Destabilization of apoprotein is insufficient to explain Cu,Zn-superoxide dismutase-linked ALS pathogenesis - Analytical Methods & Tools for Lyophilization

Destabilization of apoprotein is insufficient to explain Cu,Zn-superoxide dismutase-linked ALS pathogenesis
approved June 7, 2005 (received for review March 26, 2005)
Published online before print July 14, 2005,
Jorge A. Rodriguez * , Bryan Francis Shaw * , Armando Durazo *, Se Hui Sohn *, Peter A. Doucette *, Aram M. Nersissian *, Kym F. Faull , Daryl K. Eggers , Ashutosh Tiwari ?, Lawrence J. Hayward ?, and Joan Selverstone Valentine *, ||
PNAS | July 26, 2005 | vol. 102
Departments of *Chemistry and Biochemistry and Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, CA 90095; Department of Chemistry, San Jose State University, San Jose, CA 95192; and ?Department of Neurology, University of Massachusetts Medical School, Worcester, MA 01655
Edited by Irwin Fridovich, Duke University Medical Center, Durham, NC
Abstract
The relative stabilities and structural properties of a representative set of 20 ALS-mutant Cu,Zn-superoxide dismutase apoproteins were examined by using differential scanning calorimetry and hydrogen-deuterium (H/D) exchange followed by MS. Contrary to recent reports from other laboratories, we found that ALS-mutant apoproteins are not universally destabilized by the disease-causing mutations. For example, several of the apoproteins with substitutions at or near the metal binding region (MBR) (MBR mutants) exhibited melting temperatures (Tm) in the range 51.6?C to 56.2?C, i.e., similar to or higher than that of the WT apoprotein (Tm = 52.5?C). The apoproteins with substitutions remote from the MBR (WT-like mutants) showed a wide range of Tms, 40.0?C to 52.4?C. The H/D exchange properties of the mutants were also wide-ranging: the MBR mutant apoproteins exhibited H/D exchange kinetics similar to the WT apoprotein, as did some of the more stable WT-like mutant apoproteins, whereas the less stable apoproteins exhibited significantly less protection from H/D exchange than the WT apoprotein. Most striking were the three mutant apoproteins, D101N, E100K, and N139K, which have apparently normal metallation properties, and differ little from the WT apoprotein in either thermal stability or H/D exchange kinetics. Thus, the ALS mutant Cu,Zn-superoxide dismutase apoproteins do not all share reduced global stability, and additional properties must be identified and understood to explain the toxicity of all of the mutant proteins.
differential scanning calorimetry | hydrogen-deuterium exchange | protein stability | protein aggregation | neurodegenerative disease
Protein misfolding and aggregation have been linked to many diseases, including Alzheimer's disease, cystic fibrosis, transmissible spongiform encephalopathies, and ALS, but the pathways followed by pathogenic proteins from translation to disease-causing states are not completely understood (1?3). In some cases, partial or complete unfolding from the native state precedes protein aggregation, and thus the stability of a protein's native state may provide one measure of its propensity to aggregate. However, many familial protein misfolding diseases are caused by proteins that are not destabilized relative to their WT counterparts (4?6), implying that additional intrinsic or extrinsic factors may be required for protein aggregation.
Our recent studies of a large number of ALS-mutant Cu,Zn-superoxide dismutase (SOD1) proteins have revealed that there is great diversity in the biophysical properties of these proteins (7?12). In contrast, Lindberg et al. (13) reported in 2002 that instability of the apoproteins of ALS-mutant SOD1 proteins is a "common denominator" among the nearly 100 known ALS-linked SOD1 mutations. More recently, Furukawa and O'Halloran (14) have reported that some of the destabilized mutant apoproteins studied by Lindberg et al. are further destabilized when the intrasubunit disulfide bond is reduced, again suggesting that protein destabilization is a universal property of ALS apo-SOD1. Both of these groups analyzed a relatively small number of ALS mutant proteins (five and two, respectively).
We have carried out a study by using differential scanning calorimetry (DSC) of the relative stabilities of 20 ALS-mutant SOD1 apoproteins that represent a more complete range of ALS mutations than those studied by the other investigators (13, 14). Here, we report that the ALS-mutant apoproteins that are severely destabilized relative to the WT apoprotein compose only a subset of the known variants. In fact, some of the ALS mutant apoproteins, in their disulfide-oxidized (SOD1S-S) or disulfide-reduced (SOD12SH) states exhibit global stability that is equal to or greater than that of WT apo-SOD1.
To assess the extent of the structural differences between the various ALS-mutant apoproteins in solution, we also carried out a study of a representative subset (12 mutant proteins) by using global hydrogen-deuterium (H/D) exchange measured by electrospray ionization-MS. H/D exchange rates are sensitive indicators of the secondary and tertiary structure in proteins as well as of their dynamic behavior (15). Again, the mutant apoproteins exhibited a wide range of behaviors. Our results indicate that the causes of SOD1-linked ALS are complex and are not simply related to apoprotein stability, although that destabilization may contribute to the toxicity of some ALS-SOD1 mutants.
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