Biomedical subjects

G Multhaup

Publications and source records attributed to G Multhaup.

At least 91 records · Page 5Linked to original sources

PubMed1990

The Drosophila PROS-28.1 gene is a member of the proteasome gene family.

In the present communication, we report the identification of a new gene family which encodes the protein subunits of the proteasome. The proteasome is a high-Mr complex possessing proteolytic activity. Screening a Drosophila lambda gt11 cDNA expression library with the proteasome-specific antibody N19-28 we isolated a clone encoding the 28-kDa No. 1 proteasome protein subunit. In accordance with the nomenclature of proteasome subunits in Drosophila, the corresponding gene is designated PROS-28.1, and it encodes an mRNA of 1.1 kb with an open reading frame of 249 amino acids (aa). Genomic Southern-blot hybridization shows PROS-28.1 to be a member of a family of related genes. Analysis of the predicted aa sequence reveals a potential nuclear targeting signal, a potential site for tyrosine kinase and a potential cAMP/cGMP-dependent phosphorylation site. The aa sequence comparison of the products of PROS-28.1 and PROS-35 with the C2 proteasome subunit of rat shows a strong sequence similarity between the different proteasome subunits. The data suggest that at least a subset of the proteasome-encoding genes belongs to a family of related genes (PROS gene family) which may have evolved from a common ancestral PROS gene.

Amino Acid Sequence↗

PubMed1990

Chicken NFI/TGGCA proteins are encoded by at least three independent genes: NFI-A, NFI-B and NFI-C with homologues in mammalian genomes.

Chicken TGGCA proteins belong to the ubiquitous, eukaryotic family of NFI-like nuclear proteins, which share an identical DNA binding specificity. They are involved in viral and cellular aspects of transcriptional regulation and they are capable of stimulating Adenovirus initiation of replication. Using microsequencing data from peptides of isolated proteins and PCR supported cloning, we have derived four cDNAs for NFI/TGGCA proteins, which are encoded by three separate chicken genes. Sequence alignments of NFI proteins from chicken and various mammalian species provide evidence for a common genetic equipment among higher eukaryotes, in which several related genes, employing each differential RNA splicing generate an unexpectedly large family of diverse NFI proteins. The extensive similarity of the amino acid sequence throughout the complete coding regions between products of the same gene type in different species indicates a uniform selection pressure on all protein parts, also on those outside the DNA-binding domain.

Amino Acid Sequence↗

PubMed1990

GABAA-receptor expressed from rat brain alpha- and beta-subunit cDNAs displays potentiation by benzodiazepine receptor ligands.

In mammalian brain, the activation of GABAA-receptors is associated with the opening of chloride channels, whose function can be allosterically modulated by drugs, in particular by ligands of the benzodiazepine receptor. Agonistic ligands potentiate while inverse agonists reduce the efficiency of GABA. We have cloned cDNAs encoding alpha 1- and beta 1-subunits of the GABAA-receptor from rat brain. When the corresponding RNAs were co-expressed in Xenopus oocytes. GABA-induced currents were recorded which were inhibited by bicuculline and potentiated by pentobarbital. GABA activated the channel in a weakly cooperative manner. Furthermore, the GABA-response was modulated by benzodiazepine receptor ligands. However, not only various agonists but also the antagonist flumazenil and the inverse agonist DMCM potentiated the GABA-response. Thus, alpha 1- and beta 1-subunits are sufficient to form GABAA-receptors which contain benzodiazepine binding sites, although in a functionally restricted form.

Amino Acid Sequence↗

PubMed1990

Cloning and expression of the 58 kd beta subunit of the inhibitory glycine receptor.

The inhibitory glycine receptor (GlyR) mediates post-synaptic inhibition in spinal cord and other regions of the CNS. Purified mammalian GlyR contains two membrane-spanning subunits 48 kd (alpha) and 58 kd (beta) plus a 93 kd receptor-associated cytoplasmic protein. Here, the primary structure of the beta subunit was deduced from cDNAs isolated from rat spinal cord and brain cDNA libraries. The predicted amino acid sequence exhibits 47% identity to the previously characterized rat alpha 1 polypeptide. Northern blot analysis revealed high levels of beta subunit transcripts in postnatal spinal cord, cerebellum, and cortex. Nuclear injection into Xenopus oocytes of a beta subunit cDNA engineered for efficient expression generated weak glycine-activated chloride currents that were insensitive to the classic GlyR antagonist, strychnine. Our data indicate a differential expression of GlyR alpha and beta subunits in the rat nervous system and support a structural role of the beta polypeptide in the native receptor complex.

