Active Protein Disulfide Isomerase Research Articles

Cellular mechanisms involved in induction of selective degeneration of orexin neurons in the hypothalamus

Narcolepsy is a kind of sleep disorder featured by selective loss of orexin neurons in the lateral hypothalamus. Several lines of evidence, including association with specific HLA haplotypes, gene polymorphism in T cell receptor and detection of autoantibodies in a subpopulation of patients, suggest that autoimmune responses play an important role in the pathogenesis of this disorder. Potential relationship with influenza virus infection has also been a matter of interest. However, these events may not be able to explain all cases of narcolepsy. Based on the structural features of orexin, in addition to the findings on the characteristics of orexin neurons obtained from studies in organotypic hypothalamic slice cultures, we proposed novel mechanisms potentially involved in selective degeneration of orexin neurons. Increase in local production of nitric oxide induced by several life style-related conditions such as shortage of sleep and intake of high fat diet leads to inactivation of protein disulfide isomerase. Consequently, abnormal aggregates of orexin and/or its precursor that possess two intra-molecular disulfide bonds accumulate within orexin neurons. In addition to the increase in endoplasmic reticulum stress, accumulation of orexin as abnormal aggregates leads to increased excitability of orexin neurons by shutdown of feedback inhibition resulting from deficits in orexin release. These mechanisms may provide a clue to understand the pathogenic mechanisms of various neurological and psychiatric disorders accompanied by a decrease of orexin.

Cystamine-mediated inhibition of protein disulfide isomerase triggers aggregation of misfolded orexin-A in the Golgi apparatus and prevents extracellular secretion of orexin-A

Orexins (orexin-A and orexin-B) are neuropeptides that are reduced in narcolepsy, a sleep disorder that is characterized by excessive daytime sleepiness, sudden sleep attacks and cataplexy. However, it remains unclear how orexins in the brain and orexin neurons are reduced in narcolepsy. Orexin-A has two closely located intramolecular disulfide bonds and is prone to misfolding due to the formation of incorrect disulfide bonds. Protein disulfide isomerase (PDI) possesses disulfide interchange activity. PDI can modify misfolded orexin-A to its native form by rearrangement of two disulfide bonds. We have previously demonstrated that sleep deprivation and a high fat diet increase nitric oxide in the brain. This increase triggers S-nitrosation and inactivation of PDI, leading to aggregation of orexin-A and reduction of orexin neurons. However, the relationship between PDI inactivation and loss of orexin neurons has not yet been fully elucidated. In the present study, we used a PDI inhibitor, cystamine, to elucidate the precise molecular mechanism by which PDI inhibition reduces the number of orexin neurons. In rat hypothalamic slice cultures, cystamine induced selective depletion of orexin-A, but not orexin-B and melanin-concentrating hormone. Moreover, cystamine triggered aggregation of orexin-A, but not orexin-B in the Golgi apparatus of hypothalamic slice cultures and in vivo mouse brains. However, cystamine did not induce endoplasmic reticulum (ER) stress, and an ER stress inducer did not trigger aggregation of orexin-A in slice cultures. Finally, we demonstrated that cystamine significantly decreased extracellular secretion of orexin-A in AD293 cells overexpressing prepro-orexin. These findings suggest that cystamine-induced PDI inhibition induces selective depletion, aggregation in the Golgi apparatus and impaired secretion of orexin-A. These effects may represent an initial step in the pathogenesis of narcolepsy.

Role of protein disulfide isomerase in activation of integrins

Integrins belong to a large family of transmembrane cell adhesion receptors that communicate biochemical and mechanical signals in a bidirectional manner across the plasma membrane. Integrins and their ligands play a crucial role in a number of physiological and pathological processes, including cell migration, cell differentiation, hemostasis, adhesion, angiogenesis, cancer, cell invasiveness and wound healing. Intracellular signals switch integrins into a ligand-competent state as a result of conformational changes within the integrin molecule. Binding of extracellular ligands induces structural changes that can transmit signals to the cell interior. Transition of integrins from an inactive to a ligand binding state involves rearrangement of the disulfide bonding pattern. The rearrangement of disulfide bonds is modulated by protein disulfide isomerase (PDI). PDI has been found on the surface of several types of cells, including endothelial cells, hepatocytes, cancer cells, pancreatic cells and B cells. PDI was identified on the platelet surface, where it plays an important role in platelet reactions such as adhesion, aggregation and secretion. PDI was found to directly interact with integrins. Disulfide-thiol exchange mediated by PDI appears to be involved in the conformational changes in integrin activation. In this report we describe the structure of integrin and the role of disulfide bond rearrangement in its activation.

