Elzbieta Wyroba

A novel potassium channel in skeletal muscle mitochondria

In this work we provide evidence for the potential presence of a potassium channel in skeletal muscle mitochondria. In isolated rat skeletal muscle mitochondria, Ca(2+) was able to depolarize the mitochondrial inner membrane and stimulate respiration in a strictly potassium-dependent manner. These potassium-specific effects of Ca(2+) were completely abolished by 200 nM charybdotoxin or 50 nM iberiotoxin, which are well-known inhibitors of large conductance, calcium-activated potassium channels (BK(Ca) channel). Furthermore, NS1619, a BK(Ca)-channel opener, mimicked the potassium-specific effects of calcium on respiration and mitochondrial membrane potential. In agreement with these functional data, light and electron microscopy, planar lipid bilayer reconstruction and immunological studies identified the BK(Ca) channel to be preferentially located in the inner mitochondrial membrane of rat skeletal muscle fibers. We propose that activation of mitochondrial K(+) transport by opening of the BK(Ca) channel may be important for myoprotection since the channel opener NS1619 protected the myoblast cell line C2C12 against oxidative injury.

Ca2+-dependent Translocation of the Calcyclin-binding Protein in Neurons and Neuroblastoma NB-2a Cells

The calcyclin-binding protein (CacyBP) binds calcyclin (S100A6) at physiological levels of [Ca2+] and is highly expressed in brain neurons. Subcellular localization of CacyBP was examined in neurons and neuroblastoma NB-2a cells at different [Ca2+] i . Immunostaining indicates that CacyBP is present in the cytoplasm of unstimulated cultured neurons in which resting [Ca2+] i is known to be ∼50 nm. When [Ca2+] i was increased to above 300 nm by KCl treatment, the immunostaining was mainly apparent as a ring around the nucleus. Such perinuclear localization of CacyBP was observed in untreated neuroblastoma NB-2a cells in which [Ca2+] i is ∼120 nm. An additional increase in [Ca2+] i to above 300 nm by thapsigargin treatment did not change CacyBP localization. However, when [Ca2+] i in NB-2a cells dropped to 70 nm, because of BAPTA/AM treatment, perinuclear localization was diminished. Ca2+-induced translocation of CacyBP was confirmed by immunogold electron microscopy and by fluorescence of NB-2a cells transfected with an EGFP-CacyBP vector. Recombinant CacyBP can be phosphorylated by protein kinase C in vitro. In untreated neuroblastoma NB-2a cells, CacyBP is phosphorylated on a serine residue(s), but exists in the dephosphorylated form in BAPTA/AM-treated cells. Thus, phosphorylation of CacyBP occurs in the same [Ca2+] i range that leads to its perinuclear translocation.

Ku80 as a Novel Receptor for Thymosin β4 That Mediates Its Intracellular Activity Different from G-actin Sequestering

Our data demonstrate that increased intracellular expression of thymosin β4(Tβ4) is necessary and sufficient to induce plasminogen activator inhibitor type 1 (PAI-1) gene expression in endothelial cells. To describe the mechanism of this effect, we produced Tβ4 mutants with impaired functional motifs and tested their intracellular location and activity. Cytoplasmic distributions of Tβ4(AcSDKPT/4A), Tβ4(KLKKTET/7A), and Tβ4(K16A) mutants fused with green fluorescent protein did not differ significantly from those of wild-type Tβ4. Overexpression of Tβ4, Tβ4(AcSDKPT/4A), and Tβ4(K16A) affected intracellular formation of actin filaments. As expected, Tβ4(K16A) uptake by nuclei was impaired. On the other hand, overexpression of Tβ4(KLKKTET/7A) resulted in developing the actin filament network typical of adhering cells, indicating that the mutant lacked the actin binding site. The mechanism by which intracellular Tβ4 induced the PAI-1 gene did not depend upon the N-terminal tetrapeptide AcSDKP and depended only partially on its ability to bind G-actin or enter the nucleus. Both Tβ4 and Tβ4(AcSDKPT/4A) induced the PAI-1 gene to the same extent, whereas mutants Tβ4(KLKKTET/7A) and Tβ4(K16A) retained about 60% of the original activity. By proteomic analysis, the Ku80 subunit of ATP-dependent DNA helicase II was found to be associated with Tβ4. Ku80 and Tβ4 consistently co-immunoprecipitated in a complex from endothelial cells. Co-transfection of endothelial cells with the Ku80 deletion mutants and Tβ4 showed that the C-terminal arm domain of Ku80 is directly involved in this interaction. Furthermore, down-regulation of Ku80 by specific short interference RNA resulted in dramatic reduction in PAI-1 expression at the level of both mRNA and protein synthesis. These data suggest that Ku80 functions as a novel receptor for Tβ4 and mediates its intracellular activity.

