Hilary C. Hawkins

Protein Dislfide-Isomerase

Protein-disulfide isomerase (PDI), a critical enzyme responsible for oxidative protein folding in the eukaryotic endoplasmic reticulum, is composed of four thioredoxin domains a, b, b′, a′, and a linker x between b′ and a′. Ero1-Lα, an oxidase for human PDI (hPDI), has been determined to have one molecular flavin adenine dinucleotide (FAD) as its prosthetic group. Oxygen consumption assays with purified recombinant Ero1-Lα revealed that it utilizes oxygen as a terminal electron acceptor producing one disulfide bond and one molecule of hydrogen peroxide per dioxygen molecule consumed. Exogenous FAD is not required for recombinant Ero1-Lα activity. By monitoring the reactivation of denatured and reduced RNase A, we reconstituted the Ero1-Lα/hPDI oxidative folding system in vitro and determined the enzymatic activities of hPDI in this system. Mutagenesis studies suggested that the a′ domain of hPDI is much more active than the a domain in Ero1-Lα-mediated oxidative folding. A domain swapping study revealed that one catalytic thioredoxin domain to the C-terminal of bb′x, whether a or a′, is essential in Ero1-Lα-mediated oxidative folding. These data, combined with a pull-down assay and isothermal titration calorimetry measurements, enabled the minimal element for binding with Ero1-Lα to be mapped to the b′xa′ fragment of hPDI.

EXPERIMENTAL AND THEORETICAL ANALYSES OF THE DOMAIN ARCHITECTURE OF MAMMALIAN PROTEIN DISULPHIDE-ISOMERASE

The high resolution structure of full-length protein disulphide-isomerase (PDI) has not been determined, but the polypeptide is generally assumed to comprise a series of consecutive domains. Models of its domain organisation have been proposed on the basis of various sequence-based criteria and, more recently, from structural studies on recombinant fragments corresponding to putative domains. We here describe direct studies of the domain architecture of full-length mammalian PDI based on limited proteolysis of the native enzyme. The results are consistent with an emerging model based on the existence of 4 consecutive domains each with the thioredoxin fold. The model was further tested by expressing recombinant fragments corresponding to alternative domain models and to truncated domains; the observed properties of these purified fragments supported the 4-domain model. A multiple alignment of many PDI-like sequences was generated to test whether domain boundaries could be predicted from any features of the alignment, such as sequence variability or hydrophilicity; neither of these parameters reliably predicted the domain boundaries determined by experiment.

Protein disulfide-isomerase

Publisher Summary Protein disulfide-isomerase is an abundant protein within the lumen of the endoplasmic reticulum of secretory cells, and functions as a catalyst in the formation of native disulfide bonds in nascent secretory and cell surface proteins. In its catalytic action in vitro, it facilitates the folding and assembly of a wide range of disulfide-bonded proteins, and individual thiol-disulfide interchange steps are accelerated by over 1000-fold. The role of PDI in co and posttranslational modification of proteins has been confirmed by its cross-linking to nascent immunoglobulins, by the requirement for PDI for efficient cotranslational disulfide formation in a reconstituted in vitro translation system, and by the phenotype of yeast lacking a functional PDI. In vertebrates, the protein is also a component of two other endoplasmic reticulum (ER) lumenal enzyme systems, prolyl-4-hydroxylase, and the microsomal triglyceride transfer protein.

Rat liver type I iodothyronine deiodinase is not identical to protein disulfide isomerase.

This study was done to test the recent hypothesis (Boado et al. (1988) Biochem. Biophys. Res. Commun. 155, 1297-1304) that type I iodothyronine deiodinase (ID-I) is identical to protein disulfide isomerase (PDI). Autoradiograms of rat liver microsomal proteins, labeled with N-bromoacetyl-[125I]triiodothyronine (BrAc[125I]T3) and separated by SDS-PAGE, show predominantly 2 radioactive bands of Mr 27 and 56 kDa. Substrates and inhibitors of ID-I inhibited labeling of the 27 kDa band but not that of the 56 kDa band. Treatment of microsomes with trypsin abolished labeling of the 27 kDa protein and destroyed the activity of ID-I but did not prevent labeling of the 56 kDa protein. Following treatment of microsomes at pH 8.0-9.5 or with 0.05% deoxycholate (DOC) PDI content and labeling of the 56 kDa protein were strongly diminished but ID-I activity and labeling of the 27 kDa protein were not affected. The latter decreased in parallel after treatment at pH greater than or equal to 10. Rat pancreas microsomes contain high amounts of PDI but show no ID-I activity. Reaction of these microsomes with BrAc[125I]T3 results in extensive labeling of a 56 kDa protein but no labeling of a 27 kDa protein. Pure PDI (Mr 56 kDa) was readily labeled by BrAc[125I]T3 but showed no deiodinase activity. These results strongly suggest that the 27 kDa band represents (a subunit of) ID-I while the 56 kDa band represents PDI. From these and other data it is concluded that PDI and ID-I are not identical proteins.

