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M Seeger

Publications and source records attributed to M Seeger.

At least 37 records · Page 2Linked to original sources
PubMed1997

Mts4, a non-ATPase subunit of the 26 S protease in fission yeast is essential for mitosis and interacts directly with the ATPase subunit Mts2.

We have isolated a fission yeast gene, mts4(+), by complementation of a temperature-sensitive mutation and show that it encodes subunit 2 (S2) of the 19 S regulatory complex of the 26 S protease. mts4(+) is an essential gene, and we show that loss of this subunit causes cells to arrest in metaphase, illustrating the importance of S2 for mitosis. The Mts4 protein is 48% identical to S2 of the human 26 S protease, and the lethal phenotype of the null mts4 allele can be rescued by the human cDNA encoding S2. We provide genetic and physical evidence to suggest that the Mts4 protein interacts with the product of the mts2(+) gene, an ATPase which has previously been shown to be subunit 4 of the 26 S protease.

Adenosine Triphosphatases↗
PubMed1997

Evidence for phosphorylation and oligomeric assembly of presenilin 1.

Pathogenic mutations in presenilin 1 (PS1) are associated with approximately 50% of early-onset familial Alzheimer disease. PS1 is endoproteolytically cleaved to yield a 30-kDa N-terminal fragment (NTF) and an 18-kDa C-terminal fragment (CTF). Using COS7 cells transfected with human PS1, we have found that phorbol 12, 13-dibutyrate and forskolin increase the state of phosphorylation of serine residues of the human CTF. Phosphorylation of the human CTF resulted in a shift in electrophoretic mobility from a single major species of 18 kDa to a doublet of 20-23 kDa. This mobility shift was also observed with human PS1 that had been transfected into mouse neuroblastoma (N2a) cells. Treatment of the phosphorylated CTF doublet with phage lambda protein phosphatase eliminated the 20- to 23-kDa doublet while enhancing the 18-kDa species, consistent with the interpretation that the electrophoretic mobility shift was due to the addition of phosphate to the 18-kDa species. The NTF and CTF eluted from a gel filtration column at an estimated mass of over 100 kDa, suggesting that these fragments exist as an oligomerized species. Upon phosphorylation of the PS1 CTF, the apparent mass of the NTF- or CTF-containing oligomers was unchanged. Thus, the association of PS1 fragments may be maintained during cycles of phosphorylation/dephosphorylation of the PS1 CTF.

Animals↗
PubMed1997

HIV-1 tat inhibits the 20 S proteasome and its 11 S regulator-mediated activation.

The proteasomal system consists of a proteolytic core, the 20 S proteasome, which associates in an ATP-dependent reaction with the 19 S regulatory complex to form the functional 26 S proteasome. In the absence of ATP, the 20 S proteasome forms a complex with the gamma-interferon-inducible 11 S regulator. Both the 20 S proteasome and the 11 S regulator have been implied in the generation of antigenic peptides. The human immunodeficiency virus (HIV)-1 Tat protein causes a number of different effects during acquired immunodeficiency syndrome (AIDS). Here we show that HIV-1 Tat protein strongly inhibits the peptidase activity of the 20 S proteasome and that it interferes with formation of the 20 S proteasome-11 S regulator complex. In addition, it slightly increases the activity of purified 26 S proteasome. These results may explain the mechanism by which HIV-1-infected cells escape cytotoxic T lymphocyte response and at least in part immunodeficiency in AIDS patients.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1997

The 26S proteasome: a dynamic structure.

The proteasomal system consists of a proteolytic core, the 20S proteasome, which associates in ATP-dependent and independent reactions with endogenous regulators providing specific substrate binding sites, chaperone function and regulation of activity to the protease. The best known regulators of the 20S proteasome are the 11S and the 19S complexes. Three subunits of the 20S proteasome and the two subunits of the 11S regulator are induced by gamma-Interferon. However, there are no indications for an influence of gamma-interferon on the subunit composition of the 19S regulator and only a few data exist about the dynamics of this complex. The analysis of 19S regulator subunits from yeast mutants reveals that the ATPases appear to be stringently organized in the 26S complex, while peripheral non-ATPases, such as S5a, might serve as subunits which shuttle substrates to the enzyme. A novel non-ATPase has been cloned, sequenced and identified in a complex besides the 19S regulator, the function of which is presently unknown. The dynamic structure of the 26S proteasome is also characterized by transient associations with components such as the modulator and isopeptidases. Certain viral proteins can also be associated with components of the proteasomal system and alter enzymatic activities.

