Effects of disease-modifying anti-rheumatic drugs (DMARDs) on the activities of rheumatoid arthritis-associated cathepsins K and S

Rheumatoid arthritis is an inflammatory and disabling joint disease affecting 0.5-1.5% of the population. Although various anti-inflammatory (NSAIDs) and disease-modifying (DMARDs) drugs are in clinical use, their precise mechanisms of action are not always defined. In this report, we discuss the effects of widely used DMARDs such as gold derivatives and chloroquine on cathepsins K and S, which have been implicated as critical mediators of inflammation and joint erosion in rheumatoid arthritis. We demonstrate that clinically potent gold derivatives inhibit cathepsins K and S in in vitro and cell-based assays. An X-ray analysis of the gold thiomalate/cathepsin K complex reveals that the inhibitor is bound to the active-site cysteine residue of the protease. Chloroquine, a lysosomotropic agent of lower clinical potency than gold derivatives, inhibits neutral pH-labile cathepsins intracellularly, but does not affect the neutral pH-stable cathepsin S. The potent inhibition of cathepsins implicated in the pathogenesis of rheumatoid arthritis by gold derivatives may explain the therapeutic efficacy of these drugs.     

Ultraviolet B radiation induces cell shrinkage and increases osmolyte transporter mRNA expression and osmolyte uptake in HaCaT keratinocytes

We have previously shown that compatible organic osmolytes, such as betaine, myo-inositol and taurine, are part of the stress response of normal human keratinocytes (NHKs) to ultraviolet B (UVB) radiation. In this regard, we tested human HaCaT keratinocytes as a surrogate cell line for NHK. HaCaT cells osmo-dependently express mRNA specific for transport proteins for betaine (BGT-1), myo-inositol (SMIT) and taurine (TAUT). Compared to normoosmotic (305 mosmol/l) controls, which strongly constitutively expressed BGT-1 mRNA, strong induction of SMIT and TAUT mRNA as well as low induction of BGT-1 mRNA expression was observed between 3 and 9 h after hyperosmotic exposure (405 mosmol/l). This expression correlated with an increased osmolyte uptake. Conversely, hypoosmotic (205 mosmol/l) stimulation led to a significant efflux of osmolytes. Exposure to UVB (290-315 nm) radiation induced cell shrinkage which was followed by an upregulation of osmolyte transporter mRNA levels and osmolyte uptake. These results demonstrate that human HaCaT keratinocytes possess an osmolyte strategy including UVB-induced cell shrinkage and following increased osmolyte uptake. However, several differences in osmolyte transporter expression and uptake were noted between NHK and HaCaT cells, indicating that the use of HaCaT cells as a surrogate cell line for NHK has limitations.     

Escherichia coli α-Hemolysin Counteracts the Anti-Virulence Innate Immune Response Triggered by the Rho GTPase Activating Toxin CNF1 during Bacteremia

The detection of the activities of pathogen-encoded virulence factors by the innate immune system has emerged as a new paradigm of pathogen recognition. Much remains to be determined with regard to the molecular and cellular components contributing to this defense mechanism in mammals and importance during infection. Here, we reveal the central role of the IL-1β signaling axis and Gr1+ cells in controlling the Escherichia coli burden in the blood in response to the sensing of the Rho GTPase-activating toxin CNF1. Consistently, this innate immune response is abrogated in caspase-1/11-impaired mice or following the treatment of infected mice with an IL-1β antagonist. In vitro experiments further revealed the synergistic effects of CNF1 and LPS in promoting the maturation/secretion of IL-1β and establishing the roles of Rac, ASC and caspase-1 in this pathway. Furthermore, we found that the α-hemolysin toxin inhibits IL-1β secretion without affecting the recruitment of Gr1+ cells. Here, we report the first example of anti-virulence-triggered immunity counteracted by a pore-forming toxin during bacteremia.     