Potential Effect of S-Nitrosylated Protein Disulfide Isomerase on Mutant SOD1 Aggregation and Neuronal Cell Death in Amyotrophic Lateral Sclerosis

Aggregation of misfolded protein and resultant intracellular inclusion body formation are common hallmarks of mutant superoxide dismutase (mSOD1)-linked familial amyotrophic lateral sclerosis (FALS) and have been associated with the selective neuronal death. Protein disulfide isomerase (PDI) represents a family of enzymatic chaperones that can fold nascent and aberrant proteins in the endoplasmic reticulum (ER) lumen. Recently, our group found that S-nitrosylated PDI could contribute to protein misfolding and subsequent neuronal cell death. However, the exact role of PDI in the pathogenesis of ALS remains unclear. In this study, we propose that PDI attenuates aggregation of mutant/misfolded SOD1 and resultant neurotoxicity associated with ER stress. ER stress resulting in PDI dysfunction therefore provides a mechanistic link between deficits in molecular chaperones, accumulation of misfolded proteins, and neuronal death in neurodegenerative diseases. In contrast, S-nitrosylation of PDI inhibits its activity, increases mSOD1 aggregation, and increases neuronal cell death. Specifically, our data show that S-nitrosylation abrogates PDI-mediated attenuation of neuronal cell death triggered by thapsigargin. Biotin switch assays demonstrate S-nitrosylated PDI both in the spinal cords of SOD1 (G93A) mice and human patients with sporadic ALS. Therefore, denitrosylation of PDI may have therapeutic implications. Taken together, our results suggest a novel strategy involving PDI as a therapy to prevent mSOD1 aggregation and neuronal degeneration. Moreover, the data demonstrate that inactivation of PDI by S-nitrosylation occurs in both mSOD1-linked and sporadic forms of ALS in humans as well as mice.

Nitric Oxide Mediates Selective Degeneration of Hypothalamic Orexin Neurons through Dysfunction of Protein Disulfide Isomerase

We addressed the role of nitric oxide (NO) in orexin neuron degeneration that has been observed under various pathological conditions. Administration of an NO donor NOC18 (50 nmol) into the third ventricle of mice resulted in a significant decrease of orexin-immunoreactive (-IR) neurons, in contrast to a modest change in melanin-concentrating hormone-IR neurons. In addition, NOC18 promoted formation of orexin-A-IR aggregates within orexin neurons. An endoplasmic reticulum stress inducer tunicamycin replicated the effect of NOC18 with regard to decrease of orexin-IR neurons and formation of aggregates. We also found that NOC18 caused an increase in S-nitrosation of protein disulfide isomerase (PDI) and a decrease in PDI activity in hypothalamic tissues. Moreover, PDI inhibitors, such as cystamine and securinine, caused a selective decrease of orexin neurons and promoted formation of orexin-A-IR aggregates. Aggregate formation in orexin-IR neurons was also induced by local injection of small interfering RNA targeting PDI. Interestingly, sleep deprivation for 7 consecutive days induced a selective decrease of orexin-IR neurons, which was preceded by aggregate formation in orexin-IR neurons and an increase in S-nitrosated PDI in the hypothalamus. Activity of neuronal NO synthase (nNOS)-positive neurons in the lateral hypothalamus as assessed by c-Fos expression was elevated in response to sleep deprivation. Finally, sleep deprivation-induced decrease of orexin-IR neurons, formation of aggregates, and S-nitrosation of PDI were not observed in nNOS knock-out mice. These results indicate that nNOS-derived NO may mediate specific pathological events in orexin neurons, including neuropeptide misfolding via S-nitrosation and inactivation of PDI.

Inactivation of protein disulfide isomerase by alkylators including alpha,beta-unsaturated aldehydes at low physiological pHs.

