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)

The selectivity underlying the recognition of oxidized calmodulin (CaM) by the 20S proteasome in complex with Hsp90 was identified using mass spectrometry. We find that degradation of oxidized CaM (CaMox) occurs in a multistep process, which involves an initial cleavage that releases a large N-terminal fragment (A1-F92) as well as multiple smaller carboxyl-terminus peptides ranging from 17 to 26 amino acids in length. These latter small peptides are enriched in methionine sulfoxides (MetO), suggesting a preferential degradation around MetO within the carboxyl-terminal domain. To confirm the specificity of CaMox degradation and to identify the structural signals underlying the preferential recognition and degradation by the proteasome/Hsp90, we have investigated how the oxidation of individual methionines affect the degradation of CaM using mutants in which all but selected methionines in CaM were substituted with leucines. Substitution of all methionines with leucines except Met144 and Met145 has no detectable effect on the structure of CaM, permitting a determination of how site-specific substitutions and the oxidation of Met144 and Met145 affects the recognition and degradation of CaM by the proteasome/Hsp90. Comparable rates of degradation are observed upon the selective oxidation of Met144 and Met145 in CaM-L7 relative to that observed upon oxidation of all nine methionines in wild-type CaM. Substitution of leucines for either Met144 or Met145 promotes a limited recognition and degradation by the proteasome that correlates with decreases in the helical content of CaM. The specific oxidation of Met144 has little effect on rates of proteolytic degradation by the proteasome/Hsp90 or the structure of CaM. In contrast, the specific oxidation of Met145 results in both large increases in the rate of degradation by the proteasome/Hsp90 and significant circular dichroic spectral shape changes that are indicative of changes in tertiary rather than secondary structure. Thus, tertiary structural changes resulting from the site-specific oxidation of a single methionine (i.e., Met145) promote the degradation of CaM by the proteasome/Hsp90, suggesting a mechanism to regulate cellular metabolism through the targeted modulation of CaM abundance in response to oxidative stress.
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PMID:Tertiary structural rearrangements upon oxidation of Methionine145 in calmodulin promotes targeted proteasomal degradation. 1675 Dec 45

Activation of death-associated protein kinase (DAPK) occurs via dephosphorylation of Ser-308 and subsequent association of calcium/calmodulin. In this study, we confirmed the existence of the alternatively spliced human DAPK-beta, and we examined the levels of DAPK autophosphorylation and DAPK catalytic activity in response to tumor necrosis factor or ceramide. It was found that DAPK is rapidly dephosphorylated in response to tumor necrosis factor or ceramide and then subsequently degraded via proteasome activity. Dephosphorylation and activation of DAPK are shown to temporally precede its subsequent degradation. This results in an initial increase in kinase activity followed by a decrease in DAPK expression and activity. The decline in DAPK expression is paralleled with increased caspase activity and cell apoptosis. These results suggest that the apoptosis regulatory activities mediated by DAPK are controlled both by phosphorylation status and protein stability.
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PMID:Control of death-associated protein kinase (DAPK) activity by phosphorylation and proteasomal degradation. 1705 2

Cyclin-dependent kinase inhibitor p27(kip1) (p27), a critical determinant for cell cycle progression, is an important regulation target of mitogenic signals. We have recently reported the existence of a molecular link between decreased p27 levels and enhanced phosphorylation of pRb protein and proliferation of immortalized lymphocytes from Alzheimer's disease (AD) patients. These cell cycle disturbances might be considered systemic manifestations, which mirror changes thought to occur in the brain, where post-mitotic neurons have been shown to display various cell cycle markers prior to degeneration. This work was undertaken to delineate the molecular mechanisms underlying the p27 down-regulation associated with AD. To this end, we evaluated the p27 protein stability in control and AD lymphoblasts. Half-life of p27 protein was markedly reduced in lymphoblasts from AD patients compared with that in control cells. The increased phosphorylation of p27 at Thr187, rather than changes in the 26S proteasome activity, is likely responsible for the enhanced degradation of p27 in AD cells. The serum-induced enhanced proliferation of AD lymphoblasts and decreased levels of p27 were abrogated by calmodulin (CaM) antagonists. The findings presented here suggest that Ca(2+)/CaM-dependent overactivation of PI3K/Akt signaling cascade in AD cells, plays an important role in regulating p27 abundance by increasing its degradation in the ubiquitin-proteasome pathway.
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PMID:Enhanced proteasome-dependent degradation of the CDK inhibitor p27(kip1) in immortalized lymphocytes from Alzheimer's dementia patients. 1744 72

