The protein phosphatase PP2A/Bα binds to the microtubule-associated proteins Tau and MAP2 at a motif also recognized by the kinase Fyn: implications for tauopathies

The predominant brain microtubule-associated proteins MAP2 and tau play a critical role in microtubule cytoskeletal organization and function. We have previously reported that PP2A/Bα, a major protein phosphatase 2A (PP2A) holoenzyme, binds to and dephosphorylates tau, and regulates microtubule stability. Here, we provide evidence that MAP2 co-purifies with and is dephosphorylated by endogenous PP2A/Bα in bovine gray matter. It co-localizes with PP2A/Bα in immature and mature human neuronal cell bodies. PP2A co-immunoprecipitates with and directly interacts with MAP2. Using in vitro binding assays, we show that PP2A/Bα binds to MAP2c isoforms through a region encompassing the microtubule-binding domain and upstream proline-rich region. Tau and MAP2 compete for binding to and dephosphorylation by PP2A/Bα. Remarkably, the protein-tyrosine kinase Fyn, which binds to the proline-rich RTPPKSP motif conserved in both MAP2 and tau, inhibits the interaction of PP2A/Bα with either tau or MAP2c. The corresponding synthetic RTPPKSP peptide, but not the phosphorylated RpTPPKSP version, competes with Tau and MAP2c for binding to PP2A/Bα. Significantly, down-regulation of PP2A/Bα and deregulation of Fyn-Tau protein interactions have been linked to enhanced tau phosphorylation in Alzheimer disease. Together, our results suggest that PP2A/Bα is part of segregated MAP2 and tau signaling scaffolds that can coordinate the action of key kinases and phosphatases involved in modulating neuronal plasticity. Deregulation of these compartmentalized multifunctional protein complexes is likely to contribute to tau deregulation, microtubule disruption, and altered signaling in tauopathies.     

Defining an adequate sample of earlywood vessels for retrospective injury detection in diffuse-porous species

Vessels of broad-leaved trees have been analyzed to study how trees deal with various environmental factors. Cambial injury, in particular, has been reported to induce the formation of narrower conduits. Yet, little or no effort has been devoted to the elaboration of vessel sampling strategies for retrospective injury detection based on vessel lumen size reduction. To fill this methodological gap, four wounded individuals each of grey alder (Alnus incana (L.) Moench) and downy birch (Betula pubescens Ehrh.) were harvested in an avalanche path. Earlywood vessel lumina were measured and compared for each tree between the injury ring built during the growing season following wounding and the control ring laid down the previous year. Measurements were performed along a 10 mm wide radial strip, located directly next to the injury. Specifically, this study aimed at (i) investigating the intra-annual duration and local extension of vessel narrowing close to the wound margin and (ii) identifying an adequate sample of earlywood vessels (number and intra-ring location of cells) attesting to cambial injury. Based on the results of this study, we recommend analyzing at least 30 vessels in each ring. Within the 10 mm wide segment of the injury ring, wound-induced reduction in vessel lumen size did not fade with increasing radial and tangential distances, but we nevertheless advise favoring early earlywood vessels located closest to the injury. These findings, derived from two species widespread across subarctic, mountainous, and temperate regions, will assist retrospective injury detection in Alnus, Betula, and other diffuse-porous species as well as future related research on hydraulic implications after wounding.     

Characterisation and Validation of Insertions and Deletions in 173 Patient Exomes

Recent advances in genomics technologies have spurred unprecedented efforts in genome and exome re-sequencing aiming to unravel the genetic component of rare and complex disorders. While in rare disorders this allowed the identification of novel causal genes, the missing heritability paradox in complex diseases remains so far elusive. Despite rapid advances of next-generation sequencing, both the technology and the analysis of the data it produces are in its infancy. At present there is abundant knowledge pertaining to the role of rare single nucleotide variants (SNVs) in rare disorders and of common SNVs in common disorders. Although the 1,000 genome project has clearly highlighted the prevalence of rare variants and more complex variants (e.g. insertions, deletions), their role in disease is as yet far from elucidated.We set out to analyse the properties of sequence variants identified in a comprehensive collection of exome re-sequencing studies performed on samples from patients affected by a broad range of complex and rare diseases (N = 173). Given the known potential for Loss of Function (LoF) variants to be false positive, we performed an extensive validation of the common, rare and private LoF variants identified, which indicated that most of the private and rare variants identified were indeed true, while common novel variants had a significantly higher false positive rate. Our results indicated a strong enrichment of very low-frequency insertion/deletion variants, so far under-investigated, which might be difficult to capture with low coverage and imputation approaches and for which most of study designs would be under-powered. These insertions and deletions might play a significant role in disease genetics, contributing specifically to the underlining rare and private variation predicted to be discovered through next generation sequencing.     

