Wilma Vree Egberts

Characterization of the hamster desmin gene: Expression and formation of desmin filaments in nonmuscle cells after gene transfer

The structural organization of the hamster gene encoding the intermediate filament (IF) protein desmin has been determined. The gene, 6.5 kb in length, contains nine exons with a total length of 2169 nucleotides. Remarkably, the intervening sequences map at positions that fully correspond to those of the vimentin gene. The derived complete primary structure for hamster desmin (468 amino acids; 53,250 daltons) reveals striking species variations in the NH2-terminal domain of desmin. A plasmid containing the complete transcription unit of the desmin gene was transfected into hamster lens cells and into human epithelial (HeLa) cells. In both nonmuscle cell lines the desmin gene was biologically active. The synthesized desmin assembled into authentic IFs, as monitored by immunofluorescence. Double immunofluorescence staining showed that the newly formed desmin filaments colocalize with preexisting vimentin filaments, but not with preexisting keratin filaments.

The Small Heat-Shock Proteins HSPB2 and HSPB3 Form Well-defined Heterooligomers in a Unique 3 to 1 Subunit Ratio

Various mammalian small heat-shock proteins (sHSPs) can interact with one another to form large polydisperse assemblies. In muscle cells, HSPB2/MKBP (myotonic dystrophy protein kinase-binding protein) and HSPB3 have been shown to form an independent complex. To date, the biochemical properties of this complex have not been thoroughly characterized. In this study, we show that recombinant HSPB2 and HSPB3 can be successfully purified from Escherichia coli cells co-expressing both proteins. Nanoelectrospray ionization mass spectrometry and sedimentation velocity analytical ultracentrifugation analysis showed that HSPB2/B3 forms a series of well defined hetero-oligomers, consisting of 4, 8, 12, 16, 20 and 24 subunits, each maintaining a strict 3:1 HSPB2/HSPB3 subunit ratio. These complexes are thermally stable up to 40 degrees C, as determined by far-UV circular dichroism spectroscopy. Surprisingly, HSPB2/B3 exerted a poor chaperone-like and thermoprotective activity, which is likely related to the low surface hydrophobicity, as revealed by its interaction with the hydrophobic probe 1-anilino-8-naphthalenesulfonic acid. Co-immunoprecipitation experiments demonstrated that the HSPB2/B3 oligomer cannot interact with HSP20, HSP27 or alphaB-crystallin, whereas the homomeric form of HSPB2, thus not in complex with HSPB3, could associate efficiently with HSP20. Taken altogether, this study provides evidence that, despite the high level of sequence homology within the sHSP family the biochemical properties of the HSPB2/B3 complex are distinctly different from those of other sHSPs, indicating that the HSPB2/B3 assembly is likely to possess cellular functions other than those of its family members.

Frequent occurrence of anti-tRNAHis autoantibodies that recognize a conformational epitope in sera of patients with myositis

OBJECTIVE To investigate the incidence of autoantibodies directed to deproteinized transfer RNA(His) (tRNA(His)) in anti-Jo-1 positive myositis patients and to determine the major B cell epitope. METHODS One hundred sixty-seven myositis sera were screened by immunoblotting and enzyme-linked immunosorbent assay for the presence of anti-Jo-1 antibody. Autoantibodies directed to deproteinized RNA were detected by immunoprecipitation. Ribonuclease cleavage experiments were performed to determine the tRNA(His)-specific features important for recognition. RESULTS Approximately one-third of the anti-Jo-1 positive sera also contained autoantibodies recognizing tRNA(His). This recognition was independent of modified bases, but the presence of stabilizing Mg2+ ions appeared to be essential for efficient immunoprecipitation. Transfer RNA(His)-specific features in the anticodon loop were not protected from ribonuclease cleavage by bound antibodies, while protection of bases located in the D and T loops was observed. CONCLUSION A significant number of anti-Jo-1 positive myositis sera contain anti-tRNA(His) activity. Formation of the major autoepitope on tRNA(His) is strongly dependent on proper folding of this molecule mediated by an interaction between D and T loops which is stabilized by either modified residues or Mg2+ ions.

Vimentin and Desmin cDNA Clones: Structural Aspects of Corresponding Proteins and Genes

Microtubules, microfilaments, and intermediate-sized filaments (IF) are the major cytoskeletal elements of most eukaryotic cells. The function of the IF is far from being fully understood. Maintenance of cell shape and/or anchorage to the substratum have been suggested to represent at least some of the functions of these fibrous structures.’ Identification and visualization of the spatial orientation in intact cells have been achieved by electron micro~copy~’~ and immunofluorescence microscopy.4~’ Mainly from the latter studies it appeared that as a rule IF proteins are tissue-specific. For instance mesenchymal cells comprise vimentin as subunit of their IF, whereas cells from an epithelial source contain cytokeratins. An exception of the aforementioned regularity is the vertebrate lens that due to its epithelial origin should produce prekeratin. Nevertheless, only vimentin is found and none of the other IF protein subunits! The reason for deviation from the “rule” is not clear yet. In view of the observation that cells from different embryonic origin start synthesizing vimentin as soon as they are subjected to culturing, it might well be that the eye lens, due to its peculiar growth pattern, arising from a monolayer of epithelial cells that are attached to a natural substratum (the lenticular capsule),’ resembles to some extent cells growing in culture (FIGURE 1). This example of regulation of IF gene expression led us to undertake investigations upon the molecular basis of this process. In order to facilitate comparative studies we performed molecular cloning of both vimentin and desmin cDNAs. The isolated clones served primarily a threefold aim: The determination via the nucleotide sequence of the primary structure of the proteins; the prediction of their secondary structure contained in a general model, presumably valid for all IF protein subunits; and the isolation of the corresponding genes from genomic DNA. Moreover, the isolated genes whose structures have been determined will be used for expression studies in heterologous cell systems.

Terminal Regions Confer Plasticity to the Tetrameric Assembly of Human HspB2 and HspB3

Heterogeneity in small heat shock proteins (sHsps) spans multiple spatiotemporal regimes—from fast fluctuations of part of the protein, to conformational variability of tertiary structure, plasticity of the interfaces, and polydispersity of the inter-converting, and co-assembling oligomers. This heterogeneity and dynamic nature of sHsps has significantly hindered their structural characterization. Atomic coordinates are particularly lacking for vertebrate sHsps, where most available structures are of extensively truncated homomers. sHsps play important roles in maintaining protein levels in the cell and therefore in organismal health and disease. HspB2 and HspB3 are vertebrate sHsps that are found co-assembled in neuromuscular cells, and variants thereof are associated with disease. Here, we present the structure of human HspB2/B3, which crystallized as a hetero-tetramer in a 3:1 ratio. In the HspB2/B3 tetramer, the four α-crystallin domains (ACDs) assemble into a flattened tetrahedron which is pierced by two non-intersecting approximate dyads. Assembly is mediated by flexible “nuts and bolts” involving IXI/V motifs from terminal regions filling ACD pockets. Parts of the N-terminal region bind in an unfolded conformation into the anti-parallel shared ACD dimer grooves. Tracts of the terminal regions are not resolved, most likely due to their disorder in the crystal lattice. This first structure of a full-length human sHsp heteromer reveals the heterogeneous interactions of the terminal regions and suggests a plasticity that is important for the cytoprotective functions of sHsps.