Humans↗
PubMed1997

Heterologous expression of biphenyl dioxygenase-encoding genes from a gram-positive broad-spectrum polychlorinated biphenyl degrader and characterization of chlorobiphenyl oxidation by the gene products.

The bphA1A2A3A4 gene cluster, encoding a biphenyl dioxygenase from Rhodococcus globerulus P6, a gram-positive microorganism able to degrade a wide spectrum of polychlorobiphenyls (PCBs), was expressed in Pseudomonas putida, thereby allowing characterization of chlorobiphenyl oxidation by this enzyme. While P6 biphenyl dioxygenase activity was observed in P. putida containing bphA1A2A3A4, no activity was detected in Escherichia coli cells containing the same gene cluster. In E. coli, transcription of genes bphB and bphCl, located downstream of bphA1A2A3A4, was shown to be driven solely by a vector promoter, which indicated that the lack of biphenyl dioxygenase activity was not due to a lack of mRNA synthesis. Radioactive labelling of bph gene products in E. coli implied inefficient translation of the bphA gene cluster or rapid degradation of the gene products. The biosynthesis of functional P6 biphenyl dioxygenase in P. putida cells containing the same plasmid construct that yielded no activity in E. coli emphasizes the importance of the host strain for heterologous expression and shows that synthesis, correct folding, and assembly of a Rhodococcus biphenyl dioxygenase can be achieved in a gram-negative organism. Dioxygenation of six mono- and dichlorinated PCB congeners by P. putida containing the P6 bphA gene cluster indicates the following ring substitution preference for this reaction (from most to least preferred): un-, meta-, para-, and ortho-substitution. No indications were found for dioxygenation of meta/para carbon pairs, or for hydroxylation of chlorinated carbons at any position of a monochlorinated ring, suggesting a strict specificity of this biphenyl dioxygenase for attack at nonhalogenated ortho/meta vicinal carbons. This contrasts the properties of an analogous enzyme from Pseudomonas sp. strain LB400, which can both dioxygenate at meta and para positions and dehalogenate substituted ortho carbons during ortho and meta dioxygenation.

Biodegradation, Environmental↗
PubMed1996

Expression of presenilin 1 and 2 (PS1 and PS2) in human and murine tissues.

Mutations in genes encoding related proteins, termed presenilin 1 (PS1) and presenilin 2 (PS2), are linked to the majority of cases with early-onset familial Alzheimer's disease (FAD). To clarify potential function(s) of presenilins and relationships of presenilin expression to pathogenesis of AD, we examined the expression of PS1 and PS2 mRNA and PS1 protein in human and mouse. Semi-quantitative PCR of reverse-transcribed RNA (RT-PCR) analysis revealed that PS1 and PS2 mRNA are expressed ubiquitously and at comparable levels in most human and mouse tissues, including adult brain. However, PS1 mRNA is expressed at significantly higher levels in developing brain. In situ hybridization studies of mouse embryos revealed widespread expression of PS1 mRNA with a neural expression pattern that, in part, overlaps that reported for mRNA encoding specific Notch homologs. In situ hybridization analysis in adult mouse brain revealed that PS1 and PS2 mRNAs are enriched in neurons of the hippocampal formation and entorhinal cortex. Although PS1 and PS2 mRNA are expressed most prominently in neurons, lower but significant levels of PS1 and PS2 transcripts are also detected in white matter glial cells. Moreover, cultured neurons and astrocytes express PS1 and PS2 mRNAs. Using PS1-specific antibodies in immunoblot analysis, we demonstrate that PS1 accumulates as approximately 28 kDa N-terminal and approximately 18 kDa C-terminal fragments in brain. Immunocytochemical studies of mouse brain reveal that PS1 protein accumulates in a variety of neuronal populations with enrichment in somatodendritic and neuropil compartments.

Adult↗
PubMed1996

Characteristics of 26 S proteases from fission yeast mutants, which arrest in mitosis.

