The oligomeric structure of high molecular weight adiponectin

There is great interest in the structure of adiponectin as its oligomeric state may specify its biological activities. It occurs as a trimer, a hexamer and a high molecular weight complex. Epidemiological data indicate that the high molecular weight form is significant with low serum levels in type 2 diabetics but to date, has not been well-defined. To resolve this issue, characterization of this oligomer from bovine serum and 3T3-L1 adipocytes by sedimentation equilibrium centrifugation and gel electrophoresis respectively, was carried out, revealing that it is octadecameric. Further studies by dynamic light scattering and electron microscopy established that bovine and possibly mouse high molecular weight adiponectin is C1q-like in structure.     

Adiponectin as a growth inhibitor in prostate cancer cells

Prostate cancer is associated with obesity. However, the molecular basis of this association is not well known. Adiponectin is a major adipose cytokine that decreases in circulation in obesity and ameliorates obesity. Here, we identify adiponectin as a novel inhibitor in prostate cancer cell growth. Adiponectin occurs in non-proteolytic (full-length adiponectin: f-adiponectin) and proteolytic (globular adiponectin) forms in various oligomeric states (trimer, hexamer, and high molecular weight complex). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay demonstrates that f-adiponectin inhibits prostate cancer cell growth drastically at subphysiological concentrations. Furthermore, velocity sedimentation analysis shows that the high molecular weight complex of f-adiponectin is the inhibitory form. Moreover, f-adiponectin suppresses leptin- and/or insulin-like growth factor-I (IGF-I)-stimulated, androgen-independent DU145 cell growth, and dihydrotestosterone-stimulated, androgen-dependent LNCaP-FGC cell growth. In addition, f-adiponectin enhances doxorubicin inhibition of prostate cancer cell growth. Therefore, f-adiponectin is a molecular mediator between prostate cancer and obesity, and may be therapeutic to prostate cancer.     

Post-translational modifications of adiponectin: mechanisms and functional implications

Adiponectin is an insulin-sensitizing adipokine with anti-diabetic, anti-atherogenic, anti-inflammatory and cardioprotective properties. This adipokine is secreted from adipocytes into the circulation as three oligomeric isoforms, including trimeric, hexameric and the HMW (high-molecular-mass) oligomeric complex consisting of at least 18 protomers. Each oligomeric isoform of adiponectin exerts distinct biological properties in its various target tissues. The HMW oligomer is the major active form mediating the insulin-sensitizing effects of adiponectin, whereas the central actions of this adipokine are attributed primarily to the hexameric and trimeric oligomers. In patients with Type 2 diabetes and coronary heart disease, circulating levels of HMW adiponectin are selectively decreased due to an impaired secretion of this oligomer from adipocytes. The biosynthesis of the adiponectin oligomers is a complex process involving extensive post-translational modifications. Hydroxylation and glycosylation of several conserved lysine residues in the collagenous domain of adiponectin are necessary for the intracellular assembly and stabilization of its high-order oligomeric structures. Secretion of the adiponectin oligomers is tightly controlled by a pair of molecular chaperones in the ER (endoplasmic reticulum), including ERp44 (ER protein of 44 kDa) and Ero1-Lalpha (ER oxidoreductase 1-Lalpha). ERp44 inhibits the secretion of adiponectin oligomers through a thiol-mediated retention. In contrast, Ero1-Lalpha releases HMW adiponectin trapped by ERp44. The PPARgamma (peroxisome-proliferator-activated receptor gamma) agonists thiazolidinediones selectively enhance the secretion of HMW adiponectin through up-regulation of Ero1-Lalpha. In the present review, we discuss the recent advances in our understanding of the structural and biological properties of the adiponectin oligomeric isoforms and highlight the role of post-translational modifications in regulating the biosynthesis of HMW adiponectin.     

