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Biomedical subjects

R Hasenbank

Publications and source records attributed to R Hasenbank.

18 recordsLinked to original sources
PubMed2001

The huntingtin interacting protein HIP1 is a clathrin and alpha-adaptin-binding protein involved in receptor-mediated endocytosis.

The huntingtin interacting protein (HIP1) is enriched in membrane-containing cell fractions and has been implicated in vesicle trafficking. It is a multidomain protein containing an N-terminal ENTH domain, a central coiled-coil forming region and a C-terminal actin-binding domain. In the present study we have identified three HIP1 associated proteins, clathrin heavy chain and alpha-adaptin A and C. In vitro binding studies revealed that the central coiled-coil domain is required for the interaction of HIP1 with clathrin, whereas DPF-like motifs located upstream to this domain are important for the binding of HIP1 to the C-terminal 'appendage' domain of alpha-adaptin A and C. Expression of full length HIP1 in mammalian cells resulted in a punctate cytoplasmic immunostaining characteristic of clathrin-coated vesicles. In contrast, when a truncated HIP1 protein containing both the DPF-like motifs and the coiled-coil domain was overexpressed, large perinuclear vesicle-like structures containing HIP1, huntingtin, clathrin and endocytosed transferrin were observed, indicating that HIP1 is an endocytic protein, the structural integrity of which is crucial for maintenance of normal vesicle size in vivo.

Adaptor Protein Complex alpha Subunits↗
PubMed1999

Self-assembly of polyglutamine-containing huntingtin fragments into amyloid-like fibrils: implications for Huntington's disease pathology.

Huntington's disease is a progressive neurodegenerative disorder caused by a polyglutamine [poly(Q)] repeat expansion in the first exon of the huntingtin protein. Previously, we showed that N-terminal huntingtin peptides with poly(Q) tracts in the pathological range (51-122 glutamines), but not with poly(Q) tracts in the normal range (20 and 30 glutamines), form high molecular weight protein aggregates with a fibrillar or ribbon-like morphology, reminiscent of scrapie prion rods and beta-amyloid fibrils in Alzheimer's disease. Here we report that the formation of amyloid-like huntingtin aggregates in vitro not only depends on poly(Q) repeat length but also critically depends on protein concentration and time. Furthermore, the in vitro aggregation of huntingtin can be seeded by preformed fibrils. Together, these results suggest that amyloid fibrillogenesis in Huntington's disease, like in Alzheimer's disease, is a nucleation-dependent polymerization.

Amino Acid Sequence↗
PubMed1998

Gridded genomic libraries of different chordate species: a reference library system for basic and comparative genetic studies of chordate genomes.

The use of genomic libraries maintained in arrayed format is becoming a more and more popular tool for the analysis of molecular evolution and comparative molecular development. Being able to use already existing reference libraries considerably reduces the work load, and if results are made publicly available, it will facilitate in silica experiments in the future. Here we describe the construction and preliminary characterization of six cosmid libraries of different chordate species, Ciona intestinalis (Hemichordate), Branchiostoma floridae (Cephalochordate), Lampetra fluviatilis (Cyclostoma), Xiphophorus maculatus, and Danio rerio (Osteichthyes) in Lawrist7 and Fugu rubripes in Lawrist4.

Animals↗
PubMed1998

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 Huntington's 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.

Adaptor Proteins, Signal Transducing↗
PubMed1997

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 Huntington's 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 Alzheimer's 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.

Amino Acid Sequence↗
PubMed1997

HIP-I: a huntingtin interacting protein isolated by the yeast two-hybrid system.

We report the discovery of the huntingtin interacting protein I (HIP-I) which binds specifically to the N-terminus of human huntingtin, both in the two-hybrid screen and in in vitro binding experiments. For the interaction in vivo, a protein region downstream of the polyglutamine stretch in huntingtin is essential. The HIP1 cDNA isolated by the two-hybrid screen encodes a 55 kDa fragment of a novel protein. Using an affinity-purified polyclonal antibody raised against recombinant HIP-I, a protein of 116 kDa was detected in brain extracts by Western blot analysis. The predicted amino acid sequence of the HIP-I fragment exhibits significant similarity to cytoskeleton proteins, suggesting that HIP-I and huntingtin play a functional role in the cell filament networks. The HIP1 gene is ubiquitously expressed in different brain regions at low level. HIP-I is enriched in human brain but can also be detected in other human tissues as well as in mouse brain. HIP-I and huntingtin behave almost identically during subcellular fractionation and both proteins are enriched in the membrane containing fractions.

