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)

Beginning with the peptide sequence Cbz-Ile-Glu(OtBu)-Ala-Leu found in PSI (3), a series of vinyl sulfones (VS) were synthesized for evaluation as inhibitors of the chymotrypsin-like activity of the 20S proteasome. Variations at the key P3 position confirmed the importance of a long side chain capped with a hydrophobic group for optimal potency, consistent with a model of binding to the S3 subsite. The tert-butyl glutamic ester initially used at P3 gave plasma unstable, insoluble compounds and was replaced with the better isostere, N-beta-neopentyl asparagine. The inhibitors were shortened by replacing the N-terminal Cbz-isoleucine with a p-tosyl group without loss of potency. Small l-amino acids were used at P2, where d-substitution was not tolerated. The resulting optimized P4-P3-P2 sequence was grafted onto a novel proteasome inhibitor warhead, 2-keto-1,3,4-oxadiazoles (KOD), to produce reversible, subnanomolar proteasome inhibitors that were 1000-fold selective versus cathepsin B (CatB), cathepsin S (CatS), and trypsin-like as well as PGPH-like proteasome activity. A number of compounds in both the VS and the KOD series exhibited growth inhibitory effects against the human prostate cancer cell line PC3 at submicromolar concentrations.
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PMID:Optimization of subsite binding to the beta5 subunit of the human 20S proteasome using vinyl sulfones and 2-keto-1,3,4-oxadiazoles: syntheses and cellular properties of potent, selective proteasome inhibitors. 1668 37

The machinery used by the cell to perform essential biological processes is made up of large molecular assemblies. One such complex, the proteasome, is the central molecular machine for removal of damaged and misfolded proteins from the cell. Here we show that for the 670-kilodalton 20S proteasome core particle it is possible to overcome the molecular weight limitations that have traditionally hampered quantitative nuclear magnetic resonance (NMR) spectroscopy studies of such large systems. This is achieved by using an isotope labelling scheme where isoleucine, leucine and valine methyls are protonated in an otherwise highly deuterated background in concert with experiments that preserve the lifetimes of the resulting NMR signals. The methodology has been applied to the 20S core particle to reveal functionally important motions and interactions by recording spectra on complexes with molecular weights of up to a megadalton. Our results establish that NMR spectroscopy can provide detailed insight into supra-molecular structures over an order of magnitude larger than those routinely studied using methodology that is generally applicable.
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PMID:Quantitative dynamics and binding studies of the 20S proteasome by NMR. 1723 59

A pair of experiments is presented for measuring intra-methyl 1H-1H dipolar cross-correlated spin relaxation rates in highly deuterated, methyl protonated proteins with significantly improved sensitivity relative to previously developed experiments that measure dynamics via 1H spin relaxation. In applications to proteins with correlation times in the macromolecular limit, these cross-correlation rates are related directly to order parameters, characterizing the amplitude of motion of methyl-containing side-chains. The experimental approach is validated by comparing extracted order parameters with those obtained via 2H and 13C spin relaxation methods for both protein L (7.5 kDa) and malate synthase G (82 kDa), with excellent correlations obtained. The methodology is applied to study Ile, Leu, and Val side-chain dynamics in a 360 kDa "half-proteasome" complex. In particular, order parameters obtained from the WT complex and from a second complex where the proteasome gating residues are deleted establish that the relative levels of dynamics in each of the two molecules are very similar. It thus becomes clear that there is no communication between gating residues and other regions of the molecule involving pico- to nanosecond time-scale dynamics of these methyl-containing side-chains.
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PMID:Probing side-chain dynamics in the proteasome by relaxation violated coherence transfer NMR spectroscopy. 1724 77

The plasma membrane ATP-binding cassette (ABC) transporter, Pdr5p, mediates resistance to many different xenobiotic compounds in yeast. We have isolated several mutated forms that fail to confer resistance to cycloheximide and itraconazole. Here, we examined two variants, the expression of which was abnormally low when cells reach the stationary phase of growth. The Pdr5(1157) variant lacked the C-terminal transmembrane domain due to the presence of a nonsense mutation at codon 1158. The second variant, Pdr5(L183P), contained a Leu183Pro substitution close to the Walker A motif in the N-terminal nucleotide-binding domain. This substitution impaired UTPase activity as well as protein stability. The Pdr5(L183P) variant induced the unfolded protein response and was targeted to the proteasome for degradation. Fluorescence microscopy showed that the highly unstable Pdr5(L183P) was mislocalized to endoplasmic reticulum (ER)-associated compartments, whereas the truncated Pdr5(1157) protein was retained in the ER. When threonine 363 (located in the first nucleotide-binding domain, close to the Walker B motif) in Pdr5(L183P) was replaced with isoleucine, this double mutant conferred partial drug resistance. These results suggest that Pdr5p requires a properly folded nucleotide-binding domain for trafficking to the plasma membrane.
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PMID:Subcellular trafficking of the yeast plasma membrane ABC transporter, Pdr5, is impaired by a mutation in the N-terminal nucleotide-binding fold. 1730 5

