Correlation between the progressive cytoplasmic expression of a novel small heat shock protein (Hsp16.2) and malignancy in brain tumors

<p>Abstract</p> <p>Background</p> <p>Small heat shock proteins are molecular chaperones that protect proteins against stress-induced aggregation. They have also been found to have anti-apoptotic activity and to play a part in the development of tumors. Recently, we identified a new small heat shock protein, Hsp16.2 which displayed increased expression in neuroectodermal tumors. Our aim was to investigate the expression of Hsp16.2 in different types of brain tumors and to correlate its expression with the histological grade of the tumor.</p> <p>Methods</p> <p>Immunohistochemistry with a polyclonal antibody to Hsp16.2 was carried out on formalin-fixed, paraffin-wax-embedded sections using the streptavidin-biotin method. 91 samples were examined and their histological grade was defined. According to the intensity of Hsp16.2 immunoreactivity, low (+), moderate (++), high (+++) or none (-) scores were given.</p> <p>Immunoblotting was carried out on 30 samples of brain tumors using SDS-polyacrylamide gel electrophoresis and Western-blotting.</p> <p>Results</p> <p>Low grade (grades 1–2) brain tumors displayed low cytoplasmic Hsp16.2 immunoreactivity, grade 3 tumors showed moderate cytoplasmic staining, while high grade (grade 4) tumors exhibited intensive cytoplasmic Hsp16.2 staining. Immunoblotting supported the above mentioned results. Normal brain tissue acted as a negative control for the experiment, since the cytoplasm did not stain for Hsp16.2. There was a positive correlation between the level of Hsp16.2 expression and the level of anaplasia in different malignant tissue samples.</p> <p>Conclusion</p> <p>Hsp16.2 expression was directly correlated with the histological grade of brain tumors, therefore Hsp16.2 may have relevance as becoming a possible tumor marker.</p

The Lipidation Profile of Aquaporin-0 Correlates with The Acyl Composition of Phosphoethanolamine Lipids in Lens Membranes

The lens fiber major intrinsic protein (otherwise known as aquaporin-0 (AQP0), MIP26 and MP26) has been examined by mass spectrometry (MS) in order to determine the speciation of acyl modifications to the side chains of lysine residues and the N-terminal amino group. The speciation of acyl modifications to the side chain of one specific, highly conserved lysine residue (K238) and the N-terminal amino group of human and bovine AQP0 revealed, in decreasing order of abundance, oleoyl, palmitoyl, stearoyl, eicosenoyl, dihomo-γ-linolenoyl, palmitoleoyl and eicosadienoyl modifications. In the case of human AQP0, an arachidonoyl modification was also found at the N-terminus. The relative abundances of these modifications mirror the fatty acid composition of lens phosphatidylethanolamine lipids. This lipid class would be expected to be concentrated in the inner leaflet of the lens fiber membrane to which each of the potential AQP0 lipidation sites is proximal. Our data evidence a broad lipidation profile that is both species and site independent, suggesting a chemical-based ester aminolysis mechanism to explain such modifications

Independent Membrane Binding Properties of the Caspase Generated Fragments of the Beaded Filament Structural Protein 1 (BFSP1) Involves an Amphipathic Helix

Background: BFSP1 (beaded filament structural protein 1) is a plasma membrane, Aqua- 21 porin 0 (AQP0/MIP)-associated intermediate filament protein expressed in the eye lens. BFSP1 is 22 myristoylated, a post-translation modification that requires caspase cleavage at D433. Bioinformatic 23 analyses suggested that the sequences 434-452 were a-helical and amphipathic. Methods and Re- 24 sults: By CD spectroscopy, we show that the addition of trifluoroethanol induced a switch from an 25 intrinsically disordered to a more a-helical conformation for the residues 434-467. Recombinantly 26 produced BFSP1 fragments containing this amphipathic helix bind to lens lipid bilayers as deter- 27 mined by surface plasmon resonance (SPR). Lastly, we demonstrate by transient transfection of non- 28 lens MCF7 cells that these same BFSP1 C-terminal sequences localise to plasma membranes and to 29 cytoplasmic vesicles. These can be co-labelled with the vital dye, lysotracker, but other cell compart- 30 ments such as the nuclear and mitochondrial membranes were negative. The N-terminal myristoy- 31 lation of the amphipathic helix appeared not to change either the lipid affinity or membrane locali- 32 sation of the BFSP1 polypeptides or fragments we assessed by SPR and transient transfection, but it 33 did appear to enhance its helical content. Conclusions: These data support the conclusion that C- 34 terminal sequences of human BFSP1 distal to the caspase site at G433 have independent membrane 35 binding properties via an adjacent amphipathic helix

