The intracellular chloride ion channel protein CLIC1 undergoes a redox-controlled structural transition.

Most proteins adopt a well defined three-dimensional structure; however, it is increasingly recognized that some proteins can exist with at least two stable conformations. Recently, a class of intracellular chloride ion channel proteins (CLICs) has been shown to exist in both soluble and integral membrane forms. The structure of the soluble form of CLIC1 is typical of a soluble glutathione S-transferase superfamily protein but contains a glutaredoxin-like active site. In this study we show that on oxidation CLIC1 undergoes a reversible transition from a monomeric to a non-covalent dimeric state due to the formation of an intramolecular disulfide bond (Cys-24-Cys-59). We have determined the crystal structure of this oxidized state and show that a major structural transition has occurred, exposing a large hydrophobic surface, which forms the dimer interface. The oxidized CLIC1 dimer maintains its ability to form chloride ion channels in artificial bilayers and vesicles, whereas a reducing environment prevents the formation of ion channels by CLIC1. Mutational studies show that both Cys-24 and Cys-59 are required for channel activity

Lu-Hf isotope evidence for the provenance of Permian detritus in accretionary complexes of western Patagonia and the northern Antarctic Peninsula region

Lu–Hf isotope data are presented for dated Permian zircon grains from six samples of the latest Palaeozoic to Jurassic low-grade metasedimentary rocks of western Patagonia and the northern Antarctic Peninsula, as well as from potential source rocks in the North Patagonian Massif. The results for the metasedimentary rocks yield εHf values mostly between −15 and +4 (130 analyses), with a dominant range (more than 85%) of −6 to +1, indicating provenance from Permian magmatic rocks that incorporated continental crust with a significant residence time. Other zircon grains record more negative εHf values indicating derivation from yet more mature crustal sources. Permian subvolcanic granites in the North Patagonian massif appear to be the closest large source area and dated zircon grains from eight samples of these granites yield initial of εHf values of −12 to +4 (45 measurements), 84% of which fall between −6 and +1, the range shown by the metasediments. However, the North Patagonian massif also contains some more juvenile Permian–Carboniferous components not seen in the metasediments, so that this may not be the primary or unique source. These granites are considered to represent the southernmost extension of the Choiyoi igneous province, which contains abundant Permian rhyolites that crop out on the eastern side of the Andes in central Argentina, for which unpublished Hf isotope data yield a very similar range to that of the metasediments. The widespread nature of the Choiyoi volcanic rocks and the predominance of Permian zircon could make this a more favoured source for the detrital grains. Hf isotope data reinforce the uniformity of the provenance of the turbidite detrital zircons and confirm the Choiyoi igneous province and the Permian granitic rocks of the North Patagonian Massif as feasible sources. They further confirm the dominantly crustal origin of the Permian magmas. A source region involving mixing of, for example, crustal materials of Panafrican/Brasiliano and Grenvillian ages, together with a minor but significant subduction-related magmatic input, is an isotopically feasible explanation and is broadly consistent with the provenance of pre-Permian crust in this region, but the proportions of such a mixture must have remained relatively constant. This supports the proposal that recently recognised but widespread Permian magmatism in Patagonia represents voluminous crustal melting in response to subducted slab break-off. The results are also consistent with the premise that the Antarctic Peninsula and southern Patagonia were closely located from Permian to Jurassic times, receiving detritus from the same source

The Intracellular Chloride Ion Channel Protein CLIC1 Undergoes a Redox-controlled Structural Transition

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Crystal structure of the soluble form of the redox-regulated chloride ion channel protein CLIC4

The structure of CLIC4, a member of the CLIC family of putative intracellular chloride ion channel proteins, has been determined at 1.8 Å resolution by X-ray crystallography. The protein is monomeric and it is structurally similar to CLIC1, belonging to the GST fold class. Differences between the structures of CLIC1 and CLIC4 are localized to helix 2 in the glutaredoxin-like N-terminal domain, which has previously been shown to undergo a dramatic structural change in CLIC1 upon oxidation. The structural differences in this region correlate with the sequence differences, where the CLIC1 sequence appears to be atypical of the family. Purified, recombinant, wild-type CLIC4 is shown to bind to artificial lipid bilayers, induce a chloride efflux current when associated with artificial liposomes and produce an ion channel in artificial bilayers with a conductance of 30 pS. Membrane binding is enhanced by oxidation of CLIC4 while no channels were observed via tip-dip electrophysiology in the presence of a reducing agent. Thus, recombinant CLIC4 appears to be able to form a redox-regulated ion channel in the absence of any partner proteins.12 page(s

Mildly acidic pH activates the extracellular molecular chaperone clusterin

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.Many features of the chaperone action of clusterin are similar to those of the intracellular small heat shock proteins (sHSPs) that, like clusterin, exist in solution as heterogeneous aggregates. Increased temperature induces dissociation of some sHSP aggregates and an enhanced chaperone action, suggesting that a dissociated form is the active chaperone species. We recently reported that clusterin aggregates dissociate at mildly acidic pH. To further explore the similarities between clusterin and the sHSPs, we tested the effects of temperature and pH on the structure of clusterin and its chaperone action. Our results demonstrate that increased temperature does not induce dissociation of clusterin aggregates, or other major structural changes, and has little effect on its chaperone action. However, we show that the chaperone action of clusterin is enhanced at mildly acidic pH. Clusterin is the first chaperone shown to be activated by reduced pH. This unique mode of activation appears to result from an increase in regions of solvent-exposed hydrophobicity, which is independent of any major changes in secondary or tertiary structure. We propose a model in which low pH-induced dissociation of clusterin aggregates increases the abundance of the heterodimeric chaperone-active species, which has greater hydrophobicity exposed to solution.Stephen Poon, Mark S. Rybchyn, Simon B. Easterbrook-Smith, John A. Carver, Greg J. Pankhurst and Mark R. Wilso

The propeptide mediates formation of stromal stores of PROMIC-1: Role in determining prostate cancer outcome

The extracellular matrix (ECM) is a reservoir of cellular binding proteins and growth factors that are critical for normal cell behavior, and aberrations in the ECM invariably accompany malignancies such as prostate cancer. Carcinomas commonly overexpress macrophage inhibitory cytokine 1 (MIC-1), a proapoptotic and antitumorigenic transforming growth factor-β superfamily cytokine. Here we show that MIC-1 is often secreted in an unprocessed propeptide containing form. It is variably processed intracellularly, with unprocessed forms being secreted from several tumor lines, including prostate carcinoma lines, PC-3 and LNCaP. Once secreted, only unprocessed proMIC-1 binds ECM, demonstrating for the first time the occurrence of extracellular stores of MIC-1. The propeptide mediates this association via its COOH-terminal 89 amino acids. Xenograft models bearing tumors secreting various engineered forms of MIC-1 show that the propeptide regulates the balance between ECM stores and circulating serum levels of mature MIC-1 in vivo. The absence of propeptide results in ∼ 20-fold increase in serum MIC-1 levels. The significance of stromal MIC-1 stores was evaluated in prostate cancer tissue cores, which show major variation in stromal levels of MIC-1. Stromal MIC-1 levels are linked to prostate cancer outcome following radical prostatectomy, with decreasing stromal levels providing an important independent predictor of disease relapse. In low-grade localized prostate cancer (Gleason sum score ≤6), the level of MIC-1 stromal stores was the best predictor of future relapse when compared with all other clinicopathologic variables. The secretion and ECM association of unprocessed proMIC-1 is likely to play a central role in modulating local bioavailability of MIC-1 which can affect patient outcome in prostate cancer and other epithelial tumors