A Proteomic Approach to Study the Effect of Thiotaurine on Human Neutrophil Activation

Thiotaurine, a thiosulfonate related to taurine and hypotaurine, is formed by a metabolic process from cystine and generated by a transulfuration reaction between hypotaurine and thiocysteine. Thiotaurine can produce hydrogen sulfide (H2S) from its sulfane sulfur moiety. H2S is a gaseous signaling molecule which can have regulatory roles in inflammatory process. In addition, sulfane sulfur displays the capacity to reversibly bind to other sulfur atoms. Thiotaurine inhibits PMA-induced activation of human neutrophils, and hinders neutrophil spontaneous apoptosis. Here, we present the results of a proteomic approach to study the possible effects of thiotaurine at protein expression level. Proteome analysis of human neutrophils has been performed comparing protein extracts of resting or PMA-activated neutrophils in presence or in absence of thiotaurine. In particular, PMA-stimulated neutrophils showed high level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression compared to the level of the same glycolytic enzyme in the resting neutrophils. Conversely, decreased expression of GAPDH has been observed when human neutrophils were incubated with 1 mM thiotaurine before activation with PMA. This result, confirmed by Western blot analysis, suggests again that thiotaurine shows a bioactive role in the mechanisms underlying the inflammatory process, influencing the energy metabolism of activated leukocytes and raises the possibility that thiotaurine, acting as a sulfur donor, could modulate neutrophil activation via persulfidation of target proteins, such as GAPDH

Opposite Roles Of Tgf-ß1-Responsive Tsc22d Proteins In Regulating Cerebellar Granule Neurons Differentiation And Commitment To Apoptosis

The TSC22D (Transforming Growth Factor ß1-stimulated clone 22 domain) protein family includes widely expressed members having a TSC box and a leucine zipper domain and controlling multiple biological processes. Among these members, TSC22D4 is involved in cerebellum granule neuron (CGN) differentiation and commitment to apoptosis. These opposite functions rely on the existence of multiple TSC22D4 forms differing in modification, subcellular localization and function. In contrast to 42 kDa and 55 kDa forms, 67 kDa and 72 kDa forms have specific posttranslational modifications and subcellular localizations: the 67 kDa form is O-GlcNAcylated and unphosphorylated, whereas the 72 kDa form is O-GlcNAcylated and phoshorylated. Moreover, while the latter form is associated with chromatin, the 67 kDa form is associated with Apoptosis Inducing Factor (AIF) in mitochondria under normal cell viability conditions. However, when CGNs are committed to apoptosis the 67 kDa form is rapidly transferred from mitochondria to nuclear matrix with a kinetics similar to that of AIF transfer from mitochondria to chromatin, suggesting that the mitochondrial release of AIF and TSC22D4 is triggered by a regulatory mechanism(s) acting upon the entire AIF-TSC22D4 complex. Besides Tsc22d4, CGNs also express Tsc22d1, the first identified member of TSC22D family, which is in turn expressed with two splice variants encoding TSC22D1-1 and TSC22D1-2 proteins. While TSC22D1-2 is consistently expressed during CGN differentiation, TSC22D1-1 is highly expressed in undifferentiated CGNs and barely detected in fully differentiated CGNs. CGN apoptosis commitment triggers TSC22D1-1 translocation from the nucleus to the cytoplasm but does not modifies the nucleo/cytoplasmic distribution of TSC22D1-2. By immunoprecipitation experiments we have found that both TSC22D1-1 and TSC22D1-2 interact with TSC22D4 and that TSC22D1-1 also interacts with large conductance Ca+2-activated K+ channels (BKCa). Our findings on TSC22D4 and TSC22D1 subcellular localization/interacting protein partners depending on CGN differentiation/functional condition will be presented

The Influence of Catechol-O-Methyltransferase (COMT) Val158Met Gene Polymorphism, Persistence, and Attentional Characteristics on Novelty Seeking