While increasing evidence shows that proteasome inhibition triggers oxidative damage, mitochondrial dysfunction and death in neuronal cells, the regulatory relationship among these events is unclear. Using mouse neuronal cells we show that the cytotoxicity induced by mild (0.25 microM) and potent (5.0 microM) doses of the proteasome inhibitor, N-Benzyloxycarbonyl-Ile-Glu (O-t-butyl)-Ala-leucinal, (PSI) involved a dose-dependent increase in caspase activation, overproduction of reactive oxygen species (ROS) and a mitochondrial dysfunction manifested by the translocation of the proapoptotic protein, Bax, from the cytoplasm to the mitochondria, membrane depolarization and the release of cytochrome c and the apoptosis inducing factor (AIF) from mitochondria to the cytoplasm and nucleus, respectively. Whereas caspase or Bax inhibition failed to prevent mitochondrial membrane depolarization and neuronal cell death, pretreatments with the antioxidant N-acetyl-L-cysteine (NAC) or overexpression of the antiapoptotic protein Bcl-xL abrogated these events in cells exposed to mild levels of PSI. These findings implicated ROS as a mediator of PSI-induced cytotoxicity. However, depletions in glutathione and Bcl-xL with potent proteasome inhibition exacerbated this response whereupon survival required the cooperative protection of NAC with Bcl-xL overexpression. Collectively, ROS induced by proteasome inhibition mediates a mitochondrial dysfunction in neuronal cells that culminates in death through caspase- and Bax-independent mechanisms.
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PMID:Reactive oxygen species induced by proteasome inhibition in neuronal cells mediate mitochondrial dysfunction and a caspase-independent cell death. 1741 63

Jasmonate and related signalling compounds have a crucial role in both host immunity and development in plants, but the molecular details of the signalling mechanism are poorly understood. Here we identify members of the jasmonate ZIM-domain (JAZ) protein family as key regulators of jasmonate signalling. JAZ1 protein acts to repress transcription of jasmonate-responsive genes. Jasmonate treatment causes JAZ1 degradation and this degradation is dependent on activities of the SCF(COI1) ubiquitin ligase and the 26S proteasome. Furthermore, the jasmonoyl-isoleucine (JA-Ile) conjugate, but not other jasmonate-derivatives such as jasmonate, 12-oxo-phytodienoic acid, or methyl-jasmonate, promotes physical interaction between COI1 and JAZ1 proteins in the absence of other plant proteins. Our results suggest a model in which jasmonate ligands promote the binding of the SCF(COI1) ubiquitin ligase to and subsequent degradation of the JAZ1 repressor protein, and implicate the SCF(COI1)-JAZ1 protein complex as a site of perception of the plant hormone JA-Ile.
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PMID:JAZ repressor proteins are targets of the SCF(COI1) complex during jasmonate signalling. 1768 17

Recent discoveries show that jasmonate ZIM-domain (JAZ) transcriptional repressors are key regulators of jasmonate hormonal response. Jasmonate promotes interaction between JAZ proteins and the SCF(COI1) ubiquitin ligase, leading to JAZ degradation via the 26S proteasome in Arabidopsis thaliana. Elimination of JAZ repressors then frees the MYC2 transcription factor to stimulate jasmonate-dependent gene expression. Although jasmonic acid and methyl jasmonate were thought to be key regulators of jasmonate responses, they were ineffective in promoting SCF(COI1)-JAZ interaction and it is the isoleucine conjugate of jasmonic acid that acts in this signal transduction pathway. The discovery of JAZ transcriptional regulators greatly advances our understanding of how jasmonate signaling regulates plant growth and response to the environment.
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PMID:JAZing up jasmonate signaling. 1826 50

