Protein disulfide isomerase is the dominant acceptor for peptides translocated into the endoplasmic reticulum

Peptides derived from cytosolic protein degradation are translocated into the lumen of the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). In the ER, class I molecules bind the peptides fitting to their respective motifs and present them on the cell surface to CD8+ T lymphocytes. However, most TAP-translocated peptides are not expected to bind to the class I molecules present in a particular cell. Recently, we have demonstrated that TAP-translocated peptides containing a photoreactive phenylalanine analogue can be cross-linked to two luminal ER-resident proteins: with low efficiency to the stress protein gp96 and with high efficiency to a 60-kDa protein (Lammert, E. et al., Eur. J. Immunol. 1997. 27: 923). Both proteins have also been labeled specifically by TAP-translocated peptides conjugated to a different photoreactive group (Marusina, K. et al., Biochemistry 1997. 36: 856). Here, we show that the 60-kDa peptide-binding protein is identical to the multifunctional protein disulfide isomerase (PDI). Since PDI is the only luminal ER-resident protein that is labeled by the photoreactive peptides with high efficiency, it might represent the dominant acceptor for TAP-translocated peptides.     

Improved folding yields of a model protein using protein disulfide isomerase

PURPOSE: To study the effects of recombinant human protein disulfide isomerase (rhPDI) concentration, reduced glutathione:oxidized glutathione ratio (GSH:GSSG) and temperature on the efficiency of oxidative folding of a model protein, recombinant human interleukin 2 (C125A mutation) (C125A rhIL-2). METHODS: C 125A rhIL-2 inclusion bodies were reduced and denatured by guanidium hydrochloride (Gdm.Cl) and 100 mM GSH. The solution was diluted 10 times into folding buffer, allowing C125A rhIL-2 to fold either in the absence or presence of rhPDI. The renatured and unfolded C125A rhIL-2 species were quantitated by reversed phase-HPLC. RESULTS: The initial folding rate of C125A rhIL-2 linearly increased with rhPDI:C125A rhIL-2 molar ratio in the first 2.5 minutes, and reached the highest rate when the rhPDI:C125A rhIL-2 ratio was 1:1. The oxidative folding of C125A rhIL-2 linearly increased as the GSH:GSSG molar ratio decreased from 10:0 to 10:3. The folding of C125A rhIL-2 was also dependent on temperature, and optimum folding was realized at 23 degrees C. CONCLUSIONS: These results demonstrate that under optimal redox potential and temperature, rhPDI enhances the oxidative folding of C125A rhIL-2. In the oxidative folding of C125A rhIL-2, rhPDI exerts its effect on folding by the acceleration of thiol/disulfide interchange.     

von Willebrand factor as a regulator of intrinsic factor X activation

Factor VIII is an important cofactor in the intrinsic activation of factor X. To function effectively as a cofactor, factor VIII must be activated. In plasma, factor VIII circulates in a complex with von Willebrand factor, and although thrombin can activate complexed factor VIII, the activation by activated factor X is inhibited by von Willebrand factor. In this study, the effect of von Willebrand factor on the generation of factor Xa by the factor IXa-VIII complex was investigated. Purified human factors VIII, IXa, and X were incubated on human umbilical vein endothelial cells or phospholipid vesicles in the presence of calcium ions, and the generation of factor Xa was followed. In the presence of von Willebrand factor, a prolonged lag-phase and a dose-dependent inhibition of factor X activation was observed. These effects were not observed when von Willebrand factor was preincubated with a monoclonal antibody directed against von Willebrand factor that blocks factor VIII binding. When factor VIII was activated with thrombin before the incubation, neither the monoclonal antibody nor von Willebrand factor had an effect on the rate of factor X activation. Preincubation of endothelial cells with the monoclonal antibody resulted in a somewhat higher rate of factor X activation. When endothelial cells from a patient with von Willebrand's disease type I were used, preincubation of the monoclonal antibody had no effect on the rate of factor X activation. We conclude that von Willebrand factor on the surface of endothelial cells can modulate the intrinsic factor X activation. This effect is greatly enhanced, however, by the addition of exogenous von Willebrand factor.(ABSTRACT TRUNCATED AT 250 WORDS)     

The acidic C-terminal domain of protein disulfide isomerase is not critical for the enzyme subunit function or for the chaperone or disulfide isomerase activities of the polypeptide

