scholarly journals The two PAN ATPases from Halobacterium display N-terminal heterogeneity and form labile complexes with the 20S proteasome

2008 ◽  
Vol 411 (2) ◽  
pp. 387-397 ◽  
Author(s):  
Hala Chamieh ◽  
Dorian Guetta ◽  
Bruno Franzetti
Keyword(s):  
Sequence Analysis ◽  
Molecular Mass ◽  
High Molecular Mass ◽  
26S Proteasome ◽  
20S Proteasome ◽  
Dependent Manner ◽  
Translational Initiation ◽  
Rna Mapping ◽  
Proteolysis Regulation

The PAN (proteasome-activating nucleotidase) proteins from archaea represent homologues of the eukaryotic 26S proteasome regulatory ATPases. In vitro the PAN complex has been previously shown to have a stimulatory effect on the peptidase activities of the 20S core. By using gradient ultracentrifugation we found that, in cellular extracts, the two PAN proteins from Halobacterium do not form stable high-molecular-mass complexes. Only PAN B was found to associate transiently with the 20S proteasome, thus suggesting that the two PAN proteins are not functionally redundant. The PAN B–20S proteasome complexes associate in an ATP-dependent manner and are stabilized upon nucleotide binding. The two PAN proteins were immunodetected in cellular extracts as N-terminal-truncated polypeptides. RNA-mapping experiments and sequence analysis indicated that this process involved transcript heterogeneities and dual translational initiation mechanisms. Taken together, our results suggest that PAN N-terminal modifications and their intracellular dynamics of assembly/association may constitute important determinants of proteolysis regulation.

scholarly journals Destabilizing missense mutations in the tumour suppressor protein p53 enhance its ubiquitination in vitro and in vivo

2006 ◽  
Vol 397 (2) ◽  
pp. 355-367 ◽  
Author(s):  
Harumi Shimizu ◽  
David Saliba ◽  
Maura Wallace ◽  
Lee Finlan ◽  
Patrick R. R. Langridge-Smith ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Covalent Modification ◽  
High Molecular Mass ◽  
Mutant Form ◽  
Dependent Manner ◽  
Ubiquitination System ◽  
Covalent Adducts ◽  
In Cells

p53 ubiquitination catalysed by MDM2 (murine double minute clone 2 oncoprotein) provides a biochemical assay to dissect stages in E3-ubiquitin-ligase-catalysed ubiquitination of a conformationally flexible protein. A mutant form of p53 (p53F270A) containing a mutation in the second MDM2-docking site in the DNA-binding domain of p53 (F270A) is susceptible to modification of long-lived and high-molecular-mass covalent adducts in vivo. Mutant F270A is hyperubiquitinated in cells as defined by immunoprecipitation and immunoblotting with an anti-ubiquitin antibody. Transfection of His-tagged ubiquitin along with p53R175H or p53F270A also results in selective hyperubiquitination in cells under conditions where wild-type p53 is refractory to covalent modification. The extent of mutant p53R175H or p53F270A unfolding in cells as defined by exposure of the DO-12 epitope correlates with the extent of hyperubiquitination, suggesting a link between substrate conformation and E3 ligase function. The p53F270A:6KR chimaeric mutant (where 6KR refers to the simultaneous mutation of lysine residues at positions 370, 372, 373, 381, 382 and 386 to arginine) maintains the high-molecular-mass covalent adducts and is modified in an MDM2-dependent manner. Using an in vitro ubiquitination system, mutant p53F270A and the p53F270A:6KR chimaeric mutant is also subject to hyperubiquitination outwith the C-terminal domain, indicating direct recognition of the mutant p53 conformation by (a) factor(s) in the cell-free ubiquitination system. These data identify an in vitro and in vivo assay with which to dissect how oligomeric protein conformational alterations are linked to substrate ubiquitination in cells. This has implications for understanding the recognition of misfolded proteins during aging and in human diseases such as cancer.

scholarly journals The yeast SEN3 gene encodes a regulatory subunit of the 26S proteasome complex required for ubiquitin-dependent protein degradation in vivo.

1995 ◽  
Vol 15 (11) ◽  
pp. 6311-6321 ◽  
Author(s):  
D J DeMarini ◽  
F R Papa ◽  
S Swaminathan ◽  
D Ursic ◽  
T P Rasmussen ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Genetic Selection ◽  
26S Proteasome ◽  
Yeast Cells ◽  
Regulatory Subunit ◽  
20S Proteasome ◽  
Amino Terminal ◽  
A Subunit