Oxygen homeostasis represents an essential organizing principle of metazoan evolution and biology. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of transcriptional responses to changes in O2 concentration. HIF-1 is a heterodimer of HIF-1alpha and HIF-1beta subunits. O2-dependent degradation of the HIF-1alpha subunit is mediated by prolyl hydroxylase, von Hippel-Lindau protein (VHL)/Elongin-C E3 ubiquitin ligase, and the proteasome. O2-independent degradation of HIF-1alpha is regulated by the competition of RACK1 and HSP90 for binding to HIF-1alpha. RACK1 binding results in the recruitment of the Elongin-C E3 ubiquitin ligase, leading to VHL-independent ubiquitination and degradation of HIF-1alpha. In this report, we show that calcineurin inhibits the ubiquitination and proteasomal degradation of HIF-1alpha. Calcineurin is a serine/threonine phosphatase that is activated by calcium and calmodulin. The phosphatase activity of calcineurin is required for its regulation of HIF-1alpha. RACK1 binds to the catalytic domain of calcineurin and is required for HIF-1alpha degradation induced by the calcineurin inhibitor cyclosporine A. Elongin-C and HIF-1alpha each bind to RACK1 and dimerization of RACK1 is required to recruit Elongin-C to HIF-1alpha. Phosphorylation of RACK1 promotes its dimerization and dephosphorylation by calcineurin inhibits dimerization. Serine 146 within the dimerization domain is phosphorylated and mutation of serine 146 impairs RACK1 dimerization and HIF-1alpha degradation. These results indicate that intracellular calcium levels can regulate HIF-1alpha expression by modulating calcineurin activity and RACK1 dimerization.
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PMID:Calcineurin promotes hypoxia-inducible factor 1alpha expression by dephosphorylating RACK1 and blocking RACK1 dimerization. 1796 24

Neprilysin 2 (NEP2) has been recently identified as a new member of the M13 subfamily of zinc-dependent metalloproteases and shares a highly homologous amino acid sequence with neprilysin (EC 3.4.24.11, NEP). NEP2 has been reported to exist as membrane-bound and soluble secreted variants. To investigate mechanisms of regulating NEP2 activity, we developed a simple and sensitive method for measuring NEP2 activity using synthetic substrates with a fluorescent probe. NEP2 only cleaved Suc-Ala-Ala-Phe-AMC, while NEP cleaved both Dansyl-D-Ala-Gly-p-nitro-Phe-Gly and Suc-Ala-Ala-Phe-AMC. Using HEK293 cells stably expressing mouse NEP2, we evaluated the effects of various reagents affecting post-translational modification and protein trafficking on extracellular NEP2 activity secreted into the culture medium. Inhibition of N-glycosylation by tunicamycin reduced both the enzymatic activity of extracellular NEP2 and the molecular size of intracellular NEP2. Disruption of the Golgi apparatus with brefeldin A markedly reduced extracellular NEP2 activity in parallel with intracellular NEP2 protein level in HEK293 cells. In contrast, the cytoskeleton disrupting reagents, nocodazole and cytochalasin B barely affected NEP2 activity. Two distinct calcium-perturbing reagents, a calcium ionophore A23187 and thapsigargin, reduced extracellular NEP2 activity. However, A23187-mediated down-regulation was not rescued by co-treatment with inhibitors of MAPK, calmodulin, or the proteasome/calpains. In conclusion, we established a simple and sensitive protocol which was able to discriminate NEP2 and NEP activity, and showed that intracellular transport and secretion of NEP2 is regulated by processes such as glycosylation, ER-Golgi transport, and intracellular calcium levels.
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PMID:Biosynthesis, processing, trafficking, and enzymatic activity of mouse neprilysin 2. 1842 24

