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P Hemmerich

Publications and source records attributed to P Hemmerich.

At least 19 recordsLinked to original sources
PubMed2006

Unexpected pathogenic mechanism of a novel mutation in the coding sequence of SPG4 (spastin).

The authors report a nucleotide substitution (c.1216A>G) in SPG4 (spastin) causing hereditary spastic paraplegia. This apparent missense mutation in the ATPase domain confers aberrant, in-frame splicing and results in destabilization of mutated transcript. Mutated protein is deficient in microtubule-severing activity but, unlike neighboring mutations, shows regular subcellular localization. The authors' data point to haploinsufficiency rather than a dominant negative effect as the disease-causing mechanism for this mutation.

Adenine↗
PubMed2001

Transcription activating property of autoantigen SG2NA and modulating effect of WD-40 repeats.

Autoantibodies to intracellular proteins have been detected in sera of patients with various forms of cancer. Nuclear autoantigen SG2NA (S, G2 phase nuclear antigen) was isolated using autoantibodies from a patient with bladder and lung cancers and its expression is enhanced in the S and G2 phases of the cell cycle. Molecular cloning revealed that the C-terminal region of SG2NA contains six WD-40 repeats, motifs that are present in a large family of proteins with diverse functions. We show that the N-terminal region of SG2NA (aa 1-391) acted as a strong transcriptional activator in both yeast and mammalian cells. In contrast, the C-terminal WD-40 repeats had an inhibitory effect on transcription activation. We performed molecular swapping experiments by substituting the WD-40 repeats of SG2NA with those of yeast Met30 and Cdc4 and showed that the WD-40 regions from either Met30 or Cdc4 were capable of reproducing transcription repression function. The SG2NA WD-40 repeats were also able to repress basal level transcription and transactivation function of a GAL4-VP16 chimera. These observations suggest that some WD-40 repeats may have, as one of their functions, a negative regulatory role in the biological activities of their own and perhaps other proteins.

Animals↗
PubMed2001

Determination of the binding constants of the centromere protein Cbf1 to all 16 centromere DNAs of Saccharomyces cerevisiae.

Cbf1p is a Saccharomyces cerevisiae chromatin protein belonging to the basic region helix-loop-helix leucine zipper (bHLHzip) family of DNA binding proteins. Cbf1p binds to a conserved element in the 5'-flanking region of methionine biosynthetic genes and to centromere DNA element I (CDEI) of S.cerevisiae centromeric DNA. We have determined the apparent equilibrium dissociation constants of Cbf1p binding to all 16 CDEI DNAs in gel retardation assays. Binding constants of full-length Cbf1p vary between 1.7 and 3.8 nM. However, the dissociation constants of a Cbf1p deletion variant that has been shown to be fully sufficient for Cbf1p function in vivo vary in a range between 3.2 and 12 nM. In addition, native polyacrylamide gel electrophoresis revealed distinct changes in the 3D structure of the Cbf1p/CEN complexes. We also show that the previously reported DNA binding stimulation activity of the centromere protein p64 functions on both the Cbf1 full-length protein and a deletion variant containing only the bHLHzip domain of Cbf1p. Our results suggest that centromeric DNA outside the consensus CDEI sequence and interaction of Cbf1p with adjacent centromere proteins contribute to the complex formation between Cbf1p and CEN DNA.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed2000

Interaction of yeast kinetochore proteins with centromere-protein/transcription factor Cbf1.

The centromere-kinetochore complex of Saccharomyces cerevisiae is a specialized chromosomal substructure that mediates attachment of duplicated chromosomes to the mitotic spindle by a regulated network of protein-DNA and protein-protein interactions. We have used in vitro assays to analyze putative molecular interactions between components of the yeast centromerekinetochore complex. Glutathione S-transferase pull-down experiments showed the direct interaction of in vitro translated p110, p64, and p58 of the essential CBF3 kinetochore protein complex with Cbf1p, a basic region helix-loop-helix zipper protein (bHLHzip) that specifically binds to the CDEI region on the centromere DNA. Furthermore, recombinant p64 and p23 each stimulated the in vitro DNA binding activity of Cbf1p. The N-terminal 70 amino acids of p23 were sufficient to mediate this effect. P64 could also promote the multimerization activity of Cbf1p in the presence of centromere DNA in vitro. These results show the direct physical interaction of Cbf1p and CBF3 subunits and provide evidence that CBF3 components can promote the binding of Cbf1p to its binding site in the yeast kinetochore. A functional comparison of the centromere binding proteins with transcription factors binding at MET16 promoters reveals the strong analogy between centromeres and the MET16 promoter.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed2000

CREB-binding protein (CBP)/p300 and RNA polymerase II colocalize in transcriptionally active domains in the nucleus.

