Ochratoxin A-induced renal cortex fibrosis and epithelial-to-mesenchymal transition: molecular mechanisms of ochratoxin A-injury and potential effects of red wine

We characterized the effect of chronic ochratoxin A (OTA) on rat kidney cortex, analyzing collagen content and collagen turnover and the major markers of epithelial-to-mesenchymal transition (EMT), such as alpha-smooth muscle actin (alphaSMA), cadherins, and MMP-9. Because OTA nephrotoxicity is mediated by free radicals, we also investigated whether antioxidants in red wine provided protection for the kidney and attenuated OTA-induced EMT. Collagen content, determined by computerized analysis of Sirius red-stained kidney sections, increased in OTA, OTA-wine, and OTA-EtOH treated rats. In kidney cortex homogenates, COL-I and COL-III mRNA levels tended to rise in OTA treated rats, but were similar to CT after OTA-wine and OTA-EtOH administration. TIMP-1 gene expression was up-regulated in OTA, OTA-wine, and OTA-EtOH treated rats. LH2b mRNA/COL-I mRNA was significantly up-regulated in OTA-wine and OTA-EtOH treated rats, compared with CT and OTA alone. TGF-beta1 signaling tended to dominate after OTA, OTA-wine, and OTA-EtOH. MMP-1 protein levels were not affected. OTA induced proMMP-9 and alphaSMA overexpression, decreases of E-cadherin and N-cadherin, and DSC-2 up-regulation. OTA-wine caused a further, unexpected decrease of E- and N-cadherins and further up-regulation of OTA-induced DSC-2, while strongly reducing the OTA-induced increases of alphaSMA and proMMP-9. Posttranslational collagen modifications, such as decreased collagen degradation through MMP inhibition and increased collagen cross-links, seem to be key mechanisms leading to OTA-induced kidney cortex fibrosis. This mechanism was not affected by red wine in these conditions. Red wine seems to have some protective role against OTA-induced EMT, although without completely blocking the process and determining a condition in which abundant cells display an intermediate translational phenotype, but there are no alphaSMA or epithelial markers.     

Tailor-made alginate bearing galactose moieties on mannuronic residues: selective modification achieved by a chemoenzymatic strategy

1-Amino-1-deoxygalactose (12%, mole) has been chemically introduced on a mannuronan sample via an N-glycosidic bond involving the uronic group of the mannuronic acid (M) residues. The unsubstituted M residues in the modified polymer were converted into guluronic moieties (G) by the use of two C-5 epimerases, resulting in an alginate-like molecule selectively modified on M residues. The molecular details of the newly formed polymer, in terms of both composition and molecular dimensions, were disclosed by use of (1)H NMR, intrinsic viscosity, and high-performance size-exclusion chromatography-multiple-angle laser light scattering (HPSEC-MALLS). Circular dichroism has revealed that the modified alginate-like polymer obtained after epimerization was able to bind calcium due to the introduction of alternating and homopolymeric G sequences. The gel-forming ability of this M-selectively modified material was tested and compared with an alginate sample containing 14% galactose introduced on G residues. Mechanical spectroscopy pointed out that the modified epimerized material was able to form stable gels and that the kinetics of the gel formation was similar to that of the unsubstituted sample. In contrast, the G-modified alginate samples showed a slower gel formation, eventually leading to gel characterized by a reduced storage modulus. The advantage of the selective modification on M residues was confirmed by measuring the Young's modulus of gel cylinders of the different samples. Furthermore, due to the high content in alternating sequences, a marked syneresis was disclosed for the modified-epimerized sample. Finally, calcium beads obtained from selectively M-modified alginate showed a higher stability than those from the G-modified alginate, as evaluated upon treatment with nongelling ions.     

