Limits...
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.

Show MeSH
Re-oligomerization of hexameric adiponectin after treatment with βME.(A) Native PAGE of adiponectin oligomers after collapse of octadecamers to hexamers at pH 4 and subsequent neutralization in Tris-buffer and treatment with various concentrations of βME. (B) Non-reducing denaturing SDS-PAGE analysis of adiponectin dimers and monomers in reassembly reactions in part A. (C) Native PAGE analysis of adiponectin oligomers after collapse of octadecamers to hexamers at pH 4 and re-neutralization by dialysis against PBS followed by subsequent treatment with either 5 mM βME or PBS as control. (D) Non-reducing denaturing SDS-PAGE analysis of redox states of adiponectin oligomers in part C. Samples were removed and treated with NEM at the various time points to quench reactions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3117782&req=5

Figure 5: Re-oligomerization of hexameric adiponectin after treatment with βME.(A) Native PAGE of adiponectin oligomers after collapse of octadecamers to hexamers at pH 4 and subsequent neutralization in Tris-buffer and treatment with various concentrations of βME. (B) Non-reducing denaturing SDS-PAGE analysis of adiponectin dimers and monomers in reassembly reactions in part A. (C) Native PAGE analysis of adiponectin oligomers after collapse of octadecamers to hexamers at pH 4 and re-neutralization by dialysis against PBS followed by subsequent treatment with either 5 mM βME or PBS as control. (D) Non-reducing denaturing SDS-PAGE analysis of redox states of adiponectin oligomers in part C. Samples were removed and treated with NEM at the various time points to quench reactions.

Mentions: To further assess if disulfide rearrangement leads to oligomerization into HMW adiponectin, we first prepared oxidized hexamers by reducing the pH of the purified octadecamers to 4 in absence of reducing agents. Subsequently, varying concentrations of βME were added to partially reduce adiponectin and allow disulfide rearrangement to occur. After amounts of reduced adiponectin reach equilibrium following addition of βME, any oligomerization that takes place in absence of net oxidation or reduction is most likely the result of disulfide rearrangement. Interestingly, 100 μM βME led to immediate reduction of small amounts of adiponectin without promoting immediate octadecamer formation (Figure 5A and 5B). However, after 60 min of incubation, the octadecamer began to assemble, and continued to increase until 240 min (Figure 5A). During this time period the concentrations of oxidized dimers and reduced monomers remained constant (Figure 5B), indicating lack of net oxidation or reduction. Similar patterns of adiponectin assembly and oxidation were observed with the addition of 500 μM βME (Figure 5A and 5B). At 5 mM βME, HMW species re-oligomerized within 5 min (Figure 5A).


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 hexameric adiponectin after treatment with βME.(A) Native PAGE of adiponectin oligomers after collapse of octadecamers to hexamers at pH 4 and subsequent neutralization in Tris-buffer and treatment with various concentrations of βME. (B) Non-reducing denaturing SDS-PAGE analysis of adiponectin dimers and monomers in reassembly reactions in part A. (C) Native PAGE analysis of adiponectin oligomers after collapse of octadecamers to hexamers at pH 4 and re-neutralization by dialysis against PBS followed by subsequent treatment with either 5 mM βME or PBS as control. (D) Non-reducing denaturing SDS-PAGE analysis of redox states of adiponectin oligomers in part C. Samples were removed and treated with NEM at the various time points to quench reactions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117782&req=5

Figure 5: Re-oligomerization of hexameric adiponectin after treatment with βME.(A) Native PAGE of adiponectin oligomers after collapse of octadecamers to hexamers at pH 4 and subsequent neutralization in Tris-buffer and treatment with various concentrations of βME. (B) Non-reducing denaturing SDS-PAGE analysis of adiponectin dimers and monomers in reassembly reactions in part A. (C) Native PAGE analysis of adiponectin oligomers after collapse of octadecamers to hexamers at pH 4 and re-neutralization by dialysis against PBS followed by subsequent treatment with either 5 mM βME or PBS as control. (D) Non-reducing denaturing SDS-PAGE analysis of redox states of adiponectin oligomers in part C. Samples were removed and treated with NEM at the various time points to quench reactions.
Mentions: To further assess if disulfide rearrangement leads to oligomerization into HMW adiponectin, we first prepared oxidized hexamers by reducing the pH of the purified octadecamers to 4 in absence of reducing agents. Subsequently, varying concentrations of βME were added to partially reduce adiponectin and allow disulfide rearrangement to occur. After amounts of reduced adiponectin reach equilibrium following addition of βME, any oligomerization that takes place in absence of net oxidation or reduction is most likely the result of disulfide rearrangement. Interestingly, 100 μM βME led to immediate reduction of small amounts of adiponectin without promoting immediate octadecamer formation (Figure 5A and 5B). However, after 60 min of incubation, the octadecamer began to assemble, and continued to increase until 240 min (Figure 5A). During this time period the concentrations of oxidized dimers and reduced monomers remained constant (Figure 5B), indicating lack of net oxidation or reduction. Similar patterns of adiponectin assembly and oxidation were observed with the addition of 500 μM βME (Figure 5A and 5B). At 5 mM βME, HMW species re-oligomerized within 5 min (Figure 5A).

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