Oxidized proteins are recognized and degraded preferentially by the proteasome. This is true for numerous proteins including calmodulin (CaM). The degradation of CaM was investigated in a human fibroblast cell line under conditions of oxidative stress. Low molecular CaM fragments or peptides were found under such conditions. In in vitro experiments it was investigated whether this CaM breakdown product formation is induced by protein oxidation or is due to a limited proteolysis-derived degradation by the 20S proteasome. Native unoxidized CaM was not degraded by 20S proteasome, oxidized CaM was degraded in a time- and H2O2 concentration-dependent manner. Peptides of similar molecular weight were detected in isolated calmodulin as in oxidatively stressed fibroblasts. The peptides were identified using isolated calmodulin. Therefore, in oxidatively stressed fibroblasts and in vitro CaM is forming oxidation-driven fragments and proteasomal cleavage peptides of approximately 30 amino acids which undergo a slow or no degradation.
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PMID:Limited degradation of oxidized calmodulin by proteasome: formation of peptides. 1845 97

Since androgen receptor (AR) plays an important role in prostate cancer development and progression, androgen-ablation has been the frontline therapy for treatment of advanced prostate cancer even though it is rarely curative. A curative strategy should involve functional and structural elimination of AR from prostate cancer cells. We have previously reported that apoptosis induced by medicinal proteasome-inhibitory compound celastrol is associated with a decrease in AR protein levels. However celastrol-stimulated events contributing to this AR decrease have not been elucidated. Here, we report that a variety of chemotherapeutic agents, including proteasome inhibitors, a topoisomerase inhibitor, DNA-damaging agents and docetaxel that cause cell death, decrease AR levels in LNCaP prostate cancer cells. This decrease in AR protein levels was not due to the suppression of AR mRNA expression in these cells. We observed that a proteolytic activity residing in cytosol of prostate cancer cells is responsible for AR breakdown and that this proteolytic activity was stimulated upon induction of apoptosis. Interestingly, proteasome inhibitor celastrol- and chemotherapeutic drug VP-16-stimulated AR breakdown was attenuated by calpain inhibitors calpastatin and N-acetyl-L-leucyl-L-leucyl-L-methioninal. Furthermore, AR proteolytic activity pulled down by calmodulin-agarose beads from celastrol-treated PC-3 cells showed immunoreactivity to a calpain antibody. Taken together, these results demonstrate calpain involvement in proteasome inhibitor-induced AR breakdown, and suggest that AR degradation is intrinsic to the induction of apoptosis in prostate cancer cells.
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PMID:Calpain-mediated androgen receptor breakdown in apoptotic prostate cancer cells. 1872 91

The proteasome is a key intracellular protease that regulates processes, such as signal transduction and protein quality control, through the selective degradation of specific proteins. Signals that target a protein for degradation, collectively known as degrons, have been defined for many proteins involved in cell signaling. However, the molecular signals involved in recognition and degradation of proteins damaged by oxidation have not been completely defined. The current study used biochemical and spectroscopic measurements to define the properties in calmodulin that initiate degradation by the 20S proteasome. Our experimental approach involved the generation of multiple calmodulin mutants with specific Met replaced by Leu. This strategy of site-directed mutagenesis permitted site-selective oxidation of Met to Met sulfoxide. We found that the oxidation-induced loss of secondary structure, as measured by circular dichroism, correlated with the rate of degradation for wild-type and mutants containing Leu substitutions in the C-terminus. However, no degradation was observed for mutants with Met to Leu substitution in the N-terminus, suggesting that oxidation-induced structural unfolding in the N-terminal region is essential for degradation by the 20S proteasome. Experiments comparing the thermodynamic stability of CaM mutants helped to further localize the critical site of oxidation-induced focal disruption between residues 51 and 72 in the N-terminal region. This work brings new biochemical and structural clarity to the concept of the degron, the portion of a protein that determines its susceptibility to degradation by the proteasome.
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PMID:Site-specific methionine oxidation initiates calmodulin degradation by the 20S proteasome. 1923 37