Amino Acid Sequence↗

PubMed1990

Foot-and-mouth disease virus protease 3C induces specific proteolytic cleavage of host cell histone H3.

In foot-and-mouth disease virus (FMDV)-infected cells, the disappearance of nuclear protein histone H3 and the simultaneous appearance of a new chromatin-associated protein termed Pi can be observed (P. R. Grigera and S. G. Tisminetzky, Virology 136:10-19, 1984). We sequenced the amino terminus of protein Pi and showed that Pi derives from histone H3 by proteolytic cleavage. The 20 N-terminal amino acid residues of histone H3 are specifically cleaved off early during infection. Truncated histone H3 remains chromatin associated. In addition, we showed that the histone H3-Pi transition is catalyzed by the FMDV 3C protease. The only known function of the viral 3C protease was, until now, the processing of the viral polyprotein. The viral 3C protease is the only FMDV protein required to induce the histone H3-Pi transition, as well as being the only viral protein capable of cleaving histone H3. No viral precursor fusion protein is needed for this specific cleavage as was reported for the processing of the poliovirus P1 precursor polyprotein by 3C/D protease. As the deleted part of the histone H3 corresponds to the presumed regulatory domain involved in the regulation of transcriptionally active chromatin in eucaryotes, it seems possible that this specific cleavage of histone H3 is related to the host cell transcription shutoff reported for several picornaviruses.

Amino Acid Sequence↗

PubMed1989

Amyloid A4 protein and its precursor in Down's syndrome and Alzheimer's disease.

In patients with Alzheimer's disease, amyloid fibrils that are aggregates of A4 protein subunits are deposited in the brain. A similar process occurs at an earlier age in persons with Down's syndrome. To investigate the deposition of amyloid in these diseases, we used a radioimmunoassay to measure levels of the amyloid precursor (PreA4) in the serum of 17 patients with Down's syndrome, 15 patients with Alzheimer's disease, and 33 normal elderly controls. The mean (+/- SD) concentration of serum PreA4 was increased 1.5-fold in patients with Down's syndrome (2.49 +/- 1.13 nmol per liter) as compared with that in controls (1.68 +/- 0.49 nmol per liter; P less than 0.007); the levels in patients with Alzheimer's disease were similar to those in controls (1.83 +/- 0.78; P less than 0.98). We also found that the concentration of PreA4 in the brain tissue of two adults with Down's syndrome (100 and 190 pmol per gram) was higher than that in the brain tissue of either 26 patients with Alzheimer's disease (64.4 +/- 17.3 pmol per gram) or 17 elderly controls with neurologic disease (68.5 +/- 26.3 pmol per gram). Immunocytochemical studies of brain tissue from 26 patients with Down's syndrome showed that the deposition of A4 protein amyloid began in these patients approximately 50 years earlier than it began in 127 normal aging subjects studied previously, although the rate of deposition was the same. We conclude that, since the gene for PreA4 is on the long arm of chromosome 21, which is present in triplicate in Down's syndrome, overexpression of this gene may lead to increased levels of PreA4 and amyloid deposition in Down's syndrome. However, since increased levels of PreA4 are not present in Alzheimer's disease, additional factors must account for the amyloid deposition in that disorder.

Adolescent↗

PubMed1989

The PreA4(695) precursor protein of Alzheimer's disease A4 amyloid is encoded by 16 exons.

Alzheimer's disease (AD) is characterized by the cerebral deposition of fibrillar aggregates of the amyloid A4 protein. Complementary DNA's coding for the precursor of the amyloid A4 protein have been described. In order to identify the structure of the precursor gene relevant clones from several human genomic libraries were isolated. Sequence analysis of the various clones revealed 16 exons to encode the 695 residue precursor protein (PreA4(695] of Alzheimer's disease amyloid A4 protein. The DNA sequence coding for the amyloid A4 protein is interrupted by an intron. This finding supports the idea that amyloid A4 protein arises by incomplete proteolysis of a larger precursor, and not by aberrant splicing.