Over the last five decades, a number of biological oriented personality theories have been proposed to explain how anatomical and functional differences in the human brain are responsible for individual differences in personality. Catechol-O-methyltransferase (COMT) gene for the Val158Met single nucleotide polymorphism (rs4680) is known to influence the activity of the enzyme responsible for dopamine metabolism and has been linked with various aspect of personality dimensions and cognitive processes. In the present study, non-clinical participants (201 women and 53 men) were adminis- tered the Temperament and Character Inventory-Revised, Tellegen Absorption Scale, Differential Attentional Processes Inventory, and Waterloo-Stanford Group Scale of Hypnotic Suggestibility, Form C. Among these participants, COMT polymorphism (parameterized as a 3-level variable: 0 = Met/Met, 1 = Val/Met, 2 = Val/Val) was assessed in 117 women and 51 men. Hypnotic Suggestibility scores were significantly correlated with scores from the Absorption, Extremely Focused Attention, and Dual Attention for Physical- Cognitive task. We failed to find the expected significant association between COMT and Hypnotic Suggestibility scores. In contrast, COMT scores were significantly correlated with scores from Novelty Seeking (r = -.15, p = .049) and its Disorderliness subscale (r = -.21, p = .006). A principal component analysis (with varimax rotation), performed on personality and attention measures, yielded a four- factor solution: Factor-1 (Moderately Focused Attention, Dual Atten- tion Cognitive-Cognitive, and Dual Attention Physical-Cognitive), Factor-2 (Novelty Seeking, Reward Dependence, and Harm Avoidance), Factor-3 (Hypnotic Suggestibility, Absorption, and Extremely Focused Attention), and Factor-4 (Persistence). These factors accounted for 18.4%, 16.3%, 16.2%, and 12.3% of the total variance, respectively. These findings guided us in the choice of the COMT, Persistence, Extremely Focused Attention, and Absorption scores as predictors of Disorderli- ness scores in separate multivariate regression analyses. Lower COMT activity, higher Absorption scores, and lower Persistence scores accounted for 18% of the total variance in the whole sample, and 10.8% in female sample. In male sample, higher Absorption and lower Persistence scores significantly predicted Disorderliness scores, accounting for 22% of the total variance. Since our male sample was relatively small, further research is needed to understand gender differences, if any, using a larger male sample

Role of TGF ß1 protein family members (TSC22D) in the control of cell proliferation/differentiation and apoptosis

The TSC22D (TGF ß1-stimulated clone 22 domain) protein family, including TSC22D1 and TSC22D4, play pivotal roles in cell proliferation, differentiation and apoptosis. TSC22D genes encode a large variety of forms arising from alternative splicing and/or post-translational modifications and having synergistic or antagonistic activities. Functional activities of mammalian TSC22D proteins have mostly focused on TSC22D1, encoding a long (TSC22D1.1) and short (TSC22D1.2) splice variants. The antagonistic activities of TSC22D1 isoforms rely on their ability to compete with each other for heterodimerization with TSC22D4. Both TSC22D1 and TSC22D4 are expressed in mouse brain (embryonal/postnatal cerebellum and areas of adult neurogenesis) and in in vitro cerebellar granule neurons (CGNs). We are investigating the specific function(s) played by the various TSC22D forms/isoforms. To this aim, we are exploiting a gene-specific silencing approach by RNA interference. A medulloblastoma cell line, arising from CGN tumors is used as model system to study the role TSC22D family members exert on cell proliferation. In these cells, the silencing of TSC22D4 leads to cell cycle arrest in G0/G1, while that of TSC22D1-1/D1-2 increases the fraction of cells undergoing apoptosis. A similar approach is used in in vitro cultured CGNS, which represent a model to study “differentiation” if cultured in depolarizing condition or “apoptosis” if treated with staurosporine. Overall, TSC22D1.1/ D1.2 display a positive effect on CGN proliferation/differentiation, while TSC22D4 is crucial during early stages of apoptosis. We conclude that TGF ß1 family members have opposite effects and play a key role in the switch proliferation/differentiation and apoptosis

A marked paucity of granule cells in the developing cerebellum of the Npc1−/− mouse is corrected by a single injection of hydroxypropyl-β-cyclodextrin

AbstractIn this study we show that postnatal development of cerebellar granule neurons (GNs) is defective in Npc1−/− mice. Compared to age-matched wild-type littermates, there is an accelerated disappearance of the external granule layer (EGL) in these mice. This is due to a premature exit from the cell cycle of GN precursors residing at the level of the EGL. As a consequence, the size of cerebellar lobules of these mice displays a 20%–25% reduction compared to that of age-matched wild-type mice. This size reduction is detectable at post-natal day 28 (PN28), when cerebellar GN development is completed while signs of neuronal atrophy are not yet apparent.Based on the analysis of EGL thickness and the determination of proliferating GN fractions at increasing developmental times (PN8–PN14), we trace the onset of this GN developmental defect during the second postnatal week. We also show that during this developmental time Shh transcripts undergo a significant reduction in Npc1−/− mice compared to age-matched wild-type mice. In light of the mitogenic activity of Shh on GNs, this observation further supports the presence of defective GN proliferation in Npc1−/− mice. A single injection of hydroxypropyl-β-cyclodextrin at PN7 rescues this defect, restoring the normal patterns of granule neuron proliferation and cerebellar lobule size.To our knowledge, these findings identify a novel developmental defect that was underappreciated in previous studies. This defect was probably overlooked because Npc1 loss-of-function does not affect cerebellar foliation and causes the internal granule layer and molecular layer to decrease proportionally, giving rise to a normally appearing, yet harmoniously smaller, cerebellum