GCN5-mediated regulation of pathological cardiac hypertrophy via activation of the TAK1-JNK/p38 signaling pathway

Pathological cardiac hypertrophy is a process of abnormal remodeling of cardiomyocytes in response to pressure overload or other stress stimuli, resulting in myocardial injury, which is a major risk factor for heart failure, leading to increased morbidity and mortality. General control nonrepressed protein 5 (GCN5)/lysine acetyltransferase 2 A, a member of the histone acetyltransferase and lysine acetyltransferase families, regulates a variety of physiological and pathological events. However, the function of GCN5 in pathological cardiac hypertrophy remains unclear. This study aimed to explore the role of GCN5 in the development of pathological cardiac hypertrophy. GCN5 expression was increased in isolated neonatal rat cardiomyocytes (NRCMs) and mouse hearts of a hypertrophic mouse model. GCN5 overexpression aggravated the cardiac hypertrophy triggered by transverse aortic constriction surgery. In contrast, inhibition of GCN5 impairs the development of pathological cardiac hypertrophy. Similar results were obtained upon stimulation of NRCMs (having GCN5 overexpressed or knocked down) with phenylephrine. Mechanistically, our results indicate that GCN5 exacerbates cardiac hypertrophy via excessive activation of the transforming growth factor β-activated kinase 1 (TAK1)-c-Jun N-terminal kinase (JNK)/p38 signaling pathway. Using a TAK1-specific inhibitor in rescue experiments confirmed that the activation of TAK1 is essential for GCN5-mediated cardiac hypertrophy. In summary, the current study elucidated the role of GCN5 in promotion of cardiac hypertrophy, thereby implying it to be a potential target for treatment.Subject terms: Heart failure, Cell signalling     

Identification of highly selective type II kinase inhibitors with chiral peptidomimetic tails

Identification of highly selective type II kinase inhibitors is described. Two different chiral peptidomimetic scaffolds were introduced on the tail region of non-selective type II kinase inhibitor GNF-7 to enhance the selectivity. Kinome-wide selectivity profiling analysis showed that type II kinase inhibitor 7a potently inhibited Lck kinase with great selectivity (IC₅₀ of 23.0 nM). It was found that 7a and its derivatives possessed high selectivity for Lck over even structurally conserved all Src family kinases. We also observed that 7a inhibited Lck activation in Jurkat T cells. Moreover, 7a was found to alleviate clinical symptoms in DSS-induced colitis mice. This study provides a novel insight into the design of selective type II kinase inhibitors by adopting chiral peptidomimetic moieties on the tail region.     

A single phenylalanine residue in β-arrestin2 critically regulates its binding to G protein–coupled receptors

Arrestins and their yeast homologs, arrestin-related trafficking adaptors (ARTs), share a stretch of 29 amino acids called the ART motif. However, the functionality of that motif is unknown. We now report that deleting this motif prevents agonist-induced ubiquitination of β-arrestin2 (β-arr2) and blocks its association with activated G protein–coupled receptors (GPCRs). Within the ART motif, we have identified a conserved phenylalanine residue, Phe116, that is critical for the formation of β-arr2–GPCR complexes. β-arr2 Phe116Ala mutant has negligible effect on blunting β₂-adrenergic receptor–induced cAMP generation unlike β-arr2, which promotes rapid desensitization. Furthermore, available structures for inactive and inositol hexakisphosphate 6–activated forms of bovine β-arr2 revealed that Phe116 is ensconced in a hydrophobic pocket, whereas the adjacent Phe117 and Phe118 residues are not. Mutagenesis of Phe117 and Phe118, but not Phe116, preserves GPCR interaction of β-arr2. Surprisingly, Phe116 is dispensable for the association of β-arr2 with its non-GPCR partners. β-arr2 Phe116Ala mutant presents a significantly reduced protein half-life compared with β-arr2 and undergoes constitutive Lys-48-linked polyubiquitination, which tags proteins for proteasomal degradation. We also found that Phe116 is critical for agonist-dependent β-arr2 ubiquitination with Lys-63-polyubiquitin linkages that are known mediators of protein scaffolding and signal transduction. Finally, we have shown that β-arr2 Phe116Ala interaction with activated β₂-adrenergic receptor can be rescued with an in-frame fusion of ubiquitin. Taken together, we conclude that Phe116 preserves structural stability of β-arr2, regulates the formation of β-arr2–GPCR complexes that inhibit G protein signaling, and promotes subsequent ubiquitin-dependent β-arr2 localization and trafficking.     

