Human recombinant resistin protein displays a tendency to aggregate by forming intermolecular disulfide linkages

Resistin, a small cysteine rich protein secreted by adipocytes, has been proposed to be a link between obesity and type II diabetes by modulating the insulin signaling pathway and thus inducing insulin resistance. Resistin protein, with 11 cysteine residues, was not significantly homologous at the amino acid level to any other known cysteine rich proteins. Resistin cDNA derived from human subcutaneous adipose tissue was expressed in Escherichia coli as an N-terminal six-His-tag fusion protein. The overexpressed recombinant resistin was purified to homogeneity from inclusion bodies, after solubilization in 8 M urea, using a metal affinity column. While MALDI-TOF mass spectrometric analysis of the purified protein generated a single peak corresponding to the estimated size of 11.3 kDa, the protein exhibited a concentration-dependent oligomerization which is evident from size exclusion chromatography. The oligomeric structure was SDS-insensitive but beta-mercaptoethanol-sensitive, pointing to the importance of disulfide linkages in resistin oligomerization. Estimation of free cysteine residues using the NBD-Cl assay revealed a concentration- and time-dependent increase in the extent of formation of disulfide linkages. The presence of intermolecular disulfide bond(s), crucial in maintaining the global conformation of resistin, was further evident from fluorescence emission spectra. Circular dichroism spectra revealed that recombinant resistin has a tendency to reversibly convert from alpha-helical to beta-sheet structure as a direct function of protein concentration. Our novel observations on the biophysical and biochemical features of human resistin, particularly those shared with prion proteins, may have a bearing on its likely physiological function.     

Production and characterization of bioactive recombinant resistin in Escherichia coli

Type 2 diabetes, characterized by peripheral target tissue resistance to insulin, is epidemic in industrialized countries and is strongly associated with obesity. The protein hormone, resistin, secreted specifically by the adipose tissues, is found to antagonize insulin action upon glucose uptake and may serve as an important role between human obesity and insulin resistance. Here, we report the production of bioactive recombinant resistin in Escherichia coli. cDNA of resistin was obtained by RT-PCR from mRNA of mouse differentiated NIH/3T3-L1 cells. The cDNA of mature resistin was inserted in the pQE-31 vector and the recombinant plasmid was transferred into E. coli JM109. After IPTG induction, the rec. resistin found in the inclusion body was dissolved in 6 M guanidine-HCl in the presence of 10 mM beta-mercaptoethanol. The His-tag containing protein was purified by Ni-NTA column to 95% homogeneity. After a quasi-static-like refolding process, the secondary structure of the rec. resistin was elucidated by circular dichroism which indicated that the protein was composed of 34.3% alpha-helix, 8.9% beta-sheet, 23.4% beta-turn, and 31.2% unordered structure. No disulfide-linked homodimers were formed in SDS-PAGE analysis under non-reducing conditions. The rec. resistin showed a dose-dependent antagonizing action against insulin in [3H]-2-deoxy-glucose transport in a broad range from 1 ng ml(-1) to 10 microg ml(-1) of resistin. A suppression of 85% of transport was achieved at the dosage of 10 microg ml(-1). This result may indicate that the rec. resistin does not need to form homodimers to establish its bioactivity. The rec. resistin will be useful for exploring the biological functions of this newly discovered hormone.     

Human IgG2 can form covalent dimers

Unlike IgA and IgM, IgG has not yet been shown to form covalent polymers. However in the presence of specific Ag, murine IgG3 has been shown to polymerize through noncovalent interactions. In contrast to the noncovalent oligomers found with murine IgG3, we have detected covalent dimers in three different recombinant human IgG2 Abs produced in myeloma cells. Both IgG2,kappa and IgG2,lambda can form dimers. In addition, analysis of pooled human gamma globulin and several normal sera revealed the presence of IgG2 dimers. The IgG2 dimers are in contrast to the noncovalent IgG dimers found in pooled sera of multiple donors resulting from idiotype/anti-idiotype (Id/anti-Id) interactions. Cyanogen bromide cleavage analysis suggests that one or more Cys residues in the gamma 2 hinge are involved in dimer assembly. The potential role of IgG2 dimers in immunity against carbohydrate Ags is discussed.     

