Different contributions of the three CXXC motifs of human protein-disulfide isomerase-related protein to isomerase activity and oxidative refolding

Human protein-disulfide isomerase (hPDI)-related protein (hPDIR), which we previously cloned from a human placental cDNA library (Hayano, T., and Kikuchi, M. (1995) FEBS Lett. 372, 210-214), and its mutants were expressed in the Escherichia coli pET system and purified by sequential nickel affinity resin chromatography. Three thioredoxin motifs (CXXC) of purified hPDIR were found to contribute to its isomerase activity with a rank order of CGHC > CPHC > CSMC, although both the isomerase and chaperone activities of this protein were lower than those of hPDI. Screening for hPDIR-binding proteins using a T7 phage display system revealed that alpha1-antitrypsin binds to hPDIR. Surface plasmon resonance experiments demonstrated that alpha1-antitrypsin interacts with hPDIR, but not with hPDI or human P5 (hP5). Interestingly, the rate of oxidative refolding of alpha1-antitrypsin with hPDIR was much higher than with hPDI or hP5. Thus, the substrate specificity of hPDIR differed from that associated with isomerase activity, and the contribution of the CSMC motif to the oxidative refolding of alpha1-antitrypsin was the most definite of the three (CSMC, CGHC, CPHC). Substitution of SM and PH in the CXXC motifs with GH increased isomerase activity and decreased oxidative refolding. In contrast, substitution of GH and PH with SM decreased isomerase activity and increased oxidative refolding. Because CXXC motif mutants lacking isomerase activity retain chaperone activity for the substrate rhodanese, it is clear that, similar to PDI and hP5, the isomerase and chaperone activities of hPDIR are independent. These results suggest that the central dipeptide of the CXXC motif is critical for both redox activity and substrate specificity.     

Balance of ATPase stimulation and nucleotide exchange is not required for efficient refolding activity of the DnaK chaperone

The DnaK system from Thermus thermophilus (DnaK(Tth)) exhibits pronounced differences in organisation and regulation to its mesophile counterpart from Escherichia coli (DnaK(Eco)). While the ATPase cycle of DnaK(Eco) is tightly regulated by the concerted action of the two cofactors DnaJ(Eco) and GrpE(Eco), the DnaK(Tth) system features an imbalance in this cochaperone mediated regulation. GrpE(Tth) considerably accelerates the ATP/ADP exchange, but DnaJ(Tth) only slightly stimulates ATPase activity, believed to be a key step for chaperone activity of DnaK(Eco). By in vitro complementation assays, we could not detect significant ATPase-stimulation of orthologous DnaJ(Tth) . DnaKEco or DnaJ(Eco). DnaK(Tth)-complexes as compared to the DnaK(Eco) system, although they were nevertheless active in luciferase refolding experiments. Assistance of protein recovery by DnaK thus seems to be uncoupled of the magnitude of DnaJ mediated ATPase-stimulation.     

Both chaperone and isomerase functions of protein disulfide isomerase are essential for acceleration of the oxidative refolding and reactivation of dimeric alkaline protease inhibitor

Oxidative refolding of the dimeric alkaline protease inhibitor (API) from Streptomyces sp. NCIM 5127 has been investigated. We demonstrate here that both isomerase and chaperone functions of the protein folding catalyst, protein disulfide isomerase (PDI), are essential for efficient refolding of denatured-reduced API (dr-API). Although the role of PDI as an isomerase and a chaperone has been reported for a few monomeric proteins, its role as a foldase in refolding of oligomeric proteins has not been demonstrated hitherto. Spontaneous refolding and reactivation of dr-API in redox buffer resulted in 45% to 50% reactivation. At concentrations <0.25 microM, reactivation rates and yields of dr-API are accelerated by catalytic amounts of PDI through its isomerase activity, which promotes disulfide bond formation and rearrangement. dr-API is susceptible to aggregation at concentrations >25 microM, and a large molar excess of PDI is required to enhance reactivation yields. PDI functions as a chaperone by suppressing aggregation and maintains the partially unfolded monomers in a folding-competent state, thereby assisting dimerization. Simultaneously, isomerase function of PDI brings about regeneration of native disulfides. 5-Iodoacetamidofluorescein-labeled PDI devoid of isomerase activity failed to enhance the reactivation of dr-API despite its intact chaperone activity. Our results on the requirement of a stoichiometric excess of PDI and of presence of PDI in redox buffer right from the initiation of refolding corroborate that both the functions of PDI are essential for efficient reassociation, refolding, and reactivation of dr-API.     