Alzheimer Disease↗

PubMed1989

The PROS-35 gene encodes the 35 kd protein subunit of Drosophila melanogaster proteasome.

The proteasome is a multicatalytic proteinase complex composed of nonidentical protein subunits. We have isolated a cDNA clone encoding the 35 kd proteasome subunit of Drosophila melanogaster and propose the designation PROS-35 for the corresponding gene. The deduced amino acid sequence reveals a region of striking homology to a tyrosine phosphorylation site of viral and cellular proteins suggesting a potential regulatory function for the 35 kd subunit within the proteinase complex. Immunocytochemical experiments reveal a tissue-dependent differential distribution of the proteasome between the nucleus and cytoplasm. In addition developmental analysis shows that the proteasome is highly expressed in the CNS of stage-16 embryos and in cardia, ventriculus and ovaries of adult flies. These data suggest a tissue- and development-dependent distribution of the proteasome in D. melanogaster.

Amino Acid Sequence↗

PubMed1988

Fibrils from brains of cows with new cattle disease contain scrapie-associated protein.

During the past two years, more than 1,000 cases of a neurological disorder of cattle, bovine spongiform encephalopathy (BSE), have been confirmed from farms throughout Great Britain. The neurological signs and brain pathology of BSE resemble those produced in other species by the pathogens of scrapie and related disorders. The discovery of fibrils similar to scrapie-associated fibrils in detergent extracts o BSE-affected brain supported the clinical and pathological diagnosis of the disease, but has been controversial. Scrapie-associated fibrils are found in brain extracts of all species affected by scrapie and diseases caused by related pathogens. They are pathological aggregates of a neuronal membrane protein termed PrP and a protease-resistant form of PrP is a molecular marker of scrapie-associated fibrils. In this report, we show the major protein of BSE fibrils is the bovine homologue of PrP as judged by its size, protease resistance, immunoreactivity, lectin binding and partial N-terminal protein sequence. This confirms that BSE is a scrapie-like disease.

Amino Acid Sequence↗

PubMed1988

Molecular pathology of scrapie-associated fibril protein (PrP) in mouse brain affected by the ME7 strain of scrapie.

Scrapie-associated fibrils (SAF) are disease-specific structures found in extracts of the brains of animals affected with scrapie. These structures are pathological aggregates of a normal host protein (PrP). Abnormal post-translational modification of PrP has been suggested to explain its aberrant properties in scrapie-affected brains and although there is a form of PrP in SAF indistinguishable in size from the protein in uninfected brain, lower-molecular-mass variants of PrP are also found in SAF fractions. We report the characterisation of the multiple forms of PrP found in SAF fractions purified from mouse brain affected by the ME7 strain of scrapie. The quantitatively major forms of PrP in SAF prepared without the use of proteinase K have the amino-terminal sequence Lys-Lys-Arg-Pro-Lys-Pro-Gly-Gly-, identical to that predicted for the amino-terminus of normal mouse brain PrP. However N-terminal cleavage of some PrP does occur in vivo within a domain of repetitive sequences at sites similar to but distinct from those cut by proteinase K in vitro. This suggests the conformation of the protein in aggregates in vivo does not differ extensively from that in detergent-treated SAF in vitro. We conclude that the size diversity of PrP in SAF is only partly due to N-terminal proteolysis and is independent of the proteolysis that occurs if proteinase K is used in the purification of SAF. Apart from proteolytic changes in the structure of PrP, we found a novel, as yet unidentified, amino-acid derivative of the arginine residue at position 3 in mouse PrP, which may predispose PrP to form SAF.

Amino Acid Sequence↗

PubMed1988

Localization of the putative precursor of Alzheimer's disease-specific amyloid at nuclear envelopes of adult human muscle.

Cloning and sequence analysis revealed the putative amyloid A4 precursor (pre-A4) of Alzheimer's disease to have characteristics of a membrane-spanning glycoprotein. In addition to brain, pre-A4 mRNA was found in adult human muscle and other tissues. We demonstrate by in situ hybridization that pre-A4 mRNA is present in adult human muscle, in cultured human myoblasts and myotubes. Immunofluorescence with antipeptide antibodies shows the putative pre-A4 protein to be expressed in adult human muscle and associated with some but not all nuclear envelopes. Despite high levels of a single 3.5-kb pre-A4 mRNA species in cultured myoblasts and myotubes, the presence of putative pre-A4 protein could not be detected by immunofluorescence. This suggests that putative pre-A4 protein is stabilized and therefore functioning in the innervated muscle tissue but not in developing, i.e. non-innervated cultured muscle cells. The selective localization of the protein on distinct nuclear envelopes could reflect an interaction with motor endplates.

