V o l u m e 294, n u m b e r 3, 155-157 F E B S 10452 ~) 1991 Federation o f European Biochemical Soci2tics 001-~5793/91/$3.50
l-/ypothesA
December
1901
~
Are prions misfolded molecular chaperones? Jean-Pierre INS£-RM
Liautard
U-OS, L ~ b o r a t o i r e d e B i o l o g i e Cellula~r~. U n i v e r s i t e d e ~¢lonlpelli,,r I L C P .
ltXJ; 3 4 0 9 5 Atontpellaer C v d e x 5. ~?an~'t,
R e c e i v e d 27 A u g u s t 1991: ~ v i s e d v e r s i o n r e c e i v e d 10 O c t o b e r 1991 A theory has been developed that could explain priori infection Prions could be molecular chaperones that are required for their own assembly. T h e theory has been deduced from an analysis of protein folding and consequences explored by computer simulations. Thermo-kmetic anzlysis
o f protein folding shows that a misfolded chaperone gives rise to new misfe!ded chaperones. Consequently such a protein could behave as a new kind o f informative molecule and replicate misfotding according to a process similar to infection. A quantitative model has been derived from this hypothesis that displays the characteristics o f prion infections, t h i s hypothesis satisfactorily explains the three ma~tifeslations infection, familial and sporadic - that are the characteristic featureS o f all prion diseases. Protein fi)lding; Prion: Thermo-kinettcs
1. I N T R O D U C T I O N
2. T H E O R E T I C A L
T h e r e is n o w c o n s i d e r a b l e e v i d e n c e t h a t t h e i n f e c t i o u s agents causing bovine spongiform encephalitis, serapie, the human Gerstmann-Straussler and Creutzfeld-Jakob syndromes and other degenerative encephalopathies are pure proteins called p,-ions [I]. However many biolog i s t s a r e u n h a p p y w i t h t h i s i d e a , a s i t d o e s n o t fit r e a d i l y with the central dogma of molecular biology. The prion h y p o t h e s i s is c r i t i c i z e d o n t w o c o u n t s : s o m e w o r k e r s h a v e f o u n d n u c l e i c a c i d i n p r i o n p a r t i c l e s [2,3] a n d o t h e r s [4] s u g g e s t t h a t p r i o n s a r e n o t t h e c a u s a t i v e a g e n t s of infection. However, many studies [5-7] have provialed e x p e r i m e n t a l e v i d e n c e f o r the r o l e o f p r i o n s in t h e diseases. If prions really are the causative agents, their action should be explainable in terms ofcurrent biological c o n c e p t s , r a t h e r t h a n r e q u i r i n g a c h a n g e in f u n d a mental thinking. I have accepted two basic assumpt i o n s , (i) t h a t p r i o n s a r e p a r t i c u l a r l y s t a b l e p r o t e i n s t h a t cause illness by an infection-like process (see [i]), and (it) t h a t p r o t e i n s s i m i l a r o r i d e n t i c a l t o p r i o n s a r e e n c o ded by mammalian g e n o m e s I!]. S t a r t i n g w i t h a n a n a lysis of protein folding mediated by a molecular chaper o n e , a t h e o r e t . c a l m o d e l is p r o p o s e d t h a t e x p h i n s t h e major features of prion diseases.
M o l e c o l a r c h a p e r o n e s a r e i n v o l v e d in d i r e c t i n g t h e folding of other proteins. The best known are the heatshock proteins, it has recently been shown that molecul a r c h a p e r o n e s c a n d i r e c t t h e i r o w n a s s e m b l y [8,9]. T h e question fol!ows naturally, what happens if a molecular c h a p e r o n e is a c c i d e n t a l l y m i s f o l d e d ? C o r r e c t f o l d i n g is g e n e r a l l y b e l i e v e d t o p r o v i d e t h e m o s t s t a b l e p r o t e i n ( s e e [! 4]), h o w e v e r t h e r e is n o p r o o f f o r t h i s h y p o t h e s i s [12]. O n t h e c o n t r a r y , t h e c o n f o r m a t i o n o f f o l d e d p r o s u b t i l i s i n is u n c h a n g e d b y r e m o v a l o f a 7 7 - r e s i d u e N - t e r m i n a l s e g m e n t , b u t i f t h i s s e g m e n t is r e m o v e d b e f o r e f o l d i n g a n e w c o n f o r m a t i o n is a d o p t e d [15] Thus mature subtilisin has only kinetic stability. Metastable conformations do not violate kinetic theories o f p r o t e i n f o l d i n g [13,16], a n d t h e v e r y e x i s t e n c e o f m o l e c u l a r c h a p e r o n e s is a s t r o n g a r g u m e n t f o r s u c h metastable conformation. Proteins requiring a molecular c h a p e r o n e m a y r e m a i n in a m e t a s t a b l e c o n f o r m a t i o n for an infinite time if they do not meet their chaperone. T h i s i m p l i e s t h a t t h e k i n e t i c s o f f o l d i n g is b l o c k e d b y art e n e r g y b a r r i e r w h i c h is r e m o v e d b y t h e m o l e c u l a r c h a p erone, much like enzyme catalysis. These observations legitimate the use of kinetic theories of protein folding to a n a l y z e t h e m e c h a n i s m s i n v o l v e d in p r o t e i n f o l d i n g promoted by molecular chaperones. Analysis can be performed using the thermo-kinetic m o d e l o f p r o t e i n f o l d i n g [13]. T h e t h e r m o d y n a m i c s of i r r e v e r s i b l e p r o c e s s e s i n d i c a t e s t h a t t h e sp,,"cd o f f o l d i n g determines the final interactions between the amino a c i d s [13]. G e n e r a l i z a t i o n t o t h e f o l d i n g c a t a l y z e d b y m o l e c u l a r c h a p e r o n e s is s t r a i g h t f o r w a r d . C o n s i d e r t h e
Correspondence address: J.-P. Liautard, I N S E R M U-65. Laboratoire de Biologic Ceilulaire. Universit6 de Montpellier !!. C.P. 100; 34095 Montpellier C ~ l © x 5, France. Fa~: (33) 67 14 33 38.
