Tyrosine phosphatase activity in mitochondria: presence of Shp-2 phosphatase in mitochondria

Tyrosine phosphorylation by unidentified enzymes has been observed in mitochondria, with recent evidence indicating that non-receptorial tyrosine kinases belonging to the Src family, which represent key players in several transduction pathways, are constitutively present in mitochondria. The extent of protein phosphorylation reflects a coordination balance between the activities of specific kinases and phophatases. The present study demonstrates that purified rat brain mitochondria possess endogenous tyrosine phosphatase activity. Mitochondrial phosphatases were found to be capable of dephosphorylating different exogenous substrates, including paranitrophenylphosphate, P-32-poly(Glu-Tyr)(4:1) and P-32-angiotensin. These activities are strongly inhibited by peroxovanadate, a well-known inhibitor of tyrosine phosphatases, but not by inhibitors of alkali or Ser/Thr phosphatases, and mainly take place in the intermembrane space and outer mitochondrial membrane. Using a combination of approaches, we identified the tyrosine phosphatase Shp-2 in mitochondria. Shp-2 plays a crucial role in a number of intracellular signalling cascades and is probably involved in several human diseases. It thus represents the first tyrosine phosphatase shown to be present in mitochondria

Biological activity of antitumoural MGBG: the structural variable

Abstract The present study aims at determining the structure-activity relationships (SAR’s) ruling the biological function of MGBG (methylglyoxal bis(guanylhydrazone)), a competitive inhibitor of S-adenosyl-l-methionine decarboxylase displaying anticancer activity, involved in the biosynthesis of the naturally occurring polyamines spermidine and spermine. In order to properly understand its biochemical activity, MGBG’s structural preferences at physiological conditions were ascertained, by quantum mechanical (DFT) calculations

Hepatoblastoma and microRNA-483 two forms and one outcome

Hepatoblastoma (HB) is the most common liver cancer in infants younger than 3 years. Its onset has been associated with other genetic syndromes and some genetic and biochemical markers has been identified recently in this neoplasia. Nevertheless the patients have a poor prognosis and the resection or transplantation remains the only effective therapeutic approach. The identification of non-invasive markers may represent an innovative approach and may contribute to a more accurate histological classification of this tumor. We previously demonstrated that some microRNAs are helpful in discriminating HB from hepatocellular carcinoma. In this study, we describe the involvement of the two forms of microRNA-483 (-3p and -5p) in a selected cohort of HB patients who underwent surgical resection or liver transplantation. Differently from other liver diseases we observed that the quantitative expression of the two forms did not significantly changed among patients. Furthermore, 3p/5p ratio was different between HB and non-HB samples, being positive in the latter and negative in HB samples. Influence of concomitant treatments in the expression of miR-483 (i.e. chemotherapy, and immunosuppressive drugs) was also evaluated and no changes were found in the follow-up. In conclusion the expression and function of miR-483-3p/5p in HB still remains unclear and further studies are needed to elucidate the possible mechanisms that regulate the different strand selection between the two forms of microRNA-483 in patients affected by HB. We deem that the analysis of microRNA-483 different forms could be useful for the molecular identification of HB patients and the discrimination with non-HB patient