The yeast Sen1 protein was discovered by virtue of its role in tRNA splicing in vitro. To help determine the role of Sen1 in vivo, we attempted to overexpress the protein in yeast cells. However, cells with a high-copy SEN1-bearing plasmid, although expressing elevated amounts of SEN1 mRNA, show little increase in the level of the encoded protein, indicating that a posttranscriptional mechanism limits SEN1 expression. This control depends on an amino-terminal element of Sen1. Using a genetic selection for mutants with increased expression of Sen1-derived fusion proteins, we identified mutations in a novel gene, designated SEN3. SEN3 is essential and encodes a 945-residue protein with sequence similarity to a subunit of an activator of the 20S proteasome from bovine erythrocytes, called PA700. Earlier work indicated that the 20S proteasome associates with a multisubunit regulatory factor, resulting in a 26S proteasome complex that degrades substrates of the ubiquitin system. Mutant sen3-1 cells have severe defects in the degradation of such substrates and accumulate ubiquitin-protein conjugates. Most importantly, we show biochemically that Sen3 is a subunit of the 26S proteasome. These data provide evidence for the involvement of the 26S proteasome in the degradation of ubiquitinated proteins in vivo and for a close relationship between PA700 and the regulatory complexes within the 26S proteasome, and they directly demonstrate that Sen3 is a component of the yeast 26S proteasome.

scholarly journals Identification of Proteins from Plasmodium falciparum That Are Homologous to Reticulocyte Binding Proteins inPlasmodium vivax

2001 ◽  
Vol 69 (2) ◽  
pp. 1084-1092 ◽  
Author(s):  
Tony Triglia ◽  
Jenny Thompson ◽  
Sonia R. Caruana ◽  
Mauro Delorenzi ◽  
Terry Speed ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Red Blood Cells ◽  
Molecular Mass ◽  
Blood Cells ◽  
Potential Role ◽  
High Molecular Mass ◽  
In Vitro Growth ◽  
Merozoite Invasion ◽  
Genome Databases

ABSTRACT Plasmodium falciparum infections can be fatal, whileP. vivax infections usually are not. A possible factor involved in the greater virulence of P. falciparum is that this parasite grows in red blood cells (RBCs) of all maturities whereasP. vivax is restricted to growth in reticulocytes, which represent only approximately 1% of total RBCs in the periphery. Two proteins, expressed at the apical end of the invasive merozoite stage from P. vivax, have been implicated in the targeting of reticulocytes for invasion by this parasite. A search of the P. falciparum genome databases has identified genes that are homologous to the P. vivax rbp-1 and -2 genes. Two of these genes are virtually identical over a large region of the 5′ end but are highly divergent at the 3′ end. They encode high-molecular-mass proteins of >300 kDa that are expressed in late schizonts and localized to the apical end of the merozoite. To test a potential role in merozoite invasion of RBCs, we analyzed the ability of these proteins to bind to mature RBCs and reticulocytes. No binding to mature RBCs or cell preparations enriched for reticulocytes was detected. We identified a parasite clone that lacks the gene for one of these proteins, showing that the gene is not required for normal in vitro growth. Antibodies to these proteins can inhibit merozoite invasion of RBCs.

scholarly journals BBX21, an Arabidopsis B-box protein, directly activates HY5 and is targeted by COP1 for 26S proteasome-mediated degradation

2016 ◽  
Vol 113 (27) ◽  
pp. 7655-7660 ◽  
Author(s):  
Dongqing Xu ◽  
Yan Jiang ◽  
Jigang Li ◽  
Fang Lin ◽  
Magnus Holm ◽  
...  
Keyword(s):  
Salt Tolerance ◽  
26S Proteasome ◽  
Light Signaling ◽  
Dependent Manner ◽  
Positive Regulator ◽  
Molecular Base ◽  
Precise Role

BBX21 (also known as SALT TOLERANCE HOMOLOG 2), a B-box (BBX)-containing protein, has been previously identified as a positive regulator of light signaling; however, the precise role of BBX21 in regulating seedling photomorphogenesis remains largely unclear. In this study, we report that CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) interacts with BBX21 in vivo and is able to ubiquitinate BBX21 in vitro. Thus, BBX21 is targeted for 26S proteasome-mediated degradation in dark-grown Arabidopsis seedlings in a COP1-dependent manner. Moreover, we show that BBX21 binds to the T/G-box in the ELONGATED HYPOCOTYL 5 (HY5) promoter and directly activates HY5 expression in the light. Transgenic seedlings overexpressing BBX21 exhibit dramatically shortened hypocotyls in the light, and this phenotype is dependent on a functional HY5. Taken together, our data suggest a molecular base underlying BBX21-mediated seedling photomorphogenesis, indicating that BBX21 is a pivotal component involved in the COP1-HY5 regulatory hub.

scholarly journals Characterization of Mediator Complexes from HeLa Cell Nuclear Extract

2001 ◽  
Vol 21 (14) ◽  
pp. 4604-4613 ◽  
Author(s):  
Gang Wang ◽  
Greg T. Cantin ◽  
Jennitte L. Stevens ◽  
Arnold J. Berk
Keyword(s):  
Hela Cell ◽  
Molecular Mass ◽  
Rna Polymerase Ii ◽  
Nuclear Extract ◽  
High Molecular Mass ◽  
Multiprotein Complexes ◽  
Hela Cell Nuclear Extract ◽  
Cell Nuclear Extract

