The B55α Regulatory Subunit of Protein Phosphatase 2A Mediates Fibroblast Growth Factor-Induced p107 Dephosphorylation and Growth Arrest in Chondrocytes

Fibroblast growth factor (FGF)-induced growth arrest of chondrocytes is a unique cell type-specific response which contrasts with the proliferative response of most cell types and underlies several genetic skeletal disorders caused by activating FGF receptor (FGFR) mutations. We have shown that one of the earliest key events in FGF-induced growth arrest is dephosphorylation of the retinoblastoma protein (Rb) family member p107 by protein phosphatase 2A (PP2A), a ubiquitously expressed multisubunit phosphatase. In this report, we show that the PP2A-B55α holoenzyme (PP2A containing the B55α subunit) is responsible for this phenomenon. Only the B55α (55-kDa regulatory subunit, alpha isoform) regulatory subunit of PP2A was able to bind p107, and this interaction was induced by FGF in chondrocytes but not in other cell types. Small interfering RNA (siRNA)-mediated knockdown of B55α prevented p107 dephosphorylation and FGF-induced growth arrest of RCS (rat chondrosarcoma) chondrocytes. Importantly, the B55α subunit bound with higher affinity to dephosphorylated p107. Since the p107 region interacting with B55α is also the site of cyclin-dependent kinase (CDK) binding, B55α association may also prevent p107 phosphorylation by CDKs. FGF treatment induces dephosphorylation of the B55α subunit itself on several serine residues that drastically increases the affinity of B55α for the PP2A A/C dimer and p107. Together these observations suggest a novel mechanism of p107 dephosphorylation mediated by activation of PP2A through B55α dephosphorylation. This mechanism might be a general signal transduction pathway used by PP2A to initiate cell cycle arrest when required by external signals.     

24S-hydroxycholesterol in plasma: A marker of cholesterol turnover in neurodegenerative diseases

Brain cholesterol is mainly involved in the cell membrane structure, in signal transduction, neurotransmitter release, synaptogenesis and membrane trafficking. Impairment of brain cholesterol metabolism was described in neurodegenerative diseases, such as Multiple Sclerosis, Alzheimer and Huntington Diseases. Since the blood–brain barrier efficiently prevents cholesterol uptake from the circulation into the brain, de novo synthesis is responsible for almost all cholesterol present there. Cholesterol is converted into 24S-hydroxycholesterol (24OHC) by cholesterol 24-hydroxylase (CYP46A1) expressed in neural cells.     

Overexpression of GlyI and GlyII genes in transgenic tomato (Solanum lycopersicum Mill.) plants confers salt tolerance by decreasing oxidative stress

The glyoxalase system plays an important role in various physiological processes in plants, including salt stress tolerance. We report the effects of overexpressing glyoxalase I and glyoxalase II genes in transgenic tomato (Solanum lycopersicum Mill.) cv. Ailsa Craig. Stable expression of both transgenes was detected in the transformed tomato plants under salt stress. The transgenic lines overexpressing GlyI and GlyII under a high NaCl concentration (800 mM) showed reduced lipid peroxidation and the production of H₂O₂ in leaf tissues. A greater decrease in the chlorophyll a+b content in wild-type (WT) compared with transgenic lines was also observed. These results suggest that the over expression of two genes, GlyI and GlyII, may enhance salt stress tolerance by decreasing oxidative stress in transformed tomato plants. This work will help our understanding of the putative role of the glyoxalase system in the tolerance to abiotic stress in tomato plants.     

Action of a rhizocephalan on the genital activity of host male crabs: characterization of a parasitic secretion inhibiting spermatogenesis

Protein fractions obtained by filtration of crude extracts of roots from the sacculinid parasite Loxothylacus panopei were tested by repeated injections on healthy male crabs Rhithropanopeus harrisii, the usual host of the rhizocephalan. Testes, and rogenic glands and different parts of the host CNS were observed; the behaviour of the animals was also noted. For the first time, proteinaceous substance(s) of about 25000–30000 daltons were characterized. They induce inhibition of spermatogenesis, and rogenic gland cytolysis and depletion of the sinus gland as in naturally infested crabs. The mode of action of sacculinid on spermatogenesis in the host is discussed.     

