Real-time imaging reveals that P2Y2 and P2Y12 receptor agonists are not chemoattractants and macrophage chemotaxis to complement C5a is phosphatidylinositol 3-kinase (PI3K)- and p38 mitogen-activated protein kinase (MAPK)-independent.

Adenosine 5'-triphosphate (ATP) has been implicated in the recruitment of professional phagocytes (neutrophils and macrophages) to sites of infection and tissue injury in two distinct ways. First, ATP itself is thought to be a chemotactic "find me" signal released by dying cells, and second, autocrine ATP signaling is implicated as an amplifier mechanism for chemotactic navigation to end-target chemoattractants, such as complement C5a. Here we show using real-time chemotaxis assays that mouse peritoneal macrophages do not directionally migrate to stable analogs of ATP (adenosine-5'-(γ-thio)-triphosphate (ATPγS)) or its hydrolysis product ADP (adenosine-5'-(ß-thio)-diphosphate (ADPßS)). HPLC revealed that these synthetic P2Y(2) (ATPγS) and P2Y(12) (ADPßS) receptor ligands were in fact slowly degraded. We also found that ATPγS, but not ADPßS, promoted chemokinesis (increased random migration). Furthermore, we found that photorelease of ATP or ADP induced lamellipodial membrane extensions. At the cell signaling level, C5a, but not ATPγS, activated Akt, whereas both ligands induced p38 MAPK activation. p38 MAPK and Akt activation are strongly implicated in neutrophil chemotaxis. However, we found that inhibitors of phosphatidylinositol 3-kinase (PI3K; upstream of Akt) and p38 MAPK (or conditional deletion of p38α MAPK) did not impair macrophage chemotactic efficiency or migration velocity. Our results suggest that PI3K and p38 MAPK are redundant for macrophage chemotaxis and that purinergic P2Y(2) and P2Y(12) receptor ligands are not chemotactic. We propose that ATP signaling is strictly autocrine or paracrine and that ATP and ADP may act as short-range "touch me" (rather than long-range find me) signals to promote phagocytic clearance via cell spreading.

Translation attenuation by minocycline enhances longevity and proteostasis in old post-stress-responsive organisms.

Aging impairs the activation of stress signaling pathways (SSPs), preventing the induction of longevity mechanisms late in life. Here, we show that the antibiotic minocycline increases lifespan and reduces protein aggregation even in old, SSP-deficient Caenorhabditis elegans by targeting cytoplasmic ribosomes, preferentially attenuating translation of highly translated mRNAs. In contrast to most other longevity paradigms, minocycline inhibits rather than activates all major SSPs and extends lifespan in mutants deficient in the activation of SSPs, lysosomal or autophagic pathways. We propose that minocycline lowers the concentration of newly synthesized aggregation-prone proteins, resulting in a relative increase in protein-folding capacity without the necessity to induce protein-folding pathways. Our study suggests that in old individuals with incapacitated SSPs or autophagic pathways, pharmacological attenuation of cytoplasmic translation is a promising strategy to reduce protein aggregation. Altogether, it provides a geroprotecive mechanism for the many beneficial effects of tetracyclines in models of neurodegenerative disease. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Kinetic and crystallographic studies on 2-(beta-D-glucopyranosyl)-5-methyl-1, 3, 4-oxadiazole, -benzothiazole, and -benzimidazole, inhibitors of muscle glycogen phosphorylase b. Evidence for a new binding site.

In an attempt to identify leads that would enable the design of inhibitors with enhanced affinity for glycogen phosphorylase (GP), that might control hyperglycaemia in type 2 diabetes, three new analogs of beta-D-glucopyranose, 2-(beta-D-glucopyranosyl)-5-methyl-1, 3, 4-oxadiazole, -benzothiazole, and -benzimidazole were assessed for their potency to inhibit GPb activity. The compounds showed competitive inhibition (with respect to substrate Glc-1-P) with K(i) values of 145.2 (+/-11.6), 76 (+/-4.8), and 8.6 (+/-0.7) muM, respectively. In order to establish the mechanism of this inhibition, crystallographic studies were carried out and the structures of GPb in complex with the three analogs were determined at high resolution (GPb-methyl-oxadiazole complex, 1.92 A; GPb-benzothiazole, 2.10 A; GPb-benzimidazole, 1.93 A). The complex structures revealed that the inhibitors can be accommodated in the catalytic site of T-state GPb with very little change of the tertiary structure, and provide a rationalization for understanding variations in potency of the inhibitors. In addition, benzimidazole bound at the new allosteric inhibitor or indole binding site, located at the subunit interface, in the region of the central cavity, and also at a novel binding site, located at the protein surface, far removed (approximately 32 A) from the other binding sites, that is mostly dominated by the nonpolar groups of Phe202, Tyr203, Val221, and Phe252.

The binding of IMP to ribonuclease A.

