Elena Tibaldi

Catalase Takes Part in Rat Liver Mitochondria Oxidative Stress Defense

Highly purified rat liver mitochondria (RLM) when exposed to tert-butylhydroperoxide undergo matrix swelling, membrane potential collapse, and oxidation of glutathione and pyridine nucleotides, all events attributable to the induction of mitochondrial permeability transition. Instead, RLM, if treated with the same or higher amounts of H2O2 or tyramine, are insensitive or only partially sensitive, respectively, to mitochondrial permeability transition. In addition, the block of respiration by antimycin A added to RLM respiring in state 4 conditions, or the addition of H2O2, results in O2 generation, which is blocked by the catalase inhibitors aminotriazole or KCN. In this regard, H2O2 decomposition yields molecular oxygen in a 2:1 stoichiometry, consistent with a catalatic mechanism with a rate constant of 0.0346 s-1. The rate of H2O2 consumption is not influenced by respiratory substrates, succinate or glutamate-malate, nor by N-ethylmaleimide, suggesting that cytochrome c oxidase and the glutathione-glutathione peroxidase system are not significantly involved in this process. Instead, H2O2 consumption is considerably inhibited by KCN or aminotriazole, indicating activity by a hemoprotein. All these observations are compatible with the presence of endogenous heme-containing catalase with an activity of 825 ± 15 units, which contributes to mitochondrial protection against endogenous or exogenous H2O2. Mitochondrial catalase in liver most probably represents regulatory control of bioenergetic metabolism, but it may also be proposed for new therapeutic strategies against liver diseases. The constitutive presence of catalase inside mitochondria is demonstrated by several methodological approaches as follows: biochemical fractionating, proteinase K sensitivity, and immunogold electron microscopy on isolated RLM and whole rat liver tissue.

Heparanase and Syndecan-1 Interplay Orchestrates Fibroblast Growth Factor-2-induced Epithelial-Mesenchymal Transition in Renal Tubular Cells

Background: FGF-2 induces EMT in PTECs, and HPSE regulates HS/syndecans. Results: The lack of HPSE prevents FGF-2-induced EMT; FGF-2 induces EMT through PI3K/AKT and produces an autocrine loop. Conclusion: HPSE is necessary for FGF-2 to produce EMT, to activate FGF-2 intracellular signaling, and to regulate its autocrine loop. Significance: HPSE is an interesting pharmacological target for the prevention of renal fibrosis. The epithelial-mesenchymal transition (EMT) of proximal tubular epithelial cells (PTECs) into myofibroblasts contributes to the establishment of fibrosis that leads to end stage renal disease. FGF-2 induces EMT in PTECs. Because the interaction between FGF-2 and its receptor is mediated by heparan sulfate (HS) and syndecans, we speculated that a deranged HS/syndecans regulation impairs FGF-2 activity. Heparanase is crucial for the correct turnover of HS/syndecans. The aim of the present study was to assess the role of heparanase on epithelial-mesenchymal transition induced by FGF-2 in renal tubular cells. In human kidney 2 (HK2) PTEC cultures, although FGF-2 induces EMT in the wild-type clone, it is ineffective in heparanase-silenced cells. The FGF-2 induced EMT is through a stable activation of PI3K/AKT which is only transient in heparanase-silenced cells. In PTECs, FGF-2 induces an autocrine loop which sustains its signal through multiple mechanisms (reduction in syndecan-1, increase in heparanase, and matrix metalloproteinase 9). Thus, heparanase is necessary for FGF-2 to produce EMT in PTECs and to sustain FGF-2 intracellular signaling. Heparanase contributes to a synergistic loop for handling syndecan-1, facilitating FGF-2 induced-EMT. In conclusion, heparanase plays a role in the tubular-interstitial compartment favoring the FGF-2-dependent EMT of tubular cells. Hence, heparanase is an interesting pharmacological target for the prevention of renal fibrosis.