The spatial organization of transcription- associated proteins is an important control mechanism of eukaryotic gene expression. Here we analyzed the nuclear distribution of the transcriptional coactivators CREB-binding protein (CBP)/p300 in situ by confocal laser scanning microscopy, and in vivo complex formation by coimmunoprecipitation. A subpopulation of CBP and p300 is targeted to active sites of transcription and partially colocalizes with hyper- and hypophosphorylated RNA polymerase II (pol II) in discrete regions of variable size throughout the nucleus. However, the coactivators were found in tight association with hypophosphorylated, but not hyperphosphorylated pol II. Transcriptional inhibition induced a relocation of CBP/p300 and pol II into speckles. Moreover, double and triple immunofluorescence analyses revealed the presence of CBP, p300, and pol II in a subset of promyelocytic leukemia (PML) bodies. Our results provide evidence for a dynamic spacial link between coactivators of transcription and the basal transcription machinery in discrete nuclear domains dependent upon the transcriptional activity of the cell. The identification of pol II in CBP/PML-containing nuclear bodies supports the idea that transcription takes place at PML bodies.

Acetyltransferases↗
PubMed2000

Antinuclear autoantibodies: fluorescent highlights on structure and function in the nucleus.

The eukaryotic nucleus is dynamically organized with respect to particular activities, such as RNA transcription, RNA processing or DNA replication. The spatial separation of metabolic activities is best reflected by the identification of functionally related proteins, in particular substructures of the nucleus. In a variety of human diseases, the integrity of such structures can be compromised, thus underlining the importance of a proper nuclear architecture for cell viability. Besides their clinical relevance, antinuclear autoantibodies (ANAs) have contributed to a large extent to the identification of subnuclear compartments, the isolation and cloning of their components (the autoantigens), as well a the characterization of their function. Although sophisticated techniques, such as confocal laser scanning microscopy (CLSM), fluorescence resonance energy transfer (FRET) and in vivo observation of cellular events have recently been established as valuable tools to study subnuclear architecture and function, cell biologists will continue to appreciate the specificity and power of ANAs for their research.

Animals↗
PubMed1999

Cloning of a centromere binding factor 3d (CBF3D) gene from Candida glabrata.

The gene encoding centromere binding factor 3d (CBF3D) of the human pathogenic yeast Candida glabrata has been isolated by hybridization of Saccharomyces cerevisiae CBF3D (ScCBF3D) DNA to a C. glabrata partial genomic library. Sequence analysis revealed a 540 bp open reading frame encoding a protein of 179 amino acids with a calculated molecular mass of 20.9 kDa. The amino acid sequence is highly homologous (78.6% identity) to ScCbf3d and 48.3% identical to the human homologue p19 (SKP1). Southern blot analysis indicates that CgCbf3d is encoded by an unique nuclear gene. The cloned CgCBF3D gene can functionally substitute the S. cerevisiae homologue in a S. cerevisiae CBF3D-deletion mutant. The GenBank Accession No. for this gene is AF 072472.

Amino Acid Sequence↗
PubMed1999

Human ribosomal protein L7 carries two nucleic acid-binding domains with distinct specificities.

Protein L7 is associated with the large subunit of eukaryotic ribosomes that can act as a co-regulator of nuclear receptor-mediated transcription. In this study we show that L7 carries in addition to the known N-terminal nucleic acid-binding domain (NBD 1) a second one (NBD 2) which maps to the 50 C-terminal amino acids of the protein. The amino acid sequence of this region does not contain any of the known nucleic acid binding motifs; thus, NBD 2 may represent a new class of nucleic acid-binding protein motifs. NBD 2 is conserved in all known eukaryotic L7 homologs, whereas NBD 1 is only present in mammalian L7. Binding studies show that NBD 2 is functionally different from NBD 1 in that it binds preferentially to 28S rRNA, suggesting that NBD 2 is involved in the attachment of protein L7 to the large ribosomal subunit. Potential functions of NBD 1 and NBD 2 in translational and nuclear receptor-mediated transcriptional control are discussed.

Amino Acid Sequence↗
PubMed1998

L7 protein is a coregulator of vitamin D receptor-retinoid X receptor-mediated transactivation.

