Liver X receptor ligands inhibit the lipopolysaccharide-induced expression of microsomal prostaglandin E synthase-1 and diminish prostaglandin E2 production in murine peritoneal macrophages

Microsomal prostaglandin E synthase (mPGES)-1, which is dramatically induced in macrophages by inflammatory stimuli such as lipopolysaccharide (LPS), catalyzes the conversion of cyclooxygenase-2 (COX-2) reaction product prostaglandin H(2) (PGH(2)) into prostaglandin E(2) (PGE(2)). The mPGES-1-derived PGE(2) is thought to help regulate inflammatory responses. On the other hand, excess PGE(2) derived from mPGES-1 contributes to the development of inflammatory diseases such as arthritis and inflammatory pain. Here, we examined the effects of liver X receptor (LXR) ligands on LPS-induced mPGES-1 expression in murine peritoneal macrophages. The LXR ligands 22(R)-hydroxycholesterol (22R-HC) and T0901317 reduced LPS-induced expression of mPGES-1 mRNA and mPGES-1 protein as well as that of COX-2 protein. However, LXR ligands did not influence the expression of microsomal PGES-2 (mPGES-2) or cytosolic PGES (cPGES) protein. Consequently, LXR ligands suppressed the production of PGE(2) in macrophages. These results suggest that LXR ligands diminish PGE(2) production by inhibiting the LPS-induced gene expression of the COX-2-mPGES-1 axis in LPS-activated macrophages.     

Regulation of mitochondrial fusion and division

In many organisms, ranging from yeast to humans, mitochondria fuse and divide to change their morphology in response to a multitude of signals. During the past decade, work using yeast and mammalian cells has identified much of the machinery required for fusion and division, including the dynamin-related GTPases--mitofusins (Fzo1p in yeast) and OPA1 (Mgm1p in yeast) for fusion and Drp1 (Dnm1p) for division. However, the mechanisms by which cells regulate these dynamic processes have remained largely unknown. Recent studies have uncovered regulatory mechanisms that control the activity, assembly, distribution and stability of the key components for mitochondrial fusion and division. In this review, we discuss how mitochondrial dynamics are controlled and how these events are coordinated with cell growth, mitosis, apoptosis and human diseases.     

A novel noninvasive method for assessing glutathione-conjugate efflux systems in the brain

Brain efflux systems export such conjugated metabolites as glutathione (GSH) and glucuronate conjugates, generated by the detoxification process, from the brain and serve to protect the brain from harmful metabolites. The intracerebral injection of a radiolabeled conjugate is a useful technique to assess brain efflux systems; however, this technique is not applicable to humans. Hence, we devised a novel noninvasive approach for assessing GSH-conjugate efflux systems using positron emission tomography. Here, we investigated whether or not a designed proprobe can deliver its GSH conjugate into the brain. Radiolabeled 6-chloro-7-methylpurine (7m6CP) was designed as the proprobe, and [(14)C]7m6CP was prepared by the reaction of 6-chloropurine with [(14)C]CH(3)I as a model of [(11)C]CH(3)I. The radiochemical yield and purity of [(14)C]7m6CP were 10-20% and greater than 99%, respectively. High brain uptake (0.8% ID/g) at 1 min was observed, followed by gradual radioactivity clearance from the brain for 5-60 min after the injection of [(14)C]7m6CP into rats. Analysis of metabolites confirmed that the presence of [(14)C]7m6CP was hardly observed, and 80% of the radioactivity was identical to its GSH conjugate for 15-60 min. The brain radioactivity was single-exponentially decreased during the period of 15-60 min post-injection of [(14)C]7m6CP, and the first-order efflux rate constant of the conjugate, estimated from the slope, was 0.0253 min(-1). These results showed that (1) [(14)C]7m6CP readily entered the brain, (2) it efficiently and specifically transformed to the GSH conjugate within the brain, and (3) after [(14)C]7m6CP disappearance, the clearance of radioactivity represented the only efflux of GSH conjugate. We conclude that 7m6CP can deliver the GSH conjugate into the brain and would be useful for assessing GSH-conjugate efflux systems noninvasively.     

