Synthetic, structural and biological studies of the ubiquitin system: the total chemical synthesis of ubiquitin

Ramage, R.; Green, Jeremy; Muir, Tom W.; Ogunjobi, O.M.; Love, Steven; Shaw, K. T.

doi:10.1042/bj2990151

Cited by 143 publications

(125 citation statements)

References 36 publications

Supporting

Mentioning

116

Contrasting

Unclassified

Order By: Relevance

“…identical over 76 residues and adopt the same fold (22,23). The interactions with the last four residues of ubiquitin/Nedd8 preceding the isopeptide bond are likely to be preserved in CSN5.…”

Section: Surroundings Of the Csn5 Zinc Catalytic Site Is Not Competenmentioning

confidence: 99%

Insights into the regulation of the human COP9 signalosome catalytic subunit, CSN5/Jab1

Echalier

Pan

Birol

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The COP9 (Constitutive photomorphogenesis 9) signalosome (CSN), a large multiprotein complex that resembles the 19S lid of the 26S proteasome, plays a central role in the regulation of the E3-cullin RING ubiquitin ligases (CRLs). The catalytic activity of the CSN complex, carried by subunit 5 (CSN5/Jab1), resides in the deneddylation of the CRLs that is the hydrolysis of the cullin-neural precursor cell expressed developmentally downregulated gene 8 (Nedd8)isopeptide bond. Whereas CSN-dependent CSN5 displays isopeptidase activity, it is intrinsically inactive in other physiologically relevant forms. Here we analyze the crystal structure of CSN5 in its catalytically inactive form to illuminate the molecular basis for its activation state. We show that CSN5 presents a catalytic domain that brings essential elements to understand its activity control. Although the CSN5 active site is catalytically competent and compatible with di-isopeptide binding, the Ins-1 segment obstructs access to its substrate-binding site, and structural rearrangements are necessary for the Nedd8-binding pocket formation. Detailed study of CSN5 by molecular dynamics unveils signs of flexibility and plasticity of the Ins-1 segment. These analyses led to the identification of a molecular trigger implicated in the active/inactive switch that is sufficient to impose on CSN5 an active isopeptidase state. We show that a single mutation in the Ins-1 segment restores biologically relevant deneddylase activity. This study presents detailed insights into CSN5 regulation. Additionally, a dynamic monomer-dimer equilibrium exists both in vitro and in vivo and may be functionally relevant.C ell signaling processes mediated by ubiquitinylation, the posttranslational covalent conjugation of ubiquitin molecules, are of prime importance for cellular activity and particularly for protein turnover. Ubiquitin-ligase enzymes (E3s) are responsible for the last step of the ubiquitinylation reaction, and the multisubunit cullin-RING E3 ubiquitin ligases (CRLs) represent the most prominent of E3 enzymes. Among the several factors that regulate CRL activity, cullin neddylation/deneddylation cycles are central (1).The Cop9 signalosome (CSN), which is an eight-subunit complex largely conserved through evolution, deneddylates CRLs and thereby regulates CRL activity. As a large number of proteins are ubiquitinylated by CRLs, the CSN complex is implicated in the control of a significant proportion of the proteome, including prooncogenes, tumor suppressors, and other important cellular protagonists (1). Not surprisingly, the CSN has been implicated in various cellular functions, ranging from cell cycles to circadian rhythm and to immunity in various organisms. Furthermore, many studies have found a strong link between the CSN and cancers (2).The CSN, a multi-protein complex of about 320 kDa, contains six proteasome Cop9 eIF3 (PCI)-based subunits and two MPR1-Pad1-N-terminal (MPN)-based subunits. The subunit 5 [CSN5; also known as c-Jun activation domain-binding protein-1 (...

show abstract

Section: Surroundings Of the Csn5 Zinc Catalytic Site Is Not Competenmentioning

confidence: 99%

Insights into the regulation of the human COP9 signalosome catalytic subunit, CSN5/Jab1

Echalier

Pan

Birol

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…This was synthesized and purified by standard methods (Albachem Ltd., Edinburgh, Scotland; see Refs. 15,16). Analytical reverse phase HPLC showed that the peptide was more than 95% pure, with a molecular mass of 8508.00 Da as determined by electrospray ionization-mass spectrometry.…”

Section: Methodsmentioning

confidence: 99%

Characteristics of the Interaction of a Synthetic Human Tristetraprolin Tandem Zinc Finger Peptide with AU-rich Element-containing RNA Substrates