Pancreatic neurons receive and integrate synaptic input from a wide variety of extrinsic nerves while providing the predominant innervation of pancreatic acini, ducts, and islets of Langerhans. Here we report the first primary cultures of adult rabbit pancreatic neurons, isolated from extrinsic nerves and secretory cells, and evaluate the neurochemical and electrical properties of these neurons. Pancreatic cultures consisted of single and clustered neurons, extended varicose processes after 3-4 days in culture, and formed interconnecting networks of neurons after 7-10 days. Isolated pancreatic islet cells, added to established neuron cultures, remained attached and viable for several weeks and received innervation by varicose nerve fibers. Histochemical staining revealed populations of neurons positive for acetylcholinesterase (75%), NADPH-diaphorase (62%), nitric oxide synthase (73%), and/or vasoactive intestinal peptide (VIP) (65%). Intracellular recordings revealed active and passive electrical properties comparable to those of neurons from intact ganglia. Several distinct populations of neurons were identified by their firing patterns (phasic vs. tonic) in response to prolonged depolarizing currents or the amplitude and duration of their after-spike hyperpolarizations. Low-amplitude, pacemaker-like potentials were observed in 25% of the neurons and, in older cultures with extensive networks of fibers, spontaneous fast excitatory postsynaptic potentials (EP-SPs) also occurred. Thus these cultures retained the salient neurochemical and electrophysiologic properties observed in pancreatic neurons from intact ganglia and offer a good model for studies of the intrinsic innervation of the pancreas.     

Occurrence of autoantibody to protein disulfide isomerase in rats with xenobiotic-induced hepatitis

Until recently, direct measurements of coronary blood flow in fishes had not been made. This review presents and compares in vivo measurements of coronary flow recorded from the school shark, Galeorhinus australis, and the coho salmon, Oncorhynchus kisutch. In both species, the coronary blood flow was phasic and strongly influenced by the cardiac cycle. Coronary blood flow in the school shark was more severely compromised by the cardiac cycle with a short retrograde flow occurring during systole. In the coho salmon, there was an anterograde coronary blood flow throughout the cardiac cycle. Differences in coronary hemodynamics are discussed in relation to differences in the myoarchitecture of the school shark and coho salmon hearts. The regulation of coronary blood flow through changes in vascular resistance (vasoactivity of the coronary vessels) is also discussed.     

Redox control of exofacial protein thiols/disulfides by protein disulfide isomerase

Protein disulfide isomerase (PDI) facilitates proper folding and disulfide bonding of nascent proteins in the endoplasmic reticulum and is secreted by cells and associates with the cell surface. We examined the consequence of over- or underexpression of PDI in HT1080 fibrosarcoma cells for the redox state of cell-surface protein thiols/disulfides. Overexpression of PDI resulted in 3.6-4. 2-fold enhanced secretion of PDI and 1.5-1.7-fold increase in surface-bound PDI. Antisense-mediated underexpression of PDI caused 38-53% decreased secretion and 10-33% decrease in surface-bound PDI. Using 5,5'-dithio-bis(2-nitrobenzoic acid) to measure surface protein thiols, a 41-50% increase in surface thiols was observed in PDI-overexpressing cells, whereas a 29-33% decrease was observed in underexpressing cells. Surface thiol content was strongly correlated with cellular (r = 0.998) and secreted (r = 0.969) PDI levels. The pattern of exofacial protein thiols was examined by labeling with the membrane-impermeable thiol reactive compound, 3-(N-maleimidylpropionyl)biocytin. Fourteen identifiable proteins on HT1080 cells were labeled with 3-(N-maleimidylpropionyl)biocytin. The intensity of labeling of 11 proteins was increased with overexpression of PDI, whereas the intensity of labeling of 3 of the 11 proteins was clearly decreased with underexpression of PDI. These findings indicated that secreted PDI was controlling the redox state of existing exofacial protein thiols or reactive disulfide bonds.     

Malpractice as a regulator of psychotherapy.

