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E Knecht

Publications and source records attributed to E Knecht.

At least 19 recordsLinked to original sources
PubMed2001

[Identification and characterization of the first Spanish familial ligand-defective apolipoprotein B homozygote].

BACKGROUND: Familial ligand-defective apolipoprotein B 100 (FDB) is an autosomal inherited disease due to mutations on apo B 100, clinically indistinguishable from familial hypercholesterolemia (FH). We described the first Spanish homozygote for FDB. METHODS: We have screened R3500Q mutation of apo B gene (PCR-SSCP analysis) in a large family with FDB and have identified the first Spanish homozygote for FDB. RESULTS: The homozygote is a 58 year-old man with coronary heart disease, no presence of xanthomata and with total cholesterol and LDL cholesterol plasma levels of 415 and 352 mg/dl. The response to statins and resins was up to 42% for total cholesterol and 51% for LDLc plasma values. The LDL receptor activity was normal in the FDB homozygote. CONCLUSIONS: We have identified and characterised the first Spanish homozygote for FDB (R3500Q mutation). Our data indicate a moderate lipoprotein phenotype in FDB homozygote, different as expected comparing to homozygous FH.

Apolipoprotein B-100↗
PubMed2000

Import of a cytosolic protein into lysosomes by chaperone-mediated autophagy depends on its folding state.

We have analyzed the folding state of cytosolic proteins imported in vitro into lysosomes, using an approach originally developed by Eilers and Schatz, (Eilers, M., and Schatz, G. (1986) Nature 322, 228-232) to investigate protein import into mitochondria. The susceptibility toward proteases of mouse dihydrofolate reductase (DHFR), synthesized in a coupled transcription-translation system with rabbit reticulocytes, decreased in the presence of its substrate analogue, methotrexate. This analogue complexes with high affinity with the in vitro synthesized DHFR and locks it into a protease-resistant folded conformation. DHFR was taken up by freshly isolated rat liver lysosomes and methotrexate reduced this uptake by about 80%. A chimeric DHFR protein, which carries the N-terminal presequence of subunit 9 of ATP synthase preprotein from Neurospora crassa fused to its N terminus, was taken up by lysosomes more efficiently. Again, methotrexate abolished the lysosomal uptake of the fusion protein, which was partially restored by washing of methotrexate from DHFR or by adding together methotrexate and dihydrofolate, the natural substrate of DHFR. Immunoblot analysis with anti-DHFR of liver lysosomes and of other fractions, isolated from rats starved for 88 h and treated with lysosomal inhibitors, suggests that DHFR is degraded by chaperone-mediated autophagy. Competition with ribonuclease A and stimulation by ATP/Mg(2+) and the heat shock cognate protein of 73 kDa show that the lysosomal uptake of the fusion protein also occurs by this pathway. It is concluded that the lysosomal uptake of cytosolic proteins by chaperone-mediated autophagy mainly occurs by passage of the unfolded proteins through the lysosomal membrane. Therefore, this mechanism is different from protein transport into peroxisomes, but similar to the import of proteins into the endoplasmic reticulum and mitochondria.

Animals↗
PubMed2000

Electrothermal atomic absorption spectrometric diagnosis of familial hypercholesterolemia.

We have developed a new nonradioactive assay to identify human low-density lipoprotein receptor defects. It is based on the incubation of cultured cells with colloidal gold-LDL conjugates and quantitation of the gold associated with the cells by electrothermal atomic absorption spectrometry. After an oxidative treatment with nitric and hydrochloric acids, the biological matrix interferes neither with the gold recovery nor with the gold measurements, which are linear, at least from 0.15 to 3 ng of gold. When cells expressing a functional LDL receptor are incubated with increasing amounts of colloidal-gold LDL conjugates, the obtained saturation curve parallels that described when [125I]LDL is used as ligand. Moreover, this new assay allows us to clearly distinguish among fibroblasts from normal subjects or from heterozygous or homozygous patients of familial hypercholesterolemia, a very common autosomal disease. The assay is easy to perform, is sensitive, and avoids the use of radioactive compounds. Therefore, it could be successfully employed in the clinical diagnosis of this disease. Furthermore, since the methodology developed here can be applied to quantify the association of other gold-conjugated ligands to cells, it could have a widespread use in a variety of clinical and basic research studies.