Adult↗

PubMed1988

Identification, transmembrane orientation and biogenesis of the amyloid A4 precursor of Alzheimer's disease.

The precursor of the Alzheimer's disease-specific amyloid A4 protein is an integral, glycosylated membrane protein which spans the bilayer once. The carboxy-terminal domain of 47 residues was located at the cytoplasmic site of the membrane. The three domains following the transient signal sequence of 17 residues face the opposite side of the membrane. The C-terminal 100 residues of the precursor comprising the amyloid A4 part and the cytoplasmic domain have a high tendency to aggregate, and proteinase K treatment results in peptides of the size of amyloid A4. This finding suggests that there is a precursor-product relationship between precursor and amyloid A4 and we conclude that besides proteolytic cleavage other events such as post-translational modification and membrane injury are primary events that precede the release of the small aggregating amyloid A4 subunit.

Alzheimer Disease↗

PubMed1988

Alzheimer's disease amyloidogenic glycoprotein: expression pattern in rat brain suggests a role in cell contact.

The cloned cDNA encoding the rat cognate of the human A4 amyloid precursor protein was isolated from a rat brain library. The predicted primary structure of the 695-amino acid-long protein displays 97% identity to its human homologue shown previously to resemble an integral membrane protein. The protein was detected in rodent brain and muscle by Western blot analysis. Using in situ hybridization and immunocytochemistry on rat brain sections, we discovered that rat amyloidogenic glycoprotein (rAG) and its mRNA are ubiquitously and abundantly expressed in neurons indicating a neuronal original for the amyloid deposits observed in humans with Alzheimer's disease (AD). The protein appears in patches on or near the plasma membranes of neurons suggesting a role for this protein in cell contact. Highest expression was seen in rat brain regions where amyloid is deposited in AD but also in areas which do not contain deposits in AD. Since amyloid deposits are rarely observed in rat brain, we conclude that high expression of AG is not the sole cause of amyloidosis.

Alzheimer Disease↗

PubMed1988

Serine 71 of the glycoprotein HEF is located at the active site of the acetylesterase of influenza C virus.

The acetylesterase of influenza C virus has been reported recently to be inhibited by diisopropylfluorophosphate (DFP) [Muchmore EA, Varki A (1987) Science 236: 1293-1295]. As this inhibitor is known to bind covalently to the serine in the active site of serine esterases, we attempted to determine the serine in the active site of the influenza C acetylesterase. Incubation of purified influenza C virus with 3H-DFP resulted in the selective labelling of the influenza C glycoprotein HEF. The labelled glycoprotein was isolated from a SDS-polyacrylamide gel. Following reduction and carboxymethylation, tryptic peptides of HEF were prepared and analyzed by reversed phase HPLC. The peptide containing the 3H-DFP was subjected to sequence analysis. The amino acids determined from the NH2-terminus were used to locate the peptide on the HEF polypeptide. Radiosequencing revealed that 3H-DFP is attached to amino acid 17 of the tryptic peptide. These results indicate that serine 71 is the active-site serine of the acetylesterase of influenza C virus.

Acetylesterase↗

PubMed1988

A4 amyloid protein deposition and the diagnosis of Alzheimer's disease: prevalence in aged brains determined by immunocytochemistry compared with conventional neuropathologic techniques.

The histologic diagnosis of Alzheimer's disease (AD) might be aided if a more sensitive marker of aberrant A4 amyloid protein deposition were available. We screened a sample of aged brains, using immunocytochemical methods to detect the A4 protein deposition, and found that, in comparison with conventional histologic techniques (silver impregnation and Congo red), immunocytochemistry is more sensitive and allows an easier demarcation between "normal" and "abnormal." If A4 protein deposition is accepted as a definitive marker for AD, then the age-related prevalence of AD increases dramatically. To what degree these prevalence rates are reflected in clinically detectable impairment of higher cortical function remains to be determined.

Aged↗

PubMed1987

Amyloid of neurofibrillary tangles of Guamanian parkinsonism-dementia and Alzheimer disease share identical amino acid sequence.