Points out that attempts to expand the malpractice and tort liability of psychologists do not address a major deterrent to claims: the reluctance of clients to sue. Other forms of regulation are needed to compensate for the inability of malpractice to regulate professional psychology. (20 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Exposure of the cryptic Arg-Gly-Asp sequence in thrombospondin-1 by protein disulfide isomerase

OBJECTIVES: Human cardiac muscle from failing heart shows a decrease in active tension development and a rise in diastolic tension at stimulation frequencies above 50-60 beats/min due to both systolic and diastolic dysfunction. We have investigated underlying changes in cellular [Ca2+]i regulation. METHODS: Single ventricular myocytes were isolated enzymatically from the explanted hearts of transplant recipients with ischemic cardiomyopathy (nhearts = 5 ncells = 15) or dilated cardiomyopathy (nhearts = 6, ncells = 19). Cells were studied during whole-cell patch clamp with fluo-3 and fura-red as [Ca2+]i indicators (36 +/- 1 degrees C). RESULTS: In current clamp mode (action potential recording), the amplitude of Ca2+ release from the sarcoplasmic reticulum (SR) decreased at stimulation frequencies above 0.5 Hz; this decrease was more pronounced for cells from dilated cardiomyopathy. Diastolic [Ca2+]i increased at 1 and 2 Hz for both groups. Action potential duration (APD90) decreased with frequency in all cells; in addition there was a drop in plateau potential of 10 +/- 1 mV for cells from ischemic cardiomyopathy and of 13 +/- 2 mV for cells from dilated cardiomyopathy. In voltage clamp mode the L-type Ca2+ current showed reversible decrease during stimulation at 1 and 2 Hz. Recovery from inactivation during a double pulse protocol was slow (75 +/- 3% at 500 ms, 89 +/- 3% at 1000 ms) and followed the decay of the [Ca2+]i transient. CONCLUSIONS: The negative force-frequency relation of the failing human heart is due to a decrease in Ca2+ release of the cardiac myocytes at frequencies > or = 0.5 Hz, more pronounced in dilated than in ischemic cardiomyopathy. Inhibition of ICaL at higher frequencies, at least partially related to an increase in diastolic [Ca2+]i, will contribute to this negative staircase because of a decrease in the trigger for Ca2+ release, and of decreased loading of the SR.     

Thioredoxin domain non-equivalence and anti-chaperone activity of protein disulfide isomerase mutants in vivo

Coexpression of the enzyme, protein disulfide isomerase (PDI), has been shown to increase soluble and secreted IgG levels from baculovirus-infected insect cells (Hsu, T.-A., Watson, S., Eiden, J. J., and Betenbaugh, M. J. (1996) Protein Expression Purif. 7, 281-288). PDI is known to include catalytic active sites in two separate thioredoxin-like domains, one near the amino terminus and another near the carboxyl terminus. To examine the role of these catalytic active sites in enhancing immunoglobulin solubility, baculovirus constructs were utilized with cysteine to serine mutations at the first cysteine of one or both of the CGHC active site sequences. Trichoplusia ni insect cells were coinfected with a baculovirus vector coding for IgG in concert with either the wild-type human PDI virus, amino-terminal mutant (PDI-N), carboxyl-terminal mutant (PDI-C), or mutant with both active sites altered (PDI-NC). Western blot analysis revealed that both immunoglobulins and PDI protein were expressed in the coinfected cells. To evaluate the effect of the PDI variants on immunoglobulin solubility and secretion, the infected cells were labeled with 35S-amino-acids for different periods, and the soluble immunoglobulins were immunoprecipitated from clarified cell lysates and culture medium using anti-IgG antibodies. Only coinfections with the wild-type PDI and PDI-N mutant led to increased immunoglobulin solubility and higher IgG secretion. In contrast, infection with the PDI-C and PDI-NC variants actually lowered immunoglobulin solubility levels below those achieved with a negative control virus. Immunoprecipitation with anti-PDI antibody revealed that heterologous PDI-C and PDI-NC were insoluble, even though PDI-N and wild-type PDI protein were detected in soluble form. The capacity for PDI-N to increase immunoglobulin solubility whereas the PDI-C mutant lowered solubility indicates that the amino- and carboxyl-terminal thioredoxin domains of PDI are functionally distinct in vivo following mutations to the active site. Furthermore, mutations at the active site of the carboxyl-terminal thioredoxin domain result in PDI variants that can act as anti-chaperones of immunoglobulin solubility in vivo as has been observed in vitro for lysozyme aggregation by wild-type PDI and PDI mutants (Puig, A., and Gilbert, H. F. (1994) J. Biol. Chem. 269, 7764-7771).     