We have isolated the 26 S protease from the fission yeast Schizosaccharomyces pombe. The affinity-purified enzyme contains the two regulatory ATPases mts2+, a homolog of human S4, and CIM5, a homolog of human MSS1 = S7. We show that mts3+, a homolog of the budding yeast NIN1 protein and human S14, is a true component of the 19 S regulatory complex from the fission yeast. The 26 S proteases purified from two thermosensitive mutants, mts2-1 and mts3-1, which arrest in cell cycle at the restrictive temperature (37 degrees C), have been compared with the wild-type enzyme after growing cells at permissive (25 degrees C) and non-permissive temperatures. We demonstrate that mutated mts2 protein is integrated into the protease complex prepared from mts2 cells, whereas mutated mts3 is not present in the 19 S regulatory complex from mts3 cells. The two mutant 26 S proteases isolated after growing cells at 37 degrees C remain stable for two hours at 37 degrees C as measured by ATP-dependent cleavage of the fluorogenic peptide sucLLVY-MCA. At the restrictive temperature, the mutant 26 S proteases do not degrade ubiquitin-[125I]lysozyme conjugates in an ATP-dependent manner, indicating that mts2+ and mts3+ are essential for ubiquitin conjugate degradation. This explains the conditional lethality of the mutants and the cell-cycle arrest in metaphase to anaphase transition. In addition, our data demonstrate that the ATPases of the 26 S enzyme are not redundant.

Adenosine Triphosphate↗
PubMed1996

Phosphorylation of GroEL, DnaK and other proteins from Thiobacillus ferrooxidans grown under different conditions.

The levels of phosphorylation of the chaperones DnaK and GroEL and other proteins varied when cells of Thiobacillus ferrooxidans were subjected to phosphate starvation. The phosphorylated amino acid of GroEL was found to be threonine. Our results show that not only heat shock, but also a nutrient starvation stress leads to phosphorylation of chaperones and, in addition, support the possible role of phosphorylation of these proteins in the sensing and regulation of stress responses in bacteria.

Bacterial Proteins↗
PubMed1996

Familial Alzheimer's disease-linked presenilin 1 variants elevate Abeta1-42/1-40 ratio in vitro and in vivo.

Mutations in the presenilin 1 (PS1) and presenilin 2 genes cosegregate with the majority of early-onset familial Alzheimer's disease (FAD) pedigrees. We now document that the Abeta1-42(43)/Abeta1-40 ratio in the conditioned media of independent N2a cell lines expressing three FAD-linked PS1 variants is uniformly elevated relative to cells expressing similar levels of wild-type PS1. Similarly, the Abeta1-42(43)/Abeta1-40 ratio is elevated in the brains of young transgenic animals coexpressing a chimeric amyloid precursor protein (APP) and an FAD-linked PS1 variant compared with brains of transgenic mice expressing APP alone or transgenic mice coexpressing wild-type human PS1 and APP. These studies provide compelling support for the view that one mechanism by which these mutant PS1 cause AD is by increasing the extracellular concentration of Abeta peptides terminating at 42(43), species that foster Abeta deposition.

Alzheimer Disease↗
PubMed1996

Protein topology of presenilin 1.

Mutations in a gene encoding a multitransmembrane protein, termed presenilin 1 (PS1), are causative in the majority of early-onset cases of AD. To determine the topology of PS1, we utilized two strategies: first, we tested whether putative transmembranes are sufficient to export a protease-sensitive substrate across a lipid bilayer; and second, we examined the binding of antibodies to specific PS1 epitopes in cultured cells selectively permeabilized with the pore-forming toxin, streptolysin-O. We document that the "loop," N-terminal, and C-terminal domains of PS1 are oriented toward the cytoplasm.

Amyloid beta-Protein Precursor↗
PubMed1996

Endoproteolysis of presenilin 1 and accumulation of processed derivatives in vivo.

