Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mutations in the presenilin genes, PS1 and PS2, cause a major portion of early onset familial Alzheimer's disease (FAD). The biological roles of the presenilins and how their pathological mutations confer FAD are unknown. In this study, we set out to examine the processing and degradation pathways of PS2. For regulated expression of PS2, we have established inducible cell lines expressing PS2 under the tight control of the tetracycline-responsive transactivator. Western blot analysis revealed that PS2 was detected as an approximately 53-55-kDa polypeptide (54-kDa PS2) as well as a high molecular mass form (HMW-PS2). Using a stably transfected, inducible cell system, we have found that PS2 is proteolytically cleaved into two stable cellular polypeptides including an approximately 20-kDa C-terminal fragment and an approximately 34-kDa N-terminal fragment. PS2 is polyubiquitinated in vivo, and the degradation of PS2 is inhibited by proteasome inhibitors, N-acetyl-L-leucinal-L-norleucinal and lactacystin. Our studies suggest that PS2 normally undergoes endoproteolytic cleavage and is degraded via the proteasome pathway.
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PMID:Endoproteolytic cleavage and proteasomal degradation of presenilin 2 in transfected cells. 911 Sep 91

The majority of cases of early-onset familial Alzheimer disease are caused by mutations in the recently identified presenilin 1 (PS 1) gene, located on chromosome 14. PS1, a 467 amino acid protein, is predicted to be an integral membrane protein containing seven putative transmembrane domains and a large hydrophilic loop between the sixth and seventh membrane-spanning domain. We produced 7 monoclonal antibodies that react with 3 non-overlapping epitopes on the N-terminal hydrophilic tail of PS1. The monoclonal antibodies can detect the full size PS1 at M(r) 47,000 (47K) and a more abundant M(r) 28,000 (28K) product in membrane from human brain and human cell lines. We examined the sub-cellular localization by using these antibodies. Immuno-electronmicroscopic and biochemical analysis indicated that PS1 is localized on cellular membrane (plasma, endoplasmic reticulum, and perinuclear) in COS-7 cells overexpressing PS1. Interestingly, the PS1 immunoreactivity in the plasma membrane was concentrated in the regions with cell-cell contact. This observation suggests a possible role of PS1 on the cell membrane as a cell adhesion molecule. To determine the protease cleaving the full length PS1 to two fragments, we treated cells with various protease inhibitors. Only proteasome inhibitor affected the PS1 processing, indicating that proteasome is a candidate protease for PS1 proteolytic cleavage. PC12 cells transiently transfected with PS1 constructs containing different Alzheimer mutations fail to generate the 28K degradation product in contrast to PC12 cells transfected with wild type PS1. Our results indicate that missense mutations in this form of familial Alzheimer disease may act via a mechanism of impaired proteolytic processing of PS1.
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PMID:[Biochemistry of presenilin 1]. 965 56

Most of early onset familial forms of Alzheimer's disease (FAD) are due to inherited mutations located on two homologous proteins, presenilins 1 and 2 (PS1 and PS2) encoded by chromosomes 14 and 1, respectively. Here we show that the expression of wild type (wt)-PS2 in human HEK293 cells increases the production of the physiological alpha-secretase-derived product, APPalpha. By contrast, APPalpha secretion is drastically reduced in cells expressing the FAD-linked N141I-PS2. We establish that wt-PS2, N141I-PS2 and their C-terminal maturation fragment are degraded by the enzymatic multicatalytic complex, proteasome. Interestingly, two selective proteasome inhibitors, Z-IE(Ot-Bu)A-Leucinal and lactacystin potentiate the APPalpha secretion observed in wtPS2-expressing cells and further amplify the N141I-PS2-induced decrease in APPalpha production. By contrast, a series of pharmacological agents unable to affect the proteasome do not modify PS2 immunoreactivities and APPalpha recoveries. Altogether, our data indicate that: 1) wtPS2 positively modulates the alpha-secretase physiological pathway of betaAPP maturation in human cells; 2) N141I mutation on PS2 drastically lowers the secretion of APPalpha; 3) Proteasome inhibitors prevent the degradation of wtPS2, N141I-PS2 and their C-terminal maturation product. This protection against proteasomal degradation directly modulates the APPalpha secretion response elicited by wt- and FAD-linked PS2 expression in human HEK293 cells.
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PMID:Alzheimer's disease-linked mutation of presenilin 2 (N141I-PS2) drastically lowers APPalpha secretion: control by the proteasome. 981 58

