The autoimmune regulator PHD finger binds to non-methylated histone H3K4 to activate gene expression

Mutations in the gene autoimmune regulator (AIRE) cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy. AIRE is expressed in thymic medullary epithelial cells, where it promotes the expression of tissue-restricted antigens. By the combined use of biochemical and biophysical methods, we show that AIRE selectively interacts with histone H3 through its first plant homeodomain (PHD) finger (AIRE-PHD1) and preferentially binds to non-methylated H3K4 (H3K4me0). Accordingly, in vivo AIRE binds to and activates promoters containing low levels of H3K4me3 in human embryonic kidney 293 cells. We conclude that AIRE-PHD1 is an important member of a newly identified class of PHD fingers that specifically recognize H3K4me0, thus providing a new link between the status of histone modifications and the regulation of tissue-restricted antigen expression in thymus.     

A polymorphism in the human serotonin 5-HT2A receptor gene may protect against systemic sclerosis by reducing platelet aggregation

Introduction

Platelet aggregation may contribute to the pathogenesis of systemic sclerosis: following activation, platelets release significant amounts of serotonin – which promotes vasoconstriction and fibrosis, and further enhances aggregation. The C+1354T polymorphism in the exonic region of the serotonin 2A receptor gene determining the His452Tyr substitution was associated with blunted intracellular responses after serotonin stimulation, and may have a role in susceptibility to scleroderma.

Methods

One hundred and fifteen consecutive systemic sclerosis patients and 140 well-matched healthy control individuals were genotyped by sequence-specific primer-PCR for the His⁴⁵²Tyr substitution of the serotonin 2A receptor gene, and associations were sought with scleroderma and its main clinical features. The functional relevance of the His⁴⁵²Tyr substitution was also assessed by evaluating the aggregation of platelet-rich plasma from His⁴⁵²/His⁴⁵² and His⁴⁵²/Tyr⁴⁵² healthy individuals after stimulation with adenosine diphosphate ± serotonin.

Results

The T allele of the C+1354T polymorphism was underrepresented in scleroderma patients compared with control individuals (5.2% versus 12.4%, P < 0.001, chi-square test and 1,000-fold permutation test) and its carriage reduced the risk for systemic sclerosis (odds ratio = 0.39, 95% confidence interval = 0.19 to 0.85, P < 0.01). Platelets from His⁴⁵²/Tyr⁴⁵² healthy subjects more weakly responded to serotonin stimulation compared with platelets from His⁴⁵²/His⁴⁵² individuals (3.2 ± 2.6-fold versus 9.6 ± 8.6-fold increase in aggregation, P = 0.017 by Kolmogorov–Smirnov test and P = 0.003 after correction for baseline adenosine diphosphate-induced aggregation values).

Conclusion

The His⁴⁵²Tyr substitution may influence susceptibility to systemic sclerosis by altering platelet aggregation in response to serotonin.     

AQP1 expression analysis in human diseases: implications for proteomic characterization

Aquaporin (AQP)1 belongs to a ubiquitous family of water channel proteins characterized by sequence similarity and the presence of two NPA (Asp-Pro-Ala) motifs existing in almost all organs and tissues. Currently, 13 human AQPs are known and they are divided into two subgroups according to their ability to transport only water molecules, such as AQP1, or also glycerol and other small solutes. The genomic, structural and functional aspects of AQP1 are briefly described. An in-depth discussion is devoted to proteomic approaches that are useful for identifying and characterizing AQP1, mainly through electrophoretic techniques combined with different extraction procedures followed by mass spectrometry analysis. Moreover, the relevance of AQP1 in human diseases is also explained. Its role in human tumors and, in particular, those of the kidney (e.g., clear cell renal carcinoma) is discussed.     

Neuropharmacology and behavior in planarians: translations to mammals

Planarians are the simplest animals to exhibit a body plan common to all vertebrates and many invertebrates, characterized by bilateral rather than radial symmetry, dorsal and ventral surfaces, and a rostrocaudal axis with a head and a tail, including specialized sense organs and an aggregate of nerve cells in the head. Neurons in planarian more closely resemble those of vertebrates than those of advanced invertebrates, exhibiting typical vertebrate features of multipolar shape, dendritic spines with synaptic boutons, a single axon, expression of vertebrate-like neural proteins, and relatively low spontaneously generated electrical activity. Here we report the most relevant contribution to the knowledge of the neuropharmacology of planarians, with particular reference to the behavioral consequences of the exposure to drugs acting on neural transmission. Neurochemical and histochemical data indicate the presence of several neurotransmitter-receptor systems in planarians. Moreover, a variety of experimental studies characterized specific behavioral patterns of these animals following the exposure to drugs acting on neural transmission. There is also evidence of the interactions between discrete neurotransmitter-receptor systems in modulating behavior in planarians. Finally, the model has proved efficacy for investigating the neurotoxicology of the dopamine neurons, and for the initial screening of the neuroprotective potential of drugs. In conclusion, these findings indicate that interactions between discrete neurotransmitter-receptor systems occur very early along phylogeny, although they may have evolved from very fundamental behaviors, such as motor activity in planarian, to more complex and integrated functions in vertebrates.     