Oxidative stress, proteasome impairment and mitochondrial dysfunction are implicated as contributors to ageing and neurodegeneration. Using mouse neuronal cells, we showed previously that the reversible proteasome inhibitor, [N-benzyloxycarbonyl-Ile-Glu (O-t-bytul)-Ala-leucinal; (PSI)] induced excessive reactive oxygen species (ROS) that mediated mitochondrial damage and a caspase-independent cell death. Herein, we examined whether this insult persists in neuronal cells recovering from inhibitor removal over time. Recovery from proteasome inhibition showed a time and dose-dependent cell death that was accompanied by ROS overproduction, caspase activation and mitochondrial membrane permeabilization with the subcellular relocalizations of the proapoptotic proteins, Bax, cytochrome c and the apoptosis inducing factor (AIF). Caspase inhibition failed to promote survival indicating that cell death was caspase-independent. Treatments with the antioxidant N-acetyl-cysteine (NAC) were needed to promote survival in cell recovering from mild proteasome inhibition while overexpression of the antiapoptotic protein Bcl-xL together with NAC attenuated cell death during recovery from potent inhibition. Whereas inhibitor removal increased proteasome function, cells recovering from potent proteasome inhibition showed excessive levels of ubiquitinated proteins that required the presence of NAC for their removal. Collectively, these results suggest that the oxidative stress and mitochondrial inhibition induced by proteasome inhibition persists to influence neuronal cell survival when proteasome function is restored.
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PMID:Persistent mitochondrial dysfunction and oxidative stress hinder neuronal cell recovery from reversible proteasome inhibition. 1829 95

Proteasome inhibitors display potent anti-neoplastic and anti-angiogenic properties both in vitro and in vivo. The mechanisms, however, by which proteasome inhibitors kill tumor cells are still fairly elusive as is the molecular basis of resistance to treatment. To address these questions, we employed a high-throughput Western blotting procedure to analyze changes in a subproteome of approximately 800 proteins in the promyelocytic leukemia cell line HL-60 upon treatment with the proteasome inhibitor PSI (Z-Ile-Glu(OtBu)-Ala-Leu-aldehyde) and correlated the changes of selected target proteins with the changes in two multidrug-resistant HL-60 variants. In total, 105 proteins were upregulated more than 1.5-fold after PSI treatment, while 79 proteins were downregulated. Activation of caspases-3 and -8, modulation of members of the Bcl-2 family as well as stimulation of stress signaling pathways was prominent during HL-60 apoptosis. We also identified changes in the abundance of proteins previously not known to be affected by proteasome inhibitors. In contrast, two multidrug-resistant HL-60 cell lines, overexpressing either MRP1 or P-glycoprotein were largely resistant to PSI-induced apoptosis and could not be resensitized by the pharmacological inhibitors of the drug efflux pumps MK571 or PSC833. Drug resistance was also independent of the upregulation of Bad. Overexpression of multidrug resistance proteins, P-glycoprotein and MRP-1 is thus not sufficient to explain resistance of HL-60 cells to treatment with proteasome inhibitor PSI, which remains more closely related to a low level of Bax expression and to the inability to activate JNK. Alternative routes to the acquisition of resistance to PSI have therefore to be considered.
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PMID:Analysis of changes in the proteome of HL-60 promyeloid leukemia cells induced by the proteasome inhibitor PSI. 1846 79

The effect of amino acid on muscle protein degradation remains unclear. Recent studies have elucidated that proteolysis in catabolic conditions occurs through ubiquitin-proteasome proteolysis pathway and that muscle-specific ubiquitin ligases (atrogin-1 and MuRF1) play an important role in protein degradation. In the present study, we examined the direct effect of 5 mM amino acids (leucine, isoleucine, valine, glutamine and arginine) on atrogin-1 and MuRF1 levels in C2C12 muscle cells and the involved intracellular signal transduction pathway. Leucine, isoleucine and valine suppressed atrogin-1 and MuRF1 mRNA levels (approximately equal to 50%) at 6 and 24 h stimulations. Arginine showed a similar effect except at 24 h-treatment for atrogin-1 mRNA. However, glutamine failed to reduce atrogin-1 and MuRF1 mRNA levels. The inhibitory effect of leucine, isoleucine or arginine on atrogin-1 mRNA level was reversed by rapamycin, although wortmannin did not reverse the effect. PD98059 and HA89 reduced basal atrogin-1 level without influencing the inhibitory effects of those amino acids. The inhibitory effect of leucine, isoleucine or arginine on MuRF1 mRNA levels was not reversed by rapamycin. Taken together, these findings indicated that leucine, isoleucine and arginine decreased atrogin-1 mRNA levels via mTOR and that different pathways were involved in the effect of those amino acids on MuRF1 mRNA levels.
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PMID:Branched-chain amino acids and arginine suppress MaFbx/atrogin-1 mRNA expression via mTOR pathway in C2C12 cell line. 1861 83


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