Oligomeric States of the Detergent-solubilized Human Serum Paraoxonase (PON1)

Josse, Denis; Ebel, Christine; Stroebel, David; Fontaine, Alice; Borges, Freádeáric; Echalier, Aude; Baud, Delphine; Renault, Freádeárique; Maire, Marc le; Chabrières, Eric; Masson, Patrick

doi:10.1074/jbc.m200108200

Cited by 70 publications

(52 citation statements)

References 52 publications

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“…Other key properties of PON1 seem to be retained in the newly evolved variants. Like HuPON1 (17), rePON1s appear to be in monomer-dimer equilibrium in the presence of 0.6 mM C 12 -maltoside, as analyzed by gel filtration (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…The two mutated regions in the newly evolved PON1s may be involved in correct folding of PON1 and in its oligomeric packing. It has been suggested that exposed hydrophobic surfaces of PON1 are involved in its oligomerization and aggregation (17). These putative hydrophobic surfaces may also lead to aggregation of wild-type PON1s when expressed in E. coli.…”

Section: Resultsmentioning

confidence: 99%

“…Such engineering entirely depends on the ability to amply express and screen many thousands of variants. It was shown, however, that PON1 could not be functionally expressed in Escherichia coli, presumably because of aggregation and the absence of glycosylation (16,17).…”

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confidence: 99%

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Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization

Aharoni

Gaidukov

Yagur

et al. 2003

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

267

221

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Serum paraoxonases (PONs) are a group of enzymes that play a key role in organophosphate (OP) detoxification and in prevention of atherosclerosis. However, their structure and mechanism of action are poorly understood. PONs seem like jacks-of-all-trades, acting on a very wide range of substrates, most of which are of no physiological relevance. Family shuffling and screening lead to the first PON variants that express in a soluble and active form in Escherichia coli. We describe variants with kinetic parameters similar to those reported for PONs purified from sera and others that show dramatically increased activities. In particular, we have evolved PON1 variants with OP-hydrolyzing activities 40-fold higher than wild type and a specificity switch of >2,000-fold, producing PONs specialized for OP rather than ester hydrolysis. Analysis of the newly evolved variants provides insights into the evolutionary relationships between different family members.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization

Aharoni

Gaidukov

Yagur

et al. 2003

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

267

221

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“…3A2 and B2) reflects the good quality of the fit versus the experimental values. It should be mentioned that the analysis of analytical centrifugation data recorded in the presence of detergent is complicated by the fact that the amount of detergent bound to the studied protein is generally unknown, and thus several hypotheses must be considered (30,80). In the presence of 0.1% DM, C HCV 169* migrates as a homogeneous species with a sedimentation coefficient of 3.1 Ϯ 0.1 S (Fig.…”

Section: Resultsmentioning

confidence: 99%

Hepatitis C Virus Core Protein Is a Dimeric Alpha-Helical Protein Exhibiting Membrane Protein Features

