Renate Hasenbank

Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo.

The mechanism by which an elongated polyglutamine sequence causes neurodegeneration in Huntingtons disease (HD) is unknown. In this study, we show that the proteolytic cleavage of a GST-huntingtin fusion protein leads to the formation of insoluble high molecular weight protein aggregates only when the polyglutamine expansion is in the pathogenic range. Electron micrographs of these aggregates revealed a fibrillar or ribbon-like morphology, reminiscent of scrapie prions and beta-amyloid fibrils in Alzheimers disease. Subcellular fractionation and ultrastructural techniques showed the in vivo presence of these structures in the brains of mice transgenic for the HD mutation. Our in vitro model will aid in an eventual understanding of the molecular pathology of HD and the development of preventative strategies.

SH3GL3 Associates with the Huntingtin Exon 1 Protein and Promotes the Formation of Polygln-Containing Protein Aggregates

The mechanism by which aggregated polygins cause the selective neurodegeneration in Huntingtons disease (HD) is unknown. Here, we show that the SH3GL3 protein, which is preferentially expressed in brain and testis, selectively interacts with the HD exon 1 protein (HDex1p) containing a glutamine repeat in the pathological range and promotes the formation of insoluble polyglutamine-containing aggregates in vivo. The C-terminal SH3 domain in SH3GL3 and the proline-rich region in HDex1p are essential for the interaction. Coimmunoprecipitations and immunofluorescence studies revealed that SH3GL3 and HDex1p colocalize in transfected COS cells. Additionally, an anti-SH3GL3 antibody was also able to coimmunoprecipitate the full-length huntingtin from an HD human brain extract. The characteristics of the interaction between SH3GL3 and huntingtin and the colocalization of the two proteins suggest that SH3GL3 could be involved in the selective neuronal cell death in HD.

Electrophoretic and immunological studies on ribosomal proteins of 100 Escherichia coli revertants from streptomycin dependence

SummaryRevertants from streptomycin dependence to independence were isolated as single step mutants from six different streptomycin dependent strains. The ribosomal proteins from 100 such mutants were analyzed by two-dimensional polyacrylamide gel electrophoresis and some of them were also examined by immunological techniques. Altered proteins were found in 40 mutants, 24 in protein S4 and 16 in protein S5. No change in any other protein was detected.Altered S5 proteins migrated into five different positions on the polyacrylamide plate and it can be concluded that the mutant proteins differ from the wild type probably by single amino acid replacements. The altered S4 proteins migrated into 17 different positions on the plate. Extensive changes of length, both shorter and longer than wild type S4 protein, are postulated for many of the mutant S4 proteins.Analysis of the ribosomal proteins of four ram mutants revealed altered S4 protein in two of them. The alterations in these mutant proteins are probably very similar to those found in streptomycin independent mutants.Among the revertants there was no apparent correlation between the protein alteration and the particular response to streptomycin.These studies suggest a strong interaction between protein S12, which confers streptomycin dependence, and protein S4 or S5, which can suppress this dependence.

The accessibility of proteins of the Escherichia coli 30S ribosomal subunit to antibody binding

SummaryThe accessibility of each of the proteins on the E. coli 30S ribosomal subunit was established by investigating whether or not immunoglobulins (IgGs) and their monovalent papain fragments (Fabs), specific for each of the 21 single ribosomal proteins, bind to the 30S subunit. The interpretation of the results of five different experimental approaches, namely Ouchterlony double diffusion and immunological “sandwich” methods, sucrose gradient and analytical ultracentrifugation, and functional inhibition tests, indicate that all 21 proteins of the 30S subunit have determinants available for antibody binding. There were quantitative differences between the degree of accessibility of the different ribosomal proteins. An attempt was made to correlate the results with the protein stoichiometric data of the small subunit proteins.

Immunological studies of Escherichia coli mutants lacking one or two ribosomal proteins

SummaryA battery of immunological tests were used to investigate mutants which had been determined as lacking one or two ribosomal proteins on the basis of two-dimensional polyacrylamide gels. Proteins which were confirmed as missing from the ribosome in one or more mutants were large subunit proteins L1, L15, L19, L24, L27, L28, L30 and L33 and small subunit proteins S1, S9, S17 and S20. Cross-reacting material (CRM) was also absent from the post-ribosomal supernatant except in the case of protein S1. Since mutants lacking protein L11 have been previously described, any one of 13 of the 52 ribosomal proteins can be missing. None of these 13 proteins, except S1, can therefore have an indispensable role in ribosome function or assembly. In several mutants in which a protein was not missing but altered, it was present as several moieties of differing charge and size.

