Ruffling of metalloporphyrins bound to IsdG and IsdI, two heme-degrading enzymes in Staphylococcus aureus

IsdG and IsdI are paralogous proteins that are intracellular components of a complex heme uptake system in Staphylococcus aureus. IsdG and IsdI were shown previously to reductively degrade hemin. Crystal structures of the apoproteins show that these proteins belong to a newly identified heme degradation family distinct from canonical eukaryotic and prokaryotic heme oxygenases. Here we report the crystal structures of an inactive N7A variant of IsdG in complex with Fe(3+)-protoporphyrin IX (IsdG-hemin) and of IsdI in complex with cobalt protoporphyrin IX (IsdI-CoPPIX) to 1.8 A or better resolution. These structures show that the metalloporphyrins are buried into similar deep clefts such that the propionic acids form salt bridges to two Arg residues. His(77) (IsdG) or His(76) (IsdI), a critical residue required for activity, is coordinated to the Fe(3+) or Co(3+) atoms, respectively. The bound porphyrin rings form extensive steric interactions in the binding cleft such that the rings are highly distorted from the plane. This distortion is best described as ruffled and places the beta- and delta-meso carbons proximal to the distal oxygen-binding site. In the IsdG-hemin structure, Fe(3+) is pentacoordinate, and the distal side is occluded by the side chain of Ile(55). However, in the structure of IsdI-CoPPIX, the distal side of the CoPPIX accommodates a chloride ion in a cavity formed through a conformational change in Ile(55). The chloride ion participates in a hydrogen bond to the side chain amide of Asn(6). Together the structures suggest a reaction mechanism in which a reactive peroxide intermediate proceeds with nucleophilic oxidation at the beta- or delta-meso carbon of the hemin.     

rac regulates its effector phospholipase Cgamma2 through interaction with a split pleckstrin homology domain

Several isoforms of phospholipase C (PLC) are regulated through interactions with Ras superfamily GTPases, including Rac proteins. Interestingly, of two closely related PLCgamma isoforms, only PLCgamma(2) has previously been shown to be activated by Rac. Here, we explore the molecular basis of this interaction as well as the structural properties of PLCgamma(2) required for activation. Based on reconstitution experiments with isolated PLCgamma variants and Rac2, we show that an unusual pleckstrin homology (PH) domain, designated as the split PH domain (spPH), is both necessary and sufficient to effect activation of PLCgamma(2) by Rac2. We also demonstrate that Rac2 directly binds to PLCgamma(2) as well as to the isolated spPH of this isoform. Furthermore, through the use of NMR spectroscopy and mutational analysis, we determine the structure of spPH, define the structural features of spPH required for Rac interaction, and identify critical amino acid residues at the interaction interface. We further discuss parallels and differences between PLCgamma(1) and PLCgamma(2) and the implications of our findings for their respective signaling roles.     

T2384, a novel antidiabetic agent with unique peroxisome proliferator-activated receptor gamma binding properties

The nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma) plays central roles in adipogenesis and glucose homeostasis and is the molecular target for the thiazolidinedione (TZD) class of antidiabetic drugs. Activation of PPARgamma by TZDs improves insulin sensitivity; however, this is accompanied by the induction of several undesirable side effects. We have identified a novel synthetic PPARgamma ligand, T2384, to explore the biological activities associated with occupying different regions of the receptor ligand-binding pocket. X-ray crystallography studies revealed that T2384 can adopt two distinct binding modes, which we have termed "U" and "S", interacting with the ligand-binding pocket of PPARgamma primarily via hydrophobic contacts that are distinct from full agonists. The different binding modes occupied by T2384 induced distinct patterns of coregulatory protein interaction with PPARgamma in vitro and displayed unique receptor function in cell-based activity assays. We speculate that these unique biochemical and cellular activities may be responsible for the novel in vivo profile observed in animals treated systemically with T2384. When administered to diabetic KKAy mice, T2384 rapidly improved insulin sensitivity in the absence of weight gain, hemodilution, and anemia characteristics of treatment with rosiglitazone (a TZD). Moreover, upon coadministration with rosiglitazone, T2384 was able to antagonize the side effects induced by rosiglitazone treatment alone while retaining robust effects on glucose disposal. These results are consistent with the hypothesis that interactions between ligands and specific regions of the receptor ligand-binding pocket might selectively trigger a subset of receptor-mediated biological responses leading to the improvement of insulin sensitivity, without eliciting less desirable responses associated with full activation of the receptor. We suggest that T2384 may represent a prototype for a novel class of PPARgamma ligand and, furthermore, that molecules sharing some of these properties would be useful for treatment of type 2 diabetes.     

Prion protein attenuates excitotoxicity by inhibiting NMDA receptors

It is well established that misfolded forms of cellular prion protein (PrP [PrP(C)]) are crucial in the genesis and progression of transmissible spongiform encephalitis, whereas the function of native PrP(C) remains incompletely understood. To determine the physiological role of PrP(C), we examine the neurophysiological properties of hippocampal neurons isolated from PrP-null mice. We show that PrP-null mouse neurons exhibit enhanced and drastically prolonged N-methyl-d-aspartate (NMDA)-evoked currents as a result of a functional upregulation of NMDA receptors (NMDARs) containing NR2D subunits. These effects are phenocopied by RNA interference and are rescued upon the overexpression of exogenous PrP(C). The enhanced NMDAR activity results in an increase in neuronal excitability as well as enhanced glutamate excitotoxicity both in vitro and in vivo. Thus, native PrP(C) mediates an important neuroprotective role by virtue of its ability to inhibit NR2D subunits.     

