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.

Genetic selection for enhanced folding in vivo targets the Cys14-Cys38 disulfide bond in bovine pancreatic trypsin inhibitor.

The periplasm provides a strongly oxidizing environment; however, periplasmic expression of proteins with disulfide bonds is often inefficient. Here, we used two different tripartite fusion systems to perform in vivo selections for mutants of the model protein bovine pancreatic trypsin inhibitor (BPTI) with the aim of enhancing its expression in Escherichia coli. This trypsin inhibitor contains three disulfides that contribute to its extreme stability and protease resistance. The mutants we isolated for increased expression appear to act by eliminating or destabilizing the Cys14-Cys38 disulfide in BPTI. In doing so, they are expected to reduce or eliminate kinetic traps that exist within the well characterized in vitro folding pathway of BPTI. These results suggest that elimination or destabilization of a disulfide bond whose formation is problematic in vitro can enhance in vivo protein folding. The use of these in vivo selections may prove a valuable way to identify and eliminate disulfides and other rate-limiting steps in the folding of proteins, including those proteins whose in vitro folding pathways are unknown.

Disulfide bond isomerization in prokaryotes.

Proteins with multiple cysteine residues often require disulfide isomerization reactions before they attain their correct conformation. In prokaryotes this reaction is catalyzed mainly by DsbC, a protein that shares many similarities in structure and mechanism to the eukaryotic protein disulfide isomerase. This review discusses the current knowledge about disulfide isomerization in prokaryotes.

Kinetic characterization of the disulfide bond-forming enzyme DsbB.

DsbB is an integral membrane protein responsible for the de novo synthesis of disulfide bonds in Escherichia coli and many other prokaryotes. In the process of transferring electrons from DsbA to a tightly bound ubiquinone cofactor, DsbB undergoes an unusual spectral transition at approximately 510 nm. We have utilized this spectral transition to study the kinetic cycle of DsbB in detail using stopped flow methods. We show that upon mixing of Dsb-B(ox) and DsbA(red), there is a rapid increase in absorbance at 510 nm (giving rise to a purple solution), followed by two slower decay phases. The rate of the initial phase is highly dependent upon DsbA concentration (k(1) approximately 5 x 10(5) M(-1) s(-1)), suggesting this phase reflects the rate of DsbA binding. The rates of the subsequent decay phases are independent of DsbA concentration (k(2) approximately 2 s(-1); k(3) approximately 0.3 s(-1)), indicative of intramolecular reaction steps. Absorbance measurements at 275 nm suggest that k(2) and k(3) are associated with steps of quinone reduction. The rate of DsbA oxidation was found to be the same as the rate of quinone reduction, suggestive of a highly concerted reaction. The concerted nature of the reaction may explain why previous efforts to dissect the reaction mechanism of DsbB by examining individual pairs of cysteines yielded seemingly paradoxical results. Order of mixing experiments showed that the quinone must be pre-bound to DsbB to observe the purple intermediate as well as for efficient quinone reduction. These results are consistent with a kinetic model for DsbB action in which DsbA binding is followed by a rapid disulfide exchange event. This is followed by quinone reduction, which is rate-limiting in the overall reaction cycle.

Gram-positive DsbE proteins function differently from Gram-negative DsbE homologs. A structure to function analysis of DsbE from Mycobacterium tuberculosis.