Structure-function studies of the adipocyte-secreted hormone Acrp30/adiponectin. Implications fpr metabolic regulation and bioactivity

Acrp30/adiponectin is an adipocyte-specific secretory protein that has recently been implicated as a mediator of systemic insulin sensitivity with liver and muscle as target organs. Acrp30 is found as two forms in serum, as a lower molecular weight trimer-dimer and a high molecular weight complex. Little is know about the regulation and significance of these Acrp30 complexes in serum and about the events that lead to the generation of the bioactive ligand. Here, we show that there is a profound sexual dimorphism of Acrp30 levels and complex distribution in serum. Female mice display significantly higher levels of the high molecular weight complex in serum than males. In both females and males, levels of the high molecular weight complex are significantly reduced in response to a systemic increase of insulin. The ratio of the two complexes is restored upon normalization of glucose levels. Structurally, we show that oligomer formation of Acrp30 critically depends on disulfide bond formation mediated by Cys-39. Mutation of Cys-39 results in trimers that are subject to proteolytic cleavage in the collagenous domain. Surprisingly, Acrp30(C39S) or wild-type Acrp30 treated with dithiothreitol are significantly more bioactive than the higher order oligomeric forms of the protein with respect to reduction of serum glucose levels. Furthermore, treatment of primary hepatocytes with trimeric and higher order forms of Acrp30 confirms that the increased bioactivity seen in vivo is reflected in an augmented potency to reduce glucose output in the presence of gluconeogenic stimuli. Combined, these results shed new light on the regulation of this complex protein and suggest a new model for in vivo activation of the protein, implicating a serum reductase activity.     

Post-translational modifications of the four conserved lysine residues within the collagenous domain of adiponectin are required for the formation of its high molecular weight oligomeric complex

Adiponectin is a multifunctional adipokine that circulates as several oligomeric complexes in the blood stream. However, the molecular basis that regulates the production of the adiponectin oligomers remains largely elusive. We have shown previously that several conserved lysine residues (positions 68, 71, 80, and 104) within the collagenous domain of adiponectin are modified by hydroxylation and glycosylation (Wang, Y., Xu, A., Knight, C., Xu, L. Y., and Cooper, G. J. (2002) J. Biol. Chem. 277, 19521-19529). Here, we investigated the potential roles of these post-translational modifications in oligomeric complex formation of adiponectin. Gel filtration chromatography revealed that adiponectin produced from mammalian cells formed trimeric, hexameric, and high molecular weight (HMW) oligomeric complexes. These three oligomeric forms were differentially glycosylated, with the HMW oligomer having the highest carbohydrate content. Disruption of hydroxylation and glycosylation by substitution of the four conserved lysines with arginines selectively abrogated the intracellular assembly of the HMW oligomers in vitro as well as in vivo. In type 2 diabetic patients, both the ratios of HMW to total adiponectin and the degree of adiponectin glycosylation were significantly decreased compared with healthy controls. Functional studies of adiponectin-null mice revealed that abrogation of lysine hydroxylation/glycosylation markedly decreased the ability of adiponectin to stimulate phosphorylation of AMP-activated protein kinase in liver tissue. Chronic treatment of db/db diabetic mice with wild-type adiponectin alleviated hyperglycemia, hypertriglyceridemia, hepatic steatosis, and insulin resistance, whereas full-length adiponectin without proper post-translational modifications and HMW oligomers showed substantially decreased activities. Taken together, these data suggest that hydroxylation and glycosylation of the lysine residues within the collagenous domain of adiponectin are critically involved in regulating the formation of its HMW oligomeric complex and consequently contribute to the insulin-sensitizing activity of adiponectin in hepatocytes.     

Characterization of chemical exchange between soluble and aggregated states of beta-amyloid by solution-state NMR upon variation of salt conditions

Alzheimer's disease (AD) is characterized by the accumulation of insoluble fibrillar aggregates of beta-amyloid peptides (Abeta), a 39-42 residue peptide, in the brain of AD patients. It is hypothesized that the disease causing form is not the fibrillar species but an oligomeric Abeta molecule, which is often referred to as the "critical oligomer" of Abeta. We show in this paper that Abeta(1-40) undergoes chemical exchange between a monomeric, soluble state and an oligomeric, aggregated state under physiological conditions. In circular dichroism spectroscopy, we observe for this intermediate an alpha-helical structure. The oligomer is assigned a molecular weight of >100 kDa by diffusion-ordered spectroscopy-solution-state NMR spectroscopy (NMR). We can show by saturation transfer difference NMR experiments that the oligomer is related to monomeric Abeta. This experiment also allows us to identify the chemical groups that are involved in interactions between mono- and oligomeric Abeta molecules. Variation of the anionic strength in the buffer induces a shift of equilibrium between mono- and oligomeric states and possibly allows for the stabilization of these intermediate structures.     