The majority of early-onset cases of familial Alzheimer's disease (FAD) are linked to mutations in two related genes, PS1 and PS2, located on chromosome 14 and 1, respectively. Using two highly specific antibodies against nonoverlapping epitopes of the PS1-encoded polypeptide, termed presenilin 1 (PS1), we document that the preponderant PS1-related species that accumulate in cultured mammalian cells, and in the brains of rodents, primates, and humans are approximately 27-28 kDa N-terminal and approximately 16-17 kDa C-terminal derivatives. Notably, a FAD-linked PS1 variant that lacks exon 9 is not subject to endoproteolytic cleavage. In brains of transgenic mice expressing human PS1, approximately 17 kDa and approximately 27 kDa PS1 derivatives accumulate to saturable levels, and at approximately 1:1 stoichiometry, independent of transgene-derived mRNA. We conclude that PS1 is subject to endoproteolytic processing in vivo.

Animals↗
PubMed1996

The effects of clathrin inactivation on localization of Kex2 protease are independent of the TGN localization signal in the cytosolic tail of Kex2p.

Localization of Kex2 protease (Kex2p) to the yeast trans-Golgi network (TGN) requires a TGN localization signal (TLS) in the Kex2p C-terminal cytosolic tail. Mutation of the TLS accelerates transport of Kex2p to the vacuole by an intracellular (SEC1-independent) pathway. In contrast, inactivation of the clathrin heavy-chain gene CHC1 results in transport of Kex2p and other Golgi membrane proteins to the cell surface. Here, the relationship of the two localization defects was assessed by examining the effects of a temperature-sensitive CHC1 allele on trafficking of wild-type (WT) and TLS mutant forms of Kex2p. Inactivation of clathrin by shifting chc1-ts cells to 37 degrees C caused WT and TLS mutant forms of Kex2p to behave identically. All forms of Kex2p appeared at the plasma membrane within 30-60 min of the temperature shift. TLS mutant forms of Kex2p were stabilized, their half-lives increasing to that of wild-type Kex2p. After inactivation of clathrin heavy chain, vacuolar protease-dependent degradation of all forms of Kex2p was blocked by a sec1 mutation, which is required for secretory vesicle fusion to the plasma membrane, indicating that transport to the cell surface was required for degradation by vacuolar proteolysis. Finally, after clathrin inactivation, all forms of Kex2p were degraded in part by a vacuolar protease-independent pathway. After inactivation of both chc1-ts and sec1-ts, Kex2 was degraded exclusively by this pathway. We conclude that the effects of clathrin inactivation on Kex2p localization are independent of the Kex2p C-terminal cytosolic tail. Although these results neither prove nor rule out a direct interaction between the Kex2 TLS and a clathrin-dependent structure, they do imply that clathrin is required for the intracellular transport of Kex2p TLS mutants to the vacuole.

Biological Transport↗
PubMed1995

Degradation of chlorobiphenyls catalyzed by the bph-encoded biphenyl-2,3-dioxygenase and biphenyl-2,3-dihydrodiol-2,3-dehydrogenase of Pseudomonas sp. LB400.

In order to characterize the metabolites produced in vivo by biphenyl-2,3-dioxygenase and biphenyl-2,3-dihydrodiol-2,3-dehydrogenase, the first two enzymes of the (polychloro)biphenyl catabolic pathway encoded by the bph locus of Pseudomonas sp. LB400, recombinant E. coli strains expressing the respective genes were constructed. Biphenyl-2,3-dioxygenase attack on 2,2'- or 2,4'-dichlorobiphenyl was shown to give rise to virtually quantitative ortho-dechlorination of these congeners by hydroxylation at the chlorinated carbon 2 and its unsubstituted neighbour. Elimination of hydrochloric acid directly leads to 2,3-dihydroxy-chlorobiphenyls and obviates the need for biphenyl-2,3-dihydrodiol-2,3-dehydrogenase for the catabolism of such congeners.

Biphenyl Compounds↗
PubMed1995

The interferon-gamma-inducible 11 S regulator (PA28) and the LMP2/LMP7 subunits govern the peptide production by the 20 S proteasome in vitro.

