.! , ......... 1.. .., ., ------ ..ll, - .:U, . 1 4. M-1 W1411.'AN.4"lW*v,1 or,-Al11 m-, R a.. .....' the refolding of the short-time denatured 3. R. J. Ellis and S. M. van der Vies, Annu. Rev. 19. H. R. B. Pelham, Cell 46, 959 (1986). Biochem. 60, 321 (1991). 20. G. C. Flynn, J. Pohl, M. T. Flocco, J. E. Rothman, enzyme, but that a fraction of the prolines 4. F. X. Schmid, Curr. Opin. Struct. Biol. 1, 36 (1991). Nature 353, 726 (1991). must be very rapidly buried during the 5. G. Fischer and F. X. Schmid, Biochemistry 29, 21. P.-0. FreskgArd, U. Carlsson, L. G. MArtensson, initiation of the refolding process (pB in 2205 (1990). B.-H. Jonsson, FEBS Lett. 289, 117 (1991). 6. A. E. Eriksson, T. A. Jones, A. Uljas, Proteins 22. R. G. Khalifah, D. J. Strader, S. H. Bryant, S. M. Fig. 3) and thus become inaccessible to Struct. Funct. Genet. 4, 274 (1988). Gibson, Biochemistry 16, 2241 (1977). PPIase. It has been reported (10-12) that 7. C. Fransson et al., FEBS Lett. 296, 90 (1992). 23. L. E. Henderson and D. Henriksson, Anal. Bio- an initiation structure of carbonic anhy- 8. J. L. Cleland and D. I. C. Wang, Biochemistry 29, chem. 51, 288 (1973). 11072 (1990). 24. G. Fischer, H. Bang, C. Mech, Biomed. Biochim. drase is rapidly formed during refolding. 9. J. Buchner et al., ibid. 30, 1586 (1991). Acta 43, 1101 (1984). When the prolines in this core of the 10. U. Carlsson, R. Aasa, L. E. Henderson, B.-H. 25. N. Takahashi, T. Hayano, M. Suzuki, Nature 337, enzyme structure (PB in Fig. 3) are cis-trans Jonsson, S. Lindskog, Eur. J. Biochem. 52, 25 473 (1989). by 1 hour of denaturation, the (1975). 26. G. Fischer, B. Wittmann-Liebold, K. Lang, T. Kief- equilibrated 11. N. Bergenhem, U. Carlsson, J.-A. Karlsson, nt. J. haber, F. X. Schmid, ibid., p. 476. isomerization of these residues to the native Pept. Protein Res. 33, 140 (1989). 27. Supported by grants from the Swedish National conformers limits the rate of reactivation 12. 0. B. Ptitsyn, FEBS Lett. 285, 176 (1991). Board for Technical Development (732-88-04392) = 4 min) in the presence of PPIase. 13. T. Kiefhaber, R. Quaas, U. Hahn, F. Schmid, (U.C. and B.-H.J.), the Swedish Natural Science (ti/2 Biochemistry 29, 3061 (1990). Research Council (K-Ku 9426-300) (B.-H.J.), Mag- However, when the prolines in this core are 14. R. K. Harrison and R. L. Stein, ibid., p. 1684. nus Bergvalls Stiftelse (B.-H.J.), and Carl Tryggers in correct conformation from the beginning 15. R. Wolfenden and A. Radzicka, Chemtracts Bio- Stiftelse (U.C.). Fellowships to P.-OF. and M.S. from of refolding and all the other prolines are chem. Mol. Biol. 2, 52 (1991). Stiftelsen Bengt Lundqvists Minne are also gratefully 16. T. Drakenberg, K.-. Dahlqvist, S. J. Fors6n, J. acknlaedged. We are indebted to Sandoz Ltd. for cis-trans equilibrated by the action of PPIase Phys. Chem. 76, 2178 (1972). their generous gift of CsA. only during the first 3 s of refolding, uncat- 17. R. Wolfenden, Science 222, 1087 (1983). alyzed isomerization of the PPIase-accessi- 18. J. Kallen et al., Nature 353, 276 (1991). 16 April 1992; accepted 27 July 1992 ble prolines (PA; Fig. 3) is rate determining (t1/2 = 5 min) in the reactivation process. Downloaded from Thus, the action of PPIase on the refold- ing HCA II reveals the presence of two Spontaneous Hypercholesterolemia and Arterial classes of prolines (accessible and buried), Lesions in Mice Lacking Apolipoprotein E as evidenced by the impact of PPIase on the refolding kinetics. For ribonuclease T1, the Sunny H. Zhang, Robert L. Reddick, Jorge A. Piedrahita, structural context of prolines is important Nobuyo Maeda* http://science.sciencemag.org/ for the efficiency of catalysis (13). That PPIase acts as a classical chaperone is clear- Apolipoprotein E (apoE) is a ligand for receptors that clear remnants of chylomicrons and ly demonstrated also by its independent very low density lipoproteins. Lack of apoE is, therefore, expected to cause accumulation effect on the kinetics and the yield of the in plasma of cholesterol-rich remnants whose prolonged circulation should be atherogenic. refolding reaction. This effect is exemplified ApoE-deficient mice generated by gene targeting were used to test this hypothesis and to in Fig. 1: after 10 s of refolding, inhibition make a mouse model for spontaneous atherosclerosis. The mutant mice had five times of PPIase still gives rise to a 100% yield and normal plasma cholesterol, and developed foam cell-rich depositions in their proximal a relatively slow tl/2 (9 min; chaperone), aortas by age 3 months. These spontaneous lesions progressed and caused severe whereas addition of PPIase after 10 s of occlusion of the coronary artery ostium by 8 months. The severe yet viable phenotype of refolding does not increase the yield but the mutants should make them