RANDOMLY REOXIDISED SOYBEAN TRYPSIN INHIBITOR AND THE POSSIBILITY OF CONFORMATIONAL BARRIERS TO DISULPHIDE ISOMERIZATION IN PROTEINS

The process by which a protein folds to its native conformation is determined by both thermodynamic and kinetic constraints. The classic work of Anfinsen [ 1,2] demonstrated that the thermodynamic stability of the native state determines the product of folding of reduced ribonuclease. However the size of proteins and the observed time scale of refolding make it clear that a random search of all possible structures does not occur [3]. Much recent theoretical and experimental work of different kinds has shown that folding generally follows a specified sequence, the course of folding being determined by the existence of centres of nucleation [4-l 11,. The recent work of Creighton [ 12151 has established an obligatory sequence for the formation of disulphide bonds during the oxidation of pancreatic trypsin inhibitor in native conditions; only 4 out of 1.5 possible isomers containing one disulphide bond, and only 4 out of 4.5 isomers containing 2 disulphide bonds actually form to any extent as intermediates. All this evidence shows that a large proportion of possible conformations are never sampled by an unfolded protein in native conditions; the conformational potential energy barriers are too great. But these conformations may be explored in denaturing media, and some, at least, can be ‘fixed’ by reoxidation in denaturing conditions, so-called ‘random’ reoxidation. If ‘randomly reoxidised proteins are returned to non-denaturing conditions, the ‘native’ potential energy surface can be explored from the ‘far side’ of the potential energy barriers. It is possible that these barriers inhibit conversion of the ‘randomly’ reoxidised proteins to the native conformation even when

A gel filtration approach to the study of ribosome-membrane interactions

1. Gel filtration on agarose can be used to investigate ribosome-membrane interactions without exposing the materials to the high, and possibly perturbing, hydrostatic pressures experienced during centrifugation procedures to separate free ribosomes from membrane vesicles. 2. After treatment of microsomes with degranulating agents, degranulated membranes are isolated from Sepharose 2B columns at the void volume, while displaced ribosomes elute at the total column volume. This provides a convenient method for monitoring degranulation in vitro. 3. Centrifugation of rough microsomes or ribosomes into dense pellets or layers, followed by resuspension, leads to preparations which will not pass rapidly or quantitatively through Sepharose 2B columns. 4. Methods are described for the isolation of degranulated microsomes and ribosomes which are eluted rapidly from Sepharose 2B at the void volume and total column volume, respectively. These materials are suitable for the investigation of ribosome-membrane binding in vitro, using a gel filtration separation to monitor binding. 5. Incubation of 3H-labelled ribosomes with degranulated microsomes in vitro, leads to specific binding, demonstrated by the elution of the bound ribosomes at the void volume.

Fluorescence studies of drug and cation interactions with microsomal membranes.

The value of non-covalently bound fluorescent molecules as extrinsic probes of membrane structure and function is now well established [l] . In the case of the inner mitochondrial membrane, analysis of the responses of fluorescent probes to changes in the state of the membrane [ 21 has led to some understanding of the physical changes accompanying energy transduction [3-61. A similar analysis has been applied to chromatophore membranes [7]. In many other systems, such as the erythrocyte membrane [8] , sarcoplasmic reticulum [9] , brain microsomes [lo] and numerous artificial membranes, the binding of the most popular probe ANS (1 -aniline-naphthalene-8-sulphonate) has been studied; in general, these studies have not been particularly revealing about the major functions of the membrane systems. Microsomal membranes of mammalian liver are complex systems whose major biological functions include i) the metabolism of drugs and other foreign compounds and ii) the binding of ribosomes and organisation of the synthesis of serum proteins. We have attempted to use a fluorescent probe to investigate these functions; it was found that the increase in probe fluorescence produced by certain drugs is a function of the ionisation state of the drugs and not a specific property of the drug-microsome interaction, and that fluorescent probes cannot detect differences in cation affinity between smooth microsomes and degranulated rough microsomes which might account for their different abilities to bind ribosomes.