嗜中性粒细胞是人抵抗素表达的主要细胞

抵抗素(resistin)是小鼠白色脂肪组织大量表达的富含半胱氨酸的 分泌型蛋白.近年研究发现,人与啮齿类动物的抵抗素组织表达分布存在很 大差异．小鼠抵抗素主要在白色脂肪组织表达,而人抵抗素主要在单核细 胞/巨噬细胞表达,且在骨髓组织中大量表达,但目前骨髓中的细胞定位还 不清楚．本研究的目的是明确成人骨髓及外周血白细胞中抵抗素表达细胞 的类型．免疫荧光法检测骨髓中抵抗素表达细胞,结果显示,抵抗素主要表 达在细胞核呈杆状和分叶核状的成熟粒细胞中,其中杆状核粒细胞表达较 高,分叶核粒细胞表达减弱．Anti-hresistin IgG-Biotin-PE单色荧光流 式细胞术分选外周血白细胞中抵抗素表达细胞后经瑞氏化学染色,结果显 示,抵抗素表达细胞主要为杆状和分叶核状的嗜中性粒细胞,还有少量嗜酸 性粒细胞,且抵抗素蛋白分布在细胞质中. RT-qPCR结果在RNA水平上证明, 人抵抗素在嗜中性粒细胞中大量表达．Anti-hresistin IgG-FITC和anti- HNL IgG-Biotin-PE 双色荧光流式细胞术进一步证明,抵抗素的主要表达细 胞为成熟的嗜中性粒细胞．嗜中性粒细胞在机体免疫防御中起重要作用, 人骨髓及外周血中抵抗素主要在成熟嗜中性粒细胞中表达,这一研究结论 为人抵抗素与炎症反应的关联性及其功能的进一步研究奠定了基础.     

Adhesive and lateral E-cadherin dimers are mediated by the same interface

E-cadherin is a transmembrane protein that mediates Ca(2+)-dependent cell-cell adhesion. To study cadherin-cadherin interactions that may underlie the adhesive process, a recombinant E-cadherin lacking free sulfhydryl groups and its mutants with novel cysteines were expressed in epithelial A-431 cells. These cysteine mutants, designed according to various structural models of cadherin dimers, were constructed to reveal cadherin dimerization by the bifunctional sulfhydryl-specific cross-linker BM[PE0]3. Cross-linking experiments with the mutants containing a cysteine at strand B of their EC1 domains did show cadherin dimerization. By their properties these dimers correspond to those which have been characterized by co-immunoprecipitation assay. Under standard culture conditions the adhesive dimer is a dominant form. Calcium depletion dissociates adhesive dimers and promotes the formation of lateral dimers. Our data show that both dimers are mediated by the amino-terminal cadherin domain. Furthermore, the interfaces involved in both adhesive and lateral dimerization appear to be the same. The coexistence of the structurally identical adhesive and lateral dimers suggests some flexibility of the extracellular cadherin region.     

Expression and purification of mouse TIMP-1 from E. coli.

Tissue inhibitors of metalloproteinases (TIMPs) constitute a family of secreted glycoproteins involved in regulating extracellular matrix degradation in both normal and malignant tissues. We have expressed a cDNA clone of mouse TIMP-1 as a 22-kDa protein with 12 cysteine residues in E. coli and purified protein that shows inhibitory activity against collagenase following renaturation by chemical means. The low specific activity and circular dichroism measurements suggest, however, that the renaturation of the mouse recombinant (non-glycosylated) protein is not efficient under the conditions we have used, indicative of either thermodynamic instability or the transition to kinetic intermediates which have very low in vitro refolding rates.     

Cys146-Cys146分子间二硫键在人瘦素二聚化过程中起主导作用

在前期研究中,已发现人瘦素（leptin）在体外再折叠过程中会形成稳定的二聚体,但其二聚化机制尚不清楚. 本研究旨在分析瘦素二聚体的结构特性,并重点研究体外再折叠过程中瘦素二聚化的机制. 相较与瘦素单体,瘦素二聚体保留了约75％免疫活性及15％受体结合活性,同时显示出明显慢的天然电泳迁移率. 圆二色性分析显示,二聚体基本保留了单体α螺旋索结构特征. 还原性及非还原性凝胶电泳分析和自由巯基测定结果表明,瘦素二聚体是由一对分子间二硫键连接2个单体而成的.为了确定瘦素二聚化过程中起主导作用的分子间二硫键,利用PCR定点突变技术构建了C96S和C146S两个突变体瘦素. 通过分析C96S及C146S突变体瘦素的体外再折叠特性及过程,并与野生型瘦素相比较,揭示C96S瘦素的二聚体显示出与野生型瘦素二聚体相似的特性,而C146S瘦素不能形成结构稳定的二聚体. 以上研究结果表明,Cys146-Cys146分子间二硫键在人瘦素二聚化过程中起主导作用.     

