Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Molecular Chaperone-like Properties of an Unfolded Protein, αs-Casein

Molecular Chaperone-like Properties of an Unfolded Protein, αs-Casein THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 22, Issue of May 28, pp. 15505–15509, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Molecular Chaperone-like Properties of an a -Casein* Unfolded Protein, (Received for publication, March 5, 1999) Jaya Bhattacharyya‡ and Kali P. Das§ From the Protein Chemistry Laboratory, Department of Chemistry, Bose Institute, Calcutta-700 009, India All molecular chaperones known to date are well or- chaperone function (9 –12). Although no common sequence has ganized, folded protein molecules whose three-dimen- been identified among chaperones of different family of pro- sional structure are believed to play a key role in the teins, some common features emerges. Chaperones have dis- mechanism of substrate recognition and subsequent as- tinct hydrophilic and hydrophobic domains to enhance solubil- sistance to folding. A common feature of all protein and ity and to bind lipophilic molecules (13–15). Many of them have nonprotein molecular chaperones is the propensity to a characteristic micelle-like-associated structure. GroEL exits form aggregates very similar to the micellar aggregates. as an associated 14-mer (7, 9). TriC has a ring-like structure of a -casein, abundant in mam- In this paper we show that 8 to 9 subunits (10). Tubulin, reported recently by us (16) to act malian milk, which has no well defined secondary and like a chaperone, associates to form microtubules. a-Crystallin, tertiary structure but exits in nature as a micellar ag- which belongs to small heat shock protein (sHSP) family (17), gregate, can prevent a variety of unrelated proteins/ has been proposed to have a micellar architecture (11, 12). enzymes against thermal-, chemical-, or light-induced Even nonprotein biological molecules such as ribosomal RNA aggregation. It also prevents aggregation of its natural (18, 19) and phospholipid (20), which can form micelle-type a -Casein interacts with substrates, the whey proteins. aggregates, were shown to function as molecular chaperones. partially unfolded proteins through its solvent-exposed It is long known that casein in bovine skim milk remains as hydrophobic surfaces. The absence of disulfide bridge large (40 –300 nm) stable micelles (21). Bovine milk contains or free thiol groups in its sequence plays important role about 78% casein, of which 65% is a-casein. More than 75% of in preventing thermal aggregation of whey proteins a-casein is Ca -sensitive and called a -casein (22). The rest is caused by thiol-disulfide interchange reactions. Our re- Ca -insensitive and is mainly k-casein. a -Casein has a mo- a -casein not only prevents the for- sults indicate that s lecular mass of 23.6 kDa (23). It is present in the milk of all mation of huge insoluble aggregates but it can also in- mammals as a random coil protein (24, 25) and is the major hibit accumulation of soluble aggregates of appreciable protein constituent of casein micelle (21). In the absence of size. Unlike other molecular chaperones, this protein can solubilize hydrophobically aggregated proteins. Ca ions, it is a highly soluble protein. Despite its unorga- This protein seems to have some characteristics of cold nized secondary and tertiary structure, there are similarities shock protein, and its chaperone-like activity increases between a -casein and other known chaperones in their tend- with decrease of temperature. ency to self-associate into micelle-like aggregate. This prompted us to test if a -casein possessed any chaperone-like behavior. In this paper we report for the first time that a From the time of synthesis through their entire lifetime in random coil protein a -casein can prevent in vitro the thermal the cell, proteins are under constant threat to structural desta- aggregation of whey proteins from milk as well as the aggre- bilization because of misfolding, stress, or other unfavorable gation of a variety of unrelated proteins/enzymes caused by interactions. Molecular chaperones recognize these unstable thermal-, chemical-, and light-induced stress. We also show nonnative conformers of proteins and bind to them instantly, that unlike other chaperones, a -casein can solubilize hydro- preventing their aggregation in the cell (1– 8). Molecular chap- phobically aggregated proteins and possesses some features erones comprise several structurally unrelated protein families similar to the cold shock proteins (CSP). and assist not only in folding of other proteins but also in EXPERIMENTAL PROCEDURES subcellular transport, oligomeric assembly, and degradation of Materials—a -Casein, bovine serum albumin (BSA), rhodanese, in- undesirable proteins (1–2, 4, 7– 8). It is not understood at sulin, carbonic anhydrase, alcohol dehydrogenase from equine lever, present whether the chaperone function of a protein is because and citrate synthase were purchased from Sigma. Whey protein isolate of the presence of a particular sequence or three-dimensional (WPI) was obtained from Le Sueur Isolates (Le Sueur, MN). Dithiothre- structure, although the role of higher levels of organization of itol (DTT) was purchased from Sisco Research Laboratory, India. Re- GroEL, TriC, a-crystallin, etc. have been emphasized for their agents used for SDS-polyacrylamide gel electrophoresis were from Fisher. All reagents used for making buffer solutions were of analytical grade. Preparation of b- and g-Crystallin—Freshly excised bovine eyes were * This work was supported by the Council of Scientific and Industrial obtained from a local slaughterhouse. The lenses were surgically re- Research CSIR grant No. 37 (0943)/97/EMR-II (to K. P. D.) and Depart- moved and homogenized in 10 mM Tris-HCl buffer, pH 8.0, containing ment of Biotechnology (DBT), Government of India. The costs of publi- 0.1 M NaCl and 0.02% (w/v) NaN . The homogenate was centrifuged at cation of this article were defrayed in part by the payment of page 3 15,000 3 g for 20 min at 4 °C. The supernatant was then loaded to a charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Sephacryl S-300 column (1.5 cm 3 90 cm). Five distinct peaks corre- ‡ Research associate under the Umbrella Program of the Department sponding to high molecular weight a (a -), low molecular weight a (a -), H L of Biotechnology. high molecular weight b (b -), low molecular weight b (b -), and g-crys- H L § To whom correspondence should be addressed: Dept. of Chemistry, Bose Institute, Main campus, 93/1 A.P.C. Rd. Calcutta-700 009, India. Tel.: 91 33 350 6619; Fax: 91 33 350 6790; E-mail: kalipada@boseinst. The abbreviations used are: CSP, cold shock proteins; BSA, bovine ernet.in. serum albumin; DTT, dithiothreitol; WPI, whey protein isolate. This paper is available on line at http://www.jbc.org 15505 This is an Open Access article under the CC BY license. 15506 Chaperone-like Properties of a -casein FIG.1. Prevention of thermal aggregation of proteins by a - casein. A, aggregation assay of alcohol dehydrogenase (0.4 mg/ml, 10 mM phosphate buffer, pH 7.0, 40 °C). Curve a, only alcohol dehydrogen- ase; curve b, plus 0.1 mg/ml a -casein. B, aggregation assay of b - s L crystallin (0.2 mg/ml, 10 mM phosphate, pH 7.0, 60 °C). Curve a, only b -crystallin; curve b, plus 0.03 mg/ml a -casein; and curve c, plus 0.06 L s mg/ml a -casein. C, aggregation assay of carbonic anhydrase (0.1 mg/ ml, 10 mM phosphate buffer, pH 7.0, 60 °C). Curve a, only carbonic anhydrase; curve b, plus 0.35 mg/ml a -casein. tallin were obtained. Pooled fractions were stored at 220 °C until use. Only b - and g-crystallin were used in the present study. Assay of Aggregation of Protein—Thermally induced aggregation of proteins was measured in a Shimadzu UV-2401PC spectrophotometer fitted with thermostatic cell holder assembly with electronic tempera- ture control. Protein solution and buffer with or without a -casein were FIG.2. Prevention of thermal aggregation of whey proteins by a -casein. A, aggregation assay of whey protein isolate (0.5 mg/ml, 10 mixed in the cuvette at room temperature and then placed in the mM phosphate buffer, pH 6.6, 70 °C). Curve 1, only whey protein isolate; thermostatic cell holder, and the apparent absorbance at 400 nm was curve 2, plus 0.4 mg/ml a -casein. B, aggregation assay of bovine serum monitored as a function of time. Aggregation of insulin was assayed as s albumin (0.5 mg/ml, 10 mM phosphate buffer, pH 6.6, temperature described by us earlier (26). Briefly, insulin was dissolved in a minimum 70 °C). Curve 1, only bovine serum albumin; curve 2, plus 0.5 mg/ml volume of 0.02 M NaOH and immediately diluted in 10 mM phosphate a -casein. buffer, pH 7.0. The aggregation of insulin B-chain was initiated by adding to insulin solution (0.35 mg/ml) DTT (from a 500 mM stock solution) to a final concentration of 20 mM. The extent of aggregation was followed by measuring apparent absorbance because of light scat- (1:0.6 molar ratio). At 1:3.5 weight ratio between carbonic tering at 400 nm. The ultraviolet light-induced aggregation of g-crys- anhydrase and a -casein, 90% protection was obtained (Fig. tallin (27, 28) was followed in a Hitachi F-4500 spectrofluorometer by 1C), and complete protection required approximately a 1:5 mo- setting both excitation and emission wavelengths at 295 nm with exci- lar ratio (data not shown). a -Casein was also effective in tation and emission slits of 10 and 5 mm, respectively. preventing thermal aggregation of a number of other proteins, Gel Filtration—The gel filtration of the samples was performed using including citrate synthase, g-crystallin, and rhodanese (data a prepacked column (1 3 30 cm) of Suparose 12 attached to a fast protein liquid chromatography system of Amersham Pharmacia Bio- not shown). tech. Protein samples with or without a -casein were first heated at the Milk contains a number of globular proteins such as b-lacto- given temperature and immediately put on ice to bring the temperature globulin, a-lactalbumin, BSA, etc., collectively called whey pro- to 25 °C. The solution was filtered through a 0.22-mm filter before 100 teins, which are highly sensitive to temperature, pH, and other ml of it was loaded onto the column equilibrated with 20 