Animals↗
PubMed2000

Subcellular localization of proteasomes and their regulatory complexes in mammalian cells.

Proteasomes can exist in several different molecular forms in mammalian cells. The core 20S proteasome, containing the proteolytic sites, binds regulatory complexes at the ends of its cylindrical structure. Together with two 19S ATPase regulatory complexes it forms the 26S proteasome, which is involved in ubiquitin-dependent proteolysis. The 20S proteasome can also bind 11S regulatory complexes (REG, PA28) which play a role in antigen processing, as do the three variable gamma-interferon-inducible catalytic beta-subunits (e.g. LMP7). In the present study, we have investigated the subcellular distribution of the different forms of proteasomes using subunit specific antibodies. Both 20S proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic and microsomal preparations isolated from rat liver. LMP7 was enriched approximately two-fold compared with core alpha-type proteasome subunits in the microsomal preparations. 20S proteasomes were more abundant than 26S proteasomes, both in liver and cultured cell lines. Interestingly, some significant differences were observed in the distribution of different subunits of the 19S regulatory complexes. S12, and to a lesser extent p45, were found to be relatively enriched in nuclear fractions from rat liver, and immunofluorescent labelling of cultured cells with anti-p45 antibodies showed stronger labelling in the nucleus than in the cytoplasm. The REG was found to be localized predominantly in the cytoplasm. Three- to six-fold increases in the level of REG were observed following gamma-interferon treatment of cultured cells but gamma-interferon had no obvious effect on its subcellular distribution. These results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells and, therefore, that the distribution is more complex than has been reported for yeast proteasomes.

Adenosine Triphosphate↗
PubMed1999

The Escherichia coli trmE (mnmE) gene, involved in tRNA modification, codes for an evolutionarily conserved GTPase with unusual biochemical properties.

The evolutionarily conserved 50K protein of Escherichia coli, encoded by o454, contains a consensus GTP-binding motif. Here we show that 50K is a GTPase that differs extensively from regulatory GTPases such as p21. Thus, 50K exhibits a very high intrinsic GTPase hydrolysis rate, rather low affinity for GTP, and extremely low affinity for GDP. Moreover, it can form self-assemblies. Strikingly, the 17 kDa GTPase domain of 50K conserves the guanine nucleotide-binding and GTPase activities of the intact 50K molecule. Therefore, the structural requirements for GTP binding and GTP hydrolysis by 50K are without precedent and justify a separate classification in the GTPase superfamily. Immunoelectron microscopy reveals that 50K is a cytoplasmic protein partially associated with the inner membrane. We prove that o454 is allelic with trmE, a gene involved in the biosynthesis of the hypermodified nucleoside 5-methylaminomethyl-2-thiouridine, which is found in the wobble position of some tRNAs. Our results demonstrate that 50K is essential for viability depending on the genetic background. We propose that combination of mutations affecting the decoding process, which separately do not reveal an obvious defect in growth, can give rise to lethal phenotypes, most likely due to synergism.

Base Sequence↗
PubMed1998

A rapid procedure suitable to assess quantitatively the endocytosis of colloidal gold and its conjugates in cultured cells.

We measured the endocytic uptake of low-density lipoproteins (LDLs) conjugated to colloidal gold in cultured cells, either by counting gold particles on electron micrographs or by inductively coupled plasma (ICP) mass spectrometry (MS). Both procedures are comparable but the latter requires a considerably shorter time and allows analysis of a much larger sample. In addition, ICP MS, compared to alternative radioactive or fluorescent procedures, offers the major advantage of using the same probe to quantify the endocytic uptake and to follow it by electron microscopy. Therefore, ICP MS analysis provides an easy, rapid, and sensitive quantification of endocytosis that complements the electron microscopic studies.

Animals↗
PubMed1997

A population of rat liver lysosomes responsible for the selective uptake and degradation of cytosolic proteins.