Cloning and characterization of a mouse cysteine proteinase

cDNA clones encoding a mouse cysteine proteinase were isolated from a cDNA library constructed from mRNA derived from the macrophage-like cell line J774. The DNA sequence predicts a protein that is closely related to, but distinct from, the lysosomal enzyme cathepsin H. Alignment of the predicted amino acid sequence with the known protein sequences for seven other cysteine proteinases suggests that the cloned DNA encodes a 334-residue protein containing both a 17-amino acid pre-region and a 96-amino acid pro-region. Consistent with this prediction, antiserum raised to a recombinant fusion protein expressed in Escherichia coli immunoprecipitated multiple forms of the cysteine proteinase in mouse peritoneal macrophages and fibroblasts. In pulse-chase experiments, a 36-kDa precursor, presumedly the pro-form, was converted intracellularly into a 28-kDa protein and subsequently into a 21-kDa protein. Indirect immunofluorescence microscopy results suggested that the cysteine proteinase was localized to lysosomes. Western blot analysis detected significantly more of the proteinase in thioglycolate-elicited peritoneal macrophages than in resident peritoneal macrophages. Northern blot analysis revealed that several cell lines failed to express mouse cysteine proteinase mRNA.     

Early effects of hypoxia/reoxygenation on VEGF,Ang-1, Ang-2 and their receptors in the rat myocardium: Implications for myocardial angiogenesis

The oxidized form of vitamin C (dehydroascorbic acid, DHA) completely and irreversibly inactivates recombinant human hexokinase type I, in a pseudo-first order fashion. The inactivation reaction occurs without saturation, indicating that DHA does not form a reversible complex with hexokinase. Further characterization of this response revealed that the inactivation does not require oxygen and that dithiothreitol, while able to prevent the DHA-mediated loss of enzyme activity, failed to restore the activity of the DHA-inhibited enzyme. Inactivation was not associated with cleavage of the peptide chain or cross-linking. The decay in enzymatic activity was however both dependent on deprotonation of a residue with an alkaline pK_a and associated with covalent binding of DHA to the protein. In addition, inactivation of hexokinase decreased or increased, respectively, in the presence of the substrates glucose or MgATP. Finally, amino acid analysis of the DHA-modified hexokinase revealed a decrease of cysteine residues.Taken together, the above results are consistent with the possibility that covalent binding of the reagent with a thiol group of cysteine is a critical event for the DHA-mediated loss of hexokinase activity.     

The sugar binding activity of MR60, a mannose-specific shuttling lectin, requires a dimeric state

MR60 is an intracellular membrane protein which has been shown to act as a mannoside specific lectin and to be identical to ERGIC-53, a protein characteristic of the endoplasmic reticulum-Golgi apparatus- intermediate compartment, acting as a shuttle. According to its primary sequence, this MR60/ERGIC-53 protein contains a luminal domain including the carbohydrate recognition domain, a stem, a transmembrane segment and a cytosolic domain. The endogenous MR60/ERGIC-53 protein is spontaneously oligomeric, (dimers and hexamers). In this paper, we study the relationship between the oligomerization state and the sugar binding capacity by using recombinant proteins. The expression of the recombinant proteins was evidenced by immunocytochemistry and by immunoprecipitation followed by SDS-PAGE analysis. The full size recombinant protein binds mannosides and is oligomeric, up to the hexameric form. Two truncated proteins lacking the transmembrane and the cytosolic domains were prepared and characterized. A long one, containing the cysteine 466 close to the C-terminal end of the recombinant protein but lacking the cysteine 475, close to the C- terminal end of the native protein, does bind mannosides and forms dimers but no higher oligomeric forms. A shorter one, lacking both the cysteines 466 and 475, does not bind mannosides and does not form dimers or higher polymers. The two cysteines in the carbohydrate recognition domain (C190 and C230) are not involved in the stabilization of oligomers. In conclusion, this study shows that the luminal moiety of MR60/ERGIC-53 contains a device allowing both its oligomeric pattern and its sugar binding capability.      