Adiponectin Oligomers and Ectopic Fat in Liver and Skeletal Muscle in Humans

We aimed at determining which circulating forms of the adipokine adiponectin that increases lipid oxidation in liver and skeletal muscle are related to ectopic fat in these depots in humans. Plasma total‐, high‐molecular weight (HMW)‐, middle‐molecular weight (MMW)‐, and low‐molecular weight (LMW) adiponectin were quantified by an enzyme‐linked immunosorbent assay. Their relationships with liver‐ and intramyocellular fat, measured using ¹H magnetic resonance spectroscopy, were investigated in 54 whites without type 2 diabetes. Liver fat, adjusted for gender, age, and total body fat, was associated only with HMW adiponectin (r = ?0.35, P = 0.012), but not with total‐, MMW‐, or LMW adiponectin. In addition, subjects with fatty liver (liver fat ≥5.56%, n = 15) had significantly lower HMW‐ (P = 0.04), but not total‐, MMW‐, or LMW adiponectin levels, compared to controls (n = 39). Similarly, intramyocellular fat correlated only with HMW (r = ?0.32, P = 0.039), but not with the other circulating forms of adiponectin. These data indicate that, among circulating forms of adiponectin, HMW is strongly related to ectopic fat, thus possibly representing the form of adiponectin regulating lipid oxidation in liver and skeletal muscle.     

Proteomic and functional characterization of endogenous adiponectin purified from fetal bovine serum

Adiponectin is a plasma protein exclusively secreted from fat tissue. Many recent pharmacological studies suggest that recombinant adiponectin has multiple therapeutic potentials for obesity-related metabolic disorders, including type 2 diabetes, dyslipidemia, insulin resistance and atherosclerosis. However, the physiological relevance of these findings remains to be further established. In the present study, we have purified endogenous adiponectin from fetal bovine serum and characterized its post-translational modifications and physiological functions in animal models. Endogenous bovine serum adiponectin consists predominantly of full-length proteins that form multiple oligomeric complexes, including trimers, hexamers and higher molecular species. Two-dimensional gel electrophoresis revealed that bovine serum adiponectin exists as multiple post-translationally modified isoforms with distinct molecular weight and isoelectric point. Further analysis using mass spectrometry and Edman degradation sequencing demonstrated that five conserved lysine residues (Lys 28, 60, 63, 72 and 96) within the collagenous domain of bovine adiponectin are hydroxylated and glycosylated by a glucosyl alpha(1-2)galactosyl group. Injection of endogenous bovine adiponectin into C57 mice potently decreased circulating glucose levels and enhanced lipid clearance after a high fat meal. Chronic administration of this protein for a period of two weeks significantly increased insulin sensitivity and glucose tolerance, and depleted hepatic lipid accumulation in high-fat fed mice. These results provide direct evidence that endogenous bovine adiponectin is a physiological hormone that can regulate lipid and glucose metabolism.     

Complex distribution, not absolute amount of adiponectin, correlates with thiazolidinedione-mediated improvement in insulin sensitivity

Adiponectin is an adipocyte-specific secretory protein that circulates in serum as a hexamer of relatively low molecular weight (LMW) and a larger multimeric structure of high molecular weight (HMW). Serum levels of the protein correlate with systemic insulin sensitivity. The full-length protein affects hepatic gluconeogenesis through improved insulin sensitivity, and a proteolytic fragment of adiponectin stimulates beta oxidation in muscle. Here, we show that the ratio, and not the absolute amounts, between these two oligomeric forms (HMW to LMW) is critical in determining insulin sensitivity. We define a new index, S(A), that can be calculated as the ratio of HMW/(HMW + LMW). db/db mice, despite similar total adiponectin levels, display decreased S(A) values compared with wild type littermates, as do type II diabetic patients compared with insulin-sensitive individuals. Furthermore, S(A) improves with peroxisome proliferator-activated receptor-gamma agonist treatment (thiazolidinedione; TZD) in mice and humans. We demonstrate that changes in S(A) in a number of type 2 diabetic cohorts serve as a quantitative indicator of improvements in insulin sensitivity obtained during TZD treatment, whereas changes in total serum adiponectin levels do not correlate well at the individual level. Acute alterations in S(A) (DeltaS(A)) are strongly correlated with improvements in hepatic insulin sensitivity and are less relevant as an indicator of improved muscle insulin sensitivity in response to TZD treatment, further underscoring the conclusions from previous clamp studies that suggested that the liver is the primary site of action for the full-length protein. These observations suggest that the HMW adiponectin complex is the active form of this protein, which we directly demonstrate in vivo by its ability to depress serum glucose levels in a dose-dependent manner.     