Numerous mutations causing early onset Alzheimer's disease have been identified in the presenilin (PS) genes, particularly the PS1 gene. Like the mutations identified within the beta-amyloid precursor protein gene, PS mutations cause the increased generation of a highly neurotoxic variant of amyloid beta-peptide. PS proteins are proteolytically processed to an N-terminal approximately 30-kDa (NTF) and a C-terminal approximately 20-kDa fragment (CTF20) that form a heterodimeric complex. We demonstrate that this complex is resistant to proteolytic degradation, whereas the full-length precursor is rapidly degraded. Degradation of the PS1 holoprotein is sensitive to inhibitors of the proteasome. Formation of a heterodimeric complex is required for the stability of both PS1 fragments, since fragments that do not co-immunoprecipitate with the PS complex are rapidly degraded by the proteasome. Mutant PS fragments not incorporated into the heterodimeric complex lose their pathological activity in abnormal amyloid beta-peptide generation even after inhibition of their proteolytic degradation. The PS1 heterodimeric complex can be attacked by proteinases of the caspase superfamily that generate an approximately 10-kDa proteolytic fragment (CTF10) from CTF20. CTF10 is rapidly degraded most likely by a calpain-like cysteine proteinase. From these data we conclude that PS1 metabolism is highly controlled by multiple proteolytic activities indicating that subtle changes in fragment generation/degradation might be important for Alzheimer's disease-associated pathology.
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PMID:Expression of Alzheimer's disease-associated presenilin-1 is controlled by proteolytic degradation and complex formation. 982 12

Unassembled immunoglobulin light chains expressed by the mouse plasmacytoma cell line NS1 (kappa(NS1)) are degraded in vivo with a half-life of 50-60 min in a way that closely resembles endoplasmic reticulum (ER)-associated degradation (). Here we show that the peptide aldehydes MG132 and PS1 and the specific proteasome inhibitor lactacystin effectively increased the half-life of kappa(NS1), arguing for a proteasome-mediated degradation pathway. Subcellular fractionation and protease protection assays have indicated an ER localization of kappa(NS1) upon proteasome inhibition. This was independently confirmed by the analysis of the folding state of kappa(NS1) and size fractionation experiments showing that the immunoglobulin light chain remained bound to the ER chaperone BiP when the activity of the proteasome was blocked. Moreover, kinetic studies performed in lactacystin-treated cells revealed a time-dependent increase in the physical stability of the BiP-kappa(NS1) complex, suggesting that additional proteins are present in the older complex. Together, our data support a model for ER-associated degradation in which both the release of a soluble nonglycosylated protein from BiP and its retrotranslocation out of the ER are tightly coupled with proteasome activity.
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PMID:Dissociation from BiP and retrotranslocation of unassembled immunoglobulin light chains are tightly coupled to proteasome activity. 1063 3