Characterisation of a mutant from Escherichia coli lacking protein L15 and localisation of protein L15 by immuno-electron microscopy

SummaryTwo mutants lacking protein L15 from the ribosome as determined by two dimensional gels were investigated using a number of different immunological methods. One strain was found to possess several protein L15 moieties which differed in net charge and in size. The other showed no evidence of L15 cross-reacting material (CRM) on the ribosome or in the supernatant. Ribosomes of this strain were used as a control in the process of the localisation of protein L15 on the surface of the large subunit of Escherichia coli ribosomes. Antigenic determinants mapped in the angle between the central protuberance and the L1 protuberance. Protein L15 has been assigned a central role in the large subunit in vitro assembly map, in peptidyltransferase activity and in the binding of erythromycin, so the significance of a mutant lacking this protein is discussed.

Mutants of Escherichia coli lacking ribosomal protein L1

Two independently isolated mutants of Escherichia coli, RD19 and MV17-10, that appeared to lack protein L1 on their ribosomes, as determined by two-dimensional gels, were subjected to a battery of immunological tests to find if L1 was indeed lacking. The tests involved Ouchterlony double diffusion, modified immunoelectrophoresis, dimer formation on sucrose gradients, and affinity chromatography. By all these criteria, protein L1 was missing from the ribosome in these mutants. Nor was any L1 cross-reacting material detectable in the supernatant. There was, however, a specific two- to fivefold increase in concentrations of protein L11 in the supernatants of the mutants, which was evidence that protein L1 acts as a feedback inhibitor of expression of the operon coding for the genes for proteins L11 and L1. Electron micrographs of ribosomes obtained from these mutants were indistinguishable from those of wild-type strains. 50 S ribosomal subunits from mutants RD19 and MV17-10 were reconstituted with purified L1 from wild-type and investigated by immunoelectron microscopy. The three-dimensional location of ribosomal protein L1 on the surface of the large subunit was determined. L1 is located on the wider lateral protuberance of the 50 S subunit. The position of protein L1 in 50 S subunits reconstituted from mutants RD19 and MV17-10 was indistinguishable from the position in subunits from wild-type.

Inhibition of protein L7/L12 binding to 50 S ribosomal cores by antibodies specific for proteins L6, L10 and L18

50 S ribosomal core particles lacking proteins L7 and L12 were treated with monovalent antibody fragments specific for individual 50 S ribosomal proteins. Antibodies to proteins L6, L10 and L18 blocked the rebinding of proteins L7 and L12 and consequently the interaction of the ribosome with elongation factor G.

Inhibition of phenylalanyl-tRNA binding and elongation factor Tu-dependent GTP hydrolysis by antibodies specific for several ribosomal proteins

Antibodies specific for 30 of the 50 S ribosomal proteins have been tested for their ability to inhibit non-enzymic Phe-tRNA binding, elongation factor Tu-dependent Phe-tRNA binding and elongation factor Tu-dependent GTP hydrolysis. Antibodies to proteins L4, L7 and L12 specifically and co-ordinately inhibited all three activities. Antibodies to proteins L14 and L23 were also inhibitory, but this inhibition paralleled that previously reported for subunit association and elongation factor G-dependent GTP hydrolysis (Highland et al. , 1974) and presumably, therefore, did not directly reflect inhibition of the interaction of Phe-tRNA with the ribosome.

Effect of borrelidin on the threonyl-tRNA-synthetase activity and the regulation of threonine-biosynthetic enzymes in Saccharomyces cerivisiae

SummaryIn yeast cells grown in the presence of Borrelidin the threonyl-tRNA-synthetase is inhibited and a specific derepression of the aspartokinase takes place. These results are compatible with the assumption that in yeast cells the threonyl-tRNA-synthetase is involved in the repression of the formation of the threonine biosynthetic enzyme.