Phylogeny and evolution of Rab7 and Rab9 proteins

BackgroundAn important role in the evolution of intracellular trafficking machinery in eukaryotes played small GTPases belonging to the Rab family known as pivotal regulators of vesicle docking, fusion and transport. The Rab family is very diversified and divided into several specialized subfamilies. We focused on the VII functional group comprising Rab7 and Rab9, two related subfamilies, and analysed 210 sequences of these proteins. Rab7 regulates traffic from early to late endosomes and from late endosome to vacuole/lysosome, whereas Rab9 participates in transport from late endosomes to the trans-Golgi network.ResultsAlthough Rab7 and Rab9 proteins are quite small and show heterogeneous rates of substitution in different lineages, we found a phylogenetic signal and inferred evolutionary relationships between them. Rab7 proteins evolved before radiation of main eukaryotic supergroups while Rab9 GTPases diverged from Rab7 before split of choanoflagellates and metazoans. Additional duplication of Rab9 and Rab7 proteins resulting in several isoforms occurred in the early evolution of vertebrates and next in teleost fishes and tetrapods. Three Rab7 lineages emerged before divergence of monocots and eudicots and subsequent duplications of Rab7 genes occurred in particular angiosperm clades. Interestingly, several Rab7 copies were identified in some representatives of excavates, ciliates and amoebozoans. The presence of many Rab copies is correlated with significant differences in their expression level. The diversification of analysed Rab subfamilies is also manifested by non-conserved sequences and structural features, many of which are involved in the interaction with regulators and effectors. Individual sites discriminating different subgroups of Rab7 and Rab9 GTPases have been identified.ConclusionPhylogenetic reconstructions of Rab7 and Rab9 proteins were performed by a variety of methods. These Rab GTPases show diversification both at the phylogenetic, expression and structural levels. The presence of many Rab7 and Rab9 isoforms suggests their functional specialization and complexity of subcellular trafficking even in unicellular eukaryotes. The identified less conserved regions in analysed Rab sequences may directly contribute to such a differentiation.

Beta-adrenergic stimulation of phagocytosis in the unicellular eukaryote paramecium aurelia☆

Bete-adrenergic agonists isoproterenol and norepinephrine enhanced phagocytosis in Paramecium. Stimulation was stereospecific, dose-dependent and inhibited by the beta-agonists propranolol and alprenolol. Phorbol ester and forskolin potentiated the stimulatory effect of catecholamines on Paramecium phagocytosis. The dansyl analogue of propranolol (DAPN) was used for fluorescent visualization of the beta-adrenergic receptor sites in Paramecium which have been found to be localized at the cell membrane and within the membrane of the nascent digestive vacuoles. The appearance of the characteristic fluorescent pattern has been blocked by 1-propranolol.

Release of Paramecium immobilization antigen to the non-nutrient medium

The instability of Paramecium aurelia surface components has been shown: after 60 min incubation of dense cell suspension ((1-2 . 10(6) cells per ml) in Tris-Ca buffer at 4 degrees C or 23 degrees C the surface coat was partially stripped off and the proteins were released to the medium. The electrophoretic analysis of the released proteins shows one major band of mol. wt. 280,000--300,000 and some minor bands. The major released protein is the immobilization antigen as proved using immunodiffusion test and antibody precipitation technique.

Stimulation of Paramecium phagocytosis by phorbol ester and forskolin

Phorbol ester (PMA) exerted a dose- and time- dependent stimulating effect on phagocytosis in axenic Paramecium aurelia. When cells were exposed to 200-800 nM PMA in the presence of latex beads, the phagocytic coefficient was enhanced 2.25 to 3.14 times, during 10 min of continuous treatment and then rapidly declined. A similar effect was observed when the cells were exposed to a forskolin treatment, which resulted in nearly a twofold increase in phagocytic activity after a 10 min pulse. Both PMA and forskolin strongly stimulated phagocytosis (i.e. fivefold and threefold, respectively) in cells in which such activity had been completely inhibited by pre-exposure to the beta-receptor antagonist 1-propranolol.