I~ublished b y Elsevier Science Publishers 8. i/.
ANALYSIS
! 55
V o l u m e 294. n u m b e r 3
FEBS LETFERS
c a s e o f a n i n t e r a c t i o n b e t w e e n t w o m o h ; c u ! e s San u n f o l d e d m o l e c u l e I, a n d a f o l d e o m o l e c u l e J. t h e m o l e c u l a r chaperone) of the same protein. The ~peed of interact i o n b e t w e e n t w o a m i n o acids,, o n e (a~) f l o r a m o l e c u l e 1 a n d t h e o t h e r (aj) f r o m t h e m o l e c u l e J is g o v e r n e d b y t h e r e l a t i o n s h i p [13]:
D e c e m b e r ;991 m
% |
ac~ o EL
/
JD o
v.,~,,
=- ~
[(H
/~'~,+.,-/~',,,..,) (~ G,,,+.-%+.)]
O)
w h e r e P~" is t h e p r o b a b i l i t y ( a c c o r d i n g t o B o i t z m a n ' s law) of finding the amino acids in a conformation all o w i n g i n t e r ' a c t i o n , n is t h e n u m b e r o f a m i n o a c i d s a r o u n d a~ t h a t i n f l u e n c e t h e sr, c c d a f a s s o c i a t i o n , a n d m i s E ( l . n ) . A s m o l e c u l e J , t h e m o ) e t u l a r c h a p e r o n e , is f o l d e d , • P ~ J +,, = 1 f o r a l l m . b u t a s ~ l o l e c u i e I c a n n o t f o l d w i t h o u t a m o l e c u l a r c h a p e r o n e , i t s P~,~÷,, is v e r y low for all m. Hence, spontaneous eo=rect folding does n o t o c c u r , a n d t h e f o l d i n g s p e e d is g o v e r n e d b y t h e m o l e c u l e J. T h u s t h e c o n f o r m a t i o n o f a, a n d i t s neighbors should be that of the corresponding residues aj a n d i t s n e i g h b o r s [13]. H e n c e . i f m o l e c u l e J is misfolded, then molecule 1 will also be misfolded, and the new misfolded molecule should have the same structure as its molecular chaperone. The (misfolded) struct u r e o f t h e c h a p e r o n e is t r a n s m i t t e d t o t h e n e w s y n thesized molecules. This result implies that, under certain specific circumstances, a k~nd of biological information can be transmitted directly by proteins without the intervention of n u c l e i c a c i d . H e n c e , t h e s t r u c t u r e o f a p r o t e i n is i n f o r mative by itself. Such proteins could be regarded as infectious organisms, replicating within a cells using, as d o v i r u s e s , t h e cells" o w n c o m p o n e n t s . M i s f o l d i n g c a n be considered as a "structural mutation" that propagates in t h e ceil. Are there evidences that prions are molecular chapcrones? First, molecular chaperones, as proteins, can be denatured, and prions lose their infectivity after denaturat i o n [10], i m p l y i n g t h a t t h e i r t e r t i a r y s t r u c t u r e is o f i m portance. Second, if preens are misfolded molecular chaperones, then they should exist in two differently f o l d e d f o r m s . L o p e z e t a l . [17] s h o w e d t h a t p r i o n s s y n thesized in vitro could adopt two topological forms as a consequence of alt:.'rnative folding. Thirdly, prion mol e c u l e s p r e s e n t a z o n e c o n t a i n i n g g i y c i n e r e p e a t s [1,18]. G l y c i n e is a v e r y m o b i l e a m i n o a c i d , w i t h a l l i t s c o n f o r m a t i o n s h a v i n g a b o u t t h e s a m e e n e r g y [12]. H e n c e t h e s e sequences have a low propensity to adopt spontaneously a definite secondary structure. Analysis according to t h e ( 3 i b r a t e t a l . [19] m e t h o d r e v e a l s t h a t a t h i r d o f t h e p r o t e i n is p o o r l y s t r u c t u r e d . T h e s e o b s e r v a t i o n s s u g g e s t a necessity of interaction with a folding partner to promote a define conformation.
