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Role of redox environment on the oligomerization of higher molecular weight adiponectin.

Briggs DB, Giron RM, Malinowski PR, Nuñez M, Tsao TS - BMC Biochem. (2011)

Bottom Line: In this study, we examined the effects of redox environment on the rate of oligomer formation and the distribution of oligomers.Reassembly of adiponectin under oxidizing conditions accelerated disulfide bonding but favored formation of hexamers over the HMW species.Based upon these observations, we propose oxidative assembly of multi-subunit adiponectin complexes in a defined and stable redox environment is favored under oxidizing conditions coupled with high rates of disulfide rearrangement.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85724, USA.

ABSTRACT

Background: Adiponectin is an adipocyte-secreted hormone with insulin-sensitizing and anti-inflammatory actions. The assembly of trimeric, hexameric, and higher molecular weight (HMW) species of adiponectin is a topic of significant interest because physiological actions of adiponectin are oligomer-specific. In addition, adiponectin assembly is an example of oxidative oligomerization of multi-subunit protein complexes in endoplasmic reticulum (ER).

Results: We previously reported that trimers assemble into HMW adiponectin via intermediates stabilized by disulfide bonds, and complete oxidation of available cysteines locks adiponectin in hexameric conformation. In this study, we examined the effects of redox environment on the rate of oligomer formation and the distribution of oligomers. Reassembly of adiponectin under oxidizing conditions accelerated disulfide bonding but favored formation of hexamers over the HMW species. Increased ratios of HMW to hexameric adiponectin could be achieved rapidly under oxidizing conditions by promoting disulfide rearrangement.

Conclusions: Based upon these observations, we propose oxidative assembly of multi-subunit adiponectin complexes in a defined and stable redox environment is favored under oxidizing conditions coupled with high rates of disulfide rearrangement.

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Re-oligomerization of adiponectin following dialysis into defined concentrations of DTT. (A) Native PAGE of adiponectin oligomers after collapse to trimers and subsequent re-oligomerization as concentrations of DTT were equilibrated from 5 mM to 0.008, 0.04, 0.2, 1, or 5 mM by dialysis over an 18-hr period. (B) Non-reducing denaturing SDS-PAGE analysis to identify the oxidation state of adiponectin as either in the disulfide-bonded dimer state or reduced monomer state in each of the different conditions. Oligomerization and oxidation states were examined at 6 and 18 hrs. The redox state of adiponectin was fixed by adding NEM to the sample at the end of reassembly reactions. 3 mer: trimer; 6 mer: hexamer; 9 mer: nonamer; 12 mer: dodecamer; 18 mer: octadecamer. The number of monomers in trimers, hexamers, and octadecamers in native gels were independently verified using native mass spectrometry and gel filtration chromatography (data not shown). The number of monomers in nonamers and dodecamers were determined by extrapolation of migration distances of known oligomers in native gels as described in Methods.
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Figure 1: Re-oligomerization of adiponectin following dialysis into defined concentrations of DTT. (A) Native PAGE of adiponectin oligomers after collapse to trimers and subsequent re-oligomerization as concentrations of DTT were equilibrated from 5 mM to 0.008, 0.04, 0.2, 1, or 5 mM by dialysis over an 18-hr period. (B) Non-reducing denaturing SDS-PAGE analysis to identify the oxidation state of adiponectin as either in the disulfide-bonded dimer state or reduced monomer state in each of the different conditions. Oligomerization and oxidation states were examined at 6 and 18 hrs. The redox state of adiponectin was fixed by adding NEM to the sample at the end of reassembly reactions. 3 mer: trimer; 6 mer: hexamer; 9 mer: nonamer; 12 mer: dodecamer; 18 mer: octadecamer. The number of monomers in trimers, hexamers, and octadecamers in native gels were independently verified using native mass spectrometry and gel filtration chromatography (data not shown). The number of monomers in nonamers and dodecamers were determined by extrapolation of migration distances of known oligomers in native gels as described in Methods.