Expression,purification and preliminary biochemical studies of the N-terminal domain of leucine-rich repeat kinase 2

Leucine-rich repeat kinase 2 gene is a key factor for Parkinson's disease and encodes for a large protein kinase LRRK2 (280 kDa) with multiple domains, including the different repeat sequences at the N-terminus such as ankyrin domain. Here, we successfully expressed and purified two kinds of LRRK2's N-terminal fragments N1 (aa12–320) and N2 (aa12–860). The purified N2 protein was identified by mass spectrometry and N1's molecular weight was determined to be 33.23 kDa. Gel filtration revealed that N1 exhibits as monomer, dimer and tetramer and N2 as oligomer in solution. N1's multiple oligomeric states were further proved by native-page and cross-linking gel experiments. Circular dichroism spectrum indicated that N1 and N2 contain both α helixes and β sheets. The polymerization character of LRRK2 N-terminal region would be speculated to relate with its biological function.     

Promotion of adiponectin multimerization by emodin: A novel AMPK activator with PPARγ-agonist activity

Adiponectin is an important insulin‐sensitizing adipokine with multiple beneficial effects on obesity‐associated medical complications. It is secreted from adipocytes into circulation as high, medium, and low molecular weight forms (HMW, MMW, and LMW). Each oligomeric form of adiponectin exerts non‐overlapping biological functions, with the HMW oligomer possessing the most potent insulin‐sensitizing activity. In this study, we reported that emodin, a natural product and active ingredient of various Chinese herbs, activates AMPK in both 3T3‐L1 adipocytes and 293T cells. Activation of AMPK by emodin promotes the assembly of HMW adiponectin and increases the ratio of HMW adiponectin to total adiponectin in 3T1‐L1 adipocytes. Emodin might activate AMPK by an indirect mechanism similar to berberine. We also found that emodin activates PPARγ and promotes differentiation and adiponectin expression during differentiation of 3T3‐L1 preadipocytes. Therefore, emodin is a novel AMPK activator with PPARγ‐agonist activity. Our results demonstrate that the effects of emodin on adiponectin expression and multimerization are the ultimate effects resulting from both AMPK activation and PPARγ activation. The dual‐activity makes emodin or the derivatives potential drug candidates for the treatment of type 2 diabetes and other obesity‐related metabolic diseases. J. Cell. Biochem. 113: 3547–3558, 2012. © 2012 Wiley Periodicals, Inc.     

Regulation and Quality Control of Adiponectin Assembly by Endoplasmic Reticulum Chaperone ERp44

Adiponectin, a collagenous hormone secreted abundantly from adipocytes, possesses potent antidiabetic and anti-inflammatory properties. Mediated by the conserved Cys³⁹ located in the variable region of the N terminus, the trimeric (low molecular weight (LMW)) adiponectin subunit assembles into different higher order complexes, e.g. hexamers (middle molecular weight (MMW)) and 12–18-mers (high molecular weight (HMW)), the latter being mostly responsible for the insulin-sensitizing activity of adiponectin. The endoplasmic reticulum (ER) chaperone ERp44 retains adiponectin in the early secretory compartment and tightly controls the oxidative state of Cys³⁹ and the oligomerization of adiponectin. Using cellular and in vitro assays, we show that ERp44 specifically recognizes the LMW and MMW forms but not the HMW form. Our binding assays with short peptide mimetics of adiponectin suggest that ERp44 intercepts and converts the pool of fully oxidized LMW and MMW adiponectin, but not the HMW form, into reduced trimeric precursors. These ERp44-bound precursors in the cis-Golgi may be transported back to the ER and released to enhance the population of adiponectin intermediates with appropriate oxidative state for HMW assembly, thereby underpinning the process of ERp44 quality control.     

