1H, 15N and 13C assignments of the calcium bound S100P

To determine the three-dimensional solution structure of the calcium bound S100P protein, the backbone and side chain resonance assignments of the S100P protein have been reported based on triple-resonance experiments using uniformly [¹³C, ¹⁵N]-labeled calcium bound protein.     

Tomato transcriptional changes in response to Clavibacter michiganensis subsp. michiganensis reveal a role for ethylene in disease development

Clavibacter michiganensis subsp. michiganensis (Cmm) is a gram-positive actinomycete, causing bacterial wilt and canker disease in tomato (Solanum lycopersicum). Host responses to gram-positive bacteria and molecular mechanisms associated with the development of disease symptoms caused by Cmm in tomato are largely unexplored. To investigate plant responses activated during this compatible interaction, we used microarray analysis to monitor changes in host gene expression during disease development. This analysis was performed at 4 d postinoculation, when bacteria were actively multiplying and no wilt symptoms were yet visible; and at 8 d postinoculation, when bacterial growth approached saturation and typical wilt symptoms were observed. Of the 9,254 tomato genes represented on the array, 122 were differentially expressed in Cmm-infected plants, compared with mock-inoculated plants. Functional classification of Cmm-responsive genes revealed that Cmm activated typical basal defense responses in the host, including induction of defense-related genes, production and scavenging of free oxygen radicals, enhanced protein turnover, and hormone synthesis. Cmm infection also induced a subset of host genes involved in ethylene biosynthesis and response. After inoculation with Cmm, Never ripe (Nr) mutant plants, impaired in ethylene perception, and transgenic plants with reduced ethylene synthesis showed significant delay in the appearance of wilt symptoms, compared with wild-type plants. The retarded wilting in Nr plants was a specific effect of ethylene insensitivity, and was not due to altered expression of defense-related genes, reduced bacterial populations, or decreased ethylene synthesis. Taken together, our results indicate that host-derived ethylene plays an important role in regulation of the tomato susceptible response to Cmm.     

Inhibitor of apoptosis proteins are substrates for the mitochondrial serine protease Omi/HtrA2

The mature serine protease Omi/HtrA2 is released from the mitochondria into the cytosol during apoptosis. Suppression of Omi/HtrA2 by RNA interference in human cell lines reduces cell death in response to TRAIL and etoposide. In contrast, ectopic expression of mature wildtype Omi/HtrA2, but not an active site mutant, induces potent caspase activation and apoptosis. In vitro assays demonstrated that Omi/HtrA2 could degrade inhibitor of apoptosis proteins (IAPs). Consistent with this observation, increased expression of Omi/HtrA2 in cells increases degradation of XIAP, while suppression of Omi/HtrA2 by RNA interference has an opposite effect. Combined, our data demonstrate that IAPs are substrates for Omi/HtrA2, and their degradation could be a mechanism by which the mitochondrially released Omi/HtrA2 activates caspases during apoptosis.     

Tricyclic 4,4-dimethyl-3,4-dihydrochromeno[3,4-d]imidazole derivatives as microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibitors: SAR and in vivo efficacy in hyperalgesia pain model

A series of substituted tricyclic 4,4-dimethyl-3,4-dihydrochromeno[3,4-d]imidazole derivatives have been synthesized and their mPGES-1 biological activity has been disclosed in detail. Structure-activity relationship (SAR) optimization provided inhibitors with excellent mPGES-1 potency and low to moderate PGE₂ release A549 cell potency. Among the mPGES-1 inhibitors studied, 7, 9 and 11l provided excellent selectivity over COX-2 (>200-fold) and >70-fold selectivity for COX-1 except 11l, which exhibited dual mPGES-1/COX-1 activity. Furthermore, the above tested mPGES-1 inhibitors demonstrated good metabolic stability in liver microsomes, high plasma protein binding (PPB) and no significant inhibition observed in clinically relevant CYP isoforms. Besides, selected mPGES-1 tool compounds 9 and 11l provided good in vivo pharmacokinetic profile and oral bioavailability (%F = 33 and 85). Additionally, the representative mPGES-1 tool compounds 9 and 11l revealed moderate in vivo efficacy in the LPS-induced thermal hyperalgesia guinea pig pain model.     