Independent Membrane Binding Properties of the Caspase Generated Fragments of the Beaded Filament Structural Protein 1 (BFSP1) Involves an Amphipathic Helix.

BACKGROUND: BFSP1 (beaded filament structural protein 1) is a plasma membrane, Aquaporin 0 (AQP0/MIP)-associated intermediate filament protein expressed in the eye lens. BFSP1 is myristoylated, a post-translation modification that requires caspase cleavage at D433. Bioinformatic analyses suggested that the sequences 434-452 were α-helical and amphipathic. METHODS AND RESULTS: By CD spectroscopy, we show that the addition of trifluoroethanol induced a switch from an intrinsically disordered to a more α-helical conformation for the residues 434-467. Recombinantly produced BFSP1 fragments containing this amphipathic helix bind to lens lipid bilayers as determined by surface plasmon resonance (SPR). Lastly, we demonstrate by transient transfection of non-lens MCF7 cells that these same BFSP1 C-terminal sequences localise to plasma membranes and to cytoplasmic vesicles. These can be co-labelled with the vital dye, lysotracker, but other cell compartments, such as the nuclear and mitochondrial membranes, were negative. The N-terminal myristoylation of the amphipathic helix appeared not to change either the lipid affinity or membrane localisation of the BFSP1 polypeptides or fragments we assessed by SPR and transient transfection, but it did appear to enhance its helical content. CONCLUSIONS: These data support the conclusion that C-terminal sequences of human BFSP1 distal to the caspase site at G433 have independent membrane binding properties via an adjacent amphipathic helix

An amphipathic helix facilitates the membrane binding properties of BFSP1 and its caspase-generated C-terminal domain

Background: BFSP1 (beaded filament structural protein 1) is a plasma membrane, Aqua- 21 porin 0 (AQP0/MIP)-associated intermediate filament protein expressed in the eye lens. BFSP1 is 22 myristoylated, a post-translation modification that requires caspase cleavage at D433. Bioinformatic 23 analyses suggested that the sequences 434-452 were a-helical and amphipathic. Methods and Re- 24 sults: By CD spectroscopy, we show that the addition of trifluoroethanol induced a switch from an 25 intrinsically disordered to a more a-helical conformation for the residues 434-467. Recombinantly 26 produced BFSP1 fragments containing this amphipathic helix bind to lens lipid bilayers as deter- 27 mined by surface plasmon resonance (SPR). Lastly, we demonstrate by transient transfection of non- 28 lens MCF7 cells that these same BFSP1 C-terminal sequences localise to plasma membranes and to 29 cytoplasmic vesicles. These can be co-labelled with the vital dye, lysotracker, but other cell compart- 30 ments such as the nuclear and mitochondrial membranes were negative. The N-terminal myristoy- 31 lation of the amphipathic helix appeared not to change either the lipid affinity or membrane locali- 32 sation of the BFSP1 polypeptides or fragments we assessed by SPR and transient transfection, but it 33 did appear to enhance its helical content. Conclusions: These data support the conclusion that C- 34 terminal sequences of human BFSP1 distal to the caspase site at G433 have independent membrane 35 binding properties via an adjacent amphipathic helix

PARP-1 Regulates Metastatic Melanoma through Modulation of Vimentin-induced Malignant Transformation