Two populations of rat liver lysosomes can be distinguished on the basis of their density. A major difference between these populations is that one contains the heat shock cognate protein of 73 kDa (hsc73) within the lysosomal lumen. The lysosomal fraction containing hsc73 exhibits much higher efficiencies in the in vitro uptake and degradation of glyceraldehyde-3-phosphate dehydrogenase and ribonuclease A, two well established substrates of the selective lysosomal pathway of intracellular protein degradation. Preloading of the lysosomal population that is devoid of lumenal hsc73 with hsc73 isolated from cytosol activated the selective transport of substrate proteins into these lysosomes. Furthermore, treatment of animals with leupeptin, an inhibitor of lysosomal cathepsins, or 88 h of starvation also increased the amount of hsc73 within their lysosomal lumen, and these in vivo treatments also activated the selective transport of substrate proteins in vitro. Thus, the hsc73 located within lysosomes appears to be required for efficient uptake of cytosolic proteins by these organelles. The difference in hsc73 content between the lysosomal populations appears to be due to differences in their ability to take up hsc73 combined with differences in the intralysosomal degradation rates of hsc73. The increased stability of hsc73 in one population of lysosomes is primarily a consequence of this lysosomal population's more acidic pH.

Adenosine Triphosphate↗
PubMed1997

Acidic cytosolic proteins are preferentially imported into rat liver lysosomes.

Previous studies have reported that lysosomes isolated from human diploid fibroblasts and from rat liver can selectively import and degrade specific proteins. We have now reinvestigated this selectivity using an in vitro assay with rat liver lysosomes and an extract of cytosolic proteins prepared from cultured cells labeled to equilibriums with [35S-]methionine. Analysis by two-dimensional gel electrophoresis and autoradiography of the cytosolic proteins bound to the lysosomal membrane and imported into the lysosomes shows that when all cytosolic proteins are simultaneously present in the in vitro assay the lysosomal uptake also occurs in a specific manner. These findings suggest that isolated lysosomes are able to discriminate among different proteins, selecting those with certain features for lysosomal degradation. Additional characterization of the cytosolic proteins which are selectively imported by lysosomes shows that a common structural feature of most, but not all, of these proteins is an acidic isoelectric point (pI <6.0) and a small or intermediate size. This observation is in agreement with earlier studies which established a relationship between the in vivo half-lives of cytosolic proteins in rat liver and their net charge, with acidic proteins, in general, being degraded more rapidly than neutral or basic proteins. The reasons for this preference are still uncertain, although a possible explanation is presented.

Acids↗
PubMed1996

Use of inductively coupled plasma-mass spectrometry for the quantitation of the binding and uptake of colloidal gold-low-density lipoprotein conjugates by cultured cells.

The binding and endocytic uptake of low-density lipoprotein (LDL) particles by cells, transiently or permanently transfected with the human LDL receptor cDNA, was investigated, under different situations, using colloidal gold-LDL conjugates. The amount of gold associated with the various cells, which bind and internalize LDL to different extents, was estimated by inductively coupled plasma-MS. In all cases, the existing differences in LDL binding and uptake were clearly detectable with this procedure. We conclude, therefore, that inductively coupled plasma-MS provides an appropriate assay system for the rapid quantitation of these processes. This procedure also recognizes differences in LDL receptor expression in human lymphocytes and, therefore, it could be of value for the differential diagnosis of LDL receptor defects in familial hypercholesterolemia in various cell types. In addition, this easily performed methodology can also be applied to a variety of other problems requiring quantitation of colloidal gold associated with cells.

Animals↗
PubMed1996

Selective uptake and degradation of c-Fos and v-Fos by rat liver lysosomes.

The transcription factor c-Fos is a short-lived protein and calpains and ubiquitin-dependent systems have been proposed to be involved in its degradation. In this report, we consider a lysosomal degradation pathway for c-Fos. Using a cell-free assay, we have found that freshly isolated lysosomes can take up and degrade c-Fos with high efficiency. v-Fos, the oncogenic counterpart of c-Fos, can also be taken up by lysosomes, yet the amount of incorporated protein is much lower. c-Fos uptake is independent of its phosphorylation state but it appears to be regulated by dimerization with differentially phosphorylated forms of c-Jun, while v-Fos escapes this regulation. Moreover, we show that c-Fos is immunologically detected in lysosomes isolated from the liver of rats treated with the protease inhibitor leupeptin. Altogether, these results suggest that lysosomes can also participate in the selective degradation of c-Fos in rat liver.

Animals↗
PubMed1996

Subpopulations of proteasomes in rat liver nuclei, microsomes and cytosol.