Both the isomerase and chaperone activities of protein disulfide isomerase are required for the reactivation of reduced and denatured acidic phospholipase A2.

The spontaneous reactivation yield of acidic phospholipase A2 (APLA2), a protein containing seven disulfide bonds, after reduction and denaturation in guanidine hydrochloride is very low. Protein disulfide isomerase (PDI) markedly increases the reactivation yield and prevents the aggregation of APLA2 during refolding in a redox buffer containing GSH and GSSG. S-methylated PDI (mPDI), with no isomerase but as nearly full chaperone activity as native PDI, has no effect on either the reactivation or aggregation of APLA2. However, the simultaneous presence of PDI and mPDI in molar ratios to APLA2 of 0.1 and 0.9 respectively fully reactivates the denatured enzyme, as does PDI alone at a ratio of 1. At ratios of 0.1 and 0.15 respectively, they completely suppress APLA2 aggregation, as does PDI alone at a ratio of 0.25. Moreover, delayed addition of PDI to the refolding buffer greatly diminished the reactivation yield of APLA2, but this deteriorating effect can be alleviated markedly by the presence of mPDI in the refolding buffer. Without GSSG, mPDI prevents the aggregation of APLA2 during refolding. It is proposed that the in vitro action of PDI as a foldase consists of both isomerase and chaperone activities, and the latter activity can be fully replaced by mPDI.     

Tissue factor coagulant function is enhanced by protein-disulfide isomerase independent of oxidoreductase activity

Protein-disulfide isomerase (PDI) switches tissue factor (TF) from coagulation to signaling by targeting the allosteric Cys186-Cys209 disulfide. Here, we further characterize the interaction of purified PDI with TF. We find that PDI enhances factor VIIa-dependent substrate factor X activation 5-10-fold in the presence of wild-type, oxidized soluble TF but not TF mutants that contain an unpaired Cys186 or Cys209. PDI-accelerated factor Xa generation was blocked by bacitracin but not influenced by inhibition of vicinal thiols, reduction of PDI, changes in redox gradients, or covalent thiol modification of reduced PDI by N-ethylmaleimide or methyl-methanethiosulfonate, which abolished PDI oxidoreductase but not chaperone activity. PDI had no effect on fully active TF on either negatively charged phospholipids or in activating detergent, indicating that PDI selectively acts upon cryptic TF to facilitate ternary complex formation and macromolecular substrate turnover. PDI activation was reduced upon mutation of TF residues in proximity to the macromolecular substrate binding site, consistent with a primary interaction of PDI with TF. PDI enhanced TF coagulant activity on microvesicles shed from cells, suggesting that PDI plays a role as an activating chaperone for circulating cryptic TF.     

Ribostamycin inhibits the chaperone activity of protein disulfide isomerase.

In the process of screening of proteins binding to ribostamycin in bovine liver using the affinity column chromatography, we found that ribostamycin inhibited the chaperone activity of protein disulfide isomerase (PDI), but it did not inhibit the isomerase activity. PDI was identified by SDS-PAGE, Western blotting, and N-terminal amino acid sequence analysis. A 100:1 molar ratio of ribostamycin to PDI was almost sufficient to completely inhibit the chaperone activity of PDI. The binding affinity of ribostamycin to purified bovine PDI was determined by the Biacore system, which gave a K(D) value of 3.19 x 10(-4) M. This suggests that ribostamycin binds to region distinct from the CGHC motif of PDI. This is the first report to describe the inhibitor of the chaperone activity of PDI.     