PARP inhibition can induce anti-neoplastic effects when used as monotherapy or in combination with chemo- or radiotherapy in various tumor settings; however, the basis for the anti-metastasic activities resulting from PARP inhibition remains unknown. PARP inhibitors may also act as modulators of tumor angiogenesis. Proteomic analysis of endothelial cells revealed that vimentin, an intermediary filament involved in angiogenesis and a specific hallmark of EndoMT (endothelial to mesenchymal transition) transformation, was down-regulated following loss of PARP-1 function in endothelial cells. VE-cadherin, an endothelial marker of vascular normalization, was up-regulated in HUVEC treated with PARP inhibitors or following PARP-1 silencing; vimentin over-expression was sufficient to drive to an EndoMT phenotype. In melanoma cells, PARP inhibition reduced pro-metastatic markers, including vasculogenic mimicry. We also demonstrated that vimentin expression was sufficient to induce increased mesenchymal/pro-metastasic phenotypic changes in melanoma cells, including ILK/GSK3-β-dependent E-cadherin down-regulation, Snail1 activation and increased cell motility and migration. In a murine model of metastatic melanoma, PARP inhibition counteracted the ability of melanoma cells to metastasize to the lung. These results suggest that inhibition of PARP interferes with key metastasis-promoting processes, leading to suppression of invasion and colonization of distal organs by aggressive metastatic cells.This work was supported by Ministerio de Ciencia e Innovación SAF2006-01094, SAF2009-13281-C02-01, Fundación La Caixa BM06-219-0 and Junta de Andalucía P07-CTS-0239 and CTS-6602 to FJO, Ministerio de Educación y Ciencia SAF2007-64597; CICYT: SAF2009-13281-C02-02; Junta de Andalucía, P06-CTS-01385 to JMRdA and grants CEIC (P10-CTS5865) and FEDER-ISCIII (PI10/00883) to JCR-M. AGdH has been funded by grants from “Fundación Científica de la Asociación Española Contra el Cáncer”, Ministerio de Ciencia y Tecnología SAF2010-16089, and “Fundación La Marató de TV3”. JCR-M has been funded by Grants CEIC (P1 = -CTS5865) and FEDER-ISCIII (PI10/00883)

PARP-1 Regulates Metastatic Melanoma through Modulation of Vimentin-induced Malignant Transformation

PARP inhibition can induce anti-neoplastic effects when used as monotherapy or in combination with chemo- or radiotherapy in various tumor settings; however, the basis for the anti-metastasic activities resulting from PARP inhibition remains unknown. PARP inhibitors may also act as modulators of tumor angiogenesis. Proteomic analysis of endothelial cells revealed that vimentin, an intermediary filament involved in angiogenesis and a specific hallmark of EndoMT (endothelial to mesenchymal transition) transformation, was down-regulated following loss of PARP-1 function in endothelial cells. VE-cadherin, an endothelial marker of vascular normalization, was up-regulated in HUVEC treated with PARP inhibitors or following PARP-1 silencing; vimentin over-expression was sufficient to drive to an EndoMT phenotype. In melanoma cells, PARP inhibition reduced pro-metastatic markers, including vasculogenic mimicry. We also demonstrated that vimentin expression was sufficient to induce increased mesenchymal/pro-metastasic phenotypic changes in melanoma cells, including ILK/GSK3-β-dependent E-cadherin down-regulation, Snail1 activation and increased cell motility and migration. In a murine model of metastatic melanoma, PARP inhibition counteracted the ability of melanoma cells to metastasize to the lung. These results suggest that inhibition of PARP interferes with key metastasis-promoting processes, leading to suppression of invasion and colonization of distal organs by aggressive metastatic cells.This work was supported by Ministerio de Ciencia e Innovación SAF2006-01094, SAF2009-13281-C02-01, Fundación La Caixa BM06-219-0 and Junta de Andalucía P07-CTS-0239 and CTS-6602 to FJO, Ministerio de Educación y Ciencia SAF2007-64597; CICYT: SAF2009-13281-C02-02; Junta de Andalucía, P06-CTS-01385 to JMRdA and grants CEIC (P10-CTS5865) and FEDER-ISCIII (PI10/00883) to JCR-M. AGdH has been funded by grants from “Fundación Científica de la Asociación Española Contra el Cáncer”, Ministerio de Ciencia y Tecnología SAF2010-16089, and “Fundación La Marató de TV3”. JCR-M has been funded by Grants CEIC (P1 = -CTS5865) and FEDER-ISCIII (PI10/00883)