Phosphocysteine in the PRL‐CNNM pathway mediates magnesium homeostasis

PRLs (p hosphatases of r egenerating l iver) are frequently overexpressed in human cancers and are prognostic markers of poor survival. Despite their potential as therapeutic targets, their mechanism of action is not understood in part due to their weak enzymatic activity. Previous studies revealed that PRLs interact with CNNM ion transporters and prevent CNNM4‐dependent Mg²⁺ transport, which is important for energy metabolism and tumor progression. Here, we report that PRL‐CNNM complex formation is regulated by the formation of phosphocysteine. We show that cysteine in the PRL catalytic site is endogenously phosphorylated as part of the catalytic cycle and that phosphocysteine levels change in response to Mg²⁺ levels. Phosphorylation blocks PRL binding to CNNM Mg²⁺ transporters, and mutations that block the PRL‐CNNM interaction prevent regulation of Mg²⁺ efflux in cultured cells. The crystal structure of the complex of PRL2 and the CBS‐pair domain of the Mg²⁺ transporter CNNM3 reveals the molecular basis for the interaction. The identification of phosphocysteine as a regulatory modification opens new perspectives for signaling by protein phosphatases.     

On cladistics and human–ape relationships

The rebuttal of our correspondence (Lehtonen et al., Journal of Biogeography, 2011, 38, 805–808) by J. R. Grehan and J. H. Schwartz (Journal of Biogeography, 2011, 38, 2397–2404) reveals logical inconsistencies in their methodology. Here, we provide a clarification of these issues.     

Synovial explant inflammatory mediator production corresponds to rheumatoid arthritis imaging hallmarks: a cross-sectional study

Introduction

Despite the widespread use of magnetic resonance imaging (MRI) and Doppler ultrasound for the detection of rheumatoid arthritis (RA) disease activity, little is known regarding the association of imaging-detected activity and synovial pathology. The purpose of this study was to compare site-specific release of inflammatory mediators and evaluate the corresponding anatomical sites by examining colour Doppler ultrasound (CDUS) and MRI scans.

Methods

RA patients were evaluated on the basis of CDUS and 3-T MRI scans and subsequently underwent synovectomy using a needle arthroscopic procedure of the hand joints. The synovial tissue specimens were incubated for 72 hours, and spontaneous release of monocyte chemoattractant protein 1 (MCP-1), interleukin 6 (IL-6), macrophage inflammatory protein 1β (MIP-1β) and IL-8 was measured by performing multiplex immunoassays. Bone marrow oedema (BME), synovitis and erosion scores were estimated on the basis of the rheumatoid arthritis magnetic resonance imaging score (RAMRIS). Mixed models were used for the statistical analyses. Parsimony was achieved by omitting covariates with P > 0.1 from the statistical model.

Results

Tissue samples from 58 synovial sites were obtained from 25 patients. MCP-1 was associated with CDUS activity (P = 0.009, approximate Spearman’s ρ = 0.41), RAMRIS BME score (P = 0.01, approximate Spearman’s ρ = 0.42) and RAMRIS erosion score (P = 0.03, approximate Spearman’s ρ = 0.31). IL-6 was associated with RAMRIS synovitis score (P = 0.04, approximate Spearman’s ρ = 0.50), BME score (P = 0.04, approximate Spearman’s ρ = 0.31) and RAMRIS erosion score (P = 0.03, approximate Spearman’s ρ = 0.35). MIP-1β was associated with CDUS activity (P = 0.02, approximate Spearman’s ρ = 0.38) and RAMRIS synovitis scores (P = 0.02, approximate Spearman’s ρ = 0.63). IL-8 associations with imaging outcome measures did not reach statistical significance.

Conclusions

The association between imaging activity and synovial inflammatory mediators underscores the high sensitivity of CDUS and MRI in the evaluation of RA disease activity. The associations found in our present study have different implications for synovial mediator releases and corresponding imaging signs. For example, MCP-1 and IL-6 were associated with both general inflammation and bone destruction, in contrast to MIP-1β, which was involved solely in general synovitis. The lack of association of IL-8 with synovitis was likely underestimated because of a large proportion of samples above assay detection limits among the patients with the highest synovitis scores.     

Spatial indicators for nature conservation from European to local scale

The paper presents an overview of the objectives and exemplary results of the FP 5 project “Spatial Indicators for European Nature Conservation” (SPIN). The SPIN project is focused on the development and testing of advanced classification methods and spatial indicators based on multisensor satellite data and GIS to accomplish monitoring and management tasks in the context of Natura 2000 and nature conservation. A representative selection of eight regional test areas covers a pan-European network and allows comparative investigations to provide accepted recommendations for regional and European nature conservation. The selected results of four case studies are presented and discussed. The range of work covers the production of regional and local habitat maps by object-oriented classification, a case-based reasoning method for change detection as a management support tool for planning and regulating local land use, the selection and application of structural indicators for the monitoring of Natura 2000 habitats and the downscaling and disaggregation of soil information. Results and the further implementation of presented methods are discussed in the conclusions.     

