Mutations in Cu͞Zn superoxide dismutase (SOD) are associated with the fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS). There is considerable evidence that mutant SOD has a gain of toxic function; however, the mechanism of this toxicity is not known. We report here that purified SOD forms aggregates in vitro under destabilizing solution conditions by a process involving a transition from small amorphous species to fibrils. The assembly process and the tinctorial and structural properties of the in vitro aggregates resemble those for aggregates observed in vivo. Furthermore, the familial ALS SOD mutations A4V, G93A, G93R, and E100G decrease protein stability, which correlates with an increase in the propensity of the mutants to form aggregates. These mutations also increase the rate of protein unfolding. Our results suggest three possible mechanisms for the increase in aggregation: (i) an increase in the equilibrium population of unfolded or of partially unfolded states, (ii) an increase in the rate of unfolding, and (iii) a decrease in the rate of folding. Our data support the hypothesis that the gain of toxic function for many different familial ALS-associated mutant SODs is a consequence of protein destabilization, which leads to an increase in the formation of cytotoxic protein aggregates.
Abstract. There is circumstantial evidence that protein denaturation occurs in cells during heat shock at hyperthermic temperatures and that denatured or damaged protein is the primary inducer of the heat shock response. However, there is no direct evidence regarding the extent of denaturation of normal cellular proteins during heat shock. Differential scanning calorimetry (DSC) is the most direct method of monitoring protein denaturation or unfolding. Due to the fundamental parameter measured, heat flow, DSC can be used to detect and quantitate endothermic transitions in complex structures such as isolated organelles and even intact cells. DSC profiles with common features are obtained for isolated rat hepatocytes, liver homogenate, and Chinese hamster lung V79 fibroblasts. Five main transitions (A-E), several of which are resolvable into subcomponents, are observed with transition temperatures (Tm) of 45-98°C. The onset temperature is ,'~40°C, but some transitions may extend as low as 37-38°C. In addition to acting as the primary signal for heat shock protein synthesis, the inactivation of critical proteins may lead to cell death. Critical target analysis implies that the rate limiting step of cell killing for V79 cells is the inactivation of a protein with Tm = 46°C within the A transition. Isolated microsomal membranes, mitochondria, nuclei, and a cytosolic fraction from rat liver have distinct DSC profiles that contribute to different peaks in the profile for intact hepatocytes. Thus, the DSC profiles for intact cells appears to be the sum of the profiles of all subcellular organelles and components. The presence of endothermic transitions in the isolated organelles is strong evidence that they are due to protein denaturation. Each isolated organelle has an onset for denaturation near 40°C and contains thermolabile proteins denaturing at the predicted Tm (46°C) for the critical target.The extent of denaturation at any temperature can be approximated by the fractional calorimetric enthalpy. After scanning to 45°C at l°C/min and immediately cooling, a relatively mild heat shock, an estimated fraction denaturation of 4-7 % is found in hepatocytes, V79 cells, and the isolated organdies other than nuclei, which undergo only 1% denaturation because of the high thermostability of chromatin. Thus, thermolabile proteins appear to be present in all cellular organdies and components, and protein denaturation is widespread and extensive after even mild heat shock.XPOSURE of mammalian cells to 42--45°C for short periods of time (referred to as heat shock or hyperthermia) is sufficient for killing and induction of the synthesis of heat shock proteins (Hsp's) during subsequent incubation at 37°C. The direct effect of heat initiating both of these responses is unknown; however, circumstantial evidence suggests the involvement of protein denaturation. Thermodynamic arguments (Johnson et al., 1974;Alexandrov, 1977), the high activation energy for cell killing (Westra and Dewey, 1971), sensitization by incorporation of amino ...