Engineering Strontium Binding Affinity in an EF-hand Motif: A Quantum Chemical and Molecular Dynamics Study

Abstract

Proteins with the ability to specifically bind strontium would potentially be of great use in the field of nuclear waste management. Unfortunately, no such peptides or proteins are known—indeed, it is uncertain whether they exist under natural conditions due to low environmental concentrations of strontium. To investigate the possibility of devising such molecules, one of us (CV), in a previous experimental study [J. Biol. Inorg. Chem. 8, 33440 (2003)], proposed starting from an EF-hand motif of the protein calmodulin and mutating some residues to change the motif's specificity for calcium into one for strontium. In this paper, which represents a theoretical complement to the experimental work, we analyzed small-molecule crystallographic structures and performed quantum chemical calculations to identify possible mutations. We then constructed seven mutant sequences of the EF-hand motif and analyzed their dynamical and binding behaviors using molecular dynamics simulations and free-energy calculations (using the MM/PBSA method). As a result of these analyzes we were able to isolate some characteristics that could lead to mutant peptides with enhanced strontium affinity.     

Recent advances in proteomic technologies applied to cardiovascular disease

In recent years, the diagnosis of cardiovascular disease (CVD) has increased its potential, also thanks to mass spectrometry (MS) proteomics. Modern MS proteomics tools permit analyzing a variety of biological samples, ranging from single cells to tissues and body fluids, like plasma and urine. This approach enhances the search for informative biomarkers in biological samples from apparently healthy individuals or patients, thus allowing an earlier and more precise diagnosis and a deeper comprehension of pathogenesis, development and outcome of CVD to further reduce the enormous burden of this disease on public health. In fact, many differences in protein expression between CVD‐affected and healthy subjects have been detected, but only a few of them have been useful to establish clinical biomarkers because they did not pass the verification and validation tests. For a concrete clinical support of MS proteomics to CVD, it is, therefore, necessary to: ameliorate the resolution, sensitivity, specificity, throughput, precision, and accuracy of MS platform components; standardize procedures for sample collection, preparation, and analysis; lower the costs of the analyses; reduce the time of biomarker verification and validation. At the same time, it will be fundamental, for the future perspectives of proteomics in clinical trials, to define the normal protein maps and the global patterns of normal protein levels, as well as those specific for the different expressions of CVD. J. Cell. Biochem. 114: 7–20, 2012. © 2012 Wiley Periodicals, Inc.     

Functional studies of MP62 during male chromatin decondensation in sea urchins

In amphibians, sperm histone transition post‐fertilization during male pronucleus formation is commanded by histone chaperone Nucleoplasmin (NPM). Here, we report the first studies to analyze the participation of a Nucleoplasmin‐like protein on male chromatin remodeling in sea urchins. In this report, we present the molecular characterization of a nucleoplasmin‐like protein that is present in non fertilized eggs and early zygotes in sea urchin specie Tetrapygus niger. This protein, named MP62 can interact with sperm histones in vitro. By male chromatin decondensation assays and immunodepletion experiments in vitro, we have demonstrated that this protein is responsible for sperm nucleosome disorganization. Furthermore, as amphibian nucleoplasmin MP62 is phosphorylated in vivo immediately post‐fertilization and this phosphorylation is dependent on CDK‐cyclin activities found after fertilization. As we shown, olomoucine and roscovitine inhibits male nucleosome decondensation, sperm histone replacement in vitro and MP62 phosphorylation in vivo. This is the first report of a nucleoplasmin‐like activity in sea urchins participating during male pronucleus formation post‐fecundation. J. Cell. Biochem. 114: 1779–1788, 2013. © 2013 Wiley Periodicals, Inc.