Biomedical subjects
Veli-Pekka Lehto
Publications and source records attributed to Veli-Pekka Lehto.
Crystallization and phasing of focal adhesion protein 52 from Gallus gallus.
Focal adhesion protein 52 (FAP52) is a multidomain adaptor protein of 448 amino acids characterized as an abundant component of focal adhesions. FAP52 binds to filamin via its N-terminal alpha-helical domain, suggesting a role in linking focal adhesions to the actin-based cytoskeleton. The recombinant protein was crystallized using the hanging-drop vapour-diffusion method, which yielded two crystal forms. Native data were collected from both crystal forms to 2.8 and 2.1 A resolution, respectively. For one of the crystal forms, initial MAD phasing was successfully performed using two data sets from xenon-derivatized crystals. The derivative data sets were collected using softer X-rays of 1.5 and 1.9 A wavelength. Preliminary structural analysis reveals the presence of a dimer in the asymmetric unit.
Laminin alpha1 chain in human renal cell carcinomas and integrin-mediated adhesion of renal cell carcinoma cells to human laminin isoforms.
In human tissues, the laminin (Ln) alpha1 chain shows a restricted and developmentally regulated distribution in basement membranes (BMs) of a subset of epithelial tissues, including those of renal proximal convoluted tubules. The present study investigated the distribution of the Ln alpha1 chain in renal cell carcinomas (RCCs) and oncocytomas as well as in xenografted tumours induced in nude mice with four characterized RCC cell lines. These cell lines were also used in cell adhesion studies with purified laminins. By immunohistochemistry it was found that the Ln alpha1 chain is widely present in the BMs of RCCs, all of the specimens presenting immunoreactivity. High-grade RCCs tended to contain more BM-confined and stromal immunoreactivity than low-grade tumours, none of the grade 3 (G3) carcinomas being negative and all of the metastatic specimens showing partial or overall BM immunoreactivity. Double immunolabelling experiments showed that in RCC BMs but not in vessel walls, the Ln alpha1 chain was co-distributed with Ln alpha5, beta1, and beta2 chains, implying the presence of Ln-1/Ln-3 and Ln-10/Ln-11. In papillary RCCs, the Ln alpha1 chain co-localized with Ln-5. The oncocytomas lacked immunoreactivity for the Ln alpha1 chain. Xenografted tumours induced in nude mice showed BM-like deposition of the Ln alpha1 chain. In cell adhesion studies, mouse and human Ln-1 were equally effective in promoting cell adhesion of all RCC cell lines. For each cell line, Ln-10 and Ln-10/11 were equally effective adhesive substrates, all cell lines adhering more avidly to these laminins than to mouse or human Ln-1. As judged by inhibition assays employing specific integrin antibodies, adhesion of normal human renal proximal tubular epithelial (RPTE) cells and RCC cells from a G1 tumour to human Ln-1 was mediated mainly by alpha(6)beta(1) integrin, while only the G1 RCC cells adhered to mouse Ln-1 by using alpha(6)beta(1) integrin. For adhesion to Ln-10, RPTE cells and RCC cells from a G1 tumour used an unidentified beta(1) integrin. Cells from G3 tumours mainly used an alpha(3)beta(1) integrin complex for adhesion to mouse Ln-1 and to human Ln-1 and Ln-10. For all cells, adhesion to the Ln-10/11 mixture was mediated by an unidentified integrin complex or by other adhesion molecules. These results show that laminin trimers containing the alpha1 chain are, in contrast to oncocytomas, abundant in the BMs of RCCs. This is in keeping with their suggested origin from renal proximal tubular epithelium known for its capacity to produce the Ln alpha1 chain. The results also show that RCC cells utilize complex, mainly integrin alpha(3)beta(1)- and integrin alpha(6)beta(1)-mediated, mechanisms for adhesion to laminins. The adhesion to Ln-1 changes from integrin alpha(6)beta(1) to integrin alpha(3)beta(1) upon increasing malignancy and, especially for Ln-10 and Ln-10/11, other adhesion molecules of non-integrin type may contribute to the adhesion.
[Is autopsy a relevant procedure in modern times?].
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Focal adhesion protein FAP52 self-associates through a sequence conserved among the members of the PCH family proteins.
FAP52 is a recently described focal adhesion-associated protein. It is a member of an emerging PCH (pombe Cdc15 homology) family of proteins characterized by a common domain organization and involvement in actin cytoskeleton organization, cytokinesis, and vesicular trafficking. Using gel filtration, surface plasmon resonance, and native polyacrylamide gel electrophoresis analysis, combined with chemical cross-linking of both native and recombinant protein, we show that FAP52 self-associates in vitro and suggest that it occurs predominantly as a trimer also in vivo. Analysis of the various domains of FAP52 by surface plasmon resonance showed that the highly alpha-helical region in the N-terminal half of the protein provides the self-association interface. Overexpression of the oligomerization domain in cultured cells was accompanied by major alterations in cellular morphology, actin organization, and the structure of focal adhesions, suggesting that an orderly coming together of FAP52 molecules is crucial for a proper actin filament organization and cytoskeletal structure. Comparison of the primary structures shows that all of the members of the PCH family have, in their N-terminal halves, a similar, highly alpha-helical region, suggesting that they all have a capacity to self-associate.
FAP52 regulates actin organization via binding to filamin.
FAP52, a focal adhesion-associated phosphoprotein, is a member of a FAP52/PACSIN/syndapin family of proteins. They share a multidomain structure and are implicated in actin-based and endocytotic functions. We show, by using both native and recombinant proteins, that FAP52 selectively binds to the actin cross-linking protein filamin (ABP-280). This was based on an affinity purification followed by a sequence determination by mass spectrometry, co-immunoprecipitation, overlay binding, and surface plasmon resonance analysis. Binding studies with deletion mutants showed that the sites of the interaction map to the highly alpha-helical N-terminal part of FAP52 and to the C-terminal region of filamin, which also contains binding sites to some transmembrane signaling proteins. In immunofluorescence and immunoelectron microscopy of cultured fibroblasts, a different overall subcellular distribution was seen for filamin and FAP52 except for a stress fiber-focal adhesion junction where they showed a notable overlap. Overexpression of the full-length and mutant forms of FAP52 led to an extensive reorganization of actin and filamin in cultured fibroblasts. Thus, the results show that FAP52 interacts with filamin, and we propose that this interaction is important in linking and coordinating the events between focal adhesions and the actin cytoskeleton.