UreG, a chaperone in the urease assembly process, is an intrinsically unstructured GTPase that specifically binds Zn2+

Bacillus pasteurii UreG, a chaperone involved in the urease active site assembly, was overexpressed in Escherichia coli BL21(DE3) and purified to homogeneity. The identity of the recombinant protein was confirmed by SDS-PAGE, protein sequencing, and mass spectrometry. A combination of size exclusion chromatography and multiangle and dynamic laser light scattering established that BpUreG is present in solution as a dimer. Analysis of circular dichroism spectra indicated that the protein contains large portions of helices (15%) and strands (29%), whereas NMR spectroscopy indicated the presence of conformational fluxionality of the protein backbone in solution. BpUreG catalyzes the hydrolysis of GTP with a kcat=0.04 min(-1), confirming a role for this class of proteins in coupling energy requirements and nickel incorporation into the urease active site. BpUreG binds two Zn2+ ions per dimer, with a KD=42 +/- 3 microm, and has a 10-fold lower affinity for Ni2+. A structural model for BpUreG was calculated by using threading algorithms. The protein, in the fully folded state, features the typical structural architecture of GTPases, with an open beta-barrel surrounded by alpha-helices and a P-loop at the N terminus. The protein dynamic behavior observed in solution is critically discussed relative to the structural model, using algorithms for disorder predictions. The results suggest that UreG proteins belong to the class of intrinsically unstructured proteins that need the interaction with cofactors or other protein partners to perform their function. It is also proposed that metal ions such as Zn2+ could have important structural roles in the urease activation process.     

Differential requirement for phospholipase D/Spo14 and its novel interactor Sma1 for regulation of exocytotic vesicle fusion in yeast meiosis

During sporulation and meiosis of budding yeast a developmental program determines the formation of the new plasma membranes of the spores. This process of prospore membrane (PSM) formation leads to the formation of meiotic daughter cells, the spores, within the lumen of the mother cell. It is initiated at the spindle pole bodies during meiosis II. Spore formation, but not meiotic cell cycle progression, requires the function of phospholipase D (PLD/Spo14). Here we show that PLD/Spo14 forms a complex with Sma1, a meiotically expressed protein essential for spore formation. Detailed analysis revealed that both proteins are required for early steps of prospore membrane assembly but with distinct defects in the respective mutants. In the Deltaspo14 mutant the initiation of PSM formation is blocked and aggregated vesicles of homogenous size are detected at the spindle pole bodies. In contrast, initiation of PSM formation does occur in the Deltasma1 mutant, but the enlargement of the membrane is impaired. During PSM growth both Spo14 and Sma1 localize to the membrane, and localization of Spo14 is independent of Sma1. Biochemical analysis revealed that Sma1 is not necessary for PLD activity per se and that PLD present in a complex with Sma1 is highly active. Together, our results suggest that yeast PLD is involved in two distinct but essential steps during the regulated vesicle fusion necessary for the assembly of the membranous encapsulations of the spores.     

Extended molecular dynamics simulation of the carbon monoxide migration in sperm whale myoglobin

We report the results of an extended molecular dynamics simulation on the migration of photodissociated carbon monoxide in wild-type sperm whale myoglobin. Our results allow following one possible ligand migration dynamics from the distal pocket to the Xe1 cavity via a path involving the other xenon binding cavities and momentarily two additional packing defects along the pathway. Comparison with recent time resolved structural data obtained by Laue crystallography with subnanosecond to millisecond resolution shows a more than satisfactory agreement. In fact, according to time resolved crystallography, CO, after photolysis, can occupy the Xe1 and Xe4 cavities. However, no information on the trajectory of the ligand from the distal pocket to the Xe1 is available. Our results clearly show one possible path within the protein. In addition, although our data refer to a single trajectory, the local dynamics of the ligand in each cavity is sufficiently equilibrated to obtain local structural and thermodynamic information not accessible to crystallography. In particular, we show that the CO motion and the protein fluctuations are strictly correlated: free energy calculations of the migration between adjacent cavities show that the migration is not a simple diffusion but is kinetically or thermodynamically driven by the collective motions of the protein; conversely, the protein fluctuations are influenced by the ligand in such a way that the opening/closure of the passage between adjacent cavities is strictly correlated to the presence of CO in its proximity. The compatibility between time resolved crystallographic experiments and molecular dynamics simulations paves the way to a deeper understanding of the role of internal dynamics and packing defects in the control of ligand binding in heme proteins.     