Purification and characterization of recombinant forms of murine Tcl1 proteins

The TCL1 gene, which is located on chromosome 14, plays a major role in human hematopoietic malignancies and encodes a 14-kDa protein whose function has not been determined. This gene is expressed in pre-B cells, in immature thymocytes, and, at low levels, in activated T cells but not in peripheral mature B cells and in normal cells. The Tcl1 protein is similar in its primary structure to a protein encoded by the mature T-cell proliferation gene (MTCP1). The MTCP1 gene is located on the X chromosome and has been shown to be involved in rare chromosomal translocations in T-cell proliferative diseases. The murine TCL1 gene resides on mouse chromosome 12 and is homologous to the human TCL1 and MTCP1 genes. Murine Tcl1 protein has 116 amino acid residues and shares 50% sequence identity with human Tcl1, while the human and mouse Mtcp1 are nearly identical, with conservative differences in only six residues. The TCL1 and MTCP1 genes appear to be members of a family of genes involved in lymphoid proliferation and T-cell malignancies. Our laboratory has undertaken the study of the Tcl1 and Mtcp1 proteins to determine the structure and the function of these related proteins. In the present report, we have produced, using a bacterial expression system, the purified murine Tcl1 protein and a mutant form of murine Tcl1 protein containing a cysteine to alanine mutation at amino acid position 85. The recombinant proteins were purified by chromatography on a Ni-NTA resin followed by reverse-phase FPLC using a buffer system at pH 7.9 and a polymer-based reverse-phase column. The murine Tcl1 recombinant protein displays limited solubility and forms disulfide-linked dimers and oligomers, while the mutant murine Tcl1 C86A protein has increased solubility and does not form higher order oligomers. The purified recombinant murine proteins were characterized by N-terminal sequence analysis, mass spectrometry, and circular dichroism spectroscopy. Initial results indicate that the mutant murine Tcl1 C86A protein is suitable for both NMR and X-ray crystallographic methods of structure determination.     

Disulfide bond interchange in Escherichia coli-derived recombinant human interferon-beta 1 under denaturing conditions

Disulfide bond interchange has been pointed out as a considerable problem in preparing recombinant proteins from Escherichia coli cells. This has been reported in the system of reducing denaturation followed by a refolding process, where incorrectly folded molecules are sometimes produced. As the possibility of disulfide bond interchange may also arise in the cytoplasm of E. coli cells, the state of sulfhydryl groups of recombinant proteins obtained from a nonreducing and nondenaturing process should be examined. The state of sulfhydryl groups of E. coli-derived recombinant human interferon-beta 1, which had been purified under nonreducing and nondenaturing conditions, was examined by using the N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) labeling technique. Among the three cysteine residues in E. coli-derived human interferon-beta 1, the 17th cysteine was identified as being unpaired, as in the natural molecule. However, it was found that three isomers of the recombinant protein could be formed when the protein was denatured with 6 M guanidine hydrochloride. These three isomers were identified as having unpaired cysteine residues at positions 17, 31, and 141, respectively. These results indicate that disulfide bond interchange occurs in E. coli-derived recombinant human interferon-beta 1 under denaturing conditions in spite of the absence of a reducing agent.     

Characterization of a region of the measles virus hemagglutinin sufficient for its dimerization

Attachment of measles virus (MV) to its cellular receptor is mediated by the viral envelope glycoprotein hemagglutinin (H). H exists at the viral surface as a disulfide-linked dimer which may associate into a tetramer. We aimed to define regions of H essential for its homo-oligomerization. To delineate these more precisely, we have generated a series of H ectodomain truncation mutants and studied their abilities to form both homotypic complexes and heterotypic complexes with full-length H. We define a "minimal unit" which is sufficient for MV H dimerization as that encompassing residues 1 to 151. This unit forms both homodimers and heterodimers with full-length H protein, although neither is transported to the cell surface even in the presence of other MV proteins. We show that cysteine residues at positions 139 and 154 are both critical in mediating covalent dimerization, not only of the truncated H mutants but also of full-length MV H protein. Even those cysteine mutants unable to form covalent intermolecular interactions are biologically active, mediating the formation of syncytia, albeit at a reduced rate. We demonstrate that this impaired capacity to mediate cell-to-cell fusion is based mainly on a reduced transport rate of the mutant molecules to the cell surface, indicating a role for covalent intermolecular interactions in efficient transport of MV H dimers to the cell surface.     