Lyn-mediated SHP-1 recruitment to CD5 contributes to resistance to apoptosis of B-cell chronic lymphocytic leukemia cells

In B-cell chronic lymphocytic leukemia (B-CLL) cells, Lyn, a tyrosine kinase belonging to the Src family, is overexpressed and atypically localized in an aberrant cytosolic complex in an active conformation, contributing to the unbalance between cell survival and pro-apoptotic signals. In this study, we demonstrate that Lyn constitutively phosphorylates the immunoreceptor tyrosine inhibitory motifs of the inhibitory cell surface co-receptor CD5, a marker of B-CLL. As a result, CD5 provides an anchoring site to Src homology 2 domain-containing phosphatase 1 (SHP-1), a known negative regulator of hematopoietic cell function, thereby triggering the negative B-cell receptor (BCR) signaling. The subsequent segregation of SHP-1 into two pools, one bound to the inhibitory co-receptor CD5 in an active form, the other in the cytosol in an inhibited conformation, proves crucial for withstanding apoptosis, as shown by the use of phosphotyrosine phosphatase-I-I, a direct inhibitor of SHP-1, or SHP-1 knockdown. These results confirm that Lyn exhibits the unique ability to negatively regulate BCR signaling, in addition to positively regulating effectors downstream of the BCR, and identify SHP-1 as a novel player in the deranged signaling network and as a potential attractive target for new therapeutic strategies in B-CLL.

CD2 molecules redistribute to the uropod during T cell scanning: Implications for cellular activation and immune surveillance

Dynamic binding between CD2 and CD58 counter-receptors on opposing cells optimizes immune recognition through stabilization of cell–cell contact and juxtaposition of surface membranes at a distance suitable for T cell receptor–ligand interaction. Digitized time-lapse differential interference contrast and immunofluorescence microscopy on living cells now show that this binding also induces T cell polarization. Moreover, CD2 can facilitate motility of T cells along antigen-presenting cells via a movement referred to as scanning. Both activated CD4 and CD8 T cells are able to scan antigen-presenting cells surfaces in the absence of cognate antigen. Scanning is critically dependent on T cell β-integrin function, as well as myosin light chain kinase. More importantly, surface CD2 molecules rapidly redistribute on interaction with a cellular substratum, resulting in a 100-fold greater CD2 density in the uropod versus the leading edge. In contrast, no redistribution is observed for CD11a/CD18 or CD45. Molecular compartmentalization of CD2, T cell receptor, and lipid rafts within the uropod prearranges the cellular activation machinery for subsequent immune recognition. This “presynapse” formation on primed T cells will likely facilitate the antigen-dependent recognition capability required for efficient immune surveillance.

Motif Analysis of Phosphosites Discloses a Potential Prominent Role of the Golgi Casein Kinase (GCK) in the Generation of Human Plasma Phospho-Proteome

By comparing the recurrent features of sequences surrounding 86 Ser/Thr residues phosphorylated in peptides from human plasma collected from literature with those generated from the whole human phosphoproteome, and from repertoires of validated substrates of the acidophilic protein kinases CK2 and Golgi casein kinase (GCK), the following conclusions can be drawn: (i) the contribution of Pro-directed and basophilic kinases to the plasma phosphoproteome is negligible, if any, while the contribution of acidophilic kinases is by far predominant; (ii) the plasma weblogo profile is closely reminiscent of that generated by GCK in its substrates, while it neatly differentiates from that generated by CK2; (iii) 58 plasma phosphosites out of 86 display the canonical consensus for GCK (S/T-x-E/pS), while that for CK2 (S/T-x-x-E/D/pS) is found in 15 peptides, all of which also conform to the GCK signature. These observations, in conjunction with a very similar situation disclosed by analyzing the phosphopeptides of the human cerebrospinal fluid collected from literature, support the view that GCK may play a major role in the phosphorylation of proteins secreted into body fluids.

