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P M Kloetzel

Publications and source records attributed to P M Kloetzel.

At least 73 records · Page 4Linked to original sources
PubMed1994

20 S proteasomes are assembled via distinct precursor complexes. Processing of LMP2 and LMP7 proproteins takes place in 13-16 S preproteasome complexes.

The non-essential mouse proteasome beta-type subunits LMP2 and LMP7 are thought to connect proteasomes to the MHC class I antigen processing pathway. Both subunits are synthesized as proproteins. We have studied the processing of both subunits, correlated with the maturation of 20 S proteasomes in mouse T cells. Our data show that proteasome assembly occurs via 13-16 S precursor complexes which possess a protein pattern distinct from that of 20 S proteasomes. Both LMP2 and LMP7 proproteins are processed within these preproteasome complexes and only their processed forms become part of active 20 S proteasomes. Our data show that the maturation and assembly of 20 S proteasomes via precursor particles is a translation-dependent gradual process, that processing of subunit proproteins takes place in these 13-16 S complexes and that subunit processing and proteasome formation occur together.

Animals↗
PubMed1994

Interferon gamma stimulation modulates the proteolytic activity and cleavage site preference of 20S mouse proteasomes.

The proteasome is a 700-kD multisubunit enzyme complex with several proteolytically active sites. The enzyme complex is involved in both ubiquitin-dependent and -independent protein degradation and may contribute to the processing of antigens presented by major histocompatibility complex (MHC) class I molecules. Here we demonstrate that treatment of mouse fibroblast cells with 20 U interferon gamma (IFN-gamma) for 3 d induces a change in the proteasome subunit composition and that the beta-type subunit LMP2, which is encoded in the MHC class II region, is incorporated into the enzyme complex. This is paralleled by reduction of the homologous delta-subunit. IFN-gamma stimulation results in a downregulation of the chymotrypsin-like Suc-LLVY-MCA peptide hydrolyzing activity of 20S proteasomes whereas the trypsin-like activity remains unaffected. When tested as a substrate a synthetic 25-mer polypeptide whose sequence covers the antigenic nonapeptide YPHFMPTNL of the MCMV pp89, 20S proteasomes of IFN-gamma-induced cells exhibit altered chymotrypsin-like cleavage site preferences. In the absence of IFN-gamma induction, the naturally processed nonamer peptide that is presented by MHC class I molecules appears as a minor cleavage product. IFN-gamma activation does not result in an increase of the final peptide but results in a different set of peptides. We hypothesize that these peptides represent precursor peptides that can be trimmed to final peptide size.

Amino Acid Sequence↗
PubMed1993

Drosophila proteasome Dm25 subunit substitutes the mouse MC3 subunit in hybrid proteasomes. The N-terminal domain is essential for subunit incorporation.

The proteasome is a multisubunit 20 S proteinase complex involved in ubiquitin-dependent and -independent intracellular protein metabolism. Individual subunits of the alpha- and beta-type share extensive sequence homology and are encoded as members of two related and evolutionarily conserved gene families. Due to the lack of viable deletion mutants of essential alpha-type proteasome subunits in higher eukaryotes, an identification and analysis of potentially homologous subunits of different species was so far not possible. It is shown here that the novel Drosophila alpha-type Dm25 subunit can be incorporated into mouse proteasomes of stably transfected NIH 3T3 cells. The Dm25 subunit is able to substitute the mouse MC3 alpha-type subunit in proteasomes, indicating a high structural and possibly also functional homology of the two subunits. In contrast and pointing at the importance of the slightly hydrophobic N-terminal region stabile expression of a Dm25 subunit, which is truncated at its N terminus and lacks PROS box I, results in a subunit which cannot be incorporated into mouse proteasomes. The ability to form hybrid proteasomes involving essential nondeletable subunits now opens the possibility for structural and also functional analysis of such subunits by mutagenesis in higher eukaryotes.

3T3 Cells↗
PubMed1993

Sequence and genomic organization of the Drosophila proteasome PROS-Dm25 gene.

The genomic region encoding the Drosophila proteasome alpha-type subunit Dm25 has been isolated and analysed with regard to its nucleotide sequence and structure. Our data show that the Dm25 coding region is interrupted by four introns and that the 5' upstream region contains no sequence motifs common with the two previously described proteasome genes.