Rac1 activity is required for cardiac myocyte alignment in response to mechanical stress

Mechanical stretch is essential for the cardiac growth. The exposure of cardiac myocytes to the mechanical stretch leads to the cell alignment in parallel to the stretch direction, determining the cell polarity, though it remains to be addressed how mechanical stretch regulates cell orientation. In the present study, we investigated the signal transduction pathways responsible for the cell orientation response to mechanical stretch, focusing on Rho family proteins. Neonatal rat cardiomyocytes were cultured on silicon chambers and exposed to artificial uniaxial cyclic stretch. The pull-down assays revealed that Rac1 was rapidly activated by stretch, but not RhoA. To analyze the roles of Rho family proteins in cardiomyocyte orientation, adenoviral vectors expressing dominant-negative (dn) RhoA and Rac1 were generated. The transfection with adenovirus vector expressing dnRac1, but not dnRhoA, inhibited stretch-induced cell alignment. In conclusion, Rac1 activity is necessary for cardiomyocyte alignment in response to directional stretch.     

Characterization of the solution structure of the M intermediate of photoactive yellow protein using high-angle solution x-ray scattering

It is widely accepted that PYP undergoes global structural changes during the formation of the biologically active intermediate PYP(M). High-angle solution x-ray scattering experiments were performed using PYP variants that lacked the N-terminal 6-, 15-, or 23-amino-acid residues (T6, T15, and T23, respectively) to clarify these structural changes. The scattering profile of the dark state of intact PYP exhibited two broad peaks in the high-angle region (0.3 A(-1) < Q < 0.8 A(-1)). The intensities and positions of the peaks were systematically changed as a result of the N-terminal truncations. These observations and the agreement between the observed scattering profiles and the calculated profiles based on the crystal structure confirm that the high-angle scattering profiles were caused by intramolecular interference and that the structure of the chromophore-binding domain was not affected by the N-terminal truncations. The profiles of the PYP(M) intermediates of the N-terminally truncated PYP variants were significantly different from the profiles of the dark states of these proteins, indicating that substantial conformational rearrangements occur within the chromophore-binding domain during the formation of PYP(M). By use of molecular fluctuation analysis, structural models of the chromophore-binding region of PYP(M) were constructed to reproduce the observed profile of T23. The structure obtained by averaging 51 potential models revealed the displacement of the loop connecting beta4 and beta5, and the deformation of the alpha4 helix. High-angle x-ray scattering with molecular fluctuation simulation allows us to derive the structural properties of the transient state of a protein in solution.     

Role of interleukin-17 in a murine community-associated methicillin-resistant Staphylococcus aureus pneumonia model

Interleukin (IL)-17 is a key member of the Th17 cytokines and has been reported to be involved in the pathomechanisms underlying various diseases, including infectious diseases. Infections with community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) have garnered worldwide attention, and the representative USA300 strain is known to cause pneumonia in healthy people, which can be lethal. However, little is known about the role of IL-17 in CA-MRSA pneumonia. In this study, we investigated the role of IL-17 in a CA-MRSA pneumonia animal model. Mortality was higher and occurred at an earlier stage of infection in the IL-17A-knockout mice than in the wild-type (P < 0.01) and IL-17A/F-knockout mice (P < 0.05); however, no significant difference in the intrapulmonary bacterial counts was observed among the three groups of mice. Moreover, the IL-17A-knockout group showed significantly higher levels of IL-17F and granulocyte-colony stimulating factor (G-CSF) and a significantly higher neutrophil count in the bronchoalveolar lavage fluid than the other groups. These results confirmed that G-CSF expression significantly increased, and significant neutrophilic inflammation occurred under conditions of IL-17A deficiency in the murine CA-MRSA pneumonia model.     