Mammalian proteasomes are composed of 14-17 different types of subunits, some of which, including major-histocompatibility-complex-encoded subunits LMP2 and LMP7, are non-essential and present in variable amounts. We have investigated the distribution of total proteasomes and some individual subunits in rat liver by quantitative immunoblot analysis of purified subcellular fractions (nuclei, mitochondria, microsomes and cytosol). Proteasomes were mainly found in the cytosol but were also present in the purified nuclear and microsomal fractions. In the nuclei, proteasomes were soluble or loosely attached to the chromatin, since they could be easily extracted by treatment with nucleases or high concentrations of salt. In the microsomes, proteasomes were on the outside of the membranes. Further subfractionation of the microsomes showed that the proteasomes in this fraction were associated with the smooth endoplasmic reticulum and with the cis-Golgi but were practically absent from the rough endoplasmic reticulum. Using monospecific antibodies for some proteasomal subunits (C8, C9, LMP2 and Z), the composition of proteasomes in nuclei, microsomes and cytosol was investigated. Although there appear not to be differences in proteasome composition in the alpha subunits (C8 and C9) in the different locations, the relative amounts of some beta subunits varied. Subunit Z was enriched in nuclear proteasomes but low in microsome-associated proteasomes, whereas LMP2, which was relatively low in nuclei, showed a small enrichment in the microsomes. These differences in subunit composition of proteasomes probably reflect differences in the function of proteasomes in distinct cell compartments.

Animals↗
PubMed1995

Degradation of proteasomes by lysosomes in rat liver.

Proteasomes are high-molecular-mass multisubunit complexes which are believed, either by themselves or as a part of the 26S proteinase complex, to play a central role in extralysosomal pathways of intracellular protein breakdown. We have addressed the degradation of proteasomes in rat liver, investigating the possible role of lysosomes. Affinity-purified antibodies against rat liver proteasomes were used for immunoblot analysis of isolated lysosomes. Although proteasomes are not found in lysosomes from normally fed rats, they were found to accumulate in lysosomes of rats treated with leupeptin (an inhibitor of lysosomal proteases) and could also be detected in lysosomes isolated from livers of starved (24 h) rats. Proteinase-K treatment of these fractions, as well as immunogold procedures, show that a proportion of the proteasomes are inside lysosomes. Comparison of the amount of proteasomes found in lysosomes by immunoblotting with their experimentally determined half life (8.3 days) is consistent with an important role of these organelles in the degradation of rat liver proteasomes. Nevertheless, these data do not exclude the possibility that some nonlysosomal degradation of proteasome components also occurs. Since proteasomes were localized in autophagic vacuoles, it is likely that they are taken up mainly by nonselective autophagy. However, using an in vitro system, it was found that, under conditions of starvation, proteasomes may also be taken up into lysosomes and degraded via the heat-shock cognate protein of 73 kDa (hsc73)-mediated transport.

Animals↗
PubMed1995

Activation of a selective pathway of lysosomal proteolysis in rat liver by prolonged starvation.

Lysosomal uptake and degradation of polypeptides such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribonuclease A (RNase A), and RNase S-peptide (residues 1-20 of RNase A) are progressively activated in rat liver by starvation before isolation of lysosomes. This pathway of proteolysis is selective, since it is stimulated by the heat shock cognate protein of 73 kDa (HSC73) and ATP-MgCl2, and lysosomal uptake of RNase A could be competed by GAPDH but not by ovalbumin. A portion of intracellular HSC73 is associated with certain lysosomes, and the amount of lysosomal HSC73 increases by 5- to 10-fold during prolonged starvation. The lysosome-associated HSC73 is primarily within the lysosomal lumen. Double immunogold labeling of lysosomes incubated in vitro with RNase A detects this protein substrate as well as HSC73 within lysosomes. More than two-thirds of the labeled lysosomes contain both RNase A and HSC73. The possible physiological significance of the activation of this selective pathway of lysosomal proteolysis in long-term starvation is discussed.

Animals↗
PubMed1994

Selective binding and uptake of ribonuclease A and glyceraldehyde-3-phosphate dehydrogenase by isolated rat liver lysosomes.