Amino Acid Sequence↗
PubMed1993

The major-histocompatibility-complex-encoded beta-type proteasome subunits LMP2 and LMP7. Evidence that LMP2 and LMP7 are synthesized as proproteins and that cellular levels of both mRNA and LMP-containing 20S proteasomes are differentially regulated.

The proteasome (high-molecular-mass multicatalytic proteinase complex) is composed of a large number of non-identical protein subunits of the alpha and beta types. The mouse beta-type subunits LMP2 and LMP7 (LMP, low-molecular-mass protein) are encoded within the mouse major histocompatibility complex (MHC II) region, and are thought to connect the proteasome to the MHC class-I antigen-processing pathway. In the present communication, we have analysed the two proteasome subunits with regard to their identity within the proteasome complex, their protein levels, their amounts of mRNA in different mouse tissues and cell lines, and have investigated the intracellular localization of LMP2 and LMP7 subunits in thymus and liver by immunocytology. Our experiments indicate that LMP2 and LMP7 subunits are synthesized as precursor proteins of 24 kDa and 30 kDa, respectively, and that only the processed 21-kDa and 23-kDa subunits are part of the 20S proteasome complex. The proportion of LMP2-subunit-containing and LMP7-subunit-containing proteasome complexes, as well as LMP2 and LMP7 mRNA levels, vary strongly and are shown to be dependent on the tissues or cell lines analysed. Furthermore, high LMP2 and LMP7 mRNA levels do not always correlate with high protein levels, suggesting a specific translational mechanism which controls proteasome subunit synthesis. Generally, mRNA levels appear to be particularly high in those tissues which are known to be involved in MHC class-I antigen presentation. Immunocytological analysis shows a strong nuclear localization of the subunits in cells of the thymus, while in the liver they appear to be evenly distributed between the two cellular compartments. Our data support the idea that both LMP2 and LMP7 proteins are non-essential proteasome subunits which are probably involved in the regulation of proteasome activities. The function of the two subunits, however, may not be restricted to the proposed role of proteasomes in antigen presentation.

Animals↗
PubMed1993

Development and tissue-specific distribution of mouse small heat shock protein hsp25.

We have investigated the developmental and tissue-specific distribution of the mouse small hsp25 by immunohistology using an antibody that specifically identifies hsp25. Our analysis shows that the relative amount of hsp25 increases during embryogenesis. Through days 13-20 of embryogenesis, hsp25 accumulation is predominant in the various muscle tissues, including the heart, the bladder, and the back muscles. hsp25 is detectable also in neurons of the spinal cord and the purkinje cells. Furthermore analysis of the closely related alpha, B-crystallin shows that in several tissues, including the bladder, the notochordal sheath and the eye lens both proteins are coexpressed. Our studies demonstrate that mammalian hsp25 accumulation is developmentally regulated during mouse embryogenesis and support the view of an important functional role of small heat shock proteins in normal cell metabolism.

Animals↗
PubMed1993

Characterization of mouse proteasome subunit MC3 and identification of proteasome subtypes with different cleavage characteristics. Proteasome subunits, proteasome subpopulations.

We have isolated and characterized a cDNA encoding the mouse proteasome subunit MC3 and identified four proteasome subtypes which differ in their peptide-hydrolyzing and polypeptide-cleavage properties. Immunoblotting data show that the 25-kD MC3 subunit is a constitutive proteasome subunit which exists in several isoforms. In addition, by immunoprecipitation of proteasomes with AbMC3, a subset of enzyme complexes could be recognized which differ in their relative subunit composition from the bulk of proteasomes. Using DEAE-column chromatography we identified three different proteasome subtypes in sol-80 mouse liver extracts and, by Trition X-100 extraction, a distinct membrane-bound subtype. The four proteasome subtypes are shown to differ in their trypsin- and chymotrypsin-like hydrolyzing activities as well as in their ability to cleave a 25mer polypeptide substrate derived from the MCMV IE pp89. Our data indicate that the enzymatic properties observed for the total proteasome population may be the summary of cleavage properties of different types of proteasome complexes.

3T3 Cells↗
PubMed1992

Isolation and characterization of the MHC linked beta-type proteasome subunit MC13 cDNA.

