Robert B. Freedman

Protein disulphide isomerase: building bridges in protein folding

Protein disulphide isomerase (PDI) has been known for many years to assist in the folding of proteins containing disulphide bonds, but the exact mechanism by which it achieves this is only now becoming clear. The active site of PDI closely resembles that of the redox protein thioredoxin, and cDNA cloning has revealed a superfamily of proteins with related active-site sequences, in organisms ranging from bacteria to higher animals and plants. Recent mutagenesis studies are now helping to unravel the catalytic mechanism of PDI, and work in yeast and other systems is clarifying the physiological roles of the multiple PDI-related proteins.

Protein disulfide isomerase: Multiple roles in the modification of nascent secretory proteins

La proteine disulfide isomerase semble avoir des roles multiples dans le fonctionnement cellulaire. Par exemple, elle catalyse la formation de disulfide dans la biosynthese des proteines de secretion. Elle est identique a la sous-unite β de la prolyl-4-hydroxylase, ce qui suggere un role dans les tissus synthetisant du collagene. La proteine disulfide isomerase pourrait aussi etre un composant du systeme de N-glycosylation cotranslationnelle

The b′ domain provides the principal peptide‐binding site of protein disulfide isomerase but all domains contribute to binding of misfolded proteins

Protein disulfide isomerase (PDI) is a very efficient catalyst of folding of many disulfide‐bonded proteins. A great deal is known about the catalytic functions of PDI, while little is known about its substrate binding. We recently demonstrated by cross‐linking that PDI binds peptides and misfolded proteins, with high affinity but broad specificity. To characterize the substrate‐binding site of PDI, we investigated the interactions of various recombinant fragments of human PDI, expressed in Escherichia coli, with different radiolabelled model peptides. We observed that the b′ domain of human PDI is essential and sufficient for the binding of small peptides. In the case of larger peptides, specifically a 28 amino acid fragment derived from bovine pancreatic trypsin inhibitor, or misfolded proteins, the b′ domain is essential but not sufficient for efficient binding, indicating that contributions from additional domains are required. Hence we propose that the different domains of PDI all contribute to the binding site, with the b′ domain forming the essential core.

Native disulphide bond formation in protein biosynthesis: evidence for the role of protein disulphide isomerase

Abstract Formation of native S:Sbonds is an early step in the post-translational modification of secretory proteins. Recent evidence indicates that this step is catalysed by protein disulphide-isomerase, an abundant enzyme in the endoplasmic reticulum of secretory cells.

Protein disulfide isomerases exploit synergy between catalytic and specific binding domains

Protein disulfide isomerases (PDIs) catalyse the formation of native disulfide bonds in protein folding pathways. The key steps involve disulfide formation and isomerization in compact folding intermediates. The high‐resolution structures of the a and b domains of PDI are now known, and the overall domain architecture of PDI and its homologues can be inferred. The isolated a and a′ domains of PDI are good catalysts of simple thiol–disulfide interchange reactions but require additional domains to be effective as catalysts of the rate‐limiting disulfide isomerizations in protein folding pathways. The b′ domain of PDI has a specific binding site for peptides and its binding properties differ in specificity between members of the PDI family. A model of PDI function can be deduced in which the domains function synergically: the b′ domain binds unstructured regions of polypeptide, while the a and a′ domains catalyse the chemical isomerization steps.

Formation and isomerization of disulfide bonds in proteins: Protein disulfide-isomerase

Publisher Summary Protein disulfide-isomerase (PDI) catalyzes the formation of native proteins from the reduced denatured state. When incubated in the presence of a thiol compound, PDI catalyzes the regain of native ribonuclease structure from the scrambled ribonuclease, with concomitant return of activity toward RNA. This assay is based on a patently nonphysiological substrate. It is very sensitive and has permitted the study of PDI activity in a number of contexts, making it possible to propose a physiological role for this activity. The chapter describes the preparation of scrambled ribonuclease from the beef pancreatic ribonuclease A, which contains a complex mixture of various molecular weight components. The substrate, scrambled ribonuclease, is essentially inactive in the hydrolytic cleavage of high-molecular-weight RNA, having about 2% of the activity of native ribonuclease. The action of PDI in catalyzing the interchange of inter- and intramolecular disulfides in scrambled ribonuclease results in the regain of the native disulfide pairing, native conformation, and concomitant return of ribonuclease activity against RNA. Thus, the activity of protein disulfide-isomerase is assayed by a time-course incubation during which aliquots are removed and ribonuclease activity toward RNA is measured. Protein disulfide-isomerase is very widely distributed and has been detected in most vertebrate tissues, although detailed studies have been confined to the enzyme from the liver.