The presence of abundant intraneuronal amyloid in the form of neurofibrillary tangles (NFT) in the brains of Guamanian parkinsonism-dementia patients and the absence of extraneuronal amyloid in the form of vascular amyloid deposits or senile plaques permit the purification of NFT without contamination with extraneuronal amyloid. Thus, we have isolated and determined the amino acid sequence of the polypeptide subunit of the amyloid fibrils of these NFT and describe their ultrastructure. The NFT, which consist of single and paired helical filaments, similar to those of Alzheimer disease, and occasionally triple helical filaments, are composed of multimeric aggregates of a polypeptide of 42 amino acids (A4 protein). The relative molecular mass of the subunit protein, 4.0-4.5 kDa, is the same as the molecular mass of the amyloid of NFT, of the amyloid plaque cores, and of vascular amyloid deposits in Alzheimer disease and Down syndrome; the sequence of 15 amino acid residues at the N-terminus of the amyloid fibrils in the NFT of Guamanian parkinsonism-dementia is identical to that of the amyloid of NFT, amyloid plaque cores, and cerebrovascular deposits in Alzheimer disease and Down syndrome. Furthermore, the heterogeneity, or variation in polypeptide length, of the N-terminus of the amyloid of Guamanian parkinsonism-dementia is the same as in Alzheimer disease and Down syndrome. Our observations indicate that the brain amyloids of these diseases have a common subunit protein, which would also indicate a common pathogenesis.

Aged↗

PubMed1986

Mammalian single-stranded DNA binding protein UP I is derived from the hnRNP core protein A1.

Antibodies induced against mammalian single-stranded DNA binding protein (ssDBP) UP I were shown to be cross-reactive with most of the basic hnRNP core proteins, the main constituents of 40S hnRNP particles. This suggested a structural relationship between both groups of proteins. Using the anti-ssDBP antibodies, a cDNA clone (pRP10) was isolated from a human liver cDNA library in plasmid expression vector pEX1. By DNA sequencing this clone was shown to encode in its 949 bp insert the last 72 carboxy terminal amino acids of the ssDBP UP I. Thereafter, an open reading frame continued for another 124 amino acids followed by a UAA (ochre) stop codon. Direct amino acid sequencing of a V8 protease peptide from hnRNP core protein A1 showed that this peptide contained at its amino terminus the last 11 amino acids of UP I followed by 19 amino acids which are encoded by the open reading frame of cDNA clone pRP10 immediately following the UP I sequence. This proves that ssDBP UP I arises by proteolysis from hnRNP core protein A1. This finding must lead to a re-evaluation of the possible physiological role of UP I and related ssDBPs. The formerly assumed function in DNA replication, although not completely ruled out, should be reconsidered in the light of a possible alternative or complementary function in hnRNA processing where UP I could either be a simple degradation product of core protein A1 (as a consequence of controlling the levels of active A1) or may continue to function as an RNA binding protein which has lost the ability to interact with the other core proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1986

The major polypeptide of scrapie-associated fibrils (SAF) has the same size, charge distribution and N-terminal protein sequence as predicted for the normal brain protein (PrP).

Scrapie-associated fibrils (SAF) are unique structures characteristic of the group of unconventional slow infections which includes scrapie and Creutzfeldt-Jakob disease. A major component of hamster fibrils has been described as a protease-resistant glycoprotein with an apparent mol. wt of 27,000-30,000 (PrP27-30). However, we report here that if fibrils are prepared by procedures designed to minimise proteolysis the PrP proteins co-purifying with hamster SAF have mol. wts of 33,000-35,000 (PrP33-35) and 26,000-29,000 (PrP26-29). We find a Lys-Lys-Arg-Pro-Lys sequence at the amino terminus of these SAF proteins, that is absent from PrP27-30, and which has recently been predicted to be the N-terminal sequence of the native PrP protein of uninfected brain. The major SAF protein (PrP33-35) and its normal brain homologue are shown to have the same apparent mol. wt and ionic charge distribution by two-dimensional gel analysis, silver staining and immunoblotting. These results support our view that PrP33-35 and the normal brain PrP protein may have the same covalent structure, and that the PrP protein is recruited into these amyloid-like SAF or into association with a non-protein component of SAF by an irreversible event initiated directly or indirectly by scrapie infection.

Amino Acid Sequence↗