Absence Of Protein Disulfide Isomerase Research Articles

Folding Mechanism of a Precursor Protein of a Peptide Hormone Mediated by an Intra-Molecular Chaperone

Prouroguanylin is a precursor of uroguanylin. The mature form of uroguanylin contains intra-molecular disulfide bonds (Cys74-Cys82 and Cys77-Cys85). The propeptide region functions as an intra-molecular chaperone in the formation of the native conformation and the disulfide pairings of uroguanylin. To elucidate the mechanism of the propeptide-mediated folding, the pathway associated with the disulfide-coupled folding of prouroguanylin was examined in detail.Prouroguanylin, when prepared using an E. coli expression system, was obtained as an inclusion body. Therefore, it was purified as a reduced/denatured protein by reversed-phase HPLC after solubilization in urea. The folding reaction was carried out 0.1 M Tris/HCl (pH 8.0) at various concentrations of glutathione in the presence and absence of protein disulfide isomerase which catalyzes the disulfide exchange reaction.Kinetic analyses of the oxidative folding revealed that two types of intermediates containing mis-bridged disulfide bonds (namely, isomers 1 and 2 in which the disulfide bonds were between Cys74-Cys85 and Cys77-Cys82 and Cys74-Cys77 and Cys82-Cys85 in the mature region, respectively) are predominantly included in the folding. However, only one type of intermediate containing mis-disulfide bonds, isomer 2, was able to proceed to the native conformation of prouroguanylin, regardless of the presence of protein disulfide isomerase.The results of these experiments will be discussed in this presentation.

N-terminal Extension of the Cholera Toxin A1-chain Causes Rapid Degradation after Retrotranslocation from Endoplasmic Reticulum to Cytosol

Cholera toxin travels from the plasma membrane to the endoplasmic reticulum of host cells, where a portion of the toxin, the A1-chain, is unfolded and targeted to a protein-conducting channel for retrotranslocation to the cytosol. Unlike most retrotranslocation substrates, the A1-chain escapes degradation by the proteasome and refolds in the cytosol to induce disease. How this occurs remains poorly understood. Here, we show that an unstructured peptide appended to the N terminus of the A1-chain renders the toxin functionally inactive. Cleavage of the peptide extension prior to cell entry rescues toxin half-life and function. The loss of toxicity is explained by rapid degradation by the proteasome after retrotranslocation to the cytosol. Degradation of the mutant toxin does not follow the N-end rule but depends on the two Lys residues at positions 4 and 17 of the native A1-chain, consistent with polyubiquitination at these sites. Thus, retrotranslocation and refolding of the wild-type A1-chain must proceed in a way that protects these Lys residues from attack by E3 ligases.

A Role for Protein Disulfide Isomerase in the Early Folding and Assembly of MHC Class I Molecules

Proper folding and assembly of major histocompatibility complex (MHC) class I complexes are essential for optimal peptide loading and subsequent antigen presentation. MHC class I folding involves the coordinated formation of multiple disulfide bonds within MHC class I molecules. However, the regulation of disulfide bond formation during the early process of MHC class I folding is uncharacterized. Here, we show that protein disulfide isomerase (PDI) catalyzes the disulfide bond formation of MHC class I molecules and thereby facilitates the assembly of MHC class I heavy chain with beta(2)-microglobulin (beta(2)m). Depletion of PDI but not ERp57 by RNAi interfered with the disulfide bond formation in the MHC class I molecules. In the absence of PDI, the association of free class I heavy chain with calnexin increased, whereas the assembly of MHC class I heavy chain-beta(2)m heterodimers was delayed. These observations suggest that PDI-catalyzed disulfide bond formation of MHC class I molecules is an event downstream of the interaction of class I molecules with calnexin and upstream of their interaction with beta(2)m. Thus, our data establish a critical function for PDI in the early assembly of MHC class I molecules.

A Hypodermally Expressed Prolyl 4-Hydroxylase from the Filarial Nematode Brugia malayi Is Soluble and Active in the Absence of Protein Disulfide Isomerase

The collagen prolyl 4-hydroxylase (P4H) class of enzymes catalyze the hydroxylation of prolines in the X-Pro-Gly repeats of collagen chains. This modification is central to the synthesis of all collagens. Most P4Hs are alpha(2)beta(2) tetramers with the catalytic activity residing in the alpha subunits. The beta subunits are identical to the enzyme protein disulfide isomerase. The nematode cuticle is a collagenous extracellular matrix required for maintenance of the worm body shape. Examination of the model nematode Caenorhabditis elegans has demonstrated that its unique P4Hs are essential for viability and body morphology. The filarial parasite Brugia malayi is a causative agent of lymphatic filariasis in humans. We report here on the cloning and characterization of a B. malayi P4H with unusual properties. The recombinant B. malayi alpha subunit, PHY-1, is a soluble and active P4H by itself, and it does not become associated with protein disulfide isomerase. The active enzyme form is a homotetramer with catalytic and inhibition properties similar to those of the C. elegans P4Hs. High levels of B. malayi phy-1 transcript expression were observed in all developmental stages examined, and its expression was localized to the cuticle-synthesizing hypodermal tissue in the heterologous host C. elegans. Although active by itself, the B. malayi PHY-1 was not able to replace enzyme function in a C. elegans P4H mutant.