valuable for investigating genetic and environmental factors on April 28, 2020 does result in a shorter t1,2 (4 min; that modify the atherogenic process. isomerase). Two mechanisms proposed for the ac- tion of PPIase are catalysis by distortion (14) and catalysis by desolvation (15-17). Atherosclerotic cardiovascular disease, the its most important functions is to serve as a The latter mechanism was suggested be- major cause of death in Western society, high affinity ligand for the apoB and cause binding of a peptide segment into a results from complex interactions among apoE(LDL) receptor and for the chylomi- hydrophobic environment, as in the bind- multiple genetic and environmental factors cron-remnant receptor, thereby allowing ing site of PPIase (18), promotes cis-trans (1). Among the factors that have been the specific uptake of apoE-containing par- isomerization by decreasing the charge sep- identified to date are changes in the genes ticles by the liver. aration of the peptide bond and thus creat- involved in lipid metabolism, including the A frequent genetic variant of human ing a peptide that has a more single-bond gene encoding apolipoprotein E (apoE) (2). apoE, apoE-2, differs from the most com- character. Hence, if this in fact is the ApoE is a glycoprotein with a molecular size mon form, apoE-3, by having cysteine at mechanism for PPIase activity, then other of approximately 34 kD that is synthesized position 158 in place of arginine. This chaperones that bind peptide chains to in the liver, brain, and other tissues in both amino acid substitution in the LDL receptor apolar sites (19, 20) might also possess humans and mice (3); it is a structural binding region reduces the binding ability isomerase activity. Because PPIase (which component of all lipoprotein particles other of apoE-2 to less than 2% relative to that of is also cyclophilin) is known to be involved than low density lipoprotein (LDL). One of apoE-3. Homozygosity for the gene Apoe2 is in T cell activation, it is possible that the associated with type III hyperlipoproteine- chaperone function of this protein is impor- S. H. Zhang and R. L. Reddick, Department of Pathol- mia, which is characterized by increased ogy, University of North Carolina at Chapel Hill, Chap- plasma triglyceride and cholesterol levels, tant for essential protein-folding processes el Hill, NC 27599-7525. in the immune response. N. Maeda, Department of Pathology and Curriculum in yellow lipid-laden xanthomatous skin nod- Genetics and of the Program in Molecular Biology and ules, and the early development of athero- Biotechnology, University of North Carolina at Chapel sclerosis (4). The complexities of the REFERENCES AND NOTES Hill, Chapel Hill, NC 27599-7525. J. A. Piedrahita, Department of Veterinary Anatomy pathogenesis of this disease are, however, 1. R. Jaenicke, Prog. Biophys. Mol. Biol. 49, 117 and Public Health, Texas A&M University, College illustrated by the fact that only about 2% of (1987). TX 77840-4458. 2. M.-J. Gething and J. Sambrook, Nature 355, 33 Station, Apoe2/Apoe2 individuals develop hyperlip- (1992). *To whom correspondence should be addressed. oproteinemia. The majority of individuals 468 SCIENCE * VOL. 258 * 16 OCTOBER 1992 with this genotype have, in fact, lower than tion of these results was obtained by agarose pre-P positions were markedly increased. normal plasma cholesterol levels (5). Thus, gel electrophoresis of whole plasma (8). In Thus, the distribution of lipids in the plas- other factors must be necessary for the normal mouse plasma, HDL, which is the ma of the mutants was shifted toward the expression of type III hyperlipoproteinemia major lipoprotein, migrates in the same region where lipoproteins of lower density associated with apoE-2. position as a globulins. VLDL and LDL, normally migrate. Three human kindreds with inherited which migrate more slowly at the pre-P and To better characterize the molecular ba- apoE deficiency have been reported (6). P positions, respectively, are minor compo- sis for the changes associated with the Individuals deficient in apoE, have tubero- nents (9). In the mutant plasma, particles absence of apoE, we determined the apo- eruptive xanthomatosis and type III hyper- that migrate at the at position were reduced lipoprotein compositions of plasma lipopro- lipoproteinemia with elevated cholesterol while those migrating between the P and teins. The lipoprotein particles were first and near normal triglyceride levels. ApoE-deficient mice were generated by inactivating the Apoe gene by targeting (7). Table 1. Plasma cholesterol and triglyceride levels in apoE-deficient mice. Mice of both sexes, 2 to Mice homozygous or heterozygous for the 5 months of age, were kept on regular mouse chow (ProLab formula 3000, 4.5% crude fat).