Hemoglobin and Its Scavenger Protein Haptoglobin Associate with ApoA-1-containing Particles and Influence the Inflammatory Properties and Function of High Density Lipoprotein

Hemoglobin (Hb) uniquely associates with proinflammatory HDL in atherogenic mice and coronary heart disease (CHD) patients. In this paper, we report that Hb and its scavenger proteins, haptoglobin (Hp) and hemopexin (Hx) are significantly increased in apoA-1-containing particles of HDL both in mouse models of hyperlipidemia and in CHD patients, when compared with wild type mice and healthy donors, respectively. We further demonstrate that the association of Hb, Hp, and Hx proteins with HDL positively correlates with inflammatory properties of HDL and systemic inflammation in CHD patients. Interestingly, HDL from Hp^−/− mice under atherogenic conditions does not accumulate Hb and is anti-inflammatory, suggesting that (i) Hp is required for the association of Hb with HDL and (ii) Hb·Hp complexes regulate the inflammatory properties of HDL. Moreover, treatment of apoE^−/− mice with an apoA-1 mimetic peptide resulted in significant dissociation of Hb·Hp complexes from HDL and improvement of HDL inflammatory properties. Our data strongly suggest that HDL can become proinflammatory via the Hb·Hp pathway in mice and humans, and dissociation of Hb·Hp·Hx complexes from apoA-1-containing particles of HDL may be a novel target for the treatment of CHD.Atherosclerosis is the leading cause of morbidity and mortality in Western society. The inverse relationship between HDL² cholesterol and the risk of atherosclerosis is well established. Although HDL cholesterol is an epidemiological predictor of risk for coronary heart disease (CHD) (1), a significant number of CHD events occur in patients with normal LDL and HDL cholesterol levels (1, 2). Based on a number of recent studies in both animal models and human samples, it appears that the anti- or proinflammatory nature of HDL may be a more sensitive indicator of the presence or absence of atherosclerosis than HDL cholesterol levels. HDL exerts anti-inflammatory functions by promoting reverse cholesterol transport and preventing the oxidation of LDL (3, 4). We have previously shown that the anti-inflammatory functions of HDL can be impaired in humans (5) rabbits (6), and mice (7) during inflammatory processes. This impaired HDL is proinflammatory in nature, as characterized by (i) decreased levels and activity of anti-inflammatory, antioxidant factors, including apolipoprotein A1 (apoA-1) and PON1 (paraoxonase 1) (8); (ii) gain of proinflammatory proteins, such as serum amyloid A and ceruloplasmin (6); (iii) increased lipid hydroperoxide (LOOH) content (9); (iv) reduced potential to efflux cholesterol (10); and (v) diminished ability to prevent LDL oxidation (11). The molecular changes and mechanisms that promote anti-inflammatory HDL conversion to proinflammatory HDL are currently not well understood.We recently reported the identification and characterization of Hb associated with proinflammatory HDL in atherogenic/hyperlipidemic mice and in human CHD patients (12). We demonstrated that under normal circumstances, a small amount of Hb is always found outside of red blood cells (RBC) in the non-lipoprotein fractions of serum (on the order of 10 μm compared with the >1 m concentration of Hb in RBC). We further demonstrated that under conditions of hyperlipidemia in mice and in CHD patients, the non-RBC Hb moves out of the non-lipoprotein fractions and associates with HDL. This HDL-associated Hb was shown to play an important role in the modulation of HDL function, suggesting that Hb is not only a novel biomarker but may also serve as a therapeutic target for atherosclerosis (12). We therefore sought to determine the molecular mechanisms that play a role in the association of Hb with HDL.Hp and Hx are plasma proteins with the highest binding affinity for Hb (K_d ≈ 1 pm) and heme (K_d < 1 pm), respectively. They are expressed mainly in the liver and belong to the family of acute phase proteins, whose synthesis is induced during inflammatory processes (13, 14). Under conditions of increased hemolysis, Hb becomes highly toxic because of the oxidative properties of heme, which participates in the Fenton reaction to produce reactive oxygen species causing cell injury (15). Under these conditions, Hb is known to be scavenged by Hp·Hx complexes that utilize specific receptor pathways, thus protecting the body against the harmful effects of excess free Hb. We set out to determine whether the Hb·Hp·Hx system (i) also participates in the association of Hb with proinflammatory HDL and (ii) plays a role in the inflammatory properties of HDL.In this paper, we demonstrate that (i) Hb·Hp·Hx complexes associate with HDL in CHD patients and mouse models of hyperlipidemia but not in healthy human donors and wild type mice, and (ii) Hb·Hp·Hx association with HDL positively correlates with proinflammatory properties of HDL. We further show that HDL from Hp^−/− mice on an atherogenic diet is anti-inflammatory and did not contain any Hb, suggesting that (i) Hp is required for the association of Hb with HDL, and (ii) Hp regulates the inflammatory properties of HDL. In contrast to HDL from Hp^−/− mice, HDL from Hx^−/− mice on normal chow was proinflammatory and associated with Hb and Hp, suggesting a novel protective role for Hx in HDL function. When apoE^−/− mice were treated in vivo with an apoA-1 mimetic peptide, 4F, Hb·Hp·Hx dissociated from HDL. Our data strongly suggest that the association of Hb·Hp·Hx with HDL plays an important role in the functional status and inflammatory properties of HDL.     