Boulant

Vanbelle

Ebel

et al. 2005

J Virol

Self Cite

130

156

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The building block of hepatitis C virus (HCV) nucleocapsid, the core protein, together with viral RNA, is composed of different domains involved in RNA binding and homo-oligomerization. The HCV core protein 1-169 (C HCV 169) and its N-terminal region from positions 1 to 117 (C HCV 117) were expressed in Escherichia coli and purified to homogeneity suitable for biochemical and biophysical characterizations. The overall conformation and the oligomeric properties of the resulting proteins C HCV 169 and C HCV 117 were investigated by using analytical centrifugation, circular dichroism, intrinsic fluorescence measurements, and limited proteolysis. Altogether, our results show that core protein (C HCV 169) behaves as a membranous protein and forms heterogeneous soluble micelle-like aggregates of high molecular weight in the absence of detergent. In contrast, it behaves, in the presence of mild detergent, as a soluble, well-folded, noncovalent dimer. Similar to findings observed for core proteins of HCV-related flaviviruses, the HCV core protein is essentially composed of ␣-helices (50%). In contrast, C HCV 117 is soluble and monodispersed in the absence of detergent but is unfolded. It appears that the folding of the highly basic domain from positions 2 to 117 (2-117 domain) depends on the presence of the 117-169 hydrophobic domain, which contains the structural determinants ensuring the binding of core with cellular membranes. Finally, our findings provide valuable information for further investigations on isolated core protein, as well as for attempts to reconstitute nucleocapsid particles in vitro.Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. It is estimated that 1% of the general population in Western Europe and North America and 380 million people globally are infected with HCV worldwide (19). A protective vaccine does not exist to date, and therapeutic options are still limited. HCV has been classified in the Hepacivirus genus within the Flaviviridae family of viruses. It contains a 9.6-kb plus-strand RNA genome (10, 59, 70) composed of a 5Ј noncoding region (5Ј NCR), a long open reading frame (ORF) encoding a polyprotein precursor of about 3,000 amino acids (aa), and a 3Ј NCR. The HCV polyprotein precursor is co-and posttranslationally processed by cellular and viral proteases to yield the mature structural and nonstructural (NS) proteins. The structural proteins include the core protein, which forms the viral nucleocapsid, and the envelope glycoproteins E1 and E2. They are separated from the NS proteins by the viroporin p7. The NS proteins include the NS2-3 autoprotease and the NS3 serine protease, an NTPase/RNA helicase located in the Cterminal two-thirds of NS3, the NS4A cofactor of NS3, the NS4B and NS5A proteins, and the NS5B RNA-dependent RNA polymerase (for reviews, see references 55 and 58). Interestingly, alternative reading frames (ARF) were recently identified in the HCV core region that has the potential to encode proteins des...

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“…These results show that PON1 Leu-9, Tyr-185, and Tyr-293 participate in PON1-HDL interactions, and the nature of the site-specific mutations impact the affinity of the PON1 form to bind to HDL. PON1 binding to HDL is known to stabilize PON1 catalytic activity at 37°C, with both protein (apoA-I) and lipid phase of HDL contributing to the overall docking and stabilization of PON1 on the HDL particle (31,74,75). We therefore next analyzed the importance of PON1 Leu-9, Tyr-185, and Tyr-293 in PON1 stabilization with HDL particles.…”

Section: Pon1 Residues Leu-9 Tyr-185 and Tyr-293 Are Functionally Imentioning

confidence: 99%

Identification of Critical Paraoxonase 1 Residues Involved in High Density Lipoprotein Interaction

Gu¹,

Huang²,

Levison³

et al. 2016

Journal of Biological Chemistry

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However, specific residues on PON1 involved in the HDL-PON1 interaction remain unclear. Unambiguous identification of protein residues involved in docking interactions to lipid surfaces poses considerable methodological challenges. Here we describe a new strategy that uses a novel synthetic photoactivatable and click chemistry-taggable phospholipid probe, which, when incorporated into HDL, was used to identify amino acid residues on PON1 that directly interact with the lipoprotein phospholipid surface. Several specific PON1 residues (Leu-9, Tyr-185, and Tyr-293) were identified through covalent crosslinks with the lipid probes using affinity isolation coupled to liquid chromatography with on-line tandem mass spectrometry. Based upon the crystal structure for PON1, the identified residues are all localized in relatively close proximity on the surface of PON1, defining a domain that binds to the HDL lipid surface. Site-specific mutagenesis of the identified PON1 residues (Leu-9, Tyr-185, and Tyr-293), coupled with functional studies, reveals their importance in PON1 binding to HDL and both PON1 catalytic activity and stability. Specifically, the residues identified on PON1 provide important structural insights into the PON1-HDL interaction. More generally, the new photoactivatable and affinity-tagged lipid probe developed herein should prove to be a valuable tool for identifying contact sites supporting protein interactions with lipid interfaces such as found on cell membranes or lipoproteins.

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Oligomeric States of the Detergent-solubilized Human Serum Paraoxonase (PON1)

Cited by 70 publications

References 52 publications

Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization

Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization

Hepatitis C Virus Core Protein Is a Dimeric Alpha-Helical Protein Exhibiting Membrane Protein Features

Identification of Critical Paraoxonase 1 Residues Involved in High Density Lipoprotein Interaction

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