1.56
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o
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|
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I
200
T~me ( ; t e r a t i o n s
300 x
401
10)
F i g . I. S i m u l a t i o n o f p r i o n d i s e a s e s a p p e a r a n c e u n d e r t h e t h r e e s p e c i fic c a s e s : ( A ) s p o r a d i c , ( B ) g e n e t i c aILd ( C ) i n f e c t i o u s . I n v a s i o n b / prions was simulated by analysing the behavior of each newly synt h e s i z e d m o l e c u l e w i t h a c o m p u t e r ( s e e t e xt ). T i m e is p r o p o r t i o n a l t o '.he n u m b e r o f i t e r a t i o n s . I n B, t h e v a l u e o f P, is f o u r - t i m e s t h e o n e u s e d in f i g u r e s A a n d C . i n C , m i s g u i d e d c h a p e r o n e s ( p r i o n s ) a r e a d d e d at time=600 iterations (arrow). For each case. simulation was repeated 5000 t i m e s a n d r e s u l t s w c r c c u m u l a t e d t o g i v e a k i n d o f " p r o b a b i l i t y c urve " t h a t g i v e s t h e p r o b a b i l i t y t o d e v e l o p t h e p r i o n d i s e a s e .
3. A M O D E L INVASION
FOR
TItE
SIMULATION
OF PRION
According to the deductions presented above, a model can be developed to simulate prion invasion. Consider the number uP, of prions (misfolded molecul a r c h a p e r o n e s ) a t t i m e t w h e n a n e w m o l e c u l e is s y n t h e sized. This number will depend on the number of prions a t t i m e t - i ( n P r - i ) . T h e r e is a p r o b a b i l i t y ( v e r y l o w b u t not zero) of spontaneous misfolding (if folding is not p e r f o r m e d o n t h e c h a p e r o n i n e ) t e r m e d P i ( i t is e a s y t o demonstrate that P, is directly proportional t o P'~ o f eqn. (1)). Otherwise, the folding will be performed on a molecular chaperone and the probability of misfoldi n s is ( n P , _ , / ( n N , - i ~ u P , - 1 ) ) , w h e r e n N r - i i s t h e n u m b e r of correctly folded molecules. The elimination of the prion, as a physiologic degradation of protein, could also be calculated. Simulation was performed with computer by iteration. Numerous simulations were performed in order to obtain a probability of appearance a s a f u n c t i o n o f t h e n u m b e r o f i t e r a t i o n s ( F i g . 1). T h e number of iterations is proportional to time. Fig. IA depicts the sporadic apparition of prions after a long lag period. Clearly, according to the model, the mutations observed in degenerative brain illnesses favor prion diseases by increasing Pi (see section 4 for a discussion of this point). The calculations correctly simulate this phen o m e n o n ( F i g . 1B). I n c r e a s i n g f o u r - t i m e s t h e p r o b a b i l i t y P~ r e s u l t s i n r e d u c t i o n o f t h e i n v a s i o n l a g p e r i o d o f about 0.33. Infection is ::quivalent to a direct increase in uP. The results of the simulation presented in Fig. IC
V o l u m e 294, n u m b e r 3
FEBS LE'I~ERS
model such an infection. After injection of nP (arrow) the invasiott appears very rapidly, without a lag period. C l e a r l y , s p e c i f i c i t y o f p r i o n ~_iseases ( i n f e c t i o u s , s p o radic, familial) are well simulated by the model. 4. A N A L Y S I S
OF PRION
DISEASES
W e s t a w a y et al. [!] su g g est s t h a t p r i o n d i s e a s e s m a y fall into three categories: infectious, familial and sporadic. The above hypothesis provides a rational unified e x p l a n a t i o n for all t h ree.