Folding of omega-conotoxins. 2. Influence of precursor sequences and protein disulfide isomerase

The peptide Ca2+ channel antagonists found in the venoms of Conus snails, omega-conotoxins, are synthesized as precursors that include a leader peptide, presumed to direct the polypeptide to the endoplasmic reticulum, and a propeptide of unknown function. In addition, the precursors are synthesized with a C-terminal Gly residue that is posttranslationally converted to a terminal amide group. In order to determine whether the precursor sequences contain information that helps direct folding of the mature sequences, the disulfide-coupled folding of mature omega-conotoxin MVIIA was compared with that of two putative precursor forms: pro-omega-MVIIA-Gly, which contains the propeptide and the C-terminal Gly residue, and omega-MVIIA-Gly, which differs from the mature form only at the C-terminus. The three forms folded with similar kinetics, but the folding efficiency of omega-MVIIA-Gly was greater than 80%, versus approximately 50% for both mature omega-MVIIA and the form containing the propeptide. The enzyme protein disulfide isomerase was found to catalyze disulfide formation and folding of all three forms similarly. The affinity of omega-MVIIA-Gly for receptors in chick brain synaptosomes was approximately 10-fold lower than that of the mature peptide, and the N-terminal propeptide of pro-omega-MVIIA-Gly was found to decrease binding further, by approximately 100-fold. These results suggest that the omega-conotoxins do not rely on the propeptide region of their precursors to facilitate folding. Rather, the mature sequence contains most of the information required to specify the native disulfide pairings and three-dimensional conformation. The C-terminal Gly may enhance the folding efficiency by forming interactions that stabilize the native conformation with respect to other disulfide-bonded forms.     

Is CTLA-4 a negative regulator for T-cell activation?

Immune responses can be a two-sided sword: although lack of immunity can be fatal, so can over-activation of the immune system. The concept of homeostatic balance in immune regulation has consequently been at the core of immune research. In this context, Yang Liu takes a cautious view of the concept that CTLA-4 is a negative regulator of T-cell activation.     

Overproduction of Mpd2p suppresses the lethality of protein disulfide isomerase depletion in a CXXC sequence dependent manner

The third multicopy suppressor gene of the PDI1 deletion from Saccharomyces cerevisiae, MPD2, was isolated and characterized. The MPD2 gene encodes a protein with a putative signal sequence, ER retention signal, and a disulfide isomerase active site like sequence. The amino acid sequence around the active site like sequence is similar to the thioredoxin-like domains of PDI and PDI related proteins, although the similarity is comparatively low. A delta-pdi1 strain over-producing Mpd2p showed slow growth and was sensitive to 1 mM dithiothreitol. Mpd2p can be detected in wild type cells and is a glycoprotein. Although the MPD2 gene was not essential for growth, overexpression of the gene partially restored the maturation defect of carboxypeptidase Y caused by the PDI1 deletion. Mutagenesis analysis revealed that Mpd2p can compensate for the loss of PDI with its CXXC sequence.     

Purification, characterization, and intracellular localization of glycosylated protein disulfide isomerase from wheat grains

Wheat (Triticum aestivum) storage proteins fold and assemble into complexes that are linked by intra- and intermolecular disulfide bonds, but it is not yet clear whether these processes are spontaneous or require the assistance of endoplasmic reticulum (ER)-resident enzymes and molecular chaperones. Aiming to unravel these processes, we have purified and characterized the enzyme protein disulfide isomerase (PDI) from wheat endosperm, as well as studied its developmental expression and intracellular localization. This ER-resident enzyme was previously shown to be involved in the formation of disulfide bonds in secretory proteins. Wheat PDI appears as a 60-kD glycoprotein and is among the most abundant proteins within the ER of developing grains. PDI is notably upregulated in developing endosperm in comparison to embryos, leaves, and roots. In addition, the increase in PDI expression in grains appears at relatively early stages of development, preceding the onset of storage protein accumulation by several days. Subcellular localization analysis and immunogold labeling of electron micrographs showed that PDI is not only present in the lumen of the ER but is also co-localized with the storage proteins in the dense protein bodies. These observations are consistent with the hypothesis that PDI is involved in the assembly of wheat storage proteins within the ER.     