Apoptosis triggered by Rv1818c, a PE family gene from Mycobacterium tuberculosis is regulated by mitochondrial intermediates in T cells

Ectopic expression of the Mycobacterium tuberculosis PE-family gene Rv1818c, triggers apoptosis in the mammalian Jurkat T cells, which is blocked by anti-apoptotic protein Bcl-2. Although complete overlap is not observed, a considerable proportion of cellular pools of ectopically expressed Rv1818c localizes to mitochondria. However, recombinant Rv1818c does not trigger release of cytochrome c from isolated mitochondria even though Rv1818c protein induced apoptosis of Jurkat T cells. Apoptosis induced by Rv1818c is blocked by the broad-spectrum caspase inhibitory peptide zVAD-FMK. Unexpectedly, Rv1818c-induced apoptosis is not blocked in a Jurkat sub-clone deficient for caspase-8 (JI 9.2) or in cells where caspase-9 function is inhibited or expression of caspase-9 reduced by siRNA, arguing against a central role for these caspases in Rv1818c-induced apoptotic signaling. Depleting cellular pools of the mitochondrial protein Smac/DIABLO substantially reduces apoptosis consistent with mitochondrial involvement in this death pathway. We present evidence that Rv1818c-induced apoptosis is blocked by the co-transfection of an endogenous inhibitor of caspase activation, XIAP in T cells. Additionally, Rv1818c is released into extracellular environment via exosomes secreted by M. tuberculosis infected BM-DC's and macrophages. Furthermore, the extracellular Rv1818c protein can be detected in T cells co-cultured with infected BM-DC's. Taken together, these data suggest that Rv1818c-induced apoptotic signaling is likely regulated in part by the Smac-dependent activation of caspases in T cells.     

Context-dependent conformation of diethylglycine residues in peptides.

Diethylglycine (Deg) residues incorporated into peptides can stabilize fully extended (C5) or helical conformations. The conformations of three tetrapeptides Boc-Xxx-Deg-Xxx-Deg-OMe (Xxx=Gly, GD4; Leu, LD4 and Pro, PD4) have been investigated by NMR. In the Gly and Leu peptides, NOE data suggest that the local conformations at the Deg residues are fully extended. Low temperature coefficients for the Deg(2) and Deg(4) NH groups are consistent with their inaccessibility to solvent, in a C5 conformation. NMR evidence supports a folded beta-turn conformation involving Deg(2)-Gly(3), stabilized by a 4-->1 intramolecular hydrogen bond between Pro(1) CO and Deg(4) NH in the proline containing peptide (PD4). The crystal structure of GD4 reveals a hydrated multiple turn conformation with Gly(1)-Deg(2) adopting a distorted type II/II' conformation, while the Deg(2)-Pro(3) segment adopts a type III/III' structure. A lone water molecule is inserted into the potential 4-->1 hydrogen bond of the Gly(1)-Deg(2) beta-turn.     

Template‐free protein structure prediction and quality assessment with an all‐atom free‐energy model

Biophysical forcefields have contributed less than originally anticipated to recent progress in protein structure prediction. Here, we have investigated the selectivity of a recently developed all‐atom free‐energy forcefield for protein structure prediction and quality assessment (QA). Using a heuristic method, but excluding homology, we generated decoy‐sets for all targets of the CASP7 protein structure prediction assessment with <150 amino acids. The decoys in each set were then ranked by energy in short relaxation simulations and the best low‐energy cluster was submitted as a prediction. For four of nine template‐free targets, this approach generated high‐ranking predictions within the top 10 models submitted in CASP7 for the respective targets. For these targets, our de‐novo predictions had an average GDT_S score of 42.81, significantly above the average of all groups. The refinement protocol has difficulty for oligomeric targets and when no near‐native decoys are generated in the decoy library. For targets with high‐quality decoy sets the refinement approach was highly selective. Motivated by this observation, we rescored all server submissions up to 200 amino acids using a similar refinement protocol, but using no clustering, in a QA exercise. We found an excellent correlation between the best server models and those with the lowest energy in the forcefield. The free‐energy refinement protocol may thus be an efficient tool for relative QA and protein structure prediction. Proteins 2009. © 2009 Wiley‐Liss, Inc.