Folic acid is necessary for proliferation and differentiation of C2C12 myoblasts.

Folic acid, a water soluble B vitamin, plays an important role in cellular metabolic activities, such as functioning as a cofactor in one-carbon metabolism for DNA and RNA synthesis as well as nucleotide and amino acid biosynthesis in the body. A lack of dietary folic acid can lead to folic acid deficiency and result in several health problems, including macrocytic anemia, elevated plasma homocysteine, cardiovascular disease, birth defects, carcinogenesis, muscle weakness, and walking difficulty. However, the effect of folic acid deficiency on skeletal muscle development and its molecular mechanisms are unknown. We, therefore, investigated the effect of folic acid deficiency on myogenesis in skeletal muscle cells and found that folic acid deficiency induced proliferation inhibition and cell cycle breaking as well as cellular senescence in C2C12 myoblasts, implying that folic acid deficiency influences skeletal muscle development. Folic acid deficiency also inhibited differentiation of C2C12 myoblasts and induced deregulation of the cell cycle exit and many cell cycle regulatory genes. It inhibited expression of muscle-specific marker MyHC as well as myogenic regulatory factor (myogenin). Moreover, immunocytochemistry and Western blot analyses revealed that DNA damage was more increased in folic acid-deficient medium-treated differentiating C2C12 cells. Furthermore, we found that folic acid resupplementation reverses the effect on the cell cycle and senescence in folic acid-deficient C2C12 myoblasts but does not reverse the differentiation of C2C12 cells. Altogether, the study results suggest that folic acid is necessary for normal development of skeletal muscle cells.

Synthesis and analysis of potential prodrugs of coenzyme A analogues for the inhibition of the histone acetyltransferase p300.

Lys-CoA (1) is a selective inhibitor of p300 histone acetyltransferase (HAT) but shows poor pharmacokinetic properties because of its multiply charged phosphates. In an effort to overcome this limitation, truncated derivatives of 1 were designed, synthesized and tested as p300HAT inhibitors as well as substrates for the CoA biosynthetic bifunctional enzyme phosphopantetheine adenylyltransferase-dephospho-CoA kinase (PPAT/DPCK). Lys-pantetheine (3) and Lys-phosphopantetheine (2) showed no detectable p300HAT inhibition whereas 3'-dephospho-Lys-CoA (5) was a modest p300 inhibitor with IC(50) of 1.6 microM (compared to IC(50) of approximately 50 nM for 1 blocking p300). Compound 2 was shown to be an efficient substrate for PPAT whereas 5 was a very poor DPCK substrate. Further analysis with 3'-dephospho-Me-SCoA (7) indicated that DPCK shows relatively narrow capacity to accept substrates with sulfur substitution. While these results suggest that truncated derivatives of 1 will be of limited value as lead agents for p300 blockade in vivo, they augur well for prodrug versions of CoA analogues that do not require 3'-phosphate substitution for efficient binding to their targets, such as the GCN-5 related N-acetyltransferases.

Structure of the GCN5 histone acetyltransferase bound to a bisubstrate inhibitor.

Histone acetyltransferases (HATs) use acetyl CoA to acetylate target lysine residues within histones and other transcription factors, such as the p53 tumor suppressor, to promote gene activation. HAT enzymes fall into subfamilies with divergence in sequence and substrate preference. Several HAT proteins have been implicated in human cancer. We have previously reported on the preparation of peptide-CoA conjugate inhibitors with distinct specificities for the p300/CBP [cAMP response element binding protein (CREB)-binding protein] or GCN5 HAT subfamilies. Here we report on the crystal structure of the GCN5 HAT bound to a peptide-CoA conjugate containing CoA covalently attached through an isopropionyl linker to Lys-14 of a 20-aa N-terminal fragment of histone H3. Surprisingly, the structure reveals that the H3 portion of the inhibitor is bound outside of the binding site for the histone substrate and that only five of the 20 aa residues of the inhibitor are ordered. Rearrangements within the C-terminal region of the GCN5 protein appear to mediate this peptide displacement. Mutational and enzymatic data support the hypothesis that the observed structure corresponds to a late catalytic intermediate. The structure also provides a structural scaffold for the design of HAT-specific inhibitors that may have therapeutic applications for the treatment of HAT-mediated cancers.