4.1. Infectious disease In order to explain the infection, one necessary condit i o n is t h a t p r i o n s a r e s t a b l e e n o u g h t o b e t a k e n i n t o t h e organism, essentially undamaged, to become a chaper o n e . T h e s t a b i l i t y o f p r i o n s f a v o r s t h i s h y p o t h e s i s [1]. All the experimental evidence for the infectious proper; ties of prions obtained to date has been from direct i n j e c t i o n o f p r o t e i n p r e p a r a t i o n [1,6]. T h e y v:ill b e u s e d as molecular' chaperones for the folding of ,tewly-synthesized prion proteins. The misfolding of the priori results in the propagation of the error, producing increasing numbers of misfolded proteins which, because of their singular stability, will not be degraded by intracellular IJroteases. However, in regenerating tissues, misfolded protein will be diluted out from dividing cells a n d e l i m i n a t e d b y cell d e a t h . C o n s e q u e n t l y , m i s f o l d e d molecular chaperonez will occur in large quantity only i n n o n - d i v i d i n g cells. T h i s o b s e r v a t i o n e x p l a i n s the brain localization of prion diseases. Furthermore, if the p r i o n m o l e c u l e is r e l a t e d t o a m o l e c u l e t h a t is i m p o r t a n t f o r t h e p h y s i o l o g y o f the cell, t h e r e m u s t b e a f e d b a c k regulatory mechanism. A decrease in the number of cotTectly-foid,.-xl c h a p e r o n e m o l e c u l e s m a y w e l l l e a d t o a n i n c r e a s e d s y n t h e s i s , g i v i n g r i s e t o still m o r e m i s f o l d e d m o l e c u l e s . T h e c o n v e r s e is a l s o t r u e , i.e. i n j e c t i o n of correctly folded molecular chaperone should partially reverse the effect of the prion. This otmns the way to treating these diseases. 4 . 2 . Sporadic apparition This concept of prion diseases has other implications. A misfolded protein that arises accidentally could initiate cell i n v a s i o n . S u c h a c c i d e n t a l m i s f o l d i n g s a r e m o r e likely with age, leading to increased probability of disease w i t h age in a h i t h e r t o h e a l t h y p e r s o n (see s i m u l a tion Fig. IA), as in human brain degenerative diseases. 4 . 3 . E x p la n a tio n
o f the genetic
transmission o f the
disease The accidental misfoiding could be due to a mutation increasing the probability of spontaneous misfolding a n d g i v i n g rise t o i n h e r i t e d d i s e a s e s w h e r e illness a p p e a r s m o r e f r e q u e n t l y i n y o u n g e r p e o p l e ( s e e F i g . ! B). Similarly, the recent onset of BSE could be due to genetic selection as well as direct infection.
December
1991
I n b r e d s t r a i n s o f m i c e in w h i c h s c r a p i e i n f e c t i o n s h a v e s h o r t ( N Z B ) a n d l o n g ( l / L n ) t i m e - c o u r s e s h a y : :t d i s t i n c t p r i o n g ~ n e a l l e l e [I,18]. T h e a m i n o a c i d a t c o d o n t 0 8 i n t h e p r i o n s e q u e n c e is l e u c i n e i n t h e N Z B strain and phenylalanine in the I/Ln strain. This change would decrease the probability of alpha-helix format i o n , l o w e r i n g P ~ , t w o - f o l d [19]. T h e h i g h l y c o n s e r v e d c o d o n a t p o s i t i o n 189 is c h a n g e d f r o m t h r e o n i n e in t h e N Z B m i c e t o v a l i n e ; n t h e l / L n s t r a i n , r e s u l t i n g in a t h r e e - f o l d i n c r e a s e i n t h e p r o b a b i l i t y o f b e t a - s h e e t fold i n g i n a r e g i o n w h i c h is o t h e r w i s e l i k e l y t o c o i l [I 9]. 5. C O N C L U S I O N Structural information can be transmitted by protein. This concept offers a good frame to explain etiology of brain degenerative diseases caused by vrions. Furtherm o r e it s u g g e s t s a t r e a t m e n t f o r t h e s e i l l n e s s e s . Acknowledgements: l that~k M. Cellier f o r m a n y helpful d i s c u s s i o n s o n h e a t - s h o c k p r o t e i n s a n d E. C a r o n f o r ~eneral d i s c u s s i o n a n d help with the manuscript.
REFERENCES [!] W e s t a w a y , D., C a r l s o n , G . A a n d P r u s i n e r , S.B. (1989) T r e n d s NeuroSci. 2. 221-227. [2] K i m b e r l i n , R . H . (1982) T r e n d s B i o c h e m . Sci. 7, 392-394. [3] N a r a n g , H . K . (1990) J. M o ! . Biol. 216, 469-473. [4] S h a r p e , A , H u n t e r , J., C h a t t i e r . P. a n d J a e n i s h , R. (1990) N a t u r e 346, 181- 183. 15] P r u s i n e r , S.B. (1982) Science 216, 136 144. [6| P r u s i n e r , S.B., S c o t t , M., F o s t e r , D., Pan, K . M . , G r o t h , D.,