Combinations of protein-disulfide isomerase domains show that there is little correlation between isomerase activity and wild-type growth

Protein-disulfide isomerase (PDI) has five domains: a, b, b', a' and c, all of which except c have a thioredoxin fold. A single catalytic domain (a or a') is effective in catalyzing oxidation of a reduced protein but not isomerization of disulfides (Darby, N. J., and Creighton, T. E. (1995) Biochemistry 34, 11725-11735). To examine the structural basis for this oxidase and isomerase activity of PDI, shuffled domain mutants were generated using a method that should be generally applicable to multidomain proteins. Domains a and a' along with constructs ab, aa', aba', ab'a' display low disulfide isomerase activity, but all show significant reactivity with mammalian thioredoxin reductase, suggesting that the structure is not seriously compromised. The only domain order that retains significant isomerase activity has the b' domain coupled to the N terminus of the a' domain. This b'a'c has 38% of the isomerase activity of wild-type PDI, equivalent to the activity of full-length PDI with one of the active sites inactivated by mutation (Walker, K. W., Lyles, M. M., and Gilbert, H. F. (1996) Biochemistry 35, 1972-1980). Individual a and a' domains, despite their very low isomerase activities in vitro, support wild-type growth of a pdi1Delta Saccharomyces cerevisiae strain yeast. Thus, most of the PDI structure is dispensable for its essential function in yeast, and high-level isomerase activity appears not required for viability or rapid growth.     

The enzymatic activity of phosphoglucose isomerase is not required for its cytokine function

PGI is a housekeeping gene encoding phosphoglucose isomerase (PGI) a glycolytic enzyme that also functions as a cytokine (autocrine motility factor (AMF)/neuroleukin/maturation factor) upon secretion from the cell and binding to its 78 kDa seven-transmembrane domain receptor (gp78/AMF-R). PGI contains a CXXC motif, characteristic of redox proteins and possibly evolutionarily related to the CC and CXC motif of the chemokine gene family. Using site-directed mutagenesis, single- and double-deletion (CXC, CC) mutants were created by deleting amino acids 331 and 332 of human PGI, respectively. The mutant proteins lost their enzymatic activity; however, neither of the deletions augmented the proteins' binding affinity to the receptor and all maintained cytokine function. The results demonstrate that the enzymatic activity of PGI is not essential for either receptor binding or cytokine function of human PGI.     

Characterization of a cDNA for human glutathione-insulin transhydrogenase (protein-disulfide isomerase/oxidoreductase)

A human liver cDNA expression library in lambda-phage gt11 was screened with monoclonal antibodies to rat liver protein-disulfide isomerase/oxidoreductase (EC 5.3.4.1/1.8.4.2), also known as glutathione-insulin transhydrogenase (GIT). The nucleotide sequence of the largest cDNA insert (hgit-1) was determined. It contained approx. 1500 basepairs, representing an estimated 65% of the glutathione-insulin transhydrogenase message. The amino-acid sequence deduced from this cDNA insert contains a 7-amino-acid long polypeptide determined by sequencing the active-site fragment isolated from the rat GIT protein. A comparison of the nucleotide sequence of hgit-1 and a previously reported nucleotide sequence of rat glutathione-insulin transhydrogenase cDNA shows that the human hgit-1 clone corresponds to the middle of the transhydrogenase message at amino-acid residue number 275 of the rat protein, and codes for 206 amino-acid residues, including one of the two active-site regions of glutathione-insulin transhydrogenase, a stop codon (TAA), a long 3'-noncoding region of over 800 bases, a polyadenylation signal (AATAA), and a 29 base poly(A) tail. There exists high homology between the human and rat enzymes (94% in the overall amino-acid sequence, with 100% in the active site region and 81% in the nucleotide sequence within the coding portion of hgit-1). As with the rat enzyme, the human enzyme shows some identity with another dithiol-disulfide-exchange protein, Escherichia coli thioredoxin. Like rat cDNA, the human hgit-1 cDNA hybridized to rat mRNA of 2500 bases on a Northern blot. The relative quantitative abundance of GIT mRNA in nine rat tissues studied using hgit-1 as a hybridization probe was found to be in the same order as previously found with the rat cDNA. Thus, the above studies indicate that glutathione-insulin transhydrogenase is a highly conserved protein and that the human hgit-1 cDNA is suitable for use as a probe for further studies on gene regulation of this enzyme.     