The vitamin D receptor (VDR) heterodimerizes with the retinoid X receptor (RXR) and requires additional protein-protein interactions to regulate the expression of target genes. Using the yeast two-hybrid system, we identified the previously described protein L7, that specifically interacted with the VDR in the presence of vitamin D. Deletion analysis indicated, that the N-terminus of L7, which harbours a basic region leucine zipper like domain, mediated interaction with the VDR. Binding assays with purified GST-L7 demonstrated, that L7 specifically pulled down the VDR, that was either expressed in yeast or endogenously contained in the cell line U937. Interestingly, L7 inhibited ligand-dependent VDR-RXR heterodimerization, when constitutively expressed in yeast. We also demonstrate that L7 repressed binding of VDR-RXR heterodimers to a vitamin D response element. Surprisingly, L7 recruited RXR to the same response element in the presence of 9-cis retinoic acid. Ligand-dependent protein-protein interaction in the yeast two-hybrid system confirmed, that binding of L7 also was targeted at the RXR. Our data suggest, that protein L7 is a coregulator of VDR-RXR mediated transactivation of genes, that modulates transcriptional activity by interfering with binding of the receptors to genomic enhancer elements.

Base Sequence↗
PubMed1998

Correlation between chlamydial infection and autoimmune response: molecular mimicry between RNA polymerase major sigma subunit from Chlamydia trachomatis and human L7.

L7 is one of the ribosomal proteins frequently targeted by autoantibodies in rheumatic autoimmune diseases. A computer search revealed a region within the immunodominant epitope of L7 (peptide II) that is highly homologous to amino acid sequence 264-286 of the RNA polymerase major sigma factor of the eubacterium Chlamydia trachomatis. Anti-L7 autoantibodies affinity purified from the immunodominant epitope were able to recognize this sequence as they reacted with purified recombinant sigma factor. Immunofluorescence labeling experiments on C. trachomatis lysates revealed a punctate staining pattern of numerous spots when incubated with the affinity-purified anti-peptide II autoantibodies. Binding of autoantibodies to peptide II was inhibited by the homologous sigma peptide. This is the first demonstration of epitope mimicry between a human and a chlamydial protein on the level of B cells. Antibody screening revealed a significant correlation between the presence of anti-L7 autoantibodies and C. trachomatis infection in patients with systemic lupus erythematosus and mixed connective tissue disease. Our results suggest that molecular mimicry is involved in the initiation of anti-L7 autoantibody response and may represent a first glance into the immunopathology of Chlamydia with respect to systemic rheumatic diseases.

Amino Acid Sequence↗
PubMed1997

Human ribosomal protein L7 binds RNA with an alpha-helical arginine-rich and lysine-rich domain.

In this study we mapped the RNA-binding domain of human ribosomal protein L7 and characterized its conformation-dependent RNA-binding specificity. Binding competition assays demonstrated preferential binding of L7 to mRNAs and rRNA, but not to tRNA. The ribohomopolymer poly(G) is bound with high affinity whereas poly(U), poly(C), or poly(A) show low affinity to L7. Furthermore, L7 binds to double-stranded but not to single-stranded DNA. Deletion mapping showed that the RNA-binding domain of L7 is represented by an arginine-rich and lysine-rich oligopeptide (ELKIKRLRKKFAQKMLRKARRK), which is reminiscent of the arginine-rich motif (ARM) found in one family of RNA-binding proteins. The isolated RNA-binding domain is capable of high-affinity binding to the Rev-responsive element (RRE) of human immunodeficiency virus type 1 in vitro. Circular dichroic studies demonstrated a concentration-dependent and ligand-induced alpha-helical transition of a synthetic peptide carrying the arginine-lysine-rich RNA-binding domain of protein L7. Peptides carrying a mutation that destroys the alpha-helical conformation do not bind RNA.

Amino Acid Sequence↗
PubMed1995

Human ribosomal protein L7 inhibits cell-free translation in reticulocyte lysates and affects the expression of nuclear proteins upon stable transfection into Jurkat T-lymphoma cells.

Eucaryotic ribosomal protein L7 carries a 'Basic-Region-Leucine-Zipper (BZIP)'-like region which mediates high-affinity binding to mRNA and 28S rRNA and formation of homodimers [P. Hemmerich et al. (1993) Nucleic Acids Res. 21, 223-231). Its biological function is unknown as yet and no direct L7-equivalent in procaryotes has been found. In this report we show that eucaryotic L7 specifically inhibits the cell-free translation of reporter mRNAs. The interaction of L7 with mRNA is an essential step in this reaction which is inhibitable by antibodies directed against the BZIP-like region of L7, and by competitors of mRNA binding. L7-mediated inhibition of cell-free translation of polyA+ RNA from Jurkat T-lymphoma cells is selective in that the synthesis of a major 46 kD protein is suppressed. Upon stable transfection of L7 cDNA into Jurkat lymphoma cells two major proteins disappear, namely one nuclear protein and one which associates with the nucleus. Our data suggest a regulatory role of protein L7 in the eucaryotic translation apparatus.

