris·HCl, pH 8.0, and 50 mM NaCl. Total fluorescence was measured after 2, 4, and 8 h of incubation at 37°C. Blanks containing DLD protein only or substrate only in buffer were run in parallel with proteolytic reactions. Bars represent fluorescence intensity after background subtraction. Each bar represents the mean ± SE of three (WT and D444V) or two (S456A, S456A/D444V, E431A, and H450A) independent experiments, each conducted in duplicate. SDS/PAGE analysis of all proteins is shown in SI Fig. 8A.

Fig. 4.

Putative catalytic dyad at the DLD homodimer interface domain. Models of monomeric WT (A) and H450A (B) DLD homodimer interface domain (residues 350–474; C-term) obtained by MMDSs (39). Close-ups of the putative S456–E431 dyad were generated with PyMOL.