Characterization of a cDNA for human glutathione-insulin transhydrogenase (protein-disulfide isomerase / oxidoreductase)

A human liver cDNA expression library in λ-phage gt11 was screened with monoclonal antibodies to rat liver protein-disulfide isomerase / oxidoreductase (EC 5.3.4.1 / 1.8.4.2), also known as glutathione-insulin transhydrogenase (GIT). The nucleotide sequence of the largest cDNA insert (hgit-1) was determined. It contained approx. 1500 basepairs, representing an estimated 65% of the glutathione-insulin transhydrogenase message. The amino-acid sequence deduced from this cDNA insert contains a 7-amino-acid long polypeptide determined by sequencing the active-site fragment isolated from the rat GIT protein. A comparison of the nucleotide sequence of hgit-1 and a previously reported nucleotide sequence of rat glutathione-insulin transhydrogenase cDNA shows that the human hgit-1 clone corresponds to the middle of the transhydrogenase message at amino-acid residue number 275 of the rat protein, and codes for 206 amino-acid residues, including one of the two active-site regions of glutathione-insulin transhydrogenase, a stop codon (TAA), a long 3′-noncoding region of over 800 bases, a polyadenylation signal (AATAA), and a 29 base poly(A) tail. There exists high homology between the human and rat enzymes (94% in the overall amino-acid sequence, with 100% in the active site region and 81% in the nucleotide sequence within the coding portion of hgit-1). As with the rat enzyme, the human enzyme shows some identity with another dithiol-disulfide-exchange protein, Escherichia coli thioredoxin. Like rat cDNA, the human hgit-1 cDNA hybridized to rat mRNA of 2500 bases on a Northern blot. The relative quantitative abundance of GIT mRNA in nine rat tissues studied using hgit-1 as a hybridization probe was found to be in the same order as previously found with the rat cDNA. Thus, the above studies indicate that glutathione-insulin transhydrogenase is a highly conserved protein and that the human hgit-1 cDNA is suitable for use as a probe for further studies on gene regulation of this enzyme.     

Mutations that destabilize the a' domain of human protein-disulfide isomerase indirectly affect peptide binding

Protein-disulfide isomerase (PDI) is a catalyst of folding of disulfide-bonded proteins and also a multifunctional polypeptide that acts as the beta-subunit in the prolyl 4-hydroxylase alpha(2)beta(2)-tetramer (P4H) and the microsomal triglyceride transfer protein alphabeta-dimer. The principal peptide-binding site of PDI is located in the b' domain, but all domains contribute to the binding of misfolded proteins. Mutations in the C-terminal part of the a' domain have significant effects on the assembly of the P4H tetramer and other functions of PDI. In this study we have addressed the question of whether these mutations in the C-terminal part of the a' domain, which affect P4H assembly, also affect peptide binding to PDI. We observed a strong correlation between P4H assembly competence and peptide binding; mutants of PDI that failed to form a functional P4H tetramer were also inactive in peptide binding. However, there was also a correlation between inactivity in these assays and indicators of conformational disruption, such as protease sensitivity. Peptide binding activity could be restored in inactive, protease-sensitive mutants by selective proteolytic removal of the mutated a' domain. Hence we propose that structural changes in the a' domain indirectly affect peptide binding to the b' domain.     

The budding yeast Rad9 checkpoint complex: chaperone proteins are required for its function

Rad9 functions in the DNA-damage checkpoint pathway of Saccharomyces cerevisiae. In whole-cell extracts, Rad9 is found in large, soluble complexes, which have functions in amplifying the checkpoint signal. The two main soluble forms of Rad9 complexes that are found in cells exposed to DNA-damaging treatments were purified to homogeneity. Both of these Rad9 complexes contain the Ssa1 and/or Ssa2 chaperone proteins, suggesting a function for these proteins in checkpoint regula-tion. Consistent with this possibility, genetic experiments indicate redundant functions for SSA1 and SSA2 in survival, G2/M-checkpoint regulation, and phosphorylation of both Rad9 and Rad53 after irradiation with ultraviolet light. Ssa1 and Ssa2 can now be considered as novel checkpoint proteins that are likely to be required for remodelling Rad9 complexes during checkpoint-pathway activation.     