Sml1p is a dimer in solution: characterization of denaturation and renaturation of recombinant Sml1p

Sml1p is a small 104-amino acid protein from Saccharomyces cerevisiae that binds to the large subunit (Rnr1p) of the ribonucleotide reductase complex (RNR) and inhibits its activity. During DNA damage, S phase, or both, RNR activity must be tightly regulated, since failure to control the cellular level of dNTP pools may lead to genetic abnormalities, such as genome rearrangements, or even cell death. Structural characterization of Sml1p is an important step in understanding the regulation of RNR. Until now the oligomeric state of Sml1p was unknown. Mass spectrometric analysis of wild-type Sml1p revealed an intermolecular disulfide bond involving the cysteine residue at position 14 of the primary sequence. To determine whether disulfide bonding is essential for Sml1p oligomerization, we mutated the Cys14 to serine. Sedimentation equilibrium measurements in the analytical ultracentrifuge show that both wild-type and C14S Sml1p exist as dimers in solution, indicating that the dimerization is not a result of a disulfide bond. Further studies of several truncated Sml1p mutants revealed that the N-terminal 8-20 residues are responsible for dimerization. Unfolding/refolding studies of wild-type and C14S Sml1p reveal that both proteins refold reversibly and have almost identical unfolding/refolding profiles. It appears that Sml1p is a two-domain protein where the N-terminus is responsible for dimerization and the C-terminus for binding and inhibiting Rnr1p activity.     

Comparative studies of resistin expression and phylogenomics in human and mouse

Resistin is a newly identified adipocytokine that has been proposed to be a link between obesity and type 2 diabetes based on animal studies. However, the role of resistin in the pathogenesis of insulin resistance associated with obesity in humans remains unclear. We comparatively and quantitatively studied the tissue distributions of resistin mRNA between human and mouse. The expression level of resistin mRNA in human adipose tissue is extremely low but detectable by real-time PCR and is about 1/250 of that in the mouse. Remarkably, resistin mRNA is abundant in human primary acute leukemia cells and myeloid cell lines U937 and HL60, but not in the Raw264 mouse myeloid cell line. Resistin expression in U937 cells was not affected by lipopolysaccharide (LPS) or by ciglitazone, a PPARgamma ligand. Phylogenomics revealed that the human resistin gene is the ortholog of its murine counterpart and is located in a region of chromosome 19p13.3, which is syntenic to mouse chromosome 8A1. In addition to the resistin-like molecule (RELM) sequences already reported, bioinformatics analysis disclosed another RELM sequence in the vicinity of RELMbeta on human chromosome 3q13.1, but this sequence is unlikely to encode an expressed gene. Therefore, only two RELMs, resistin and RELMbeta, exist in humans, instead of the three RELMs, resistin, RELMalpha, and RELMbeta, that exist in mice. This finding provides a possible answer to the question of why only two RELMs have been cloned in humans and suggests that the RELM family is not well conserved in evolution and may function differently between species. Therefore, caution should be exercised in interpreting resistin as a link between obesity and insulin resistance in humans. The high expression of resistin in human leukemia cells suggests a hitherto unidentified biological function of resistin in leukocytes.     

Biophysical analyses of human resistin: oligomer formation suggests novel biological function

Resistin, a small secreted peptide initially identified as a link between obesity and diabetes in mice, was shown to be involved in mediating inflammation in humans. We had shown earlier that recombinant human resistin has a tendency to form aggregates by formation of inter/intramolecular disulfide linkages and that it undergoes a concentration-dependent conformational change in secondary structure from alpha-helical to beta-sheet form. Here we report that this change in secondary structural conformation is due to the increase in the oligomeric form of human resistin as a function of protein concentration. Gel filtration analysis under different conditions further demonstrated that recombinant human resistin exists as a mixture of oligomer and trimer but is converted to a mixture of monomer and oligomer in the presence of 100 mM NaCl. We show that while the trimeric form of human resistin is stable to urea-induced denaturation, it is highly susceptible to NaCl and NaF, indicating the importance of ionic interactions in stabilization of trimer. In addition, urea was able to destabilize the oligomers indicating the involvement of hydrophobic interactions in oligomerization. Ionic as well as hydrophobic interactions stabilize the monomeric human resistin. Our data suggest that human resistin exists predominantly as oligomer and trimer in vitro. The oligomeric form of human resistin shows more potent effect on stimulation of proinflammatory cytokines. Therefore, it is very tempting to propose that the structural conformation of resistin may be involved in maintaining the very fine balance in regulation of macrophage function for successful response to a variety of pathological conditions.     