Efficient and Stable TiO2:Pt–Cu(In,Ga)Se2 Composite Photoelectrodes for Visible Light Driven Hydrogen Evolution

Novel thin film composite photocathodes based on device‐grade Cu(In,Ga)Se₂ chalcopyrite thin film absorbers and transparent conductive oxide Pt‐implemented TiO₂ layers on top are presented for an efficient and stable solar‐driven hydrogen evolution. Thin films of phase‐pure anatase TiO₂ are implemented with varying Pt‐concentrations in order to optimize simultaneously i) conductivity of the films, ii) electrocatalytic activity, and iii) light‐guidance toward the chalcopyrite. Thereby, high incident‐photon‐to‐current‐efficiencies of more than 80% can be achieved over the full visible light range. In acidic electrolyte (pH 0.3), the most efficient Pt‐implemented TiO₂–Cu(In,Ga)Se₂ composite electrodes reveal i) photocurrent densities up to 38 mA cm^?2 in the saturation region (?0.4 V RHE, reversible hydrogen electrode), ii) 15 mA cm^?2 at the thermodynamic potential for H₂‐evolution (0 V RHE), and iii) an anodic onset potential shift for the hydrogen evolution (+0.23 V RHE). It is shown that the gradual increase of the Pt‐concentration within the TiO₂ layers passes through an efficiency‐ and stability‐maximum of the device (5 vol% of Pt precursor solution). At this maximum, optimized light‐incoupling into the device‐grade chalcopyrite light‐absorber as well as electron conductance properties within the surface layer are achieved while no degradation are observed over more than 24 h of operation.     

Pitfalls in histone propionylation during bottom‐up mass spectrometry analysis

Despite their important role in regulating gene expression, posttranslational histone modifications remain technically challenging to analyze. For identification by bottom‐up MS, propionylation is required prior to and following trypsin digestion. Hereby, more hydrophobic peptides are generated enabling RP HPLC separation. When histone dynamics are studied in a quantitative manner, specificity, and efficiency of this chemical derivatization are crucial. Therefore we examined eight different protocols, including two different propionylation reagents. This revealed amidation (up to 70%) and methylation (up to 9%) of carboxyl groups as a side reaction. Moreover, incomplete (up to 85%) as well as a specific propionylation (up to 63%) can occur, depending on the protocol. These results highlight the possible pitfalls and implications for data analysis when doing bottom‐up MS on histones.     

Novel mechanistic link between focal adhesion remodeling and glucose-stimulated insulin secretion

Actin cytoskeleton remodeling is well known to be positively involved in glucose-stimulated pancreatic β cell insulin secretion. We have observed glucose-stimulated focal adhesion remodeling at the β cell surface and have shown this to be crucial for glucose-stimulated insulin secretion. However, the mechanistic link between such remodeling and the insulin secretory machinery remained unknown and was the major aim of this study. MIN6B1 cells, a previously validated model of primary β cell function, were used for all experiments. Total internal reflection fluorescence microscopy revealed the glucose-responsive co-localization of focal adhesion kinase (FAK) and paxillin with integrin β1 at the basal cell surface after short term stimulation. In addition, blockade of the interaction between β1 integrins and the extracellular matrix with an anti-β1 integrin antibody (Ha2/5) inhibited short term glucose-induced phosphorylation of FAK (Tyr-397), paxillin (Tyr-118), and ERK1/2 (Thr-202/Tyr-204). Pharmacological inhibition of FAK activity blocked glucose-induced actin cytoskeleton remodeling and glucose-induced disruption of the F-actin/SNAP-25 association at the plasma membrane as well as the distribution of insulin granules to regions in close proximity to the plasma membrane. Furthermore, FAK inhibition also completely blocked short term glucose-induced activation of the Akt/AS160 signaling pathway. In conclusion, these results indicate 1) that glucose-induced activation of FAK, paxillin, and ERK1/2 is mediated by β1 integrin intracellular signaling, 2) a mechanism whereby FAK mediates glucose-induced actin cytoskeleton remodeling, hence allowing docking and fusion of insulin granules to the plasma membrane, and 3) a possible functional role for the Akt/AS160 signaling pathway in the FAK-mediated regulation of glucose-stimulated insulin secretion.     