REGULATION OF MEMBRANE BAND 3 TYR-PHOSPHORYLATION BY PROTEOLYSIS OF P72 AND POSSIBLE INVOLVEMENT IN SENESCENCE PROCESS

Erythrocyte senescence is characterized by exposure of cell surface epitopes on cell membrane proteins leading to immune mediated removal of red blood cells. One mechanism for antigen formation is tyrosine phosphorylation (Tyr-P) of the transmembrane protein band 3 by Syk kinase. Our aim was to test the hypothesis that proteolytic activation of Syk kinase by conversion from 72 kDa (p72Syk) to the 36 kDa (p36Syk) isoform enhances its phosphorylating activity independently of the association of Syk kinase with the cytoskeleton. Tyr-P assay was conducted using quantification of 32P uptake into the cytoplasmic domain of band 3 after addition of p72Syk or p36Syk. Effect of prephosphorylation of erythrocyte membrane band 3 protein by p36Syk on p72Syk-mediated phosphorylation and the effect of addition of a protease inhibitor (leupeptin) on p72Syk-mediated phosphorylation were studied by autoradiographic visualization of 32P uptake. Tyr-P by Syk isoforms of membrane skeletal and soluble fractions of band 3 was visualized by immunoblotting. It was found that p36Syk had a higher band 3 tyrosine phosphorylating activity compared with p72Syk. Pre-phosphorylation with p36Syk or p72Syk increased band 3 phosphorylating activity. Protease inhibition treatment reduced p72Syk but not p36Syk band 3 tyrosine phosphorylating activity significantly. Both soluble and membrane skeletal fractions of band 3 protein were equally tyrosine phosphorylated by each Syk isoform. In conclusion, we confirmed the hypothesis that proteolytic cleavage of p72Syk is an important regulatory step for band 3 Tyr-P and its independence of the association of band 3 with the cytoskeleton

Effect of peroxides on spermine transport in rat brain and liver mitochondria.

The polyamine spermine is transported into the matrix of various types of mitochondria by a specific uniporter system identified as a protein channel. This mechanism is regulated by the membrane potential; other regulatory effectors are unknown. This study analyzes the transport of spermine in the presence of peroxides in both isolated rat liver and brain mitochondria, in order to evaluate the involvement of the redox state in this mechanism, and to compare its effect in both types of mitochondria. In liver mitochondria peroxides are able to inhibit spermine transport. This effect is indicative of redox regulation by the transporter, probably due to the presence of critical thiol groups along the transport pathway, or in close association with it, with different accessibility for the peroxides and performing different functions. In brain mitochondria, peroxides have several effects, supporting the hypothesis of a different regulation of spermine transport. The fact that peroxovanadate can inhibit tyrosine phosphatases in brain mitochondria suggests that mitochondrial spermine transport is regulated by tyrosine phosphorylation in this organ. In this regard, the evaluation of spermine transport in the presence of Src inhibitors suggests the involvement of Src family kinases in this process. It is possible that phosphorylation sites for Src kinases are present in the channel pathway and have an inhibitory effect on spermine transport under regulation by Src kinases. The results of this study suggest that the activity of the spermine transporter probably depends on the redox and/or tyrosine phosphorylation state of mitochondria, and that its regulation may be different in distinct organs

The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin.

Uromodulin is the most abundant protein in the urine. It is exclusively produced by renal epithelial cells and it plays key roles in kidney function and disease. Uromodulin mainly exerts its function as an extracellular matrix whose assembly depends on a conserved, specific proteolytic cleavage leading to conformational activation of a Zona Pellucida (ZP) polymerisation domain. Through a comprehensive approach, including extensive characterisation of uromodulin processing in cellular models and in specific knock-out mice, we demonstrate that the membrane-bound serine protease hepsin is the enzyme responsible for the physiological cleavage of uromodulin. Our findings define a key aspect of uromodulin biology and identify the first in vivo substrate of hepsin. The identification of hepsin as the first protease involved in the release of a ZP domain protein is likely relevant for other members of this protein family, including several extracellular proteins, as egg coat proteins and inner ear tectorins

Olfactory neuroepithelium alterations and cognitive correlates in schizophrenia

BACKGROUND: Few studies have investigated alterations of olfactory neuroepithelium (ONE) as a biomarker of schizophrenia, and none its association with cognitive functioning. METHOD: Fresh ONE cells from twelve patients with schizophrenia and thirteen healthy controls were collected by nasal brushing, cultured in proper media and passed twelve times. Markers of cell proliferation (BrdU incorporation, Cyclin-D1 and p21 protein level) were quantified.Cognitive function was measured using Brief Neuropsychological Examination-2. PRIMARY OUTCOME: proliferation of ONE cells from schizophrenic patients at passage 3. Secondary outcome: association between alteration of cell proliferation and cognitive function. RESULTS: Fresh ONE cells from patients showed a faster cell proliferation than those from healthy controls at passage 3. An opposite trend was observed at passage 9, ONE cells of patients with schizophrenia showing slower cell proliferation as compared to healthy controls. In schizophrenia, overall cognitive function (Spearman's rho -0.657, p\u202f<\u202f0.01), verbal memory - immediate recall, with interference at 10\u202fs and 30\u202fs (Spearman's rho from -0.676 to 0.697, all p\u202f<\u202f0.01) were inversely associated with cell proliferation at passage 3. CONCLUSION: Fresh ONE cells collected by nasal brushing might eventually represent a tool for diagnosing schizophrenia based upon markers of cell proliferation, which can be easily implemented as single-layer culture. Cell proliferation at passage 3 can be regarded as a promising proxy of cognitive functioning in schizophrenia. Future studies should replicate these findings, and may assess whether ONE alterations are there before onset of psychosis, serving as an early sign in patients with at risk mental state