Protein disulfide isomerase isomerizes non-native disulfide bonds in human proinsulin independent of its peptide-binding activity

Protein disulfide isomerase (PDI) supports proinsulin folding as chaperone and isomerase. Here, we focus on how the two PDI functions influence individual steps in the complex folding process of proinsulin. We generated a PDI mutant (PDI-aba'c) where the b' domain was partially deleted, thus abolishing peptide binding but maintaining a PDI-like redox potential. PDI-aba'c catalyzes the folding of human proinsulin by increasing the rate of formation and the final yield of native proinsulin. Importantly, PDI-aba'c isomerizes non-native disulfide bonds in completely oxidized folding intermediates, thereby accelerating the formation of native disulfide bonds. We conclude that peptide binding to PDI is not essential for disulfide isomerization in fully oxidized proinsulin folding intermediates.     

High enzymatic activity and chaperone function are mechanistically related features of the dimeric E. coli peptidyl-prolyl-isomerase FkpA.

We have recently described the existence of a chaperone activity for the dimeric peptidyl-prolyl cis/trans isomerase FkpA from the periplasm of Escherichia coli that is independent of its isomerase activity. We have now investigated the molecular mechanism of these two activities in vitro in greater detail. The isomerase activity with a protein substrate (RNaseT1) is characterized by a 100-fold higher k(cat)/K(M) value than with a short tetrapeptide substrate. This enhanced activity with a protein is due to an increased affinity towards the protein substrate mediated by a polypeptide-binding site that is distinct from the active site. The chaperone activity is also mediated by interaction of folding and unfolding intermediates with a binding site that is most likely identical to the polypeptide-binding site which enhances catalysis. Both activities are thus mechanistically related, being based on the transient interaction with this high-affinity polypeptide-binding site. Only the isomerase activity, but not the chaperone activity, with the substrate citrate synthase can be inhibited by FK520. Experiments with the isolated domains of FkpA imply that both the isomerase and the chaperone site are located on the highly conserved FKBP domain. The additional amino-terminal domain mediates the dimerization and thus places the two active sites of the FKBP domains in juxtaposition, such that they can simultaneously interact with a protein, and this is required for full catalytic activity.     

The acidic C-terminal domain stabilizes the chaperone function of protein disulfide isomerase

Protein disulfide isomerase (PDI, EC 5.3.4.1) is a chaperone and catalyzes the formation and rearrangement of disulfide bonds in proteins. Domain c-(463-491), containing 18 acidic residues, is an interesting and important C-terminal extension of PDI. In this study, the PDI mutant abb'a', in which domain c is truncated, was used to investigate the relationship between the C-terminal structure and chaperone function. Reactivation and light-scattering experiments show that both wild-type PDI and abb'a' interact with lactate dehydrogenase (LDH, EC 1.1.1.27), which tends to self-aggregate during reactivation. The interaction enhances reactivation of LDH and reduces aggregation. According to these results, it seems as if domain c might be dispensable to the chaperone function of PDI. However, abb'a' is prone to self-aggregation and causes increased aggregation of LDH during thermal denaturation. In contrast, wild-type PDI remains active as a chaperone under these conditions and prevents self-aggregation of LDH. Furthermore, measurements of intrinsic fluorescence and difference absorbance during denaturation show that abb'a' is much more labile to heat or guanidine hydrochloride denaturation than wild-type PDI. This suggests that domain c is required for the stabilization and maintenance of the chaperone function of PDI under extreme conditions.     