Mentions: We previously showed the main function of disulfide bonds in oxidative oligomerization of adiponectin is to stabilize intermediate oligomers and that fully oxidized hexamers oligomerized to HMW adiponectin at a much slower rate than reduced trimers [21]. This led us to hypothesize that adiponectin oligomerization into HMW or hexameric isoforms could be modulated by the prevailing redox environment. Specifically, our model predicts that oxidizing conditions will accelerate disulfide bonding and favor the formation of fully oxidized hexamers that are unable to assemble further into HMW adiponectin. To test this hypothesis, we performed re-oligomerization experiments in which the rate of disulfide formation was slowed by decreasing the rate of DTT removal. Following collapse to trimers by reduction with 5 mM DTT and lowering of pH to 4, reassembly of HMW adiponectin was examined as reactions were exchanged into varying concentrations of DTT in PBS at pH 7.4. As reactions were dialyzed into increasing concentrations of DTT, decreased formation of both octadecameric and hexameric adiponectin were observed, with hexamer formation affected to a greater extent than octadecamer formation (Figure 1A). The increased susceptibility of hexamers to reduction could be explained by the inherent stability of reduced octadecamer [21]. Alternatively, the inter-trimer disulfide bonds between hexamers are more susceptible to reduction than those in the HMW complex [18]. Formation of octadecamers and hexamers was accompanied by oxidation of reduced monomers to disulfide-bonded dimers (Figure 1B). As the concentration of DTT increased, the amount of reduced monomer increased at the expense of oxidized dimer, indicating reduced rates of disulfide formation (Figure 1B). This increase in reduced monomers compared to oxidized dimers is accompanied by a decrease in the conversion of trimers to larger species (Figure 1A). These results demonstrate that decreased rate of disulfide bonding led to decreased formation of both HMW and hexameric adiponectin formation. Complete removal of DTT did not inhibit HMW adiponectin formation at the expense of hexamers (Figure 1A). These results do not support the notion that modulating the rate of disulfide bond formation could result in preferential formation of HMW adiponectin over hexamers or vice versa.


Role of redox environment on the oligomerization of higher molecular weight adiponectin.

Briggs DB, Giron RM, Malinowski PR, Nuñez M, Tsao TS - BMC Biochem. (2011)