We have cloned and analysed the second mouse MHC-linked proteasome subunit, designated MC13, which appears to be homologous to the human RING10 proteasome protein. The isolated cDNA has an ORF encoding a protein of 276 amino acids with a molecular weight of ca. 30 kDa. Sequence alignment reveals that the subunit MC13 and several other mammalian proteasome subunits are encoded by a second proteasome gene family. This second gene family encodes subunits of the beta-type, reveals striking sequence similarities with the beta-subunit of archaebacterial proteasomes and is related to, but distinct from, the genes encoding the so-called alpha-type subunits.

Amino Acid Sequence↗
PubMed1992

Molecular characterization of the genomic regions of the Drosophila alpha-type subunit proteasome genes PROS-Dm28.1 and PROS-Dm35.

The proteasome (multicatalytic proteinase) consists of a large number of non-identical protein subunits which are encoded by the evolutionarily conserved PROS gene family. Using the PROS-Dm35 and PROS-Dm28.1 cDNAs as probes, we have isolated the corresponding genomic DNA clones of Drosophila melanogaster. In situ hybridization shows that the members of the PROS gene family are not organized in a single gene cluster and that, in contrast to the PROS-Dm35 gene, the PROS-Dm28.1 gene is localized on the X chromosome. Analysis of the genomic organization of the PROS-Dm28.1 and PROS-Dm35 genes reveals that both genes are interrupted by two small introns whereby the relative positions of the introns within the two coding regions are not conserved. Neither gene possesses a distinct transcriptional start site as shown by nuclease S1 analysis. Since the promoter regions also do not contain a TATA box, PROS genes appear to be typical house-keeping genes. A putative heat-shock element in the promoter region of the PROS-Dm35 gene was shown to be inactive on stress induction when fused to a reporter gene and tested in transient transfections assays. In addition, promoter deletion analysis demonstrates that the promoter region between positions -605 and -330 contains sequence elements important for PROS-Dm35 gene activity and that deletions beyond position -150 result in an almost complete inhibition of transcription.

Amino Acid Sequence↗
PubMed1992

The 70 kD heat shock protein (hsp 70) in soil invertebrates: a possible tool for monitoring environmental toxicants.

The expression of hsp 70 after heat shock or exposure to heavy metals/molluscicides was investigated by fluorography or immunoblot in three diplopods (Glomeris marginata, Cylindroiulus punctatus, Tachypodoiulus niger), two slugs (Deroceras reticulatum, Arion ater), and one isopod (Oniscus asellus). In O. asellus, hsp 70 expression occurred after heat shock and also after lead treatment, whereby a solution of 100 mg/kg Pb2+ was sufficient. Animals of the same species taken from a heavy metal polluted site in the vicinity of a lead/zinc smelter also showed the presence of hsp 70. The comparison of laboratory and field experiments demonstrated the suitability of O. asellus for monitoring tests. In contrast, the blot pattern after contamination with 1,000 mg/kg Pb2+ (in the mentioned diplopods) or different concentrations of the molluscicide Cloethocarb (BASF, FRG) (slugs) showed no differences compared to the respective control group.

Animals↗
PubMed1991

Molecular interaction of the proteasome (multicatalytic proteinase). Evidence that the proteasome is not a constituent of the '26 S' multienzyme complex.

On the basis of recent reports that suggested that proteasomes, via an ATP-dependent process, become integral components of a '26 S' complex possessing 3-carboxypropionyl-Leu-Leu-Val-Tyr 4-methylcoumarin-7-ylamide-hydrolysing activity, we have investigated the molecular interaction of proteasomes in ATP-stabilized fraction II (proteins absorbed on DEAE-matrix and eluted with 0.5 M-KCl) of rabbit reticulocytes and mouse liver. Analysis of the various extracts by (NH4)2SO4 fractionation, velocity-gradient centrifugation, non-denaturing PAGE and SDS/PAGE and immunoblotting with proteasome-specific antisera failed to identify the proteasome as part of a higher-molecular-mass '26 S' multienzyme complex. In all instances proteasomes are identified in their 'free' 650 kDa '20 S' form. In addition to the proteasome and independent of the presence of MgATP, we isolated a high-molecular-mass proteinase whose electrophoretic migration behaviour and sedimentation rate correspond to that of the previously described '26 S' proteinase. This '26 S' proteinase possesses a strong 3-carboxypropionyl-Leu-Leu-Val-Tyr 4-methylcoumarin-7-ylamide-hydrolysing activity and is composed of several non-identical polypeptides in the molecular-mass range 20-150 kDa. Despite its similarity to proteasomal enzyme activity, protein analysis and immunoblotting experiments demonstrate that neither the intact proteasome nor subunits thereof are components of the '26 S' proteinase complex.