Protease-resistant core form of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry1Ie: monomeric and oligomeric forms in solution

Cry1Ie is encoded by cry1Ie, a gene that is silent in Bacillus thuringiensis strains but can be over-expressed in Escherichia coli. A protease-resistant core form of Cry1Ie with a molecular weight of approx. 55 kDa was found among the products of trypsin digestion. The oligomer and monomer of the protease-resistant core form were purified separately. The oligomer fraction contained a small quantity of dimer and a large amount of aggregates larger than tetramers. The 50% lethal concentrations (LC₅₀) of the full-length Cry1Ie and both the monomer and oligomer fractions of the protease-resistant core form against Plutella xylostella were 14, 21 and 1400 μg/ml, respectively.     

Impaired multimerization of human adiponectin mutants associated with diabetes. Molecular structure and multimer formation of adiponectin

Adiponectin is an adipocyte-derived hormone, which has been shown to play important roles in the regulation of glucose and lipid metabolism. Eight mutations in human adiponectin have been reported, some of which were significantly related to diabetes and hypoadiponectinemia, but the molecular mechanisms of decreased plasma levels and impaired action of adiponectin mutants were not clarified. Adiponectin structurally belongs to the complement 1q family and is known to form a characteristic homomultimer. Herein, we demonstrated that simple SDS-PAGE under non-reducing and non-heat-denaturing conditions clearly separates multimer species of adiponectin. Adiponectin in human or mouse serum and adiponectin expressed in NIH-3T3 or Escherichia coli formed a wide range of multimers from trimers to high molecular weight (HMW) multimers. A disulfide bond through an amino-terminal cysteine was required for the formation of multimers larger than a trimer. An amino-terminal Cys-Ser mutation, which could not form multimers larger than a trimer, abrogated the effect of adiponectin on the AMP-activated protein kinase pathway in hepatocytes. Among human adiponectin mutations, G84R and G90S mutants, which are associated with diabetes and hypoadiponectinemia, did not form HMW multimers. R112C and I164T mutants, which are associated with hypoadiponectinemia, did not assemble into trimers, resulting in impaired secretion from the cell. These data suggested impaired multimerization and/or the consequent impaired secretion to be among the causes of a diabetic phenotype or hypoadiponectinemia in subjects having these mutations. In conclusion, not only total concentrations, but also multimer distribution should always be considered in the interpretation of plasma adiponectin levels in health as well as various disease states.     

N-terminal Domain of Prion Protein Directs Its Oligomeric Association

The self-association of prion protein (PrP) is a critical step in the pathology of prion diseases. It is increasingly recognized that small non-fibrillar β-sheet-rich oligomers of PrP may be of crucial importance in the prion disease process. Here, we characterize the structure of a well defined β-sheet-rich oligomer, containing ∼12 PrP molecules, and often enclosing a central cavity, formed using full-length recombinant PrP. The N-terminal region of prion protein (residues 23–90) is required for the formation of this distinct oligomer; a truncated form comprising residues 91–231 forms a broad distribution of aggregated species. No infectivity or toxicity was found using cell and animal model systems. This study demonstrates that examination of the full repertoire of conformers and assembly states that can be accessed by PrP under specific experimental conditions should ideally be done using the full-length protein.     

Nox5 forms a functional oligomer mediated by self-association of its dehydrogenase domain