Amino Acid Sequence↗
PubMed1983

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

Two 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.

Bacterial Proteins↗
PubMed1983

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

A 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.

Bacterial Proteins↗
PubMed1981

Expression of the L11-L1 operon in mutants of Escherichia coli lacking the ribosomal proteins L1 or L11.

Using a variety of immunological techniques, the supernatant levels of ribosomal proteins were measured in mutants lacking the ribosomal proteins L1 or L11, and in wild-type strains. There was a 2.5--5-fold elevation of protein L11 level in the supernatant of strains lacking protein L1, compared to wild-type. In contrast, there was no elevation, but rather a diminution, in the corresponding L1 level in strains lacking protein L11, compared to wild-type. These results are consistent with a model for the control of expression of the L11-L1 operon in which protein L1 is an inhibitor of expression of that operon, but protein L11 is not. The supernatant concentrations of other proteins were indistinguishable in all strains.

Bacterial Proteins↗
PubMed1975

Identification of a ribosomal protein necessary for thiostrepton binding to Escherichia coli ribosomes.

A ribosomal protein necessary for thiostrepton binding to Escherichia coli ribosomes has been identified using the following criteria: 1. A loss in the thiostrepton binding ability of the ribosome was correlated with the selective removal of ribosomal protein L11. This was achieved by a comparison of the thiostrepton binding ability of 50 S ribosomal subunits treated with 1 M NH4C1 and 50% ethanol at 37 degrees which still contained protein L11, and subunits treated successively at 0 degrees and 37 degrees in the same medium, from which protein L11 had been removed. 2. The thiostrepton binding ability of a ribosomal core containing only seven proteins, produced by treatment of 50 S subunits with 4 M LiC1, was fully restored by the rebinding of protein L11, obtained by Sephadex G-100 fractionation of the 1 M LiC1 split protein fraction from 50 S subunits. In addition, treatment of the 1 M LiC1 split protein fraction with an IgG specific for protein L11, uniquely inhibited the restoration of activity. 3. Thiostrepton binding to the 4 M LiC1 core, reconstituted with the 1 M LiC1 split protein fraction, was blocked by treatment with a monovalent antibody fragment (Fab) prepared against protein L11, but not by treatment with antibodies specific for the proteins of the 4 M LiC1 core. We conclude, therefore, that protein L11 is required for the ribosomal binding of thiostrepton.

Anti-Bacterial Agents↗
PubMed1974

Coordinate inhibition of elongation factor G function and ribosomal subunit association by antibodies to several ribosomal proteins.

We previously showed that treatment of Escherichia coli ribosomes with antibodies specific for proteins L7 and L12 inhibits both EF-G.GDP binding and ribosome-dependent GTP hydrolysis (Highland et al. (1973) Proc. Nat. Acad. Sci. USA 70, 142-150; and Kischa et al. (1971) Nature New Biol. 233, 62-63). We now report that antibodies to six additional proteins also inhibit GTP hydrolysis, but do not inhibit EF-G.GDP binding. Moreover, inhibition by these antibodies is dependent on the state of association of the treated ribosomes. When 70S couples are treated, only antibodies to proteins L14 and L23 are inhibitory and then only partially. However, when separated ribosomal subunits are treated individually and then mixed with the complementary untreated subunits, inhibition by antibodies L14 and L23 is complete, and antibodies to proteins L19, L27, S9, and S11 now show an inhibitory effect. In addition, treatment of subunits with any of these six antibodies (but not those to L7 or L12) results in inhibition of reassociation, which is presumably responsible for the inhibition of hydrolysis. These data suggest that the area of interaction between EF-G and the ribosome is restricted to proteins L7 and L12, and that antibodies to proteins L14, L19, L23, L27, S9, and S11, but not L7 and L12, block the physical association of the subunits.

Antibodies↗
PubMed1973

Identity of the ribosomal proteins involved in the interaction with elongation factor G.

Rabbit antibodies produced against 50 of the 55 individually purified ribosomal proteins of Escherichia coli were tested for their ability to interfere with the formation of the ribosome.EF-G.GDP complex. Only antibodies produced against proteins L7 and L12 inhibited complex formation, and they did so completely. These two proteins were previously shown to be immunologically indistinguishable and necessary for the interaction between ribosomes and EF-G. The present data are consistent with the view that the interaction between ribosomes and EF-G that results in GTP hydrolysis occurs on, and is limited to, proteins L7 and L12 on the surface of the 50S ribosomal subunit.

Animals↗