Catalysis by protein-disulphide isomerase of the unfolding and refolding of proteins with disulphide bonds

Abstract Purified protein-disulphide isomerase has been examined for effects on the pathway and kinetics of the unfolding and refolding which accompanies disulphide bond breakage and reformation in bovine pancreatic trypsin inhibitor and bovine ribonuclease A. The intermediates of the pathways were not altered, although some interconversions which normally are not significant became so in the presence of the isomerase. The rate of every step involving both substantial protein conformational changes and protein disulphide bond formation, breakage or rearrangement was found to be increased significantly, but only when the conformational changes were rate-determining. The protein-disulphide isomerase appears to be a true catalyst of protein unfolding and refolding involving disulphide bond breakage, formation or rearrangement.

A major fraction of endoplasmic reticulum-located glutathione is present as mixed disulfides with protein.

The tripeptide glutathione is the most abundant thiol/disulfide component of the eukaryotic cell and is known to be present in the endoplasmic reticulum lumen. Accordingly, the thiol/disulfide redox status of the endoplasmic reticulum lumen is defined by the status of glutathione, and it has been assumed that reduced and oxidized glutathione form the principal redox buffer. We have determined the distribution of glutathione between different chemical states in rat liver microsomes by labeling with the thiol-specific label monobromobimane and subsequent separation by reversed phase high performance liquid chromatography. More than half of the microsomal glutathione was found to be present in mixed disulfides with protein, the remainder being distributed between the reduced and oxidized forms of glutathione in the ratio of 3:1. The high proportion of the total population of glutathione that was found to be in mixed disulfides with protein has significant implications for the redox state and buffering capacity of the endoplasmic reticulum and, hence, for the formation of disulfide bonds in vivo.

Activity of lipase in water-in-oil microemulsions

The lipase-catalysed hydrolysis rates of several nitrophenyl alkanoate esters of varying alkyl chain length (C4–C16) have been measured both in aqueous solution and in water-in-oil (w/o) microemulsions (which are known to contain discrete droplets). Lipase retains its activity in w/o microemulsions of water, heptane and sodium bis-2-ethylhexyl sulphosuccinate (AOT); the observed rates are consistent with the intrinsic activity of the enzyme (i.e. kcat/Km) being the same as in water. However, the observed conversion rates for C4 and C6 substrates are slower in the microemulsion system because of substrate partitioning to the oil-continuous phase, which results in a reduced concentration in the aqueous pseudophase. This conclusion is reached by comparing lipase and non-enzymic-(i.e. buffer) catalysed rates in both solution media. Again for the C4 and C6 substrate, partition coefficients for the substrates in the limit of high molar ratio of H2O:AOT, as determined from the kinetic results, show good agreement with measured values in heptane + water mixtures. This suggests that lipase functions effectively in the water pseudophase of the microemulsion. Lipase in the microemulsion can also catalyse the hydrolysis of longer chain alkanoates (up to C16). It can be inferred from the kinetics that such substrates partition to the interface where the lipase must also be active. In the case of AOT microemulsions, the pH profile of enzyme activity is not significantly altered compared with bulk water. The lipase retains > 60% activity in the microemulsion after incubation at 35 °C for 6 days. In w/o microemulsions of water, heptane, chloroform and cetyltrimethylammonium bromide (CTAB), the observed hydrolysis rates are significantly reduced and the intrinsic activity is reduced by a factor of twenty as compared with the AOT system. This is thought to be caused by inhibitory binding of CTAB to the protein.

Rapid monitoring of recombinant protein products: a comparison of current technologies

Specific measurement of recombinant protein titer in a complex environment during industrial bioprocessing has traditionally relied on labor-intensive and time-consuming immunoassays. In recent years, however, developments in analytical technology have resulted in improved methods for protein product monitoring during bioprocessing. The choice of product-monitoring technology for a particular bioprocess will depend on a variety of assay factors and instrument-specific factors. In this article, we have compiled an overview of the advantages and disadvantages of the most commonly used technologies used: electrochemiluminescence, optical biosensors, rapid chromatography and nephelometry. The advantages of each technology for measuring both small and large recombinant therapeutic proteins are compared with a conventional enzyme-linked immunosorbent assay (ELISA) technique.