Ribonuclease A (RNase A) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are selectively taken up and degraded by isolated rat liver lysosomes by very similar processes. The uptake and degradation of both of these proteins are stimulated by the heat shock cognate protein of 73 kDa and ATP/Mg2+. Both binding and uptake of RNase A and GAPDH by lysosomes are saturable, and uptake of RNase A and GAPDH requires a protease-sensitive component within the lysosomal membrane. GAPDH competes for binding and uptake of RNase A by lysosomes and vice versa while another protein, ovalbumin, does not compete. RNase S-peptide (amino acids 1-20 of RNase A) also competes for RNase A binding and uptake by lysosomes, while RNase S-protein (amino acids 21-124 of RNase A) does not compete. The uptake of RNase A by lysosomes appears to involve an intermediate step in which approximately 2 kDa of the polypeptide's COOH terminus remains outside lysosomes while the remainder is inside the lysosomal lumen.

Amino Acid Sequence↗
PubMed1994

Changes in proteasome localization during the cell cycle.

We have investigated proteasome localization in synchronized cells using polyclonal anti-proteasome antibodies. Proteasomes were localized in the nucleus and cytoplasm at all phases of the cycle, but changes in localization were observed which explain the different immunofluorescence patterns found in asynchronous cells. In the nucleus, the intensity of staining in early S phase was low and showed a punctate distribution which changed to a more diffuse and intense labeling during S to G1. In the cytoplasm, proteasomes were concentrated in the perinuclear region at G1 and at the start of S phase and gradually moved towards the periphery of the cell as the cell cycle progressed to G2. No cell cycle-dependent changes were detected in the rate of synthesis or level of proteasomes. An apparent colocalization of proteasomes with elements of the cytoskeleton mainly observed in G2 was investigated further in PtK2 cells. The overall distribution of proteasomes and cell cycle-dependent changes in PtK2 cells were similar to those in L-132 cells. Double-label immunofluorescence studies using anti-proteasome and anti-cytokeratin (TROMA-1) antibodies showed that proteasomes do colocalize with intermediate filaments of the cytokeratin type, mainly during G2. In mitosis, proteasomes were found by immunogold electron microscopy to be localized around the chromosomes in both PtK2 and L-132 cells. Cell cycle-dependent changes in the localization of proteasomes suggest that they may have a regulatory function related to the cell cycle, for example, in the degradation of proteins which control its progression.

Animals↗
PubMed1993

Uptake and degradation of glyceraldehyde-3-phosphate dehydrogenase by rat liver lysosomes.

The molecular mechanisms involved in the degradation of individual cellular proteins are probably unique and characteristic. We have investigated in rat liver the degradation of glyceraldehyde-3-phosphate dehydrogenase, an abundant cytosolic enzyme of the glycolytic pathway. Immunoblot analysis of isolated liver lysosomes from rats treated with lysosomal inhibitors show that this protein is degraded, at least in part, by a lysosomal pathway. This pathway was further investigated by incubating the enzyme with lysosomes in a cell-free system, followed by proteolysis measurements, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of lysosomes, and electron microscopic immunocytochemistry. We postulate that the degradative mechanism of glyceraldehyde-3-phosphate dehydrogenase includes a temperature-dependent lysosomal pathway, different from classical nonspecific macroautophagy. The postulated pathway involves: binding of the enzyme to the lysosomal membrane, entry into the lysosomal matrix, and degradation. This cell-free system, which can also incorporate in vitro synthesized proteins, should allow further advances toward clarifying the complex signals that regulate protein degradation as well as its close interrelationship with protein synthesis.

Animals↗
PubMed1993

Mitosis and protein synthesis 4 turnover of short- and long-lived proteins during the cell cycle of synchronized L-132 and HeLa S-3 cells.

Turnover rates of short- and long-lived proteins throughout the cell cycle were measured in two established mammalian cell lines, L-132 and HeLa S-3, using both selection and induction synchronization. Short-lived, newly synthesized proteins turned over at the same rate during all stages of the cell cycle when this rate was expressed as a percentage of total labelling of proteins present at the start of the chase. Since fewer proteins were made in M-phase, the absolute turnover rate was probably reduced to a small degree during division itself, indicating a close co-ordination between synthesis and degradation. In contrast, long-lived proteins showed a considerable reduction in their rate of turnover specifically during M-phase. One possible explanation for the reduced degradation of long-lived protein is the suppression of lysosomal activity during division, with no apparent effect on the turnover of short-lived proteins which is due to a non-lysosomal system.

Cell Cycle↗