Alpha-ketoglutarate dehydrogenase complex-dependent succinylation of proteins in neurons and neuronal cell lines

Abstract

Reversible post-translation modifications of proteins are common in all cells and appear to regulate many processes. Nevertheless, the enzyme(s) responsible for the alterations and the significance of the modification are largely unknown. Succinylation of proteins occurs and causes large changes in the structure of proteins; however, the source of the succinyl groups, the targets, and the consequences of these modifications on other proteins remain unknown. These studies focused on succinylation of mitochondrial proteins. The results demonstrate that the α-ketoglutarate dehydrogenase complex (KGDHC) can serve as a trans-succinylase that mediates succinylation in an α-ketoglutarate-dependent manner. Inhibition of KGDHC reduced succinylation of both cytosolic and mitochondrial proteins in cultured neurons and in a neuronal cell line. Purified KGDHC can succinylate multiple proteins including other enzymes of the tricarboxylic acid cycle leading to modification of their activity. Inhibition of KGDHC also modifies acetylation by modifying the pyruvate dehydrogenase complex. The much greater effectiveness of KGDHC than succinyl-CoA suggests that the catalysis owing to the E2k succinyltransferase is important. Succinylation appears to be a major signaling system and it can be mediated by KGDHC. Reversible post-translation modifications of proteins are common and may regulate many processes. Succinylation of proteins occurs and causes large changes in the structure of proteins. However, the source of the succinyl groups, the targets, and the consequences of these modifications on other proteins remains unknown. The results demonstrate that the mitochondrial α-ketoglutarate dehydrogenase complex (KGDHC) can succinylate multiple mitochondrial proteins and alter their function. Succinylation appears to be a major signaling system and it can be mediated by KGDHC.

Keywords: Alzheimer's disease; acetylation; brain metabolism; mitochondria; succinylation; α-ketoglutarate dehydrogenase complex.

Figure 1. Succinylation and acetylation of proteins in the cytosol and mitochondria are sensitive to inhibition of KGDHC

Figure 1A. Characterization of the mitochondrial and cytosolic fractions. The media of cultured neurons was changed to BSS and the cells were incubated with or without 100 μM-CESP in BSS buffer for one hour at 37°C. Mitochondrial and cytosolic fractions were prepared. Ten μg protein were loaded on each lane and were separated on 4–20 % Tris-glycine gel and transferred to blotting membrane. Figure 1B. Succinylation is sensitive to inhibition of KGDHC. Aliquots (10 μg protein) were added to each lane. Similar results were observed in three independent experiments. Figure 1C. Acetylation is sensitive to inhibition of KGDHC. Aliquots (10 μg protein) were added to each lane.

FIGURE 2. KG dependent succinylation of proteins by KGDHC

Figure 2A. KG dependent succinylation of ICDH by KGDHC. Various combinations of purified KGDHC, purified ICDH, KG or SC were used to test whether KGDHC could donate succinyl groups to other proteins or if they could be non-enzymatically succinylated. The details are in the Methods section. KGDHC, ICDH and/or SC were mixed together in media in which KGDHC generated SC. Ten μg protein was added to each lane of a 4–20 % TG gel. Each condition was run in triplicate with similar results. Figure 2B. KG dependent succinylation of the 70 kDa protein and smaller succinylation with SC. KGDHC increases the succinyl-lysine of the 70 kDa protein band in KG dependent manner. The bars are the quantitation of the signals for the 70 kDa bands in Figure 2A from multiple experiments. Figure 2C. KG dependent succinylation of ICDH and KGDHC. The conditions are described above for 2A except the Western blot was probed with antibodies against KGDHC (E1k; E2k) and ICDH. The quantification of the intensities are shown below the Western.

FIGURE 3. SC inhibits isocitrate dehydrogenase (ICDH) activity

Figure 3A. The inhibitory effects of SC varied with the amount of ICDH. The treatments were at 30°C for 50 min. The final ICDH activities were 4, 8, 16 or 32 mU/well. Values are means of two independent experiments. Figure 3B. SC concentration dependent changes in ICDH. The amount of enzyme was kept at 8 mU and the amount of succinyl Co A was varied. ICDH was either not incubated or incubated for 40 min with the indicated concentration of SC and incubated at 30°C for 0 or 40 min. After incubation, the ICDH activity was assayed in a 96 well plate.

FIGURE 4

Succinylation alters the activity of KGDHC. KGDHC was incubated with SC (100 μM) for either 0 min or 30 min in KGDHC lysate buffer. KGDHC (7.8 mU) was diluted in KGDHC lysate buffer and were read at 340–460 nm (cut off 455 nm) at 30°C for 30 min.

FIGURE 5

PDHC is succinylated in KG dependent manner. See methods for the treatment paradigm. For Western blots, 10 μg of protein were added on each lane.

FIGURE 6

Succinylation of fumarate hydratase. See methods for the treatment paradigm.

FIGURE 7

E2k as a succinyltransferase to proteins. The picture shows the three components of KGDHC: E1k (α-ketoglutarate dehy-drogenase), E2k (dihyrolipoyul succinyltransferase (DLST) and E3 dihyrdolipoyl dehydrogen-ase. The results suggest that E2k can transfer succinyl groups to other proteins.