Catalysis of the Oxidative Folding of Bovine Pancreatic Ribonuclease A by Protein Disulfide Isomerase

The major oxidative folding pathways of bovine pancreatic ribonuclease A at pH 8.0 and 25 °C involve a pre-equilibrium steady state among ensembles of intermediates with zero, one, two, three and four disulfide bonds. The rate-determining steps are the reshuffling of the unstructured three-disulfide ensemble to two native-like three-disulfide species, des-[65-72] and des-[40-95], that convert to the native structure during oxidative formation of the fourth disulfide bond. Under the same regeneration conditions, with oxidized and reduced DTT, used previously for kinetic oxidative-folding studies of this protein, the addition of 4 μM protein disulfide isomerase (PDI) was found to lead to catalysis of each disulfide-formation step, including the rate-limiting rearrangement steps in which the native-like intermediates des-[65-72] and des-[40-95] are formed. The changes in the distribution of intermediates were also determined in the presence and absence of PDI at three different temperatures (with the DTT redox system) as well as at 25 °C (with the glutathione redox system). The results indicate that the acceleration of the formation of native protein by PDI, which we observed earlier, is due to PDI catalysis of each of the intermediate steps without changing the overall pathways or folding mechanism.

Effects of macromolecular crowding on protein folding and aggregation.

We have studied the effects of polysaccharide and protein crowding agents on the refolding of oxidized and reduced hen lysozyme in order to test the prediction that association constants of interacting macromolecules in living cells are greatly increased by macromolecular crowding relative to their values in dilute solutions. We demonstrate that whereas refolding of oxidized lysozyme is hardly affected by crowding, correct refolding of the reduced protein is essentially abolished due to aggregation at high concentrations of crowding agents. The results show that the protein folding catalyst protein disulfide isomerase is particularly effective in preventing lysozyme aggregation under crowded conditions, suggesting that crowding enhances its chaperone activity. Our findings suggest that the effects of macromolecular crowding could have major implications for our understanding of how protein folding occurs inside cells.

Protein disulphide isomerase assisted folding of human glucose-6-phosphate dehydrogenase

Glucose 6-phosphate dehydrogenase (G6PD) plays a key role to maintain the redox state within the red cells. The molecular basis of human G6PD deficiency rely on the presence of point mutations along its 59.2 kDa subunit [l]. The intracellular active enzyme behave mostly as a homodimer. Over 300 different G6PD deficient variants has been already reported and, in spite of their low intracellular activity some of them have normal Km values for glucose-6-phosphate and NADP. We have assumed that the alteration of the conformational stability of the protein could be driven by point mutations in the G6PD molecule and therefore a defective folded state could be incompatible with its normal activity. In order to test our hypothesis in this system we have studied the kinetics of the assisted protein disulfide isomerase (PDI) refolding of the normal G6PD for a subsequent comparison of these data with equivalent data from the variants. In this paper we report the experimental conditions of the PDI catalyzed refolding of the recombinant human G6PD. Human G6PD was expressed and purified to homogeneity as previously described [2] . Purification of bovine PDI was carried out by the method of Hillson et al., [3]. Reduced, denatured G6PD was obtained as previously reported by Creighton [41 with DTT and guanidine hydrochloride (Gdn-HCI). Excess of DTI and Gdn-HC1 was removed from the denatured G6PD by centrifugal gel filtration (Sephadex G25) in 0.1 YO acetic acid. Renaturation of the G6PD activity was monitored spectrophotometrically at 340 nm. As shown in Table 1, the presence of PDI in the folding mixture favoured the oxidative renaturation of G6PD increasing the in vitro rate of this event with respect the absence of PDI. As G6PD monomers do not show any activity, the refolding of the protein must be accompanied by the assembly of the monomers into dimers, allowing the detection of the G6PD activity. NADP, both in the presence and absence of PDI, increased the refolding rate, in agreement with previous studies on the unfolding of G6PD where NADP was shown to drive a conformational drift of the enzyme [5]. As illustrated in Figure 1, this effect

Redox conditions for stimulation of in vitro folding and assembly of the glycoprotein hormone chorionic gonadotropin

The formation of native disulfide bonds during in vitro protein folding can be limiting in obtaining biologically active proteins. Thus, optimization of redox conditions can be critical in maximizing the yield of renatured, recombinant proteins. We have employed a folding model, that of the beta subunit of human chorionic gonadotropin (hCG- beta), to investigate in vitro oxidation conditions that facilitate the folding of this protein, and have compared the in vitro rates obtained with the rate of folding that has been observed in intact cells. Two steps in the folding pathway of hCG-beta were investigated: the rate-limiting events in the folding of this protein, and the assembly of hCG-beta with, hCG-alpha. The rates of these folding events were determined with and without protein disulfide isomerase (PDI) using two different types of redox reagents: cysteamine and its oxidized equivalent, cystamine, and reduced and oxidized glutathione. Rates of the rate-limiting folding events were twofold faster in cysteamine/cystamine redox buffers than in glutathione buffers in the absence of PDI. Optimal conditions for hCG-beta folding were attained in a 2 mM glutathione buffer, pH 7.4, that contained 1 mg/mL PDI and in 10 microM cysteamine/cystamine, pH 8.7, without PDI. Under these conditions, the half-time of the ratelimiting folding event was 16 to 20 min and approached the rate observed in intact cells (4 to 5 min). Moreover, folding of the beta subunit under these conditions yields a functional protein, based on its ability to assemble with the alpha subunit. The rates of assembly of hCG-beta with hCG-alpha in the cysteamine/cystamine or glutathione/PDI redox buffers were comparable (t(1/2/sb> = 9 to 12 min)). These studies show that rates of folding and assembly events that involve disulfide bond formation can be optimized by a simple buffer system composed of cysteamine and cystamine.