Molecular characterization and function analysis of MT-10 and MT-20 metallothionein isoforms from Mytilus galloprovincialis.

Structure and function of molluscan metallothioneins (MTs) are still poorly understood. The sea mussel Mytilus galloprovincialis displays two MT isoforms which differ in both primary sequences and physiological functions. MT-10 is the constitutive isoform, whereas MT-20 is mainly induced by cadmium (Cd). Both MTs were produced as recombinant proteins and showed identical Cd content and similar Cd-binding properties. Conversely, circular dichroism disclosed marked differences in the secondary conformations of the two Cd(7)-MTs. The possible relapses of these structural differences on protein stability and function were assessed. MT-10 presented a higher thermal stability and a more compact structure than MT-20, as it was inferred by absorption and emission spectroscopy studies. Moreover, the kinetics of Cd-release clearly indicated that MT-10 is much more sensitive to oxidation than is MT-20. The observed differences between MT-10 and MT-20 are discussed in terms of the different physiological roles exerted by the two isoforms in mussel.

Expression, purification and preliminary characterization of mussel (Mytilus galloprovincialis) metallothionein MT20.

Metallothioneins are rather ubiquitous metal-binding proteins induced by stressing or physiological stimuli. Two major metallothionein isoforms have been identified in mussel: MT10 and MT20. Nevertheless the high sequence homology, the two isoforms exhibit different expression and inducibility in vivo. We cloned and produced in Escherichia coli the MT20 isoform from Mytilus galloprovincilis. cDNA was subcloned into pGEX-6P.1 vector, in frame with a sequence encoding a glutathione-S-transferase (GST) tail. Recombinant protein was purified to electrophoretic homogeneity by affinity chromatography. After enzymatic cleavage of the GST tail the MT moiety was recovered with a final yield of about 5 mg of protein per litre of bacterial culture. The metal-binding ability of MT20 was assessed by absorption spectroscopy upon addition of cadmium equivalents and the metal release was checked as a function of the environment pH. Moreover the protein was analysed for the propensity to polymerization, typical of this class of protein, before and after exposure to reducing and alkylating agents.

Cyclooxygenase 2 expression in vessels and nerves in reversal reaction leprosy.

Tissue expression of cyclooxygenase (COX)2, an inducible enzyme synthesizing eicosanoids in inflammation, was studied in reversal reaction (RR) leprosy in comparison with nonreactionary leprosy. COX2 was consistently expressed in cells of the mononuclear-macrophage lineage across the leprosy spectrum. Only in RR, the following two additional sites showed COX2 expression in the dermis and subcutis: 1) microvessels and 2) nerve bundles and isolated nerve fibers. The same sites also express vascular endothelial growth factor (VEGF). This is in keeping with experimental models relating VEGF to COX2 expression, with VEGF enhancing prostaglandin production through COX2 stimulation and prostaglandin synthase expression. We postulate that selective COX2 inhibitors, which are currently used in several inflammatory conditions, could be considered for RR treatment to reduce acute symptoms caused by tissue edema and possibly prevent long-term nerve damage, the main complication of RR.

Modifications of nuclear architecture and chromatin organization in ataxia telangiectasia cells are coupled to changes of gene transcription.

Ataxia telangiectasia (AT) is a rare genetic disorder caused by mutations of ATM gene. ATM kinase is a "master controller" of DNA-damage response and signal transducer of external stimuli. The complex role of ATM may explain the pleiotropic phenotype characteristic of AT syndrome, only partially. In our hypothesis, the multi-faceted phenotype of AT patients might depend on specific chromatin reorganization, which then reflects on the cellular transcription. We analyzed three lymphoblastoid cell-lines isolated from AT patients and one healthy control. The three-dimensional reconstruction disclosed marked changes of nuclear morphology and architecture in AT cells. When chromatin condensation was analyzed by differential scanning calorimetry, a remodeling was observed at the level of fiber folding and nucleosome conformation. Despite the structural differences, chromatin did not exhibit modifications of the average acetylation status in comparison to the control. Moreover, AT cells presented significant alterations in the transcription of genes involved in cell-cycle regulation and stress response. In AT3RM cells, the average chromatin decondensation went with the upregulation of c-fos, c-jun, and c-myc and downregulation of metallothioneins, p21 and p53. AT9RM and AT44RM cells were instead characterized by an increased chromatin condensation and presented a different transcription unbalance. Whereas in AT44RM all the considered genes were downregulated, in AT3RM the three oncogenes and metallothioneins were upregulated, but p53 and p21 were downregulated.

Short report: Cyclooxygenase 2 expression in vessels and nerves in reversal reaction leprosy

Tissue expression of cyclooxygenase (COX)2, an inducible enzyme synthesizing eicosanoids in inflammation, was studied in reversal reaction (RR) leprosy in comparison with nonreactionary leprosy. COX2 was consistently expressed in cells of the mononuclear-macrophage lineage across the leprosy spectrum. Only in RR, the following two additional sites showed COX2 expression in the dermis and subcutis: 1) microvessels and 2) nerve bundles and isolated nerve fibers. The same sites also express vascular endothelial growth factor (VEGF). This is in keeping with experimental models relating VEGF to COX2 expression, with VEGF enhancing prostaglandin production through COX2 stimulation and prostaglandin synthase expression. We postulate that selective COX2 inhibitors, which are currently used in several inflammatory conditions, could be considered for RR treatment to reduce acute symptoms caused by tissue edema and possibly prevent long-term nerve damage, the main complication of RR.

Fish and molluscan metallothioneins.