Differential scanning calorimetry (DSC) was used to assay thermal transitions that might be responsible for cell death and other responses to hyperthermia or heat shock, such as induction of heat shock proteins (HSP), in whole Chinese hamster lung V79 cells. Seven distinct peaks, six of which are irreversible, with transition temperatures from 49.5 degrees C to 98.9 degrees C are detectable. These primarily represent protein denaturation with minor contributions from DNA and RNA melting. The onset temperature of denaturation, 38.7 degrees C, is shifted to higher temperatures by prior heat shock at 43 degrees and 45 degrees C, indicative of irreversible denaturation occurring at these temperatures. Thus, using DSC it is possible to demonstrate significant denaturation in a mammalian cell line at temperatures and times of exposure sufficient to induce hyperthermic damage and HSP synthesis. A model was developed based on the assumption that the rate limiting step of hyperthermic cell killing is the denaturation of a critical target. A transition temperature of 46.3 degrees C is predicted for the critical target in V79 cells. No distinct transition is detectable by DSC at this temperature, implying that the critical target comprises a small fraction of total denaturable material. The short chain alcohols methanol, ethanol, isopropanol, and t-butanol are known hyperthermic sensitizers and ethanol is an inducer of HSP synthesis. These compounds non-specifically lower the denaturation temperature of cellular protein. Glycerol, a hyperthermic protector, non-specifically raises the denaturation temperature for proteins denaturing below 60 degrees C. Thus, there is a correlation between the effect of these compounds on protein denaturation in vivo and their effect on cellular sensitivity to hyperthermia.
Eukaryotic cytochromes c contain a buried water molecule (Wat166) next to the heme that is associated through a network of hydrogen bonds to three invariant residues: tyrosine 67, asparagine 52, and threonine 78. Single-site mutations to two of these residues (Y67F, N52I, N52A) and the double-site mutation (Y67F/N52I) were introduced into Saccharomyces cerevisiae iso-1-cytochrome c to disrupt the hydrogen bonding network associated with Wat166. The N52I and Y67F/N52I mutations lead to a loss of Wat166 while N52A and Y67F modifications lead to the addition of a new water molecule ( Water in and around proteins is recognized as being important to protein structure, function and stability (4 -6). Surface and bound water molecules have been identified in protein structures using crystallographic methods and NMR spectroscopy. Eukaryotic cytochromes c, the paradigms of electron transfer proteins, are ideally suited for investigating the structural and functional purposes of water-protein interactions (7). Crystallographic studies performed on the oxidized and reduced states of both tuna and yeast iso-1-cytochrome c proteins have indicated that a conserved and internally bound water molecule (Wat166), 1 along with the surrounding hydrogen bond network are central to the structural transition of cytochrome c between oxidation states (8, 9). The water molecule is adjacent to the heme and the hydrogen bonding network which is composed of conserved residues Asn 52 , Tyr 67 , and Thr 78 , which are also hydrogen bonded in ferrocytochrome c to the Met 80 sulfur which is one of the two heme iron ligands. The oxidation-reduction or redox potential that determines the direction of electron flow between electron transfer proteins is dependent upon the heme ligands and the surrounding peptide (10). In addition, the functional properties of cytochrome c are dependent on the oxidation state of the protein, and knowledge of the energetics of protein stability with respect to oxidation state is central to our understanding of function (11). For example, the addition of an electron to ferricytochrome c results in modified functional properties including a significant increase in stability (12).Several studies using classical genetic procedures or site directed mutagenesis have shown that the hydrogen bond network and Wat166 modulate redox potential and the stability of the protein (13-20). The high resolution three dimensional structures of the reduced and oxidized states of yeast iso-1-cytochromes c carrying mutations at position 52 and/or 67 have been recently reported (1-3). When compared to the wild type protein structure, these mutants show significant changes in their hydrogen bonding networks adjacent to the heme as well as either the displacement of the conserved internally bound Wat166 or the addition of a second internally bound water molecule.Recent investigations have shown that depending on the amino acid substitutions, varying degrees of change in the free energy of unfolding are observed for the two redox states of cyto...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.