MBNL142 and MBNL143 gene isoforms, overexpressed in DM1-patient muscle, encode for nuclear proteins interacting with Src family kinases

Myotonic dystrophy type-1 (DM1) is the most prevalent form of muscular dystrophy in adults. This disorder is an RNA-dominant disease, caused by expansion of a CTG repeat in the DMPK gene that leads to a misregulation in the alternative splicing of pre-mRNAs. The longer muscleblind-like-1 (MBNL1) transcripts containing exon 5 and the respective protein isoforms (MBNL142–43) were found to be overexpressed in DM1 muscle and localized exclusively in the nuclei. In vitro assays showed that MBNL142–43 bind the Src-homology 3 domain of Src family kinases (SFKs) via their proline-rich motifs, enhancing the SFK activity. Notably, this association was also confirmed in DM1 muscle and myotubes. The recovery, mediated by an siRNA target to Ex5-MBNL142–43, succeeded in reducing the nuclear localization of both Lyn and MBNL142–43 proteins and in decreasing the level of tyrosine phosphorylated proteins. Our results suggest an additional molecular mechanism in the DM1 pathogenesis, based on an altered phosphotyrosine signalling pathway.

Astaxanthin Improves Human Sperm Capacitation by Inducing Lyn Displacement and Activation

Astaxanthin (Asta), a photo-protective red pigment of the carotenoid family, is known for its multiple beneficial properties. In this study, the effects of Asta on isolated human sperm were evaluated. Capacitation involves a series of transformations to let sperm acquire the correct features for potential oocyte fertilization, including the generation of a controlled amount of reactive oxygen species (ROS), cholesterol depletion of the sperm outer membrane, and protein tyrosine phosphorylation (Tyr-P) process in the head region. Volunteers, with normal spermiogram values, were divided in two separate groups on the basis of their ability to generate the correct content of endogenous ROS. Both patient group (PG) and control group (CG) were analysed for Tyr-phosphorylation (Tyr-P) pattern and percentages of acrosome-reacted cells (ARC) and non-viable cells (NVC), in the presence or absence of Asta. In addition, the involvement of ROS on membrane reorganization and the presence of Lyn, a Src family kinase associated with lipid rafts, were investigated. Results show that Lyn is present in the membranes of human sperm, mainly confined in midpiece in resting conditions. Following capacitation, Lyn translocated to the head concomitantly with raft relocation, thus allowing the Tyr-P of head proteins. Asta succeeded to trigger Lyn translocation in PG sperm thus bypassing the impaired ROS-related mechanism for rafts and Lyn translocation. In this study, we showed an interdependence between ROS generation and lipid rafts and Lyn relocation leading the cells to undergo the successive acrosome reaction (AR). Asta, by ameliorating PG sperm functioning, may be utilised to decrease male idiopathic infertility.

Viral proteins and Src family kinases: Mechanisms of pathogenicity from a “liaison dangereuse”

To complete their life cycle and spread, viruses interfere with and gain control of diverse cellular processes, this most often occurring through interaction between viral proteins (VPs) and resident protein partners. Among the latter, Src family kinases (SFKs), a class of non-receptor tyrosine kinases that contributes to the conversion of extracellular signals into intracellular signaling cascades and is involved in virtually all cellular processes, have recently emerged as critical mediators between the cells infrastructure and the viral demands. In this scenario, structural or ex novo synthesized VPs are able to bind to the different domains of these enzymes through specific short linear motifs present along their sequences. Proline-rich motifs displaying the conserved minimal consensus PxxP and recognizing the SFK Src homology (SH)3 domain constitute a cardinal signature for the formation of multiprotein complexes and this interaction may promote phosphorylation of VPs by SFKs, thus creating phosphotyrosine motifs that become a docking site for the SH2 domains of SFKs or other SH2 domain-bearing signaling molecules. Importantly, the formation of these assemblies also results in a change in the activity and/or location of SFKs, and these events are critical in perturbing key signaling pathways so that viruses can utilize the cells machinery to their own benefit. In the light of these observations, although VPs as such, especially those with enzyme activity, are still regarded as valuable targets for therapeutic strategies, multiprotein complexes composed of viral and host cell proteins are increasingly becoming objects of investigation with a view to deeply characterize the structural aspects that favor their formation and to develop new compounds able to contrast viral diseases in an alternative manner.