Re-oligomerization of adiponectin following dialysis into defined concentrations of DTT. (A) Native PAGE of adiponectin oligomers after collapse to trimers and subsequent re-oligomerization as concentrations of DTT were equilibrated from 5 mM to 0.008, 0.04, 0.2, 1, or 5 mM by dialysis over an 18-hr period. (B) Non-reducing denaturing SDS-PAGE analysis to identify the oxidation state of adiponectin as either in the disulfide-bonded dimer state or reduced monomer state in each of the different conditions. Oligomerization and oxidation states were examined at 6 and 18 hrs. The redox state of adiponectin was fixed by adding NEM to the sample at the end of reassembly reactions. 3 mer: trimer; 6 mer: hexamer; 9 mer: nonamer; 12 mer: dodecamer; 18 mer: octadecamer. The number of monomers in trimers, hexamers, and octadecamers in native gels were independently verified using native mass spectrometry and gel filtration chromatography (data not shown). The number of monomers in nonamers and dodecamers were determined by extrapolation of migration distances of known oligomers in native gels as described in Methods.
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Figure 1: Re-oligomerization of adiponectin following dialysis into defined concentrations of DTT. (A) Native PAGE of adiponectin oligomers after collapse to trimers and subsequent re-oligomerization as concentrations of DTT were equilibrated from 5 mM to 0.008, 0.04, 0.2, 1, or 5 mM by dialysis over an 18-hr period. (B) Non-reducing denaturing SDS-PAGE analysis to identify the oxidation state of adiponectin as either in the disulfide-bonded dimer state or reduced monomer state in each of the different conditions. Oligomerization and oxidation states were examined at 6 and 18 hrs. The redox state of adiponectin was fixed by adding NEM to the sample at the end of reassembly reactions. 3 mer: trimer; 6 mer: hexamer; 9 mer: nonamer; 12 mer: dodecamer; 18 mer: octadecamer. The number of monomers in trimers, hexamers, and octadecamers in native gels were independently verified using native mass spectrometry and gel filtration chromatography (data not shown). The number of monomers in nonamers and dodecamers were determined by extrapolation of migration distances of known oligomers in native gels as described in Methods.
Mentions: We previously showed the main function of disulfide bonds in oxidative oligomerization of adiponectin is to stabilize intermediate oligomers and that fully oxidized hexamers oligomerized to HMW adiponectin at a much slower rate than reduced trimers [21]. This led us to hypothesize that adiponectin oligomerization into HMW or hexameric isoforms could be modulated by the prevailing redox environment. Specifically, our model predicts that oxidizing conditions will accelerate disulfide bonding and favor the formation of fully oxidized hexamers that are unable to assemble further into HMW adiponectin. To test this hypothesis, we performed re-oligomerization experiments in which the rate of disulfide formation was slowed by decreasing the rate of DTT removal. Following collapse to trimers by reduction with 5 mM DTT and lowering of pH to 4, reassembly of HMW adiponectin was examined as reactions were exchanged into varying concentrations of DTT in PBS at pH 7.4. As reactions were dialyzed into increasing concentrations of DTT, decreased formation of both octadecameric and hexameric adiponectin were observed, with hexamer formation affected to a greater extent than octadecamer formation (Figure 1A). The increased susceptibility of hexamers to reduction could be explained by the inherent stability of reduced octadecamer [21]. Alternatively, the inter-trimer disulfide bonds between hexamers are more susceptible to reduction than those in the HMW complex [18]. Formation of octadecamers and hexamers was accompanied by oxidation of reduced monomers to disulfide-bonded dimers (Figure 1B). As the concentration of DTT increased, the amount of reduced monomer increased at the expense of oxidized dimer, indicating reduced rates of disulfide formation (Figure 1B). This increase in reduced monomers compared to oxidized dimers is accompanied by a decrease in the conversion of trimers to larger species (Figure 1A). These results demonstrate that decreased rate of disulfide bonding led to decreased formation of both HMW and hexameric adiponectin formation. Complete removal of DTT did not inhibit HMW adiponectin formation at the expense of hexamers (Figure 1A). These results do not support the notion that modulating the rate of disulfide bond formation could result in preferential formation of HMW adiponectin over hexamers or vice versa.

Bottom Line: In this study, we examined the effects of redox environment on the rate of oligomer formation and the distribution of oligomers.Reassembly of adiponectin under oxidizing conditions accelerated disulfide bonding but favored formation of hexamers over the HMW species.Based upon these observations, we propose oxidative assembly of multi-subunit adiponectin complexes in a defined and stable redox environment is favored under oxidizing conditions coupled with high rates of disulfide rearrangement.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85724, USA.

ABSTRACT

Background: Adiponectin is an adipocyte-secreted hormone with insulin-sensitizing and anti-inflammatory actions. The assembly of trimeric, hexameric, and higher molecular weight (HMW) species of adiponectin is a topic of significant interest because physiological actions of adiponectin are oligomer-specific. In addition, adiponectin assembly is an example of oxidative oligomerization of multi-subunit protein complexes in endoplasmic reticulum (ER).

Results: We previously reported that trimers assemble into HMW adiponectin via intermediates stabilized by disulfide bonds, and complete oxidation of available cysteines locks adiponectin in hexameric conformation. In this study, we examined the effects of redox environment on the rate of oligomer formation and the distribution of oligomers. Reassembly of adiponectin under oxidizing conditions accelerated disulfide bonding but favored formation of hexamers over the HMW species. Increased ratios of HMW to hexameric adiponectin could be achieved rapidly under oxidizing conditions by promoting disulfide rearrangement.

Conclusions: Based upon these observations, we propose oxidative assembly of multi-subunit adiponectin complexes in a defined and stable redox environment is favored under oxidizing conditions coupled with high rates of disulfide rearrangement.

Show MeSH