Mirenda, C., Torchia, M., Yang. S.L., Sgrban. D., Carlson. G.A., Hopp¢, P.C., Westaway, O. and DeArmond, S.J. (1990) Cell 63. 673-686. [71 Safar, J., Wang, W., Padgett, M.P., Ceroni. M_, Piccardo, P., gopf, D., Gadjusek. D.C. and Gibbs, C.J. (1990) Proc. Natl. Acad. Sci. USA ST, 6373 6377. [8] Cheng, M.Y.. H trtl. F.U. and Horwich, A.L. (I 9901 Nature ?,48. 455-458. [9] Lissin, N . M . , V t : n y a m i n o y , S Y. a n d G i r s h o v i c h , A.S. (1990) N a t u r e 348, 339-342. [10l P r u s i n e r , S.B., G r o t h , D . F . , B o i t o n . D . C . , K e n t , S.B. a n d H o o d . L.E. (1984) Cell 38, 127-134. [11] M c L a c h l a n 0 A. ( 1 9 7 6 ) J . M o l . Biol. 103, 271 298. [121 Glaelis, C. a n d Y o n , I. (1982) in: P r o t e i n F o l d i n g , A c a d e m i c Press. N e w Y o r k . [13l L i a u t a r d , J.P. (1990) C . R . A c a d . Sci. 311, 385 389. [141 K i m , P.S. a n d B a l d w i n , R . L . (1990) A n n u . Rev. B i o c h e m . 59, [! 5] Z h u , X., O h t a , Y . , J o r d a n , F. a n d I n o u y e , M. (1989} N a t u r e 339, 483-484. [161 B r o o k s , C., K a r p l u s , M. a n d Pettit, M. (1988) in: Proteins: A Theoretical Perspective o f Dynamics, Structure, a n d T h e r m o d y n a m i c s , p. 129, J o h n W i l e y a n d S o n , N e w Y o r k . [17] L o p e 2 , C., Y o s t , C . , P r d s i n e r , S.B_, M y e r s , R. a n d t i n g a p p a , V. (1990) Science 248. 226-229. [18] C a r l s o n , G , A . , H s i a o , K., O e s c h , B., W e s t a w a y , D. a n d P r u s i n e r , S.B. ( 1 9 9 1 ) T r e n d s G e n e t . 7, 6 1 - 6 5 . [19] G i b r a t , J . F . , G a m i e r , J. a n d R o b s o n , B. (1987) J. M o l . Biol. 198. 425-443.
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l-/ypothesA
December
1901
~
Are prions misfolded molecular chaperones? Jean-Pierre INS£-RM
Liautard
U-OS, L ~ b o r a t o i r e d e B i o l o g i e Cellula~r~. U n i v e r s i t e d e ~¢lonlpelli,,r I L C P .
ltXJ; 3 4 0 9 5 Atontpellaer C v d e x 5. ~?an~'t,
R e c e i v e d 27 A u g u s t 1991: ~ v i s e d v e r s i o n r e c e i v e d 10 O c t o b e r 1991 A theory has been developed that could explain priori infection Prions could be molecular chaperones that are required for their own assembly. T h e theory has been deduced from an analysis of protein folding and consequences explored by computer simulations. Thermo-kmetic anzlysis
o f protein folding shows that a misfolded chaperone gives rise to new misfe!ded chaperones. Consequently such a protein could behave as a new kind o f informative molecule and replicate misfotding according to a process similar to infection. A quantitative model has been derived from this hypothesis that displays the characteristics o f prion infections, t h i s hypothesis satisfactorily explains the three ma~tifeslations infection, familial and sporadic - that are the characteristic featureS o f all prion diseases. Protein fi)lding; Prion: Thermo-kinettcs
1. I N T R O D U C T I O N
2. T H E O R E T I C A L
T h e r e is n o w c o n s i d e r a b l e e v i d e n c e t h a t t h e i n f e c t i o u s agents causing bovine spongiform encephalitis, serapie, the human Gerstmann-Straussler and Creutzfeld-Jakob syndromes and other degenerative encephalopathies are pure proteins called p,-ions [I]. However many biolog i s t s a r e u n h a p p y w i t h t h i s i d e a , a s i t d o e s n o t fit r e a d i l y with the central dogma of molecular biology. The prion h y p o t h e s i s is c r i t i c i z e d o n t w o c o u n t s : s o m e w o r k e r s h a v e f o u n d n u c l e i c a c i d i n p r i o n p a r t i c l e s [2,3] a n d o t h e r s [4] s u g g e s t t h a t p r i o n s a r e n o t t h e c a u s a t i v e a g e n t s of infection. However, many studies [5-7] have provialed e x p e r i m e n t a l e v i d e n c e f o r the r o l e o f p r i o n s in t h e diseases. If prions really are the causative agents, their action should be explainable in terms ofcurrent biological c o n c e p t s , r a t h e r t h a n r e q u i r i n g a c h a n g e in f u n d a mental thinking. I have accepted two basic assumpt i o n s , (i) t h a t p r i o n s a r e p a r t i c u l a r l y s t a b l e p r o t e i n s t h a t cause illness by an infection-like process (see [i]), and (it) t h a t p r o t e i n s s i m i l a r o r i d e n t i c a l t o p r i o n s a r e e n c o ded by mammalian g e n o m e s I!]. S t a r t i n g w i t h a n a n a lysis of protein folding mediated by a molecular chaper o n e , a t h e o r e t . c a l m o d e l is p r o p o s e d t h a t e x p h i n s t h e major features of prion diseases.