Nox5 belongs to the calcium-regulated subfamily of NADPH oxidases (Nox). Like other calcium-regulated Noxes, Nox5 has an EF-hand-containing calcium-binding domain at its N-terminus, a transmembrane heme-containing region, and a C-terminal dehydrogenase (DH) domain that binds FAD and NADPH. While Nox1-4 require regulatory subunits, including p22phox, Nox5 activity does not depend on any subunits. We found that inactive point mutants and truncated forms of Nox5 (including the naturally expressed splice form, Nox5S) inhibit full-length Nox5, consistent with formation of a dominant negative complex. Oligomerization of full-length Nox5 was demonstrated using co-immunoprecipitation of coexpressed, differentially tagged forms of Nox5 and occurred in a manner independent of calcium ion. Several approaches were used to show that the DH domain mediates oligomerization: Nox5 could be isolated as a multimer when the calcium-binding domain and/or the N-terminal polybasic region (PBR-N) was deleted, but deletion of the DH domain eliminated oligomerization. Further, a chimera containing the transmembrane domain of Ciona intestinalis voltage sensor-containing phosphatase (CiVSP) fused to the Nox5 DH domain formed a co-immunoprecipitating complex with, and functioned as a dominant inhibitor of, full-length Nox5. Radiation inactivation of Nox5 overexpressed in HEK293 cells and endogenously expressed in human aortic smooth muscle cells indicated molecular masses of ～350 and ～300 kDa, respectively, consistent with a tetramer being the functionally active unit. Thus, Nox5 forms a catalytically active oligomer in the membrane that is mediated by its dehydrogenase domain. As a result of oligomerization, the short, calcium-independent splice form, Nox5S, may function as an endogenous inhibitor of calcium-stimulated ROS generation by full-length Nox5.     

Hepatitis C virus NS3 helicase forms oligomeric structures that exhibit optimal DNA unwinding activity in vitro

HCV NS3 helicase exhibits activity toward DNA and RNA substrates. The DNA helicase activity of NS3 has been proposed to be optimal when multiple NS3 molecules are bound to the same substrate molecule. NS3 catalyzes little or no measurable DNA unwinding under single cycle conditions in which the concentration of substrate exceeds the concentration of enzyme by 5-fold. However, when NS3 (100 nm) is equimolar with the substrate, a small burst amplitude of approximately 8 nm is observed. The burst amplitude increases as the enzyme concentration increases, consistent with the idea that multiple molecules are needed for optimal unwinding. Protein-protein interactions may facilitate optimal activity, so the oligomeric properties of the enzyme were investigated. Chemical cross-linking indicates that full-length NS3 forms higher order oligomers much more readily than the NS3 helicase domain. Dynamic light scattering indicates that full-length NS3 exists as an oligomer, whereas NS3 helicase domain exists in a monomeric form in solution. Size exclusion chromatography also indicates that full-length NS3 behaves as an oligomer in solution, whereas the NS3 helicase domain behaves as a monomer. When NS3 was passed through a small pore filter capable of removing protein aggregates, greater than 95% of the protein and the DNA unwinding activity was removed from solution. In contrast, only approximately 10% of NS3 helicase domain and approximately 20% of the associated DNA unwinding activity was removed from solution after passage through the small pore filter. The results indicate that the optimally active form of full-length NS3 is part of an oligomeric species in vitro.     

Adiponectin inhibits cell proliferation by interacting with several growth factors in an oligomerization-dependent manner

Adiponectin, an adipocyte-specific secretory protein, is present in serum as three oligomeric complexes. Apart from its roles as an anti-diabetic and anti-atherogenic hormone, adiponectin has been implicated as an important regulator of cell growth and tissue remodeling. Here we show that some of these functions might be mediated by the specific interactions of adiponectin with several important growth factors. Among six different growth factors examined, adiponectin was found to bind with platelet-derived growth factor BB (PDGF-BB), basic fibroblast growth factor (FGF), and heparin-binding epidermal growth factor-like growth factor (HB EGF) with distinct affinities. The bindings of adiponectin with these growth factors are oligomerization-dependent. PDGF-BB bound to the high molecular weight (HMW) and middle molecular weight (MMW) complexes, but not to the low molecular weight (LMW) complex of adiponectin. Basic FGF preferentially interacted with the HMW form, whereas HB EGF bound to all three forms with comparable affinities. These three growth factors did not compete with each other for their bindings to adiponectin, suggesting the involvement of distinct binding sites. The interactions of adiponectin with PDGF-BB, basic FGF, and HB EGF precluded the bindings to their respective membrane receptors and attenuated the DNA synthesis and cell proliferation induced by these growth factors. Small interfering RNA-mediated down-regulation of adiponectin receptors did not affect the suppressive effects of adiponectin on cell proliferation stimulated by these growth factors. These data collectively suggest that the oligomeric complexes of adiponectin can modulate the biological actions of several growth factors by controlling their bioavailability at a pre-receptor level and that this effect might partly account for the anti-atherogenic, anti-angiogenic, and anti-proliferative functions of adiponectin.