Enhanced in vitro refolding of fibroblast growth factor 15 with the assistance of SUMO fusion partner

Fibroblast growth factor 15 (Fgf15) is the mouse orthologue of human FGF19. Fgf15 is highly expressed in the ileum and functions as an endocrine signal to regulate liver function, including bile acid synthesis, hepatocyte proliferation and insulin sensitivity. In order to fully understand the function of Fgf15, methods are needed to produce pure Fgf15 protein in the prokaryotic system. However, when expressed in Escherichia coli (E. coli), the recombinant Fgf15 protein was insoluble and found only in inclusion bodies. In the current study, we report a method to produce recombinant Fgf15 protein in E. coli through the use of small ubiquitin-related modifier (SUMO) fusion tag. Even though the SUMO has been shown to strongly improve protein solubility and expression levels, our studies suggest that the SUMO does not improve Fgf15 protein solubility. Instead, proper refolding of Fgf15 protein was achieved when Fgf15 was expressed as a partner protein of the fusion tag SUMO, followed by in vitro dialysis refolding. After refolding, the N-terminal SUMO tag was cleaved from the recombinant Fgf15 fusion protein by ScUlp1 (Ubiquitin-Like Protein-Specific Protease 1 from S. cerevisiae). With or without the SUMO tag, the refolded Fgf15 protein was biologically active, as revealed by its ability to reduce hepatic Cyp7a1 mRNA levels in mice. In addition, recombinant Fgf15 protein suppressed Cyp7a1 mRNA levels in a dose-dependent manner. In summary, we have developed a successful method to express functional Fgf15 protein in prokaryotic cells.     

Archaeal group II chaperonin mediates protein folding in the cis-cavity without a detachable GroES-like co-chaperonin.

Group II chaperonins of archaea and eukaryotes are distinct from group I chaperonins of bacteria. Whereas group I chaperonins require the co-chaperonin Cpn-10 or GroES for protein folding, no co-chaperonin has been known for group II. The protein folding mechanism of group II chaperonins is not yet clear. To understand this mechanism, we examined protein refolding by the recombinant alpha or beta-subunit chaperonin homo-oligomer (alpha16mer and beta16mer) from a hyperthermoplilic archaeum, Thermococcus strain KS-1, using a model substrate, green fluorescent protein (GFP). The alpha16mer and beta16mer captured the non-native GFP and promoted its refolding without any co-chaperonin in an ATP dependent manner. A non-hydrolyzable ATP analog, AMP-PNP, induced the GFP refolding mediated by beta16mer but not by the alpha16mer. A mutant alpha-subunit chaperonin homo-oligomer (trap-alpha) could capture the non-native protein but lacked the ability to refold it. Although trap-alpha suppressed ATP-dependent refolding of GFP mediated by alpha16mer or beta16mer, it did not affect the AMP-PNP-dependent refolding. This indicated that the GFP refolding mediated by beta16mer with AMP-PNP was not accessible to the trap-alpha. Gel filtration chromatography and a protease protection experiment revealed that this refolded GFP, in the presence of AMP-PNP, was associated with beta16mer. After the completion of GFP refolding mediated by beta16mer with AMP-PNP, addition of ATP induced an additional refolding of GFP. Furthermore, the beta16mer preincubated with AMP-PNP showed the ability to capture the non-native GFP. These suggest that AMP-PNP induced one of two chaperonin rings (cis-ring) to close and induced protein refolding in this ring, and that the other ring (trans-ring) could capture the unfolded GFP which was refolded by adding ATP. The present data indicate that, in the group II chaperonin of Thermococcus strain KS-1, the protein folding proceeds in its cis-ring in an ATP-dependent fashion without any co-chaperonin.