ABSTRACT A number of mammalian multiprotein complexes containing homologs ofSaccharomyces cerevisiae Mediator subunits have been described recently. High-molecular-mass complexes (1 to 2 MDa) sharing several subunits but apparently differing in others include the TRAP/SMCC, NAT, DRIP, ARC, and human Mediator complexes. Smaller multiprotein complexes (∼500 to 700 kDa), including the murine Mediator, CRSP, and PC2, have also been described that contain subsets of subunits of the larger complexes. To evaluate whether these different multiprotein complexes exist in vivo in a single form or in multiple different forms, HeLa cell nuclear extract was directly resolved over a Superose 6 gel filtration column. Immunoblotting of column fractions using antisera specific for several Mediator subunits revealed one major size class of high-molecular-mass (∼2-MDa) complexes containing multiple mammalian Mediator subunits. No peak was apparent at ∼500 to 700 kDa, indicating that either the smaller complexes reported are much less abundant than the higher-molecular-mass complexes or they are subcomplexes generated by dissociation of larger complexes during purification. Quantitative immunoblotting indicated that there are about 3 × 105to 6 × 105 molecules of hSur2 Mediator subunit per HeLa cell, i.e., the same order of magnitude as RNA polymerase II and general transcription factors. Immunoprecipitation of the ∼2-MDa fraction with anti-Cdk8 antibody indicated that at least two classes of Mediator complexes occur, one containing CDK8 and cyclin C and one lacking this CDK-cyclin pair. The ∼2-MDa complexes stimulated activated transcription in vitro, whereas a 150-kDa fraction containing a subset of Mediator subunits inhibited activated transcription.

Purification and Characterization of a Novel Metalloproteinase, Acurhagin, from Agkistrodon acutus Venom

2002 ◽  
Vol 87 (04) ◽  
pp. 641-650 ◽  
Author(s):  
Wen-Jeng Wang ◽  
Tur-Fu Huang
Keyword(s):  
Platelet Aggregation ◽  
Molecular Mass ◽  
Hydrophobic Interaction Chromatography ◽  
Inhibitory Effect ◽  
High Molecular Mass ◽  
Dependent Manner ◽  
Peptide Fragments ◽  
Crude Venom ◽  
Reducing Conditions ◽  
Sds Page

SummaryAcurhagin, a high-molecular mass hemorrhagic metalloproteinase, was purified from the crude venom of Agkistrodon acutus using anionexchange and hydrophobic interaction chromatography. Acurhagin is a monomer with a molecular mass of 51.4 kDa under non-reducing conditions on SDS-PAGE and 48,133 Da by mass spectrometry. Partial amino acid sequence of its metalloproteinase domain is homologous to other high-molecular mass metalloproteinases from snake venoms. It preferentially cleaved Aα. chain of fibrinogen, followed by Bβ chain, while γ chains was minimally affected. Monitored by RP-HPLC, it extensively degraded fibrinogen into various peptide fragments. In aqueous solution, acurhagin autoproteolyzed to a 30 kDa fragment at 37° C. The N-terminal sequence of the 30 kDa fragment of acurhagin showed a high homology to those proteins consisting of disintegrinlike and cysteine-rich domains. Caseinolytic assay showed that the proteinase activity of acurhagin was slightly enhanced by Ca2+ and Mg2+, but completely inhibited by Zn2+. When treated with metal chelators, acurhagin was completely inactivated. Furthermore, acurhagin exerts an inhibitory effect on ADP-induced platelet aggregation of plateletrich plasma in an incubation-time dependent manner. It also impairs collagen- and ristocetin-induced platelet aggregation by cleaving collagen and vWF, respectively.

scholarly journals Properties of the yeast nuclear histone deacetylase

1994 ◽  
Vol 303 (3) ◽  
pp. 723-729 ◽  
Author(s):  
M M Sanchez del Pino ◽  
G Lopez-Rodas ◽  
R Sendra ◽  
V Tordera
Keyword(s):  
Molecular Mass ◽  
Histone Deacetylase ◽  
Trichostatin A ◽  
High Mobility ◽  
Inhibitory Effect ◽  
High Molecular Mass ◽  
Core Histones ◽  
Nuclear Histone

A nuclear histone deacetylase from yeast was partially purified and some of its characteristics were studied. Histone deacetylase activity was stimulated in vitro by high-mobility-group nonhistone chromatin proteins 1 and 2 and ubiquitin and inhibited by spermine and spermidine, whereas n-butyrate had no significant inhibitory effect. Like the mammalian enzyme, partially purified histone deacetylase from yeast was strongly inhibited by trichostatin A. However, in crude extract preparations the yeast enzyme was not inhibited and treatment with trichostatin in vivo did not show any effect, either on the histone acetylation level or on cell viability. At low ionic strength, the enzyme can be isolated as a complex of high molecular mass that is much less inhibited by trichostatin A than is partially purified histone deacetylase activity. Furthermore, radiolabelled oligonucleosomes were more efficiently deacetylated by the complex than by the low-molecular-mass form of the enzyme. The histone deacetylase activity was separated from a polyamine deacetylase activity and its specificity studied. Using h.p.l.c.-purified core histone species as substrate, histone deacetylase from yeast is able to deacetylate all core histones with a slight preference for H3. Our results support the idea that the yeast histone deacetylase may act as a high-molecular-mass complex in vivo.

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