M o l e c o l a r c h a p e r o n e s a r e i n v o l v e d in d i r e c t i n g t h e folding of other proteins. The best known are the heatshock proteins, it has recently been shown that molecul a r c h a p e r o n e s c a n d i r e c t t h e i r o w n a s s e m b l y [8,9]. T h e question fol!ows naturally, what happens if a molecular c h a p e r o n e is a c c i d e n t a l l y m i s f o l d e d ? C o r r e c t f o l d i n g is g e n e r a l l y b e l i e v e d t o p r o v i d e t h e m o s t s t a b l e p r o t e i n ( s e e [! 4]), h o w e v e r t h e r e is n o p r o o f f o r t h i s h y p o t h e s i s [12]. O n t h e c o n t r a r y , t h e c o n f o r m a t i o n o f f o l d e d p r o s u b t i l i s i n is u n c h a n g e d b y r e m o v a l o f a 7 7 - r e s i d u e N - t e r m i n a l s e g m e n t , b u t i f t h i s s e g m e n t is r e m o v e d b e f o r e f o l d i n g a n e w c o n f o r m a t i o n is a d o p t e d [15] Thus mature subtilisin has only kinetic stability. Metastable conformations do not violate kinetic theories o f p r o t e i n f o l d i n g [13,16], a n d t h e v e r y e x i s t e n c e o f m o l e c u l a r c h a p e r o n e s is a s t r o n g a r g u m e n t f o r s u c h metastable conformation. Proteins requiring a molecular c h a p e r o n e m a y r e m a i n in a m e t a s t a b l e c o n f o r m a t i o n for an infinite time if they do not meet their chaperone. T h i s i m p l i e s t h a t t h e k i n e t i c s o f f o l d i n g is b l o c k e d b y art e n e r g y b a r r i e r w h i c h is r e m o v e d b y t h e m o l e c u l a r c h a p erone, much like enzyme catalysis. These observations legitimate the use of kinetic theories of protein folding to a n a l y z e t h e m e c h a n i s m s i n v o l v e d in p r o t e i n f o l d i n g promoted by molecular chaperones. Analysis can be performed using the thermo-kinetic m o d e l o f p r o t e i n f o l d i n g [13]. T h e t h e r m o d y n a m i c s of i r r e v e r s i b l e p r o c e s s e s i n d i c a t e s t h a t t h e sp,,"cd o f f o l d i n g determines the final interactions between the amino a c i d s [13]. G e n e r a l i z a t i o n t o t h e f o l d i n g c a t a l y z e d b y m o l e c u l a r c h a p e r o n e s is s t r a i g h t f o r w a r d . C o n s i d e r t h e
Correspondence address: J.-P. Liautard, I N S E R M U-65. Laboratoire de Biologic Ceilulaire. Universit6 de Montpellier !!. C.P. 100; 34095 Montpellier C ~ l © x 5, France. Fa~: (33) 67 14 33 38.
I~ublished b y Elsevier Science Publishers 8. i/.
ANALYSIS
! 55
V o l u m e 294. n u m b e r 3
FEBS LETFERS
c a s e o f a n i n t e r a c t i o n b e t w e e n t w o m o h ; c u ! e s San u n f o l d e d m o l e c u l e I, a n d a f o l d e o m o l e c u l e J. t h e m o l e c u l a r chaperone) of the same protein. The ~peed of interact i o n b e t w e e n t w o a m i n o acids,, o n e (a~) f l o r a m o l e c u l e 1 a n d t h e o t h e r (aj) f r o m t h e m o l e c u l e J is g o v e r n e d b y t h e r e l a t i o n s h i p [13]:
D e c e m b e r ;991 m
% |
ac~ o EL
/
JD o
v.,~,,
=- ~
[(H
/~'~,+.,-/~',,,..,) (~ G,,,+.-%+.)]