The binding of inosine 5' phosphate (IMP) to ribonuclease A has been studied by kinetic and X-ray crystallographic experiments at high (1.5 A) resolution. IMP is a competitive inhibitor of the enzyme with respect to C>p and binds to the catalytic cleft by anchoring three IMP molecules in a novel binding mode. The three IMP molecules are connected to each other by hydrogen bond and van der Waals interactions and collectively occupy the B1R1P1B2P0P(-1) region of the ribonucleolytic active site. One of the IMP molecules binds with its nucleobase in the outskirts of the B2 subsite and interacts with Glu111 while its phosphoryl group binds in P1. Another IMP molecule binds by following the retro-binding mode previously observed only for guanosines with its nucleobase at B1 and the phosphoryl group in P(-1). The third IMP molecule binds in a novel mode towards the C-terminus. The RNase A-IMP complex provides structural evidence for the functional components of subsite P(-1) while it further supports the role inferred by other studies to Asn71 as the primary structural determinant for the adenine specificity of the B2 subsite. Comparative structural analysis of the IMP and AMP complexes highlights key aspects of the specificity of the base binding subsites of RNase A and provides a structural explanation for their potencies. The binding of IMP suggests ways to develop more potent inhibitors of the pancreatic RNase superfamily using this nucleotide as the starting point.

Atypical antidepressants extend lifespan of Caenorhabditis elegans by activation of a non-cell-autonomous stress response.

Oxidative stress has long been associated with aging and has recently been linked to psychiatric disorders, including psychosis and depression. We identified multiple antipsychotics and antidepressants that extend Caenorhabditis elegans lifespan and protect the animal from oxidative stress. Here, we report that atypical antidepressants activate a neuronal mechanism that regulates the response to oxidative stress throughout the animal. While the activation of the oxidative stress response by atypical antidepressants depends on synaptic transmission, the activation by reactive oxygen species does not. Lifespan extension by atypical antidepressants depends on the neuronal oxidative stress response activation mechanism. Neuronal regulation of the oxidative stress response is likely to have evolved as a survival mechanism to protect the organism from oxidative stress, upon detection of adverse or dangerous conditions by the nervous system.

Endothelial and macrophage-specific deficiency of P38α MAPK does not affect the pathogenesis of atherosclerosis in ApoE-/- mice.

BACKGROUND: The p38α Mitogen-Activated Protein Kinase (MAPK) regulates stress- and inflammation-induced cellular responses. Factors implicated in the development of atherosclerosis including modified low-density lipoprotein (LDL), cytokines and even shear stress induce p38 activation in endothelial cells and macrophages, which may be important for plaque formation. This study investigates the effects of endothelial- and macrophage-specific deficiency of p38α in atherosclerosis development, in Apolipoprotein E deficient (ApoE(-/-)) mice. METHODOLOGY/PRINCIPAL FINDINGS: ApoE(-/-) mice with macrophage or endothelial cell-specific p38α deficiency were fed a high cholesterol diet (HCD) for 10 weeks and atherosclerosis development was assessed by histological and molecular methods. Surprisingly, although p38α-deficiency strongly attenuated oxidized LDL-induced expression of molecules responsible for monocyte recruitment in endothelial cell cultures in vitro, endothelial-specific p38α ablation in vivo did not affect atherosclerosis development. Similarly, macrophage specific deletion of p38α did not affect atherosclerotic plaque development in ApoE(-/-) mice. CONCLUSIONS: Although previous studies implicated p38α signaling in atherosclerosis, our in vivo experiments suggest that p38α function in endothelial cells and macrophages does not play an important role in atherosclerotic plaque formation in ApoE deficient mice.

Endothelial cell-specific NF-kappaB inhibition protects mice from atherosclerosis.

Atherosclerosis is a progressive disorder of the arterial wall and the underlying cause of cardiovascular diseases such as heart attack and stroke. Today, atherosclerosis is recognized as a complex disease with a strong inflammatory component. The nuclear factor-kappaB (NF-kappaB) signaling pathway regulates inflammatory responses and has been implicated in atherosclerosis. Here, we addressed the function of NF-kappaB signaling in vascular endothelial cells in the pathogenesis of atherosclerosis in vivo. Endothelium-restricted inhibition of NF-kappaB activation, achieved by ablation of NEMO/IKKgamma or expression of dominant-negative IkappaBalpha specifically in endothelial cells, resulted in strongly reduced atherosclerotic plaque formation in ApoE(-/-) mice fed with a cholesterol-rich diet. Inhibition of NF-kappaB abrogated adhesion molecule induction in endothelial cells, impaired macrophage recruitment to atherosclerotic plaques, and reduced expression of cytokines and chemokines in the aorta. Thus, endothelial NF-kappaB signaling orchestrates proinflammatory gene expression at the arterial wall and promotes the pathogenesis of atherosclerosis.

Binding of beta-D-glucopyranosyl bismethoxyphosphoramidate to glycogen phosphorylase b: kinetic and crystallographic studies.

In an attempt to identify a new lead molecule that would enable the design of inhibitors with enhanced affinity for glycogen phosphorylase (GP), beta-D-glucopyranosyl bismethoxyphosphoramidate (phosphoramidate), a glucosyl phosphate analogue, was tested for inhibition of the enzyme. Kinetic experiments showed that the compound was a weak competitive inhibitor of rabbit muscle GPb (with respect to alpha-D-glucose-1-phosphate (Glc-1-P)) with a Ki value of 5.9 (+/-0.1) mM. In order to elucidate the structural basis of inhibition, we determined the structure of GPb complexed with the phosphoramidate at 1.83 A resolution. The complex structure reveals that the inhibitor binds at the catalytic site and induces significant conformational changes in the vicinity of this site. In particular, the 280s loop (residues 282-287) shifts 0.4-4.3 A (main-chain atoms) to accommodate the phosphoramidate, but these conformational changes do not lead to increased contacts between the inhibitor and the protein that would improve ligand binding.