Sphingosine 1-phosphate (S1P) lyase deficiency increases sphingolipid formation via recycling at the expense of de novo biosynthesis in neurons

Sphingosine 1-phosphate lyase (S1P lyase) irreversibly cleaves sphingosine 1-phosphate (S1P) in the final step of sphingolipid catabolism. As sphingoid bases and their 1-phosphate are not only metabolic intermediates but also highly bioactive lipids that modulate a wide range of physiological processes, it would be predicted that their elevation might induce adjustments in other facets of sphingolipid metabolism and/or alter cell behavior. Indeed, we have previously reported that S1P lyase deficiency causes neurodegeneration and other adverse symptoms. We next asked the question whether and how S1P lyase deficiency affects the metabolism of (glyco)sphingolipids and cholesterol, two lipid classes that might be involved in the neurodegenerative processes observed in S1P lyase-deficient mice. As predicted, there was a considerable increase in free and phosphorylated sphingoid bases upon elimination of S1P lyase, but to our surprise, rather than increasing, the mass of (glyco)sphingolipids persisted at wild type levels. This was discovered to be due to reduced de novo sphingoid base biosynthesis and a corresponding increase in the recycling of the backbones via the salvage pathway. There was also a considerable increase in cholesterol esters, although free cholesterol persisted at wild type levels, which might be secondary to the shifts in sphingolipid metabolism. All in all, these findings show that accumulation of free and phosphorylated sphingoid bases by loss of S1P lyase causes an interesting readjustment of the balance between de novo biosynthesis and recycling to maintain (glyco)sphingolipid homeostasis. These changes, and their impact on the metabolism of other cellular lipids, should be explored as possible contributors to the neurodegeneration in S1P lyase deficiency.     

The Mechanism of Pre-transfer Editing in Yeast Mitochondrial Threonyl-tRNA Synthetase

Accurate translation of mRNA into protein is a fundamental biological process critical for maintaining normal cellular functions. To ensure translational fidelity, aminoacyl-tRNA synthetases (aaRSs) employ pre-transfer and post-transfer editing activities to hydrolyze misactivated and mischarged amino acids, respectively. Whereas post-transfer editing, which requires either a specialized domain in aaRS or a trans-protein factor, is well described, the mechanism of pre-transfer editing is less understood. Here, we show that yeast mitochondrial threonyl-tRNA synthetase (MST1), which lacks an editing domain, utilizes pre-transfer editing to discriminate against serine. MST1 misactivates serine and edits seryl adenylate (Ser-AMP) in a tRNA-independent manner. MST1 hydrolyzes 80% of misactivated Ser-AMP at a rate 4-fold higher than that for the cognate threonyl adenylate (Thr-AMP) while releasing 20% of Ser-AMP into the solution. To understand the mechanism of pre-transfer editing, we solved the crystal structure of MST1 complexed with an analog of Ser-AMP. The binding of the Ser-AMP analog to MST1 induces conformational changes in the aminoacylation active site, and it positions a potential hydrolytic water molecule more favorably for nucleophilic attack. In addition, inhibition results reveal that the Ser-AMP analog binds the active site 100-fold less tightly than the Thr-AMP analog. In conclusion, we propose that the plasticity of the aminoacylation site in MST1 allows binding of Ser-AMP and the appropriate positioning of the hydrolytic water molecule.     

Estimation of an early meaningful time point of bone parameter changes in application to an osteoporotic rat model with in vivo microcomputed tomography measurements

The commonly used preclinical animal model of postmenopausal osteoporosis is the mature ovariectomized rat, whereby cessation of ovarian oestrogen production consequently results in bone volume reduction. The study aim was to precisely define the time course of structural changes resulting from ovariectomy and thereby reduce the time animals have to be treated to judge the effects of osteoporosis treatment. For this purpose, we assessed architectural changes by microcomputed tomography (μCT) during 10 weeks following ovariectomy or sham surgery at two-week intervals. Moreover, the trabecular microarchitecture of the lumbar vertebrae was assessed after necropsy. Besides this, serum biomarkers of bone turnover were determined. These data were in a new approach additionally correlated to femur mRNA expression profiles. We selected the osteoblast marker genes osteocalcin and type I collagen as well as the two osteoclast marker genes cathepsin k and tartrate-resistant acid phosphatase 5. The gene expression analysis suggested an activation of osteoblasts as well as octeoclasts. The significantly induced serum levels of osteocalcin and collagen degradation fragments also revealed this higher rate of bone turnover. Our results indicate that as soon as four weeks after ovariectomy the bone volume fraction exhibited a decline of 30% and 50% of the connectivity density. In addition, significant decreases of trabecular number and thickness as well as of the bone volume fraction were only observed in vertebrae of ovariectomized animals. Interestingly, changes of trabecular morphology were also found in the sham animals as a consequence of senescence.