Yeast Ku as a regulator of chromosomal DNA end structure

During telomere replication in yeast, chromosome ends acquire an S-phase-specific overhang of the guanosine-rich strand. Here it is shown that in cells lacking Ku, a heterodimeric protein involved in nonhomologous DNA end joining, these overhangs are present throughout the cell cycle. In vivo cross-linking experiments demonstrated that Ku is bound to telomeric DNA. These results show that Ku plays a direct role in establishing a normal DNA end structure on yeast chromosomes, conceivably by functioning as a terminus-binding factor. Because Ku-mediated DNA end joining involving telomeres would result in chromosome instability, our data also suggest that Ku has a distinct function when bound to telomeres.     

Unfolding of Plasmodium falciparum triosephosphate isomerase in urea and guanidinium chloride: evidence for a novel disulfide exchange reaction in a covalently cross-linked mutant

The conformational stability of Plasmodium falciparum triosephosphate isomerase (TIMWT) enzyme has been investigated in urea and guanidinium chloride (GdmCl) solutions using circular dichroism, fluorescence, and size-exclusion chromatography. The dimeric enzyme is remarkably stable in urea solutions. It retains considerable secondary, tertiary, and quaternary structure even in 8 M urea. In contrast, the unfolding transition is complete by 2.4 M GdmCl. Although the secondary as well as the tertiary interactions melt before the perturbation of the quaternary structure, these studies imply that the dissociation of the dimer into monomers ultimately leads to the collapse of the structure, suggesting that the interfacial interactions play a major role in determining multimeric protein stability. The Cm(urea)/Cm(GdmCl) ratio (where Cm is the concentration of the denaturant required at the transition midpoint) is unusually high for triosephosphate isomerase as compared to other monomeric and dimeric proteins. A disulfide cross-linked mutant protein (Y74C) engineered to form two disulfide cross-links across the interface (13-74') and (13'-74) is dramatically destablized in urea. The unfolding transition is complete by 6 M urea and involves a novel mechanism of dimer dissociation through intramolecular thiol-disulfide exchange.     

Active site mutations in yeast protein disulfide isomerase cause dithiothreitol sensitivity and a reduced rate of protein folding in the endoplasmic reticulum

Aspects of protein disulfide isomerase (PDI) function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a', each of which contains an active-site CXXC motif. The relative importance of the two domains was analyzed by rendering each one inactive by mutation to SGAS. Such mutations had no significant effect on growth. The domains however, were not equivalent since the rate of folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a' domain. To investigate the relevance of PDI redox potential, the G and H positions of each CGHC active site were randomly mutagenized. The resulting mutant PDIs were ranked by their growth phenotype on medium containing increasing concentrations of DTT. The rate of CPY folding in the mutants showed the same ranking as the DTT sensitivity, suggesting that the oxidative power of PDI is an important factor in folding in vivo. Mutants with a PDI that cannot perform oxidation reactions on its own (CGHS) had a strongly reduced growth rate. The growth rates, however, did not correlate with CPY folding, suggesting that the protein(s) required for optimal growth are dependent on PDI for oxidation. pdi1-deleted strains overexpressing the yeast PDI homologue EUG1 are viable. Exchanging the wild-type Eug1p C(L/I)HS active site sequences for C(L/I)HC increased the growth rate significantly, however, further highlighting the importance of the oxidizing function for optimal growth.     

Alterations in the enzyme activity and protein contents of protein disulfide isomerase in rat tissues during fasting and refeeding

Elevated level of cellular lipid peroxidation can increase the incidence of vascular disease. The mechanism by which ketosis causes accelerated cellular damage and vascular disease in diabetes is not known. This study was undertaken to test the hypothesis that elevated levels of ketone bodies increase lipid peroxidation in endothelial cells. Human umbilical venous endothelial cells (HUVEC) were cultured for 24 h at 37 degrees C with ketone bodies (acetoacetate, beta-hydroxybutyrate). Acetoacetate, but not beta-hydroxybutyrate, caused an increase in lipid peroxidation and growth inhibition in cultured HUVEC. To determine whether ketone bodies generate oxygen radicals, studies using cell-free buffered solution were performed. They showed a significant superoxide dismutase (SOD) inhibitable reduction of cytochrome C by acetoacetate, but not by beta-hydroxybutyrate, suggesting the generation of superoxide anion radicals by acetoacetate. Additional studies show that Fe2+ potentiates oxygen radical generation by acetoacetate. Thus, elevated levels of ketone body acetoacetate can generate oxygen radicals and cause lipid peroxidation in endothelial cells, providing a possible mechanism for the increased incidence of vascular disease in diabetes.