O)
w h e r e P~" is t h e p r o b a b i l i t y ( a c c o r d i n g t o B o i t z m a n ' s law) of finding the amino acids in a conformation all o w i n g i n t e r ' a c t i o n , n is t h e n u m b e r o f a m i n o a c i d s a r o u n d a~ t h a t i n f l u e n c e t h e sr, c c d a f a s s o c i a t i o n , a n d m i s E ( l . n ) . A s m o l e c u l e J , t h e m o ) e t u l a r c h a p e r o n e , is f o l d e d , • P ~ J +,, = 1 f o r a l l m . b u t a s ~ l o l e c u i e I c a n n o t f o l d w i t h o u t a m o l e c u l a r c h a p e r o n e , i t s P~,~÷,, is v e r y low for all m. Hence, spontaneous eo=rect folding does n o t o c c u r , a n d t h e f o l d i n g s p e e d is g o v e r n e d b y t h e m o l e c u l e J. T h u s t h e c o n f o r m a t i o n o f a, a n d i t s neighbors should be that of the corresponding residues aj a n d i t s n e i g h b o r s [13]. H e n c e . i f m o l e c u l e J is misfolded, then molecule 1 will also be misfolded, and the new misfolded molecule should have the same structure as its molecular chaperone. The (misfolded) struct u r e o f t h e c h a p e r o n e is t r a n s m i t t e d t o t h e n e w s y n thesized molecules. This result implies that, under certain specific circumstances, a k~nd of biological information can be transmitted directly by proteins without the intervention of n u c l e i c a c i d . H e n c e , t h e s t r u c t u r e o f a p r o t e i n is i n f o r mative by itself. Such proteins could be regarded as infectious organisms, replicating within a cells using, as d o v i r u s e s , t h e cells" o w n c o m p o n e n t s . M i s f o l d i n g c a n be considered as a "structural mutation" that propagates in t h e ceil. Are there evidences that prions are molecular chapcrones? First, molecular chaperones, as proteins, can be denatured, and prions lose their infectivity after denaturat i o n [10], i m p l y i n g t h a t t h e i r t e r t i a r y s t r u c t u r e is o f i m portance. Second, if preens are misfolded molecular chaperones, then they should exist in two differently f o l d e d f o r m s . L o p e z e t a l . [17] s h o w e d t h a t p r i o n s s y n thesized in vitro could adopt two topological forms as a consequence of alt:.'rnative folding. Thirdly, prion mol e c u l e s p r e s e n t a z o n e c o n t a i n i n g g i y c i n e r e p e a t s [1,18]. G l y c i n e is a v e r y m o b i l e a m i n o a c i d , w i t h a l l i t s c o n f o r m a t i o n s h a v i n g a b o u t t h e s a m e e n e r g y [12]. H e n c e t h e s e sequences have a low propensity to adopt spontaneously a definite secondary structure. Analysis according to t h e ( 3 i b r a t e t a l . [19] m e t h o d r e v e a l s t h a t a t h i r d o f t h e p r o t e i n is p o o r l y s t r u c t u r e d . T h e s e o b s e r v a t i o n s s u g g e s t a necessity of interaction with a folding partner to promote a define conformation.
1.56
o CL
o
I
|
0
1 O0
I
200
T~me ( ; t e r a t i o n s
300 x
401
10)
F i g . I. S i m u l a t i o n o f p r i o n d i s e a s e s a p p e a r a n c e u n d e r t h e t h r e e s p e c i fic c a s e s : ( A ) s p o r a d i c , ( B ) g e n e t i c aILd ( C ) i n f e c t i o u s . I n v a s i o n b / prions was simulated by analysing the behavior of each newly synt h e s i z e d m o l e c u l e w i t h a c o m p u t e r ( s e e t e xt ). T i m e is p r o p o r t i o n a l t o '.he n u m b e r o f i t e r a t i o n s . I n B, t h e v a l u e o f P, is f o u r - t i m e s t h e o n e u s e d in f i g u r e s A a n d C . i n C , m i s g u i d e d c h a p e r o n e s ( p r i o n s ) a r e a d d e d at time=600 iterations (arrow). For each case. simulation was repeated 5000 t i m e s a n d r e s u l t s w c r c c u m u l a t e d t o g i v e a k i n d o f " p r o b a b i l i t y c urve " t h a t g i v e s t h e p r o b a b i l i t y t o d e v e l o p t h e p r i o n d i s e a s e .
3. A M O D E L INVASION
FOR
TItE
SIMULATION
OF PRION
According to the deductions presented above, a model can be developed to simulate prion invasion. Consider the number uP, of prions (misfolded molecul a r c h a p e r o n e s ) a t t i m e t w h e n a n e w m o l e c u l e is s y n t h e sized. This number will depend on the number of prions a t t i m e t - i ( n P r - i ) . T h e r e is a p r o b a b i l i t y ( v e r y l o w b u t not zero) of spontaneous misfolding (if folding is not p e r f o r m e d o n t h e c h a p e r o n i n e ) t e r m e d P i ( i t is e a s y t o demonstrate that P, is directly proportional t o P'~ o f eqn. (1)). Otherwise, the folding will be performed on a molecular chaperone and the probability of misfoldi n s is ( n P , _ , / ( n N , - i ~ u P , - 1 ) ) , w h e r e n N r - i i s t h e n u m b e r of correctly folded molecules. The elimination of the prion, as a physiologic degradation of protein, could also be calculated. Simulation was performed with computer by iteration. Numerous simulations were performed in order to obtain a probability of appearance a s a f u n c t i o n o f t h e n u m b e r o f i t e r a t i o n s ( F i g . 1). T h e number of iterations is proportional to time. Fig. IA depicts the sporadic apparition of prions after a long lag period. Clearly, according to the model, the mutations observed in degenerative brain illnesses favor prion diseases by increasing Pi (see section 4 for a discussion of this point). The calculations correctly simulate this phen o m e n o n ( F i g . 1B). I n c r e a s i n g f o u r - t i m e s t h e p r o b a b i l i t y P~ r e s u l t s i n r e d u c t i o n o f t h e i n v a s i o n l a g p e r i o d o f about 0.33. Infection is ::quivalent to a direct increase in uP. The results of the simulation presented in Fig. IC
V o l u m e 294, n u m b e r 3
FEBS LE'I~ERS
model such an infection. After injection of nP (arrow) the invasiott appears very rapidly, without a lag period. C l e a r l y , s p e c i f i c i t y o f p r i o n ~_iseases ( i n f e c t i o u s , s p o radic, familial) are well simulated by the model. 4. A N A L Y S I S
OF PRION
DISEASES
W e s t a w a y et al. [!] su g g est s t h a t p r i o n d i s e a s e s m a y fall into three categories: infectious, familial and sporadic. The above hypothesis provides a rational unified e x p l a n a t i o n for all t h ree.