Tyrosine phosphorylation modulates peroxiredoxin-2 activity in normal and diseased red cells

: Peroxiredoxin-2 (Prx2) is the third most abundant cytoplasmic protein in red blood cells. Prx2 belongs to a well-known family of antioxidants, the peroxiredoxins (Prxs), that are widely expressed in mammalian cells. Prx2 is a typical, homodimeric, 2-Cys Prx that uses two cysteine residues to accomplish the task of detoxifying a vast range of organic peroxides, H2O2, and peroxynitrite. Although progress has been made on functional characterization of Prx2, much still remains to be investigated on Prx2 post-translational changes. Here, we first show that Prx2 is Tyrosine (Tyr) phosphorylated by Syk in red cells exposed to oxidation induced by diamide. We identified Tyr-193 in both recombinant Prx2 and native Prx2 from red cells as a specific target of Syk. Bioinformatic analysis suggests that phosphorylation of Tyr-193 allows Prx2 conformational change that is more favorable for its peroxidase activity. Indeed, Syk-induced Tyr phosphorylation of Prx2 enhances in vitro Prx2 activity, but also contributes to Prx2 translocation to the membrane of red cells exposed to diamide. The biologic importance of Tyr-193 phospho-Prx2 is further supported by data on red cells from a mouse model of humanized sickle cell disease (SCD). SCD is globally distributed, hereditary red cell disorder, characterized by severe red cell oxidation due to the pathologic sickle hemoglobin. SCD red cells show Tyr-phosphorylated Prx2 bound to the membrane and increased Prx2 activity when compared to healthy erythrocytes. Collectively, our data highlight the novel link between redox related signaling and Prx2 function in normal and diseased red cells

Wnt5a induces ROR1 to complex with HS1 to enhance migration of chronic lymphocytic leukemia cells.

ROR1 (receptor tyrosine kinase-like orphan receptor 1) is a conserved, oncoembryonic surface antigen expressed in chronic lymphocytic leukemia (CLL). We found that ROR1 associates with hematopoietic-lineage-cell-specific protein 1 (HS1) in freshly isolated CLL cells or in CLL cells cultured with exogenous Wnt5a. Wnt5a also induced HS1 tyrosine phosphorylation, recruitment of ARHGEF1, activation of RhoA and enhanced chemokine-directed migration; such effects could be inhibited by cirmtuzumab, a humanized anti-ROR1 mAb. We generated truncated forms of ROR1 and found its extracellular cysteine-rich domain or kringle domain was necessary for Wnt5a-induced HS1 phosphorylation. Moreover, the cytoplamic, and more specifically the proline-rich domain (PRD), of ROR1 was required for it to associate with HS1 and allow for F-actin polymerization in response to Wnt5a. Accordingly, we introduced single amino acid substitutions of proline (P) to alanine (A) in the ROR1 PRD at positions 784, 808, 826, 841 or 850 in potential SH3-binding motifs. In contrast to wild-type ROR1, or other ROR1P→︀A mutants, ROR1P(841)A had impaired capacity to recruit HS1 and ARHGEF1 to ROR1 in response to Wnt5a. Moreover, Wnt5a could not induce cells expressing ROR1P(841)A to phosphorylate HS1 or activate ARHGEF1, and was unable to enhance CLL-cell motility. Collectively, these studies indicate HS1 plays an important role in ROR1-dependent Wnt5a-enhanced chemokine-directed leukemia-cell migration