Modifier of cell adhesion protein (MOCA; previously called presenilin [PS] binding protein) is a DOCK180-related molecule, which interacts with PS1 and PS2, is localized to brain areas involved in Alzheimer's disease (AD) pathology, and is lost from the soluble fraction of sporadic Alzheimer's disease (AD) brains. Because PS1 has been associated with gamma-secretase activity, MOCA may be involved in the regulation of beta-amyloid precursor protein (APP) processing. Here we show that the expression of MOCA decreases both APP and amyloid beta-peptide secretion and lowers the rate of cell-substratum adhesion. In contrast, MOCA does not lower the secretion of amyloid precursor-like protein (APLP) or several additional type 1 membrane proteins. The phenotypic changes caused by MOCA are due to an acceleration in the rate of intracellular APP degradation. The effect of MOCA expression on the secretion of APP and cellular adhesion is reversed by proteasome inhibitors, suggesting that MOCA directs nascent APP to proteasomes for destruction. It is concluded that MOCA plays a major role in APP metabolism and that the effect of MOCA on APP secretion and cell adhesion is a downstream consequence of MOCA-directed APP catabolism. This is a new mechanism by which the expression of APP is regulated.
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PMID:A novel mechanism for the regulation of amyloid precursor protein metabolism. 1209 89

The gamma-secretase complex catalyzes intramembrane proteolysis of a number of transmembrane proteins, including amyloid precursor protein, Notch, ErbB4, and E-cadherin. gamma-Secretase is known to contain four major protein constituents: presenilin (PS), nicastrin, Aph-1, and Pen-2, all of which are integral membrane proteins. There is increasing evidence that the formation of the complex and the stability of the individual components are tightly controlled in the cell, assuring correct composition of functional complexes. In this report, we investigate the topology, localization, and mechanism for destabilization of Pen-2 in relation to PS function. We show that PS1 regulates the subcellular localization of Pen-2: in the absence of PS, Pen-2 is sequestered in the endoplasmic reticulum (ER) and not transported to post-ER compartments, where the mature gamma-secretase complexes reside. PS deficiency also leads to destabilization of Pen-2, which is alleviated by proteasome inhibitors. In keeping with this, we show that Pen-2, which adopts a hairpin structure with the N and C termini facing the luminal space, is ubiquitylated prior to degradation and presumably retrotranslocated from the ER to the cytoplasm. Collectively, our data suggest that failure to become incorporated into the gamma-secretase complex leads to degradation of Pen-2 through the ER-associated degradation-proteasome pathway.
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PMID:Pen-2 is sequestered in the endoplasmic reticulum and subjected to ubiquitylation and proteasome-mediated degradation in the absence of presenilin. 1472 71

Mutations in presenilin proteins (PS1 and PS2) lead to early-onset Alzheimer's disease. PS proteins are endoproteolytically cleaved into two main fragments: the NTF (PS N-terminal fragment) and the CTF (PS C-terminal fragment). The two fragments are believed to constitute the core catalytic enzyme activity called gamma-secretase, which is responsible for cleaving beta-amyloid precursor protein to release Abeta. Thus, studying factors that modulate PS fragment levels could provide important information about gamma-secretase. Previously, we demonstrated that the protein, ubiquilin-1, interacts both in vivo and in vitro with PS and that overexpression of ubiquilin-1 or -2 leads to increased accumulation of full-length PS proteins. Using wild-type HEK-293 cells (human embryonic kidney 293 cells) and PS-inducible cells, we now show that overexpression of either ubiquilin-1 or -2 decreases the PS NTF and CTF levels. Conversely, siRNA (small interfering RNA)-mediated knockdown of ubiquilin-1 and -2 proteins increased the PS NTF and CTF levels. We considered that ubiquilin might alter PS fragment accumulation by acting as a shuttle factor escorting PS fragments to the proteasome for degradation. However, through proteasome inhibition studies, we show that this does not occur. Instead, our results suggest that ubiquilin regulates PS fragment production. We also examined whether other components of the gamma-secretase complex are affected by ubiquilin expression. Interestingly, overexpression of ubiquilin resulted in a decrease in Pen-2 and nicastrin levels, two essential components of the gamma-secretase complex. In contrast, knockdown of ubiquilin-1 and -2 protein expression by RNAi (RNA interference) increased Pen-2 and nicastrin levels. Finally, we show that inhibition of the proteasome results in decreased PS fragment production and that reversal of proteasome inhibition restores PS fragment production, suggesting that the proteasome may be involved in PS endoproteolysis. These studies implicate ubiquilin as an important factor in regulating PS biogenesis and metabolism.
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PMID:Ubiquilin regulates presenilin endoproteolysis and modulates gamma-secretase components, Pen-2 and nicastrin. 1597 90