Mycobacterium tuberculosis, a Gram-positive bacterium, encodes a secreted Dsb-like protein annotated as Mtb DsbE (Rv2878c, also known as MPT53). Because Dsb proteins in Escherichia coli and other bacteria seem to catalyze proper folding during protein secretion and because folding of secreted proteins is thought to be coupled to disulfide oxidoreduction, the function of Mtb DsbE may be to ensure that secreted proteins are in their correctly folded states. We have determined the crystal structure of Mtb DsbE to 1.1 A resolution, which reveals a thioredoxin-like domain with a typical CXXC active site. These cysteines are in their reduced state. Biochemical characterization of Mtb DsbE reveals that this disulfide oxidoreductase is an oxidant, unlike Gram-negative bacteria DsbE proteins, which have been shown to be weak reductants. In addition, the pK(a) value of the active site, solvent-exposed cysteine is approximately 2 pH units lower than that of Gram-negative DsbE homologs. Finally, the reduced form of Mtb DsbE is more stable than the oxidized form, and Mtb DsbE is able to oxidatively fold hirudin. Structural and biochemical analysis implies that Mtb DsbE functions differently from Gram-negative DsbE homologs, and we discuss its possible functional role in the bacterium.

Disulfide bond formation involves a quinhydrone-type charge-transfer complex.

The chemistry of disulfide exchange in biological systems is well studied. However, the detailed mechanism of how oxidizing equivalents are derived to form disulfide bonds in proteins is not clear. In prokaryotic organisms, it is known that DsbB delivers oxidizing equivalents through DsbA to secreted proteins. DsbB becomes reoxidized by reducing quinones that are part of the membrane-bound electron-transfer chains. It is this quinone reductase activity that links disulfide bond formation to the electron transport system. We show here that purified DsbB contains the spectral signal of a quinhydrone, a charge-transfer complex consisting of a hydroquinone and a quinone in a stacked configuration. We conclude that disulfide bond formation involves a stacked hydroquinone-benzoquinone pair that can be trapped on DsbB as a quinhydrone charge-transfer complex. Quinhydrones are known to be redox-active and are commonly used as redox standards, but, to our knowledge, have never before been directly observed in biological systems. We also show kinetically that this quinhydrone-type charge-transfer complex undergoes redox reactions consistent with its being an intermediate in the reaction mechanism of DsbB. We propose a simple model for the action of DsbB where a quinhydrone-like complex plays a crucial role as a reaction intermediate.

Cell-type specific targeting and gene expression using a variant of polyoma VP1 virus-like particles.

The variant VP1-Z of the polyomavirus coat protein VP1 has been recently described as an engineered fusion protein of VP1 and the antibody binding domain protein Z. This construct is able to specifically bind and functionally present antibodies on the surface of virus-like particles of VP1-Z. Here we demonstrate that with the binding of Herceptin, an antibody directed against the receptor tyrosine kinase ErbB2, a cell type-specific targeting was established. ErbB2-positive cell lines were transduced with different plasmids encoding eGFP or beta-galactosidase. With both reporter systems functional gene expression in transduced cells could be observed. The transduction was strictly dependent on the use of a ternary complex formed of VLPs of VP1-Z, Herceptin, and the reporter plasmid DNA. The use of single components or ErbB2-negative cell lines did not result in functional gene transfer. The transduction was also completely dependent on the use of chloroquine, a lysosomotropic reagent. This indicates that the complex is internalized by ErbB2-mediated endocytosis.

Assessment of cell type specific gene transfer of polyoma virus like particles presenting a tumor specific antibody Fv fragment.

Application of delivery systems in cancer therapy is restricted as a result of the lack of cell specificity of the respective vectors. Recently, a vector system based on virus-like particles (VLPs) of modified polyoma-VP1 was described which were able to bind specifically a tumor-specific antibody fragment, thus directing the vector system towards tumor cells. The functional gene transfer using the VP1 variant VP1-E8C, coupled with the antibody fragment of the tumor-specific antibody B3 is described in this paper. The specific targeting of the antigen expressing cells was highly efficient as determined by fluorescence microscopy. However, only a low percentage of these cells showed a functional gene transfer. This discrepancy could be accounted for by a rather low capacity of the virus like particles to transport DNA and the mechanism of their internalization by the target cells, which led to a lysosomal degradation of the particles. These limitations could be surmounted partially in cell culture experiments, and the principles suitable for applying this vector system in vivo are discussed.