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.     

The nuclear factor-kappa B RelA transcription factor is constitutively activated in human pancreatic adenocarcinoma cells

To assess the efficiency of nasally administered cartilage-specific collagens as vaccination against development of arthritis and to ameliorate already established chronic arthritis, experimental models which develop chronic arthritis, pristane-induced arthritis (PIA), and homologous collagen-induced arthritis (CIA) in the rat were selected. Cartilage-specific collagens type IX (CIX) and type II (CII) were used for vaccination intranasally. A single dose of 250 microg CII instilled intranasally in rats with established PIA ameliorated the disease. For the prevention of disease, the same dose given before immunization was found to be most effective. Most importantly, the disease was more severe if this dose was given three times. For treatment of PIA, CIX was found to be more effective than CII, whereas for treatment of CIA only CII was effective. The amelioration of CIA was associated with a marked suppression of delayed type hypersensitivity and the flare reaction to CII and lower levels of IgG2b anti-CII antibodies in serum, i.e., with suppression of the TH1 rather than the TH2 response to CII. These findings, that cartilage proteins, if given intranasally, can both prevent and ameliorate established chronic arthritis in rats, are of significant importance for possible use in rheumatoid arthritis. The identification of two different cartilage-specific proteins (CII and CIX) effective against a disease induced with a well-defined nonimmunogenic adjuvant such as pristane will be of value for enhancing the effectiveness of the treatment.     

Cyclic GMP-dependent protein kinase and cellular signaling in the nervous system

Nitric oxide (NO) and natriuretic peptide hormones play key roles in a surprising number of neuronal functions, including learning and memory. Most data suggest that they exert converging actions by elevation of intracellular cyclic GMP (cGMP) levels through activation of soluble and particulate guanylyl cyclases. However, cGMP is only the starting point for multiple signaling cascades, which are now beginning to be defined. A primary action of elevated cGMP levels is the stimulation of cGMP-dependent protein kinase (PKG), the major intracellular receptor protein for cGMP, which phosphorylates substrate proteins to exert its actions. It has become increasingly clear that PKG mediates some of the neuronal effects of cGMP, but how is not yet clear. One clear illustration of this pathway has been reported in striatonigral nerve terminals, where NO mediates phosphorylation of the protein phosphatase regulator dopamine- and cyclic AMP-regulated phosphoprotein having a molecular mass of 32,000 (DARPP-32) by PKG. There are remarkably few PKG substrates in brain whose identities are known. A survey of these proteins and those known from other tissues that might also be found in the nervous system reveals the key molecular sites where cGMP and PKG signaling is likely to be regulating neural function. These potential substrates are critically placed to have profound effects on the protein phosphorylation network through regulation of protein phosphatases, intracellular calcium levels, and the function of many ion channels and neurotransmitter receptors. The brain also contains a rich diversity of specific PKG substrates whose identities are not yet known. Their future identification will provide exciting new leads that will permit better understanding of the role of PKG signaling in both basic and higher orders of brain function.