Mutational effects on thermostable superoxide dismutase from Aquifex pyrophilus: understanding the molecular basis of protein thermostability

We designed two mutants of superoxide dismutase (SOD), one is thermostable and the other is thermolabile, which provide valuable insight to identify amino acid residues essential for the thermostability of the SOD from Aquifex pyrophilus (ApSOD). The mutant K12A, in which Lys12 was replaced by Ala, had increased thermostability compared to that of the wild type. The T(1/2) value of K12A was 210 min and that of the wild type was 175 min at 95 degrees C. However, the thermostability of the mutant E41A, which has a T(1/2) value of 25 min at 95 degrees C, was significantly decreased compared to the wild type of ApSOD. To explain the enhanced thermostability of K12A and thermolabile E41A on the structural basis, the crystal structures of the two SOD mutants have been determined. The results have clearly shown the general significance of hydrogen bonds and ion-pair network in the thermostability of proteins.     

Isolation and characterization of cultured human periodental ligament fibroblast-specific cDNAs

The molecular mechanisms that control the function of periodontal ligament (PDL) fibroblasts remain unclear. We speculated that the character of differentiating PDL fibroblasts is defined by the altered expansion of specific genes not found in neighboring gingival fibroblasts in the periodontium. To expand this set, subtractive hybridization was applied between cultured human PDL and gingival fibroblasts to identify genes differentially expressed in PDL. Consequently five candidate clones, PDLs (periodontal ligament specific) 5, -17, -22, -25, and -31 were identified and characterized by homology search, Northern analysis, and in situ hybridization. Although the mRNAs of these clones were expressed by bone marrow cells and rarely by gingival fibroblasts, the highest expression was detected in the PDL cells, which were uniformly distributed throughout the whole PDL. Amongst the five candidate clones, we focused on PDLs17, because it is a hypothetical protein whose biological function has not been reported yet in the database. Polyclonal antiserum raised against PDLs17 peptide was made, and stained the PDL fibroblasts, osteoblast-like cells and stromal cells in the bone marrow, but not gingival fibroblasts. The results suggest that clones, PDLs5, -17, -22, -25, and -31 may be used as PDL fibroblast-specific markers, and that PDLs17 could act as an important factor in the differentiation process of PDL fibroblasts.     

An improved synthesis of the saponin, polyphyllin D

Polyphyllin D, namely diosgenyl alpha-L-rhamnopyranosyl-(1 --> 2)- [(alpha-L-arabinofuranosyl)-(1 --> 4)]-[beta-D-glucopyranoside, was synthesized from diosgenyl-beta-D-glucopyranoside in four steps and in 30% overall yield, taking advantage of regioselective pivaloylation and alpha-L-rhamnopyranosylation reactions.     

Alpha(v) integrin, p38 mitogen-activated protein kinase, and urokinase plasminogen activator are functionally linked in invasive breast cancer cells

We reported previously that endogenous p38 MAPK activity is elevated in invasive breast cancer cells and that constitutive p38 MAPK activity is important for overproduction of uPA in these cells (Huang, S., New, L., Pan, Z., Han, J., and Nemerow, G. R. (2000) J. Biol. Chem. 275, 12266-12272). However, it is unclear how elevated endogenous p38 MAPK activity is maintained in invasive breast cancer cells. In the present study, we found that blocking alpha(v) integrin functionality with a function-blocking monoclonal antibody or down-regulating alpha(v) integrin expression with alpha(v)-specific antisense oligonucleotides significantly decreased the levels of active p38 MAPK and inhibited cell-associated uPA expression in invasive breast cancer MDA-MB-231 cells. These results suggest a function link between alpha(v) integrin, p38 MAPK activity, and uPA expression in invasive tumor cells. We also found that vitronectin/alpha(v) integrin ligation specifically induced p38 MAPK activation and uPA up-regulation in invasive MDA-MB-231 cells but not in non-invasive MCF7 cells. Finally, using a panel of melanoma cells, we demonstrated that the cytoplasmic tail of alpha(v) integrin subunit is required for alpha(v) integrin ligation-induced p38 MAPK activation.