Amino Acid Sequence↗
PubMed1994

Characterization of eukaryotic protein L7 as a novel autoantigen which frequently elicits an immune response in patients suffering from systemic autoimmune disease.

Autoantibodies targeted against cellular proteins and nucleic acids are a common feature of autoimmune diseases. In this study, we show that ribosomal protein L7 is a novel autoantigen in patients suffering from systemic lupus erythematosus (SLE) and other connective tissue diseases. From 24 patients diagnosed as having SLE, 18 produce antibodies which precipitate in vitro translated L7 protein. The anti-L7 titer appears to correlate with the active state of the disease. Anti-L7 autoantibodies were also detected in 7 of 13 patients with mixed connective tissue disease (MCTD), 2 of 7 patients with rheumatoid arthritis (RA), 1 of 4 patients with Sjögren's syndrome (SS) and in 1 patient with progressive systemic sclerosis (PSS). Anti-L7 autoantibodies belong to the IgG-class and detect specifically at least two epitopes on the L7 molecule, as shown by immunoprecipitation and immunoblotting. The epitope(s) of the highly conserved C-terminal region are preferentially recognized. Utilizing rabbit anti-L7 serum, autoimmune sera and affinity-purified anti-L7 autoantibodies in immunoblotting, and rabbit and chicken anti-L7 antibodies in indirect immunofluorescence, we detect L7 protein in the nuclei and in the cytoplasm of various cell-lines. Yet unlike most integral structural components of ribosomes, L7 is absent from nucleoli.

Adolescent↗
PubMed1993

Structural and functional properties of ribosomal protein L7 from humans and rodents.

By subtractive screening of a library made from mRNA of lipopolysaccharide (LPS)-stimulated mouse B lymphocytes we isolated cDNA-clones encoding the ribosomal protein L7. Human L7 mRNA was cloned from activated T-lymphocytes. Although no specific function of L7 in the translation apparatus is known as yet, it should be a critical one as indicated by its high degree of structural conservation during evolution and its regulated expression in lymphoid cells. Human and rodent L7 proteins carry sequences similar to the basic-region-leucine-zipper(BZIP)-motif of DNA-binding eucaryotic transcription factors. We show here that the region of L7 carrying the latter motif mediates L7-dimerization and stable binding to DNA and RNA. A preferential binding to RNA-structures is demonstrated.

Amino Acid Sequence↗
PubMed1984

Structure of a novel flavin chromophore from Avena coleoptiles, the possible 'blue light' photoreceptor.

A yellow chromophore has been isolated from Avena coleoptiles grown in the dark. It had previously been shown by Zenk [Zenk, M. H. (1967) Z. Pflanzenphysiol. 56, 122 - 140] to be a flavin of still unidentified structure, and had been suggested to be the 'blue light' photoreceptor in this organism. The structure of this flavin (F1-X) has been identified as 5'-malonylriboflavin by a combination of physicochemical techniques, and by its identity with a sample obtained synthetically. The 5'-malonylester linkage is relatively labile towards hydrolysis and photolysis, it isomerizes to an equilibrium mixture containing probably the 4' isomer. The electronic spectra of 5'-malonylriboflavin (absorption and fluorescence) are practically identical to those of normal riboflavin (vitamin B2).

Carrier Proteins↗
PubMed1983

Reaction of 2-thio-FAD-reconstituted p-hydroxybenzoate hydroxylase with hydrogen peroxide. Formation of a covalent flavin-protein linkage.

Hydrogen peroxide reacts with 2-thio-FAD-reconstituted p-hydroxybenzoate hydroxylase to yield a long wavelength intermediate (lambda max = 360, 620 nm) which can be isolated in stable form on removal of excess H2O2. The blue flavin derivative slowly decays in a second peroxide-dependent reaction to yield a new flavin product lacking long wavelength absorbance (lambda max = 408, 472 nm). This final peroxide-modified enzyme binds p-hydroxybenzoate with a 10-fold lower affinity than does the native enzyme; furthermore, substrate binding leads to the inhibition of enzyme reduction by NADPH. Trichloroacetic acid treatment of the final peroxide-modified enzyme results in the quantitative conversion of the bound flavin to free FAD. However, gel filtration of the modified enzyme in guanidine hydrochloride at neutral pH leads to the co-elution of protein and modified flavin. The nondenatured peroxide product reacts rapidly with hydroxylamine to yield 2-NHOH-substituted FAD. These observations indicate that the secondary reaction of peroxide with the blue intermediate from 2-thio-FAD p-hydroxybenzoate hydroxylase results in the formation of an acid-labile covalent flavin-protein linkage within the enzyme active site, involving the flavin C-2 position.

4-Hydroxybenzoate-3-Monooxygenase↗