Assessment of some tools for the characterization of the human osteoarthritic cartilage proteome.

Since the proteome of osteoarthritic articular cartilage has been poorly investigated as yet, we adapted proteomic technologies to the study of the proteins secreted or released by fresh human osteoarthritic cartilage in culture. Fresh cartilage explants were obtained from three donors undergoing surgery for knee joint replacement. The explants were dissected out, minced, and incubated in serum-free culture medium. After 48 h, proteins in the medium were identified by two-dimensional or off-gel electrophoresis coupled to tandem mass spectrometry, or by using an antibody-based protein microarray designed to detect angiogenic factors, growth factors, chemokines, and cytokines. We identified a series of 43 proteins. Some of these proteins were already described as secretion products of chondrocytes, such as YKL-39 or osteoprotegerin, while several other were known proteins but have never been reported previously in cartilage, such as the serum amyloid P-component, the vitamin D binding protein, the pigment epithelium derived factor, the pulmonary and activation-regulated chemokine, lyl-1, thrombopoietin, fibrinogen, angiogenin, gelsolin, and osteoglycin/mimecan. While this study enabled the identification of novel proteins secreted or released by human osteoarthritic cartilage, the goal of the present work was essentially to describe the technical approach necessary for a systematic study of osteoarthritic cartilages from a large population of donors, in order to be able to select the good markers and/or targets for this poorly explored disease.     

Characterization of G3BPs: Tissue specific expression,chromosomal localisation and rasGAP120 binding studies

The G3BP (ras‐GTPase‐Activating Protein SH3‐Domain‐Binding Protein) family of proteins has been implicated in both signal transduction and RNA‐metabolism. We have previously identified human G3BP‐1, G3BP‐2, and mouse G3BP‐2. Here, we report the cloning of mouse G3BP‐1, the discovery of two alternatively spliced isoforms of mouse, and human G3BP‐2 (G3BP‐2a and G3BP‐2b), and the chromosomal localisation of human G3BP‐1 and G3BP‐2, which map to 5q14.2‐5q33.3 and 4q12‐4q24 respectively. We mapped the rasGAP¹²⁰ interactive region of the G3BP‐2 isoforms and show that both G3BP‐2a and G3BP‐2b use an N‐terminal NTF2‐like domain for rasGAP¹²⁰ binding rather than several available proline‐rich (PxxP) motifs found in members of the G3BPs. Furthermore, we have characterized the protein expression of both G3BP‐1 and G3BP‐2a/b in adult mouse tissues, and show them to be both tissue and isoform specific. J. Cell. Biochem. 84: 173–187, 2002. © 2001 Wiley‐Liss, Inc.     

The plant hormone auxin: insight in sight.

Physiological experiments conducted over the last 60 years indicate that the plant hormone auxin regulates a diverse set of developmental processes via changes in cell division, cell elongation and cell differentiation. Recent studies using transgenic plants with altered auxin levels support these conclusions and promise to provide more detailed information on the role of auxin during plant development. Although it is possible that all auxin responses are mediated by the same primary biochemical events, the studies described in this review are more consistent with multiple modes of auxin action. The development of molecular and genetic approaches to the study of hormone action should resolve this issue. The accelerated rate of progress in this field suggests that real insight into the mechanism of auxin action may be forthcoming.     

A multidimensional <Superscript>1</Superscript>H NMR lipidomics workflow to address chemical food safety issues

Introduction

Although it is still at a very early stage compared to its mass spectrometry (MS) counterpart, proton nuclear magnetic resonance (NMR) lipidomics is worth being investigated as an original and complementary solution for lipidomics. Dedicated sample preparation protocols and adapted data acquisition methods have to be developed to set up an NMR lipidomics workflow; in particular, the considerable overlap observed for lipid signals on 1D spectra may hamper its applicability.

Objectives

The study describes the development of a complete proton NMR lipidomics workflow for application to serum fingerprinting. It includes the assessment of fast 2D NMR strategies, which, besides reducing signal overlap by spreading the signals along a second dimension, offer compatibility with the high-throughput requirements of food quality characterization.

Method

The robustness of the developed sample preparation protocol is assessed in terms of repeatability and ability to provide informative fingerprints; further, different NMR acquisition schemes—including classical 1D, fast 2D based on non-uniform sampling or ultrafast schemes—are evaluated and compared. Finally, as a proof of concept, the developed workflow is applied to characterize lipid profiles disruption in serum from β-agonists diet fed pigs.

Results

Our results show the ability of the workflow to discriminate efficiently sample groups based on their lipidic profile, while using fast 2D NMR methods in an automated acquisition framework.

Conclusion

This work demonstrates the potential of fast multidimensional ¹H NMR—suited with an appropriate sample preparation—for lipidomics fingerprinting as well as its applicability to address chemical food safety issues.