Death-associated protein kinase 1 (DAPK-1) is a Ca(2+)/CaM-regulated kinase involved in multiple cellular signalling pathways that trigger cell survival, apoptosis, and autophagy. An alternatively spliced product expressed from the dapk1 locus, named s-DAPK-1, does not contain the kinase domain but has part of the DAPK-1 ankyrin-repeat and a novel polypeptide tail extension which is processed proteolytically in vivo. Cleavage of this polypeptide tail from s-DAPK-1 can regulate the ability of the protein to mimic one of the biological functions of DAPK-1 in promoting membrane blebbing. The full-length DAPK-1 protein is a relatively long-lived protein whose half-life is regulated by stress-activated signals from TNFR1 or HSP90 that can promote DAPK-1 protein degradation. Transfection of s-DAPK-1 into cells can also have a direct effect on DAPK-1 protein itself by promoting DAPK-1 de-stabilization. This effect does not require the novel polypeptide tail-extension of s-DAPK-1, as the core ankyrin-repeat containing region of s-DAPK-1 is sufficient to promote DAPK-1 protein de-stabilization. Conversely, the minimal domain on full-length DAPK-1 that responds to the effect of s-DAPK-1 is not the ankyrin-repeat domain but the core kinase domain of DAPK-1. The de-stabilization of DAPK-1 by s-DAPK-1 is not dependent upon the proteasome. However, s-DAPK-1 itself is a very short-lived protein which is regulated by a proteasomal-dependent pathway. Together, these data identify a novel function of s-DAPK-1 in controlling the half-life of DAPK-1 protein itself and indicate that the degradation of each gene product is controlled by two distinct degradation pathways.
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PMID:The alternative splice variant of DAPK-1, s-DAPK-1, induces proteasome-independent DAPK-1 destabilization. 1926 29

Voltage-gated sodium channels (VGSCs), classically known to play a central role in excitability and signalling in nerves and muscles, have also been found to be expressed in a range of 'non-excitable' cells, including lymphocytes, fibroblasts and endothelia. VGSC abnormalities are associated with various diseases including epilepsy, long-QT syndrome 3, Brugada syndrome, sudden infant death syndrome and, more recently, various human cancers. Given their pivotal role in a wide range of physiological and pathophysiological processes, regulation of functional VGSC expression has been the subject of intense study. An emerging theme is post-translational regulation and macro-molecular complexing by protein-protein interactions and intracellular trafficking, leading to changes in functional VGSC expression in plasma membrane. This partially involves endoplasmic reticulum associated degradation and ubiquitin-proteasome system. Several proteins have been shown to associate with VGSCs. Here, we review the interactions involving VGSCs and the following proteins: p11, ankyrin, syntrophin, beta-subunit of VGSC, papin, ERM and Nedd4 proteins. Protein kinases A and C, as well as Ca(2+)-calmodulin dependent kinase II that have also been shown to regulate intracellular trafficking of VGSCs by changing the balance of externalization vs. internalization, and an effort is made to separate these effects from the short-term phosphorylation of mature proteins in plasma membrane. Two further modulatory mechanisms are reciprocal interactions with the cytoskeleton and, late-stage, activity-dependent regulation. Thus, the review gives an updated account of the range of post-translational molecular mechanisms regulating functional VGSC expression. However, many details of VGSC subtype-specific regulation and pathophysiological aspects remain unknown and these are highlighted throughout for completeness.
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PMID:Protein-protein interactions involving voltage-gated sodium channels: Post-translational regulation, intracellular trafficking and functional expression. 1940 Nov 47


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