Inhibition of SIRT1 by a small molecule induces apoptosis in breast cancer cells

Overexpression of SIRT1, a NAD⁺-dependent class III histone deacetylases (HDACs), is implicated in many cancers and therefore could become a promising antitumor target. Here we demonstrate a small molecule SIRT1 inhibitor, ILS-JGB-1741(JGB1741) with potent inhibitory effects on the proliferation of human metastatic breast cancer cells, MDA-MB 231. The molecule has been designed using medicinal chemistry approach based on known SIRT1 inhibitor, sirtinol. The molecule showed a significant inhibition of SIRT1 activity compared to sirtinol. Studies on the antitumor effects of JGB on three different cancer cell lines, K562, HepG2 and MDA-MB 231 showed an IC₅₀ of 1, 10 and 0.5 μM, respectively. Further studies on MDA-MB 231 cells showed a dose-dependent increase in K9 and K382 acetylation of H3 and p53, respectively. Results also demonstrated that JGB1741-induced apoptosis is associated with increase in cytochrome c release, modulation in Bax/Bcl2 ratio and cleavage of PARP. Flowcytometric analysis showed increased percentage of apoptotic cells, decrease in mitochondrial membrane potential and increase in multicaspase activation. In conclusion, the present study indicates the potent apoptotic effects of JGB1741 in MDA-MB 231 cells.     

Engineering the recruitment of phosphotyrosine binding domain-containing adaptor proteins reveals distinct roles for RET receptor-mediated cell survival

The RET receptor tyrosine kinase is important for several different biological functions during development. The recruitment at the phosphorylated Tyr(1062) site in RET of a number of different phosphotyrosine binding (PTB) domain-containing adaptor proteins, including Shc and Frs2, plays a dominant role for the multiple different biological functions of the RET receptor during development, including stimulation of cell survival. Here, we demonstrate that a competitive recruitment of Shc as opposed to Frs2 mediates the survival signaling arising from RET activation. Based on results from a peptide array, we have genetically engineered the PTB domain binding site of RET to rewire its recruitment of the PTB proteins Shc and Frs2. An engineered RET that has a competitive interaction with Shc at the expense of Frs2, but not a RET receptor that only recruits Frs2, activates cell survival signaling pathways and is protective from cell death in neuronal SK-N-MC cells. Thus, cell type-specific functions involve a competitive recruitment of different PTB adaptor molecules by RET that activate selective signaling pathways.     

Activation of p21-activated kinase 2 and its association with Nef are conserved in murine cells but are not sufficient to induce an AIDS-like disease in CD4C/HIV transgenic mice

A well conserved feature of human immunodeficiency virus, type 1 (HIV-1) and simian immunodeficiency virus (SIV) Nef is the interaction with and activation of the human p21-activated kinase 2 (PAK2). The conservation of this interaction in other species and its significance for Nef pathogenesis in vivo are poorly documented. In the present study, we measured these parameters in Nef-expressing thymocytes, macrophages, and dendritic cells of a transgenic (Tg) mouse model of AIDS (CD4C/HIV). We found that Nef binds to and activates PAK2, but not PAK1 and -3, in these three cell subsets. Nef associates with only a small fraction of PAK2. The Nef-PAK2 complex also comprises beta-PIX-COOL. The impact of the Nef-PAK2 association on disease development was also analyzed in Tg mice expressing 10 different Nef mutant alleles. CD4C/HIV Tg mice expressing Nef alleles defective in Nef-PAK2 association (P69A, P72A/P75A, R105A/R106A, Delta56-66, or G2A (myristoylation site)) failed to develop disease of the non-lymphoid organs (kidneys and lungs). Among these, only Tg mice expressing Nef(P69A) and Nef(G2A) showed some depletion of CD4(+) T cells, although a down-regulation of the CD4 surface protein was documented in all these Tg lines, except those expressing Nef(Delta56-66). Among other Tg mice expressing Nef mutants having conserved the Nef-PAK2 association (RD35AA, D174K, P147A/P150A, Delta8-17, and Delta25-65), only Tg mice expressing Nef(Delta8-17) develop kidney and lung diseases, but all showed partial CD4(+) T cell depletion despite some being defective for CD4 down-regulation (RD35AA and D174K). Therefore, Nef can activate murine PAK2 and associate with a small fraction of it, as in human cells. Such activation and binding of PAK2 is clearly not sufficient but may be required to induce a multiorgan AIDS-like disease in Tg mice.