Blackshear

Lai²,

Kennington³

et al. 2003

Journal of Biological Chemistry

116

130

View full text Add to dashboard Cite

Tristetraprolin (TTP) and its two known mammalian family members are tandem CCCH zinc finger proteins that can bind to AU-rich elements (AREs) in cellular mRNAs and destabilize those transcripts, apparently by initiating their deadenylation. Previous studies have shown that the ϳ70-amino acid tandem zinc finger domain of TTP is required and sufficient for RNA binding, and that the integrity of both zinc fingers is also required. However, little is known about the kinetics or structure of the peptide-RNA interaction, in part because of difficulties in obtaining soluble recombinant protein or peptides. We characterized the binding of a synthetic 73-amino acid peptide from human TTP to the tumor necrosis factor (TNF) ARE by gel mobility shift analyses and fluorescence anisotropy experiments. Both types of studies yielded a peptide-RNA dissociation constant of ϳ10 nM. Surprisingly, we found that the "footprint" from the TNF ARE required for peptide binding was only ϳ9 bases and that two molecules of peptide could bind to probes containing as little as 19 bases. An identical recombinant peptide exhibited gel shift characteristics similar to those of the synthetic peptide. NMR analysis of the 15 N-labeled recombinant peptide suggested that its first zinc finger was structured in solution but that the second was not. The titration of oligonucleotides representing 17, 13, and even 9 bases of the TNF ARE caused an essentially identical, dramatic shift of existing resonances, and the appearance of new resonances in the peptide spectra, so that all amino acids could be assigned. These data suggest that this TTP peptide-RNA complex is structured in solution and might be amenable to NMR structure determination. Tristetraprolin (TTP)1 is the prototype of a small family of eukaryotic CCCH tandem zinc finger proteins (see Ref. 1 for recent review). In mammals, there are three known members of this protein family, whereas fish and frogs express a fourth member that seems to consist of a group of closely related proteins (2-4). The characteristic tandem zinc finger domain of these proteins consists of two zinc fingers with 18 amino acids between fingers, strict intrafinger spacing of Cx8Cx5Cx3H, and a characteristic sequence motif leading into each finger of R(K)YKTEL or a closely related variant.Studies in TTP knockout mice and cells derived from them revealed that TTP acts in normal physiology to regulate the synthesis and secretion of two clinically important cytokines, tumor necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (5-9). The increased secretion of these cytokines in the TTP knockout mice was secondary to increased steady state levels of their mRNAs, which in turn was caused by excessive stabilization of the transcripts. TTP was found to confer instability on these two transcripts by binding to the so-called class II AREs contained within the 3Ј-untranslated regions of these mRNAs (7, 10 -13). The tandem zinc finger domain was found to be the RNA binding domain, and the invariant cys...

show abstract

“…The Crystal Structure of Plant ATG12 and its Biological Implication in Autophagy 27 respectively. α-helices are colored red, and β-strands are colored yellow.…”

Section: Methodsmentioning

confidence: 99%

The Crystal Structure of Plant ATG12 and its Biological Implication in Autophagy

Suzuki

Yoshimoto²,

Fujioka³

et al. 2005

Autophagy

111

View full text Add to dashboard Cite

Previously published online as a Autophagy E-publication: http://www.landesbioscience.com/journals/autophagy/abstract.php?id=1859 KEY WORDSAtATG12, autophagy, crystal structure, posttranslational modifier, ubiquitin-like protein ABBREVIATIONS AtArabidopsis thaliana PE phosphatidylethanolamine PAS pre-autophagosomal structure GST glutathione S-transferase MIRAS multiple isomorphous replacement with anomalous scattering ACKNOWLEDGEMENTSWe thank Dr. T. Hanada for valuable comments and suggestions on the manuscript. This work was supported by Grant-in-Aids for Scientific Research on Priority Areas and National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan. This work was carried out under the NIBB Cooperative Research Program (4-148). Research PaperThe Crystal Structure of Plant ATG12 and its Biological Implication in Autophagy ABSTRACTAtg12 is a post-translational modifier that is activated and conjugated to its single target, Atg5, by a ubiquitin-like conjugation system. The Atg12-Atg5 conjugate is essential for autophagy, the bulk degradation process of cytoplasmic components by the vacuolar/ lysosomal system. Here, we demonstrate that the Atg12 conjugation system exists in Arabidopsis and is essential for plant autophagy as well as in yeast and mammals. We also report the crystal structure of Arabidopsis thaliana (At) ATG12 at 1.8 Å resolution. Despite no obvious sequence homology with ubiquitin, the structure of AtATG12 shows a ubiquitin fold strikingly similar to those of mammalian homologs of Atg8, the other ubiquitinlike modifier essential for autophagy, which is conjugated to phosphatidylethanolamine. Two types of hydrophobic patches are present on the surface of AtATG12: one is conserved in both Atg12 and Atg8 orthologs, while the other is unique to Atg12 orthologs. Considering that they share Atg7 as an E1-like enzyme, we suggest that the first hydrophobic patch is responsible for the conjugation reaction, while the latter is involved in Atg12-specific functions.

show abstract

Synthetic, structural and biological studies of the ubiquitin system: the total chemical synthesis of ubiquitin

Cited by 143 publications

References 36 publications

Insights into the regulation of the human COP9 signalosome catalytic subunit, CSN5/Jab1

Insights into the regulation of the human COP9 signalosome catalytic subunit, CSN5/Jab1

Characteristics of the Interaction of a Synthetic Human Tristetraprolin Tandem Zinc Finger Peptide with AU-rich Element-containing RNA Substrates

The Crystal Structure of Plant ATG12 and its Biological Implication in Autophagy

Contact Info

Product

Resources

About