Identification of the role of bone morphogenetic protein (BMP) and transforming growth factor‐β (TGF‐β) signaling in the trajectory of serotonergic differentiation in a rapid assay in mouse embryonic stem cells in vitro

The mechanism by which extracellular molecules control serotonergic cell fate remains elusive. Recently, we showed that noggin, which inactivates bone morphogenetic proteins (BMPs), induces serotonergic differentiation of mouse embryonic (ES) and induced pluripotent stem cells with coordinated gene expression along the serotonergic lineage. Here, we created a rapid assay for serotonergic induction by generating knock‐in ES cells expressing a naturally secreted Gaussia luciferase driven by the enhancer of Pet‐1/Fev, a landmark of serotonergic differentiation. Using these cells, we performed candidate‐based screening and identified BMP type I receptor kinase inhibitors LDN‐193189 and DMH1 as activators of luciferase. LDN‐193189 induced ES cells to express the genes encoding Pet‐1, tryptophan hydroxylase 2, and the serotonin transporter, and increased serotonin release without altering dopamine release. In contrast, TGF‐β receptor inhibitor SB‐431542 selectively inhibited serotonergic differentiation, without changing overall neuronal differentiation. LDN‐193189 inhibited expression of the BMP signaling target gene Id, and induced the TGF‐β target gene Lefty, whereas the opposite effect was observed with SB‐431542. This study thus provides a new tool to investigate serotonergic differentiation and suggests that inhibition of BMP type I receptors and concomitant activation of TGF‐β receptor signaling are implicated in serotonergic differentiation.

     

Site-specific Interaction Mapping of Phosphorylated Ubiquitin to Uncover Parkin Activation

Damaged mitochondria are eliminated through autophagy machinery. A cytosolic E3 ubiquitin ligase Parkin, a gene product mutated in familial Parkinsonism, is essential for this pathway. Recent progress has revealed that phosphorylation of both Parkin and ubiquitin at Ser⁶⁵ by PINK1 are crucial for activation and recruitment of Parkin to the damaged mitochondria. However, the mechanism by which phosphorylated ubiquitin associates with and activates phosphorylated Parkin E3 ligase activity remains largely unknown. Here, we analyze interactions between phosphorylated forms of both Parkin and ubiquitin at a spatial resolution of the amino acid residue by site-specific photo-crosslinking. We reveal that the in-between-RING (IBR) domain along with RING1 domain of Parkin preferentially binds to ubiquitin in a phosphorylation-dependent manner. Furthermore, another approach, the Fluoppi (fluorescent-based technology detecting protein-protein interaction) assay, also showed that pathogenic mutations in these domains blocked interactions with phosphomimetic ubiquitin in mammalian cells. Molecular modeling based on the site-specific photo-crosslinking interaction map combined with mass spectrometry strongly suggests that a novel binding mechanism between Parkin and ubiquitin leads to a Parkin conformational change with subsequent activation of Parkin E3 ligase activity.     

Effects of C-terminal Truncation of Chaperonin GroEL on the Yield of In-cage Folding of the Green Fluorescent Protein

Chaperonin GroEL from Escherichia coli consists of two heptameric rings stacked back-to-back to form a cagelike structure. It assists in the folding of substrate proteins in concert with the co-chaperonin GroES by incorporating them into its large cavity. The mechanism underlying the incorporation of substrate proteins currently remains unclear. The flexible C-terminal residues of GroEL, which are invisible in the x-ray crystal structure, have recently been suggested to play a key role in the efficient encapsulation of substrates. These C-terminal regions have also been suggested to separate the double rings of GroEL at the bottom of the cavity. To elucidate the role of the C-terminal regions of GroEL on the efficient encapsulation of substrate proteins, we herein investigated the effects of C-terminal truncation on GroE-mediated folding using the green fluorescent protein (GFP) as a substrate. We demonstrated that the yield of in-cage folding mediated by a single ring GroEL (SR1) was markedly decreased by truncation, whereas that mediated by a double ring football-shaped complex was not affected. These results suggest that the C-terminal region of GroEL functions as a barrier between rings, preventing the leakage of GFP through the bottom space of the cage. We also found that once GFP folded into its native conformation within the cavity of SR1 it never escaped even in the absence of the C-terminal tails. This suggests that GFP molecules escaped through the pore only when they adopted a denatured conformation. Therefore, the folding and escape of GFP from C-terminally truncated SR1·GroES appeared to be competing with each other.