Transketolase and 2',3'-cyclic-nucleotide 3'-phosphodiesterase type I isoforms are specifically recognized by IgG autoantibodies in multiple sclerosis patients

The presence of autoantibodies in multiple sclerosis (MuS) is well known, but their target antigens have not been clearly identified. In the present study, IgG autoreactivity to neural antigens of normal human white matter separated by bidimensional electrophoresis was assessed in serum and cerebrospinal fluid of 18 MuS and 20 control patients. Broad IgG autoreactivity was detected by two-dimensional immunoblotting in all cases to neural antigens, most of which were identified by mass spectrometry. The comparative analysis of MuS and non-MuS reactive spots showed that a restricted number of neural protein isoforms were specifically recognized by MuS IgG. Almost all MuS patients had cerebrospinal fluid IgG directed to isoforms of one of the oligodendroglial molecules, transketolase, 2',3'-cyclic-nucleotide 3'-phosphodiesterase type I, collapsin response mediator protein 2, and tubulin beta 4. Interestingly 50% of MuS IgG recognized transketolase, which was mostly localized on oligodendrocytes in human white matter from normal and MuS samples. IgG autoreactivity to cytoskeletal proteins (radixin, sirtuin 2, and actin-interacting protein 1) was prevalent in secondary progressive MuS patients. Among the proteins recognized by serum IgG, almost all MuS patients specifically recognized a restricted number of neuronal/cytoskeletal proteins, whereas 2',3'-cyclic-nucleotide 3'-phosphodiesterase type I was the oligodendroglial antigen most frequently recognized (44%) by MuS seric IgG. Our immunomics approach shed new light on the autoimmune repertoire present in MuS patients revealing novel oligodendroglial and/or neuronal putative autoantigens with potential important pathogenic and diagnostic implications.     

Cell death in fetal oocytes: many players for multiple pathways

We devised a short-term culture system allowing us to define novel characteristics of programmed cell death (PCD) of fetal oocytes and to underscore new aspects of this process. Mouse fetal oocytes cultured in conditions allowing meiotic progression underwent apoptotic degeneration as revealed by TUNEL staining, DNA ladder, Annexin V binding, PARP cleavage and, usually, caspase activation. TEM observations show, however, recurrent atypical apoptotic morphologies characterized by the absence of chromatin margination and nuclear fragmentation; oocytes with autophagic and necrotic features are also observed. Moreover, under the fluorescence microscope a subpopulation of TUNEL(+) oocytes appear morphologically healthy and do not show detectable caspase activity. Finally, caspase inhibitors are able to slow down, but not to abolish, oocyte cell death, whereas calpain inhibitor I significantly reduces the number of TUNEL(+) oocytes after 4 days of culture, and rapamycin (mTOR inhibitor) increases such numbers both at day 3 and 4. These observations together with results showing expression in cultured oocytes undergoing cell death of apoptosis inducing factor and Beclin 1, two important players of caspase independent and autophagic cell death, respectively, demonstrate that fetal oocytes possess and are able to activate several players of various forms of cell death. However, causal correlation among different cell death pathways in such oocytes remains to be determined and stimuli causing the activation of these pathways in vitro and in vivo also clarified.     

A role for <Emphasis Type="Italic">Haemophilus ducreyi</Emphasis> Cu,ZnSOD in resistance to heme toxicity

The Cu,Zn superoxide dismutase (Cu,ZnSOD) from Haemophilus ducreyi is the only enzyme of this class which binds a heme molecule at its dimer interface. To explore the role of the enzyme in this heme-obligate bacterium, a sodC mutant was created by insertional inactivation. No difference in growth rate was observed during heme limitation. In contrast, under heme rich conditions growth of the sodC mutant was impaired compared to the wild type strain. This growth defect was abolished by supplementation of exogenous catalase. Genetic complementation of the sodC mutant in trans demonstrated that the enzymatic property or the heme-binding activity of the protein could repair the growth defect of the sodC mutant. These results indicate that Cu,ZnSOD protects Haemophilus ducreyi from heme toxicity.     

Secretion without Golgi

A growing number of proteins devoid of signal peptides have been demonstrated to be released through the non-classical pathways independent of endoplasmic reticulum and Golgi. Among them are two potent proangiogenic cytokines FGF1 and IL1alpha. Stress-induced transmembrane translocation of these proteins requires the assembly of copper-dependent multiprotein release complexes. It involves the interaction of exported proteins with the acidic phospholipids of the inner leaflet of the cell membrane and membrane destabilization. Not only stress, but also thrombin treatment and inhibition of Notch signaling stimulate the export of FGF1. Non-classical release of FGF1 and IL1alpha presents a promising target for treatment of cardiovascular, oncologic, and inflammatory disorders.     

Mechanism of inhibition of wt-dihydrofolate reductase from E. coli by tea epigallocatechin-gallate

Dihydrofolate reductase (DHFR) is a ubiquitous enzyme involved in major biological process, including DNA synthesis and cancer inhibition, and its modulation is the object of extensive structural, kinetic, and pharmacological studies. In particular, earlier studies showed that green tea catechins are powerful inhibitors of bovine liver and chicken liver DHFR. In this article, we report the results of inhibition kinetics for the enzyme from another source (DHFR from E. coli) exerted by (-)-epigallocatechingallate (EGCG). Using different analytical techniques, we reported that EGCG acts as a bisubstrate inhibitor on the bacterial DHFR. Moreover, the combined approach of biosensor, kinetic, and molecular modelling analysis disclosed the ability of EGCG to bind to the enzyme both on substrate (DHF) and cofactor (NADPH) site. Collectively, our data have confirmed the selectivity of antifolate compounds with respect to the different source of enzyme (bacterial or mammalian DHFR) and the possible role of tea catechins as chemopreventive agents.