Studies on the surface coat of Paramecium aurelia

SummaryCorrelations between the presence of surface coat and immobilization antigen of Paramecium tetraurelia were studied. Supravital, partial removal of the surface coat resulted in accelerated response of monobacterially and axenically grown cells to the homologous antiserum. Ciliates pretreated with trypsin or pronase (0.5mg/ml for 45 min at 0–4° C) were immobilized approximately twice as fast as untreated control cells. The probable localization of at least part, of the immobilization antigen within the surface coat of P. tetraurelia is discussed.

Association of Plasminogen Activator Inhibitor Type 2 (PAI-2) with Proteasome within Endothelial Cells Activated with Inflammatory Stimuli

Quiescent endothelial cells contain low concentrations of plasminogen activator inhibitor type 2 (PAI-2). However, its synthesis can be rapidly stimulated by a variety of inflammatory mediators. In this study, we provide evidence that PAI-2 interacts with proteasome and affects its activity in endothelial cells. To ensure that the PAI-2·proteasome complex is formed in vivo, both proteins were coimmunoprecipitated from endothelial cells and identified with specific antibodies. The specificity of this interaction was evidenced after (a) transfection of HeLa cells with pCMV-PAI-2 and coimmunoprecipitation of both proteins with anti-PAI-2 antibodies and (b) silencing of the PAI-2 gene using specific small interfering RNA (siRNA). Subsequently, cellular distribution of the PAI-2·proteasome complexes was established by immunogold staining and electron microscopy analyses. As judged by confocal microscopy, both proteins appeared in a diffuse cytosolic pattern, but they also could be found in a dense perinuclear and nuclear location. PAI-2 was not polyubiquitinated, suggesting that it bound to proteasome not as the substrate but rather as its inhibitor. Consistently, increased PAI-2 expression (a) abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-2 and pd2EGFP-N1, (b) prevented degradation of p53, as evidenced both by confocal microscopy and Western immunoblotting, and (c) inhibited proteasome cleavage of specific fluorogenic substrate. This suggests that PAI-2, in endothelial cells induced with inflammatory stimuli, can inhibit proteasome and thus tilt the balance favoring proapoptotic signaling.

Phagosome maturation in unicellular eukaryote Paramecium: the presence of RILP, Rab7 and LAMP-2 homologues

Phagosome maturation is a complex process enabling degradation of internalised particles. Our data obtained at the gene, protein and cellular level indicate that the set of components involved in this process and known up to now in mammalian cells is functioning in unicellular eukaryote. Rab7-interacting partners: homologues of its effector RILP (Rab-interacting lysosomal protein) and LAMP-2 (lysosomal membrane protein 2) as well as alpha7 subunit of the 26S proteasome were revealed in Paramecium phagolysosomal compartment. We identified the gene/transcript fragments encoding RILP-related proteins (RILP1 and RILP2) in Paramecium by PCR/RT-PCR and sequencing. The deduced amino acid sequences of RILP1 and RILP2 show 60.5% and 58.3% similarity, respectively, to the region involved in regulating of lysosomal morphology and dynein-dynactin recruitment of human RILP. RILP colocalised with Rab7 in Paramecium lysosomes and at phagolysosomal membrane during phagocytosis of both the latex beads and bacteria. In the same compartment LAMP-2 was present and its expression during latex internalisation was 2.5-fold higher than in the control when P2 protein fractions (100,000 x g) of equal load were quantified by immunoblotting. LAMP-2 cross-reacting polypeptide of approximately106 kDa was glycosylated as shown by fluorescent and Western analysis of the same blot preceded by PNGase F treatment. The alpha7 subunit of 26S proteasome was detected close to the phagosomal membrane in the small vesicles, in some of which it colocalised with Rab7. Immunoblotting confirmed presence of RILP-related polypeptide and a7 subunit of 26S proteasome in Paramecium protein fractions. These results suggest that Rab7, RILP and LAMP-2 may be involved in phagosome maturation in Paramecium.