Antigenic peptides presented on major histocompatibility complex (MHC) class I molecules to cytotoxic T cells are generated in the cytosol by the 20 S proteasome. Upon stimulation of antigen presenting cells with interferon-gamma, two constitutive subunits of the 20 S proteasome are replaced by the MHC-encoded subunits low molecular mass polypeptide (LMP) 2 and LMP 7. In addition the expression of the two subunits of the 11 S regulator of the 20 S proteasome (PA28) are increased. As the function of LMP2 and LMP7 in antigen presentation is still controversial, we tested whether these subunits might operate by modifying proteasome activation through the 11 S regulator. We strongly overexpressed the two LMP subunits separately or together by transfection in murine fibroblasts. Isolated 20 S proteasomes from LMP transfectants were applied in digests of a 25-mer peptide in the presence or absence of a purified preparation of 11 S regulator from rabbit erythrocytes. Analysis of the cleavage products by high performance liquid chromatography and electrospray mass spectroscopy revealed marked differences in the peptide product profile in dependence on the LMP2 and LMP7 content. While the 11 S regulator did not preferentially activate LMP2 or 7 containing proteasomes, the binding of the 11 S regulator to any of the proteasome preparations markedly changed both the quality and quantity of peptides produced. These results suggest that the 11 S regulator increases the spectrum of peptides which can be generated in antigen presenting cells.

Amino Acid Sequence↗
PubMed1995

Conversion of chlorobiphenyls into phenylhexadienoates and benzoates by the enzymes of the upper pathway for polychlorobiphenyl degradation encoded by the bph locus of Pseudomonas sp. strain LB400.

Metabolism of 21 chlorobiphenyls by the enzymes of the upper biphenyl catabolic pathway encoded by the bph locus of Pseudomonas sp. strain LB400 was investigated by using recombinant strains harboring gene cassettes containing bphABC or bphABCD. The enzymes of the upper pathway were generally able to metabolize mono- and dichlorinated biphenyls but only partially transform most trichlorinated congeners investigated: 14 of 15 mono- and dichlorinated and 2 of 6 trichlorinated congeners were converted into benzoates. All mono- and at least 8 of 12 dichlorinated congeners were attacked by the bphA-encoded biphenyl dioxygenase virtually exclusively at ortho and meta carbons. This enzyme exhibited a high degree of selectivity for the aromatic ring to be attacked, with the order of ring preference being non- > ortho- > meta- > para-substituted for mono- and dichlorinated congeners. The influence of the chlorine substitution pattern of the metabolized ring on benzoate formation resembled its influence on the reactivity of initial dioxygenation, suggesting that the rate of benzoate formation may frequently be determined by the rate of initial attack. The absorption spectra of phenylhexadienoates formed correlated with the presence or absence of a chlorine substituent at an ortho position.

Benzoates↗
PubMed1994

Clathrin-dependent localization of alpha 1,3 mannosyltransferase to the Golgi complex of Saccharomyces cerevisiae.

Posttranslational modification of yeast glycoproteins with alpha 1,3-linked mannose is initiated within a Golgi compartment analogous to the medial Golgi cisternae of higher eukaryotes. We have characterized the synthesis, posttranslational modification, and localization of the yeast alpha 1,3 mannosyltransferase (Mnn1p) using antibodies prepared against a segment of this protein expressed in bacteria. Mnn1p is initially synthesized as a 98.5-kD, type II integral membrane glycoprotein that is modified with both N- and O-linked oligosaccharides. It is subject to a slow, incremental increase in molecular mass that is dependent upon protein transport to the Golgi complex. Self-modification of Mnn1p with alpha 1,3 mannose epitopes, primarily on O-linked oligosaccharides, is at least partly responsible for the incremental increase in molecular mass. Mnn1p is a resident protein of the Golgi complex and colocalizes with guanosine diphosphatase to at least two physically distinct Golgi compartments by sucrose gradient fractionation, one of which may be a late Golgi compartment that also contains the Kex2 endopeptidase. Surprisingly, we found that a significant fraction of Mnn1p is mislocalized to the plasma membrane in a clathrin heavy chain temperature sensitive mutant while guanosine diphosphatase remains intracellular. A mutant Mnn1p that lacks the NH2-terminal cytoplasmic tail is properly localized to the Golgi complex, indicating that clathrin does not mediate Mnnlp Golgi retention by a direct interaction with the Mnn1p cytoplasmic tail. These results indicate that clathrin plays a broader role in the localization of Golgi proteins than anticipated.

Amino Acid Sequence↗