Composite bounds on the elastic modulus of bone

Advances in diagnosis and treatment of some bone disorders can be made by understanding the linkage between mineral content and mechanical function. Bone is approximately half by volume a hydrated protein network, and the remainder is a biomineral analogue of hydroxyapatite. In the current work, paired measurements of mechanical properties, using nanoindentation, and of bone mineral volume fraction, computed from quantitative back-scattered electron imaging, were made on six different types of normal and outlier bone samples. Local elastic modulus was plotted against mineral fraction and compared with predictions of engineering bounds for a two-phase composite material. Experimental data spanning the composite bounds showed no one-to-one relationship between mechanical stiffness and bone composition, excluding the possibility of any single, simple composites model for bone at nanometer length-scales.     

Maternal supply of omega-3 polyunsaturated fatty acids alter mechanisms involved in oocyte and early embryo development in the mouse

Despite the well-known benefits of omega-3 (n-3) polyunsaturated fatty acid (PUFA) supplementation on human health, relatively little is known about the effect of n-3 PUFA intake on fertility. More specifically, the aim of this study was to determine how oocyte and preimplantation embryo development might be influenced by n-3 PUFA supply and to understand the possible mechanisms underlying these effects. Adult female mice were fed a control diet or a diet relatively high in the long-chain n-3 PUFAs for 4 wk, and ovulated oocytes or zygotes were collected after gonadotropin stimulation. Oocytes were examined for mitochondrial parameters (active mitochondrial distribution, mitochondrial calcium and membrane potential) and oxidative stress, and embryo developmental ability was assessed at the blastocyst stage following 1) in vitro fertilization (IVF) or 2) culture of in vivo-derived zygotes. This study demonstrated that exposure of the oocyte during maturation in the ovary to an environment high in n-3 PUFA resulted in altered mitochondrial distribution and calcium levels and increased production of reactive oxygen species. Despite normal fertilization and development in vitro following IVF, the exposure of oocytes to an environment high in n-3 PUFA during in vivo fertilization adversely affected the morphological appearance of the embryo and decreased developmental ability to the blastocyst stage. This study suggests that high maternal dietary n-3 PUFA exposure periconception reduces normal embryo development in the mouse and is associated with perturbed mitochondrial metabolism, raising questions regarding supplementation with n-3 PUFAs during this period of time.     

Comet assay to determine DNA damage induced by food deprivation in rats

The aim of this work was to evaluate, by comet assay, the possible inducing of DNA lesions in peripheral blood mononuclear cells of rats subjected to acute or chronic food deprivation. Wistar male rats were subjected to 72 h of partial (50%), or total acute food deprivation, and then allowed to recover for different time periods (24, 48 and 72 h). In other experiments, comet scores were determined in peripheral blood mononuclear cells of rats subjected to chronic food deprivation (25% and 50%) for 50 days. Blood aliquots were obtained before, during and after food deprivation. Comet assay was carried out, the comet units photographed and scored (class 0 up to 3). Acute and chronic food-deprived rats presented peripheral blood mononuclear cells with DNA lesions (comet classes 1, 2 and 3) and a significant increase (p<0.05) in the number of comet units compared with its basal level. The increase was proportional to acute food deprivation time, but after being taken off, it progressively returned to basal level after 48 h (partial group) or 72 h (total group). Chronic food-deprived rats presented a progressive increase of comet score up to 5 days, and a decrease thereafter to reach a basal level. Possible mechanisms of DNA lesions are discussed.     

Targeted disruption of the Pak5 and Pak6 genes in mice leads to deficits in learning and locomotion

PAK6 is a member of the group B family of PAK serine/threonine kinases, and is highly expressed in the brain. The group B PAKs, including PAK4, PAK5, and PAK6, were first identified as effector proteins for the Rho GTPase Cdc42. They have important roles in filopodia formation, the extension of neurons, and cell survival. Pak4 knockout mice die in utero, and the embryos have several abnormalities, including a defect in the development of motor neurons. In contrast, Pak5 knockout mice do not have any noticeable abnormalities. So far nothing is known about the biological function of Pak6. To address this, we have deleted the Pak6 gene in mice. Since Pak6 and Pak5 are both expressed in the brain, we also generated Pak5/Pak6 double knockout mice. These mice were viable and fertile, but had several locomotor and behavioral deficits. Our results indicate that Pak5 and Pak6 together are not required for viability, but are required for a normal level of locomotion and activity as well as for learning and memory. This is consistent with a role for the group B PAKs in the nervous system.     

Metastatic breast cancer induces an osteoblast inflammatory response

Breast cancer preferentially metastasizes to the skeleton, a hospitable environment that attracts and allows breast cancer cells to thrive. Growth factors released as bone is degraded support tumor cell growth, and establish a cycle favoring continued bone degradation. While the osteoclasts are the direct effectors of bone degradation, we found that osteoblasts also contribute to bone loss. Osteoblasts are more than intermediaries between tumor cells and osteoclasts. We have presented evidence that osteoblasts contribute through loss of function induced by metastatic breast cancer cells. Metastatic breast cancer cells suppress osteoblast differentiation, alter morphology, and increase apoptosis. In this study we show that osteoblasts undergo an inflammatory stress response in the presence of human metastatic breast cancer cells. When conditioned medium from cancer cells was added to human osteoblasts, the osteoblasts were induced to express increased levels of IL-6, IL-8, and MCP-1; cytokines known to attract, differentiate, and activate osteoclasts. Similar findings were seen with murine osteoblasts and primary murine calvarial osteoblasts. Osteoblasts are co-opted into creating a microenvironment that exacerbates bone loss and are prevented from producing matrix proteins for mineralization. This is the first study implicating osteoblast produced IL-6, IL-8 (human; MIP-2 and KC mouse), and MCP-1 as key mediators in the osteoblast response to metastatic breast cancer cells.