Metallothioneins (MTs) are noncatalytic peptides involved in storage of essential ions, detoxification of nonessential metals, and scavenging of oxyradicals. They exhibit an unusual primary sequence and unique 3D arrangement. Whereas vertebrate MTs are characterized by the well-known dumbbell shape, with a beta domain that binds three bivalent metal ions and an alpha domain that binds four ions, molluscan MT structure is still poorly understood. For this reason we compared two MTs from aquatic organisms that differ markedly in primary structure: MT 10 from the invertebrate Mytilus galloprovincialis and MT A from Oncorhyncus mykiss. Both proteins were overexpressed in Escherichia coli as glutathione S-transferase fusion proteins, and the MT moiety was recovered after protease cleavage. The MTs were analyzed by gel electrophoresis and tested for their differential reactivity with alkylating and reducing agents. Although they show an identical cadmium content and a similar metal-binding ability, spectropolarimetric analysis disclosed significant differences in the Cd7-MT secondary conformation. These structural differences reflect the thermal stability and metal transport of the two proteins. When metal transfer from Cd7-MT to 4-(2-pyridylazo)resorcinol was measured, the mussel MT was more reactive than the fish protein. This confirms that the differences in the primary sequence of MT 10 give rise to peculiar secondary conformation, which in turn reflects its reactivity and stability. The functional differences between the two MTs are due to specific structural properties and may be related to the different lifestyles of the two organisms.

Modifications of chromatin structure and gene expression following induced alterations of cellular shape.

In higher eukaryotes cellular shape is a dynamic element which can be altered by external and internal factors (i.e. surface interactions, temperature, ionic strength). Our question was: might modifications of cell shape reflect on nuclear morphology and architecture and hence on chromatin function, in order to represent a mechanism of cell regulation? We altered the shape of cultured fibroblasts by coating the growth substratum with synthetic polymers, which alternatively increased and decreased the adhesiveness. By means of Fluorescence microscopy we analysed the modifications of cell and nucleus architecture induced by the different substrata. Then we used differential scanning calorimetry to investigate if a remodelling of chromatin structure was associated with the induced morphological changes. Finally, we evaluated if the observed modifications of chromatin condensation affect the transcriptional profile. At this stage of the work we focused on just four genes (c-myc, c-fos, c-jun and collagen) and we analysed their expression by dot blot hybridization and RT-PCR. The results confirm that mechanical factors external to the cell, such as the physico-chemical features of the substratum, are able to modulate gene transcription through a remodelling of chromatin structure. Therefore the work supports our starting hypothesis of a regulatory pathway connecting in sequence cellular morphomety/nuclear architecture/chromatin structure/gene expression.

Histone deacetylase inhibitors enhance retinoid response in human breast cancer cell lines.

Solid tumors develop resistance to retinoids during carcinogenesis. One of the strategies to overcome this resistance may include the combination of these molecules with other differentiating, cytotoxic or chromatin-remodelling agents. We analysed the anti-proliferative activity of two histone-deacetylase inhibitors (HDACIs), Trichostatin A (TSA) and sodium phenylbutyrate (PB), alone or combined with retinoids, all-trans retinoic acid (ATRA) and Ro 41-5253, on two human breast cancer cell lines: the hormone-dependent MCF-7 and the hormone-independent MDA-MB-231. These lines responded differently to retinoids: MCF-7 were sensitive, whilst MDA-MB-231 were rather resistant. When the retinoids were combined with HDACIs, these molecules potentiated the retinoid activity on growth inhibition, especially for the association Ro 41-5253 and TSA. By FACS analysis, we observed that the anti-proliferative effects were only partially due to pro-apopotic mechanisms, suggesting a cell-cycle block. The efficacy of the retinoids/HDACIs combinations could represent a new strategy in breast cancer chemotherapy, allowing inhibition of both ER + and ER- cell populations.

A structural characterization of in situ chromatin on cell lines isolated from patients affected by ataxia telangiectasia.

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by numerous clinical and cellular features. The pleiotropic nature of the AT syndrome attests to the multiple roles of ATM, the protein codified by the gene altered in AT patients. We investigated if different mutations of ATM could reflect on different alterations of nuclear architecture and chromatin organization. We selected three lymphoblastoid cell lines isolated from AT patients affected by different mutations of ATM gene and one healthy control. We characterized the in situ chromatin structure of each cell line by a biophysical approach: (1) we evaluated the rearrangements of the chromatin domains at the level of single cell by quantitative fluorescence microscopy; (2) we analysed the changes of the average chromatin condensation by differential scanning calorimetry. The results show that the three different ATM mutations produce significant modifications of both nuclear architecture and chromatin condensation.

Expression, purification, and characterization of metallothionein-A from rainbow trout.

Recombinant metallothionein A (MT-A) from rainbow trout has been successfully produced in milligram quantities in Escherichia coli. cDNA has been subcloned into pGEX-6P.1 vector, in-frame with a sequence encoding an N-terminal glutathione-S-transferase (GST) tail. Purification to electrophoretic homogeneity has been obtained by affinity chromatography using GSH-Sepharose. After enzymatic cleavage of GST tail, the MT-A moiety shows a molecular weight, corresponding to the expected one (6630 Da). The final yield of the entire expression and purification process was about 5 mg of pure metallothionein per liter of bacterial culture. The effects of different reducing and alkylating agents have been evaluated at the level of the formation of higher molecular weight aggregates. To investigate the metal-binding ability of the recombinant MT-A, we carried out a spectrophotometrical titration with cadmium ions. Finally, we checked the metal dissociation by recording the UV absorbance of the protein as a function of the environmental pH.