Bcl-2 and Bax exert opposing effects on Ca2+ signaling, which do not depend on their putative pore-forming region

Recent work has shown that Bcl-2 and other anti-apoptotic proteins partially deplete the endoplasmic reticulum (ER) Ca(2+) store and that this alteration of Ca(2+) signaling reduces cellular sensitivity to apoptotic stimuli. We expressed in HeLa cells Bcl-2, Bax, and Bcl-2/Bax chimeras in which the putative pore-forming domains of the two proteins (alpha 5-alpha 6) were mutually swapped, comparing the effects on Ca(2+) signaling of the two proteins and relating them to defined molecular domains. The results showed that only Bcl-2 reduces ER Ca(2+) levels and that this effect does not depend on the alpha 5-alpha 6 helices of this oncoprotein. Soon after its expression, Bax increased ER Ca(2+) loading, with ensuing potentiation of mitochondrial Ca(2+) responses. Then the cells progressed into an apoptotic phenotype (which included drastic reductions of cytosolic and mitochondrial Ca(2+) responses and alterations of organelle morphology). These results provide a coherent scenario that high-lights a primary role of Ca(2+) signals in deciphering apoptotic stimuli.     

Chondrocyte protein with a poly-proline region (CHPPR) is a novel mitochondrial protein and promotes mitochondrial fission

We have recently identified a chondrocyte protein with a poly-proline region, referred to as CHPPR, and showed that this protein is expressed intracellularly in chick embryo chondrocytes. Conventional fluorescence and confocal localization of CHPPR shows that CHPPR is sorted to mitochondria. Furthermore, immunoelectron microscopy of CHPPR transfected cells demonstrates that this protein is mostly associated with the mitochondrial inner membranes. Careful analysis of CHPPR expressing cells reveals, instead of the regular mitochondrial tubular network, the presence of a number of small spheroid mitochondria. Here we show that the domain responsible for network-spheroid transition spans amino acid residues 182-309 including the poly-proline region. Functional analyses of mitochondrial activity rule out the possibility of mitochondrial damage in CHPPR transfected cells. Since cartilage expresses high levels of CHPPR mRNA when compared to other tissues and because CHPPR is associated with late stages of chondrocyte differentiation, we have investigated mitochondrial morphology in hypertrophic chondrocytes by MitoTracker Orange labeling. Confocal microscopy shows that these cells have spheroid mitochondria. Our data demonstrate that CHPPR is able to promote mitochondrial fission with a sequence specific mechanism suggesting that this event may be relevant to late stage of chondrocyte differentiation.     

Sorting of GFP Tagged NtSyr1, an ABA Related Syntaxin

Exocytosis molecular mechanisms in plant cells are not fully understood. The full characterization of molecular determinants, such as SNAREs, for the specificity in vesicles delivery to the plasma membrane should shed some light on these mechanisms. Nicotiana tabacum Syntaxin 1 (NtSyr1 or SYP121) is a SNARE protein required for ABA control of ion channels and appears involved in the exocytosis of exogenous markers.NtSyr1 is mainly localized on the plasma membrane, but when over expressed the protein also appears on endomembranes. Since NtSyr1 is a tail-anchored protein inserted into the target membrane post-translationally, it is not clear whether its initial anchoring site is the ER or the plasma membrane.In this study, we investigated the sorting events of NtSyr1 in vivo using its full-length cDNA or its C-terminal domain, fused to a GFP tag and transiently expressed in protoplasts or in the leaves of Nicotiana tabacum cv. SR1. Five chimeras were produced of which two were useful to investigate the protein sorting within the endomembrane system. One (GFP-H3M) had a dominant negative effect on exocytosis; the other one (SP1-GFP) resulted in a slow targeting to the same localization of the full-length chimera (GFP-SP1). The insertion of signal peptides on SP1-GFP further characterized the insertion site for this protein. Our data indicates that NtSyr1 is firstly anchored on ER membrane and then sorted to plasma membrane.Key Words: syntaxins, SNAREs, GFP tagging, exocytosis, secretion, protoplasts, dominant negative mutant