Amino Acid Sequence↗
PubMed1991

Subunit of the '20S' proteasome (multicatalytic proteinase) encoded by the major histocompatibility complex.

Cytotoxic T lymphocytes recognize fragments (peptides) of protein antigens presented by major histocompatibility complex (MHC) class I molecules. In general, the peptides are derived from cytosolic proteins and are then transported to the endoplasmic reticulum where they assemble with the MHC class I heavy chains and beta 2-microglobulin to form stable and functional class I molecules. The proteases involved in the generation of these peptides are unknown. One candidate is the proteasome, a nonlysosomal proteinase complex abundantly present in the cytosol. Proteasomes have several proteolytically active sites and are complexes of high relative molecular mass (Mr about 600K), consisting of about 20-30 subunits with Mrs between 15 and 30K. Here we show that at least one of these subunits is encoded by the mouse MHC in the region between the K locus and the MHC class II region, and inducible by interferon-gamma. This raises the intriguing possibility that the MHC encodes not only the MHC class I molecules themselves but also proteases involved in the formation of MHC-binding peptides.

Animals↗
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

Molecular analysis of alpha ecdysone induced 16S complexes in Drosophila Schneider's S3 cells.

The molecular organization of alpha ecdysone induced small heat shock proteins (small hsps) in Schneider's S3 tissue culture cells was analysed. Sucrose gradient centrifugation of cytoplasmic extracts and nondenaturing gel electrophoresis shows that hormone induced small hsps form 16S particles which differ in the relative molar ratios of the small hsps composing the 16S particles. The 16S particles possess a buoyant density in Cs2SO4 of rho = 1.34 g/cm3 which is indicative of RNP complexes with an RNA:protein ratio of 1:4. The RNA component of the 16S particles was identified by cDNA cloning using a cDNA library established from alpha ecdysone induced pupal 16S material. Northern hybridization using the 16S RNP specific partial cDNA clone Ec3 identifies a single alpha ecdysone inducible 300 nt RNA species. Our data suggest that the small hsps may unfold their so far unresolved function in form of RNP complexes.

Animals↗
PubMed1990

Cell-specific accumulation of Drosophila proteasomes (MCP) during early development.

The proteasome (MCP) is a high relative molecular mass multicatalytic proteinase complex composed of nonidentical protein subunits. We have investigated the cellular distribution of the enzyme complex during Drosophila embryogenesis using the proteasome specific antibodies N19-35 and N19-28 for immunocytology. Antibody staining of whole-mount embryos shows that during embryogenesis proteasomes are present in proliferating cells and that their accumulation and turnover is differentially regulated. Our data suggest that the proteasome may serve different proteolytic processes and that the enzyme may be involved in cell-specific proteolytic events required for cell proliferation and morphogenesis during early Drosophila development.

Animals↗
PubMed1990

Developmental expression of Drosophila melanogaster small heat-shock proteins.

We have investigated the developmental expression of the small heat-shock proteins (hsps) during embryogenesis and in adult flies by immunocytology using an antibody that specifically identifies the small hsps. Antibody staining of unstressed early embryos reveals a predominantly cytoplasmic, homogeneous distribution of the small hsps throughout the embryo. At 6h of development small hsp expression can be identified in large, neuroblast-like cells within the extended germ band and in the brain of the embryo. During germ band contraction these cells appear to migrate to the midline where they align pairwise in a segmental pattern. After germ band contraction is complete a high level of small hsp expression can be observed in the midline glia (MECs) and in a cluster of six non-neuronal cells within the midline. In contrast to several other genes that are known to be important for embryogenesis and are expressed in the central nervous system (CNS) of embryos, CNS-specific expression of the small hsps is not restricted to the embryo but is also observed in the adult fly. In adult flies strong small hsp expression is observed in the brain, the thoracic ganglion and the leg nerves. Since the small hsps seem to be expressed predominantly in the glia of the nervous system, our data suggest a protective or stabilizing function of the small hsps within the nervous system during normal fly development, which is independent of the stress response.

Animals↗