Protein disulfide isomerase (PDI) is known to contain the thioredoxin box motif with a low pKa cysteine residue. To investigate the reactivity of PDI with thiol modifiers at low physiological pHs, either the reduced (PDIred) or oxidized form (PDIoxid) of PDI was exposed to various alkylating ragents. When PDI was incubated with iodoacetamide at pH 6.3 for 30 min at 38 degrees C, a remarkable inactivation (>90%) of PDIred was caused by iodoacetamide (IC50=8 microM). However, PDIoxid was only slightly inactivated (approximately 18%) by iodoacetamide. Similarly, PDIred was significantly inactivated by N-ethylmaleimide (NEM), but PDIoxid was not. When the inactivation by these alkylators was analyzed by pseudo-first order kinetics, NEM (k3=1.75x10(-2) s(-1); K(i)=124 microM) was observed to be more potent than iodoacetamide (k3=9.1x10(-3) s(-1); K(i)=311 microM). Interestingly, the inactivation of PDIred by iodoacetamide was greater at pH 6.3 than pH 7.0, in contrast to a similar inactivation potency of NEM at both pHs. Moreover, the maximal inactivation of PDIred or PDIoxid by iodoacetamide was mainly observed around pH 6.0. In addition, PDIred was found to be inactivated by acrolein (IC50=10 microM) at pH 6.3, and this inactivation was also greater at pH 6.3 than at pH 7. Based on these results, we suggest that PDIred is susceptible to inactivation by alkylators including endogenous alpha,beta-unsaturated aldehydes at low physiological pHs.

New formulae for folding catalysts make them multi‐purpose enzymes

Whereas protein disulfide isomerase (PDI) and prolyl isomerase (PPI) are considered as efficient protein folding catalysts, very few large scale processes use them because of economical and technical limitations. PDI and PPI were successfully immobilized on cross-linked agarose beads. PDI inactivation during coupling reaction was overcome by oxidizing active site thiols with dimethylsulfoxide and led to a 64% active enzyme. Alternatively, PPI and PDI biotinylation resulted in 100% and 55–66% active enzymes respectively. The use of these modified catalysts suppresses post-refolding purification and enables the design of biochemical reactors. Several other possible applications are also discussed. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 645–649, 1997.

New formulae for folding catalysts make them multi-purpose enzymes

Whereas protein disulfide isomerase (PDI) and prolyl isomerase (PPI) are considered as efficient protein folding catalysts, very few large scale processes use them because of economical and technical limitations. PDI and PPI were successfully immobilized on cross-linked agarose beads. PDI inactivation during coupling reaction was overcome by oxidizing active site thiols with dimethylsulfoxide and led to a 64% active enzyme. Alternatively, PPI and PDI biotinylation resulted in 100% and 55-66% active enzymes respectively. The use of these modified catalysts suppresses post-refolding purification and enables the design of biochemical reactors. Several other possible applications are also discussed. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 645-649, 1997.

The role of the thiol/disulfide centers and peptide binding site in the chaperone and anti-chaperone activities of protein disulfide isomerase.

The complexity of protein folding is often aggravated by the low solubility of the denatured state. The inefficiency of the oxidative refolding of reduced, denatured lysozyme results from a kinetic partitioning of the unfolded protein between pathways leading to aggregation and pathways leading to the native structure. Protein disulfide isomerase (PDI), a resident foldase of the endoplasmic reticulum, catalyzes the in vitro oxidative refolding of reduced, disulfide-containing proteins, including denatured lysozyme. Depending on the concentrations of foldase and denatured substrate and the order in which they are added to initiate folding, PDI can exhibit either a chaperone activity or an anti-chaperone activity (Puig, A., and Gilbert, H. F. (1994) J. Biol. Chem 269, 7764-7771). PDI's chaperone activity leads to quantitative recovery of native lysozyme. Its anti-chaperone activity diverts substrate away from productive folding and facilitates disulfide cross-linking of lysozyme into large, inactive aggregates that specifically incorporate PDI. A mutant PDI (NmCm-PDI), in which both the N- and C-terminal active site cysteines have been changed to serines, loses all chaperone activity and behaves as an anti-chaperone at all substrate and PDI concentrations tested. The dithiol/disulfide sites of PDI are essential for the chaperone activity observed at high PDI concentrations, but they are not required for the anti-chaperone activity found at low PDI concentrations. Inactivation of PDI's peptide/protein binding site by a specific photoaffinity label (Noiva, R., Freedman, R. B., and Lennarz, W. J. (1993) J. Biol. Chem. 268, 19210-19217) inhibits the disulfide isomerase and chaperone activity, but the protein still retains its anti-chaperone activity. In a glutathione redox buffer, lysozyme-PDI aggregates are disulfide cross-linked; however, disulfide cross-linking is not required for aggregate formation or for the incorporation of PDI into the aggregates. Although both the peptide binding site and the catalytic active sites of PDI are required for chaperone and disulfide isomerase activity, neither of these sites are involved in PDI's anti-chaperone activity. PDI's anti-chaperone activity could serve as a quality control device by providing an efficient mechanism to retain misfolded proteins in the endoplasmic reticulum (Marquardt, T., and Helenius, A. (1992) J. Cell. Biol. 117, 505-513).