Mammalian metabolism of ingested cysteine is conducted principally within the liver. The liver tightly regulates its intracellular cysteine pool to keep levels high enough to meet the many catabolic and anabolic pathways for which cysteine is needed, but low enough to prevent toxicity. One of the enzymes the liver uses to regulate cysteine levels is CDO (cysteine dioxygenase). Catalysing the irreversible oxidation of cysteine, CDO protein is up-regulated in the liver in response to the dietary intake of cysteine. In the present study, we have evaluated the contribution of the ubiquitin-26 S proteasome pathway to the diet-induced changes in CDO half-life. In the living rat, inhibition of the proteasome with PS1 (proteasome inhibitor 1) dramatically stabilized CDO in the liver under dietary conditions that normally favour its degradation. Ubiquitinated CDO intermediates were also seen to accumulate in the liver. Metabolic analyses showed that PS1 had a significant effect on sulphoxidation flux secondary to the stabilization of CDO but no significant effect on the intracellular cysteine pool. Finally, by a combination of in vitro hepatocyte culture and in vivo whole animal studies, we were able to attribute the changes in CDO stability specifically to cysteine rather than the metabolite 2-mercaptoethylamine (cysteamine). The present study represents the first demonstration of regulated ubiquitination and degradation of a protein in a living mammal, inhibition of which had dramatic effects on cysteine catabolism.
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PMID:Regulation of cysteine dioxygenase degradation is mediated by intracellular cysteine levels and the ubiquitin-26 S proteasome system in the living rat. 1626 2

PS (presenilin)-dependent gamma-secretase occurs as a high-molecular-mass complex composed of either PS1 or PS2 associated with Nct (nicastrin), PEN2 (presenilin enhancer 2 homologue) and APH1 (anterior pharynx defective 1 homologue). Numerous reports have documented the very complicated physical and functional cross-talk between these proteins that ultimately governs the biological activity of the gamma-secretase, but very few studies examined the fate of the components of the complex. We show that, in both HEK-293 cells and the TSM1 neuronal cell line, the immunoreactivities of overexpressed myc-tagged-APH1a and -PEN2 were enhanced by the proteasome inhibitors ZIE and lactacystin, whereas a broad range of protease inhibitors had no effect. By contrast, proteasome inhibitors were totally unable to affect the cellular expression of endogenous APH1aL and PEN2 in HEK-293 cells, TSM1 and primary cultured cortical neurons. To explain this apparent discrepancy, we examined the degradation of myc-tagged-APH1a and -PEN2, in vitro, by cell extracts containing endogenous proteasome and by purified 20S proteasome. Strikingly, myc-tagged-APH1a and -PEN2 resist proteolysis by endogenous proteasome and purified 20S proteasome. We also show that endogenous PEN2 expression was drastically higher in wild-type than in PS- and Nct-deficient fibroblasts and was enhanced by proteasome inhibitors only in the two deficient cell systems. However, here again, purified 20S proteasome appeared unable to cleave endogenous PEN2 present in PS-deficient fibroblasts. The levels of endogenous APH1aL-like immunoreactivity were not modified by proteasome inhibitors and were unaffected by PS deficiency. Altogether, our results indicate that endogenous PEN2 and APH1aL do not undergo proteasomal degradation under physiological conditions in HEK-293 cells, TSM1 cells and fibroblasts and that the clearance of PEN2 in PS- and Nct-deficient fibroblasts is not mediated by 20S proteasome. Whether the 26S proteasome participates to PEN2 proteolysis in deficient fibroblasts remains to be established.
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PMID:Catabolism of endogenous and overexpressed APH1a and PEN2: evidence for artifactual involvement of the proteasome in the degradation of overexpressed proteins. 1630 45


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