A simplified method for the efficient purification and refolding of recombinant human TRAIL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) belongs to the TNF cytokine superfamily that specifically induces apoptosis in a broad spectrum of human cancer cell lines but not in most healthy cells. The antitumor potential of recombinant human TRAIL (rhTRAIL) has attracted great attention among biologists and oncologists. However, attempts to express rhTRAIL in Escherichia coli often results in limited yield of bioactive protein due to the formation of inclusion bodies (IBs), which are dense insoluble particulate protein aggregates inside cells. We describe herein a highly simplified method to produce pure bioactive rhTRAIL using E. coli. The method is straightforward and requires only basic laboratory equipment, with highly efficient purification and high yield of renaturation, and may also be applied to produce other proteins that form IBs in E. coli.     

Cationic polypeptides are required for antibacterial activity of human airway fluid

In a search for direct evidence leading to the biological relevance of airway secretions in innate host defense, we characterized the antibacterial function of cationic polypeptides within minimally manipulated nasal fluid. In this study, we show that cationic antimicrobial polypeptides are responsible for most of the bactericidal activity of whole nasal fluid. The removal of cationic polypeptides using a cation-exchange resin ablated the activity of nasal fluid against Escherichia coli, Listeria monocytogenes, and Pseudomonas aeruginosa. By using a novel proteomic approach, we identified a dozen cationic peptides and proteins within nasal fluid, all of which either are known antimicrobial polypeptides or have other proposed roles in host defense. Of the three most abundant cationic polypeptides in nasal fluid, lysozyme was more effective than either lactoferrin or secretory leukoprotease inhibitor in restoring the antibacterial activity of the cationic polypeptide-depleted fluid against a mucoid cystic fibrosis isolate of P. aeruginosa.     

TCRbeta transmembrane tyrosines are required for pre-TCR function.

The pre-TCR promotes thymocyte development in the alphabeta lineage. Productive rearrangement of the TCRbeta locus triggers the assembly of the pre-TCR, which includes the pTalpha chain and CD3 epsilongammadeltazeta subunits. This complex receptor signals the up-regulation of CD4 and CD8 expression, thymocyte proliferation/survival, and the cessation of TCRbeta rearrangements (allelic exclusion). In this study, we investigate the function of two conserved tyrosine residues located in the TCRbeta chain transmembrane region of the pre-TCR. We show that replacement of both tyrosines with alanine and expression of the mutant receptor in RAG-1(null) thymocytes prevents surface expression and abolishes pre-TCR function relative to wild-type receptor. Replacement of both tyrosines with phenylalanines (YF double mutant) generates a complex phenotype in which thymocyte survival and proliferation are severely disrupted, differentiation is moderately disrupted, and allelic exclusion is unaffected. We further show that the YF double mutant receptor is expressed on the cell surface and associates with pTalpha and CD3epsilon at the same level as does wild-type TCRbeta, while association of the YF double mutant with CD3zeta is slightly reduced relative to wild type. These data demonstrate that pre-TCR signaling pathways leading to proliferation and survival, differentiation, and allelic exclusion are differently sensitive to subtle mutation-induced alterations in pre-TCR structure.     

Hsp90 chaperone complexes are required for the activity and stability of yeast protein kinases Mik1, Wee1 and Swe1.

The Wee1 protein kinase negatively regulates entry into mitosis by mediating the inhibitory tyrosine phosphorylation of Cdc2-cyclin B kinase. The stability and activity of Wee1 from the fission yeast Schizosaccharomyces pombe is critically dependent on functional Hsp90 chaperones. Here we identify two related tyrosine protein kinases, Mik1 from fission yeast and its Saccharomyces cerevisiae homolog Swe1, as Hsp90 substrates and show that the kinase domain is sufficient to mediate this interaction. Morphological and biochemical defects arising from overexpression of the kinases in fission yeast are suppressed in the conditional Hsp90 mutant swo1-26. A subset of all three kinases is associated with the Hsp90 cochaperones cyclophilin 40 and p23. Under conditions of impaired chaperone function or treatment with the Hsp90 inhibitory drug geldanamycin, intracellular levels of the kinases are reduced and the proteins become rapidly degraded by the proteasome machinery, indicating that Wee1, Mik1 and Swe1 require Hsp90 heterocomplexes for their stability and maintenance of function.