4.1. Infectious disease In order to explain the infection, one necessary condit i o n is t h a t p r i o n s a r e s t a b l e e n o u g h t o b e t a k e n i n t o t h e organism, essentially undamaged, to become a chaper o n e . T h e s t a b i l i t y o f p r i o n s f a v o r s t h i s h y p o t h e s i s [1]. All the experimental evidence for the infectious proper; ties of prions obtained to date has been from direct i n j e c t i o n o f p r o t e i n p r e p a r a t i o n [1,6]. T h e y v:ill b e u s e d as molecular' chaperones for the folding of ,tewly-synthesized prion proteins. The misfolding of the priori results in the propagation of the error, producing increasing numbers of misfolded proteins which, because of their singular stability, will not be degraded by intracellular IJroteases. However, in regenerating tissues, misfolded protein will be diluted out from dividing cells a n d e l i m i n a t e d b y cell d e a t h . C o n s e q u e n t l y , m i s f o l d e d molecular chaperonez will occur in large quantity only i n n o n - d i v i d i n g cells. T h i s o b s e r v a t i o n e x p l a i n s the brain localization of prion diseases. Furthermore, if the p r i o n m o l e c u l e is r e l a t e d t o a m o l e c u l e t h a t is i m p o r t a n t f o r t h e p h y s i o l o g y o f the cell, t h e r e m u s t b e a f e d b a c k regulatory mechanism. A decrease in the number of cotTectly-foid,.-xl c h a p e r o n e m o l e c u l e s m a y w e l l l e a d t o a n i n c r e a s e d s y n t h e s i s , g i v i n g r i s e t o still m o r e m i s f o l d e d m o l e c u l e s . T h e c o n v e r s e is a l s o t r u e , i.e. i n j e c t i o n of correctly folded molecular chaperone should partially reverse the effect of the prion. This otmns the way to treating these diseases. 4 . 2 . Sporadic apparition This concept of prion diseases has other implications. A misfolded protein that arises accidentally could initiate cell i n v a s i o n . S u c h a c c i d e n t a l m i s f o l d i n g s a r e m o r e likely with age, leading to increased probability of disease w i t h age in a h i t h e r t o h e a l t h y p e r s o n (see s i m u l a tion Fig. IA), as in human brain degenerative diseases. 4 . 3 . E x p la n a tio n
o f the genetic
transmission o f the
disease The accidental misfoiding could be due to a mutation increasing the probability of spontaneous misfolding a n d g i v i n g rise t o i n h e r i t e d d i s e a s e s w h e r e illness a p p e a r s m o r e f r e q u e n t l y i n y o u n g e r p e o p l e ( s e e F i g . ! B). Similarly, the recent onset of BSE could be due to genetic selection as well as direct infection.
December
1991
I n b r e d s t r a i n s o f m i c e in w h i c h s c r a p i e i n f e c t i o n s h a v e s h o r t ( N Z B ) a n d l o n g ( l / L n ) t i m e - c o u r s e s h a y : :t d i s t i n c t p r i o n g ~ n e a l l e l e [I,18]. T h e a m i n o a c i d a t c o d o n t 0 8 i n t h e p r i o n s e q u e n c e is l e u c i n e i n t h e N Z B strain and phenylalanine in the I/Ln strain. This change would decrease the probability of alpha-helix format i o n , l o w e r i n g P ~ , t w o - f o l d [19]. T h e h i g h l y c o n s e r v e d c o d o n a t p o s i t i o n 189 is c h a n g e d f r o m t h r e o n i n e in t h e N Z B m i c e t o v a l i n e ; n t h e l / L n s t r a i n , r e s u l t i n g in a t h r e e - f o l d i n c r e a s e i n t h e p r o b a b i l i t y o f b e t a - s h e e t fold i n g i n a r e g i o n w h i c h is o t h e r w i s e l i k e l y t o c o i l [I 9]. 5. C O N C L U S I O N Structural information can be transmitted by protein. This concept offers a good frame to explain etiology of brain degenerative diseases caused by vrions. Furtherm o r e it s u g g e s t s a t r e a t m e n t f o r t h e s e i l l n e s s e s . Acknowledgements: l that~k M. Cellier f o r m a n y helpful d i s c u s s i o n s o n h e a t - s h o c k p r o t e i n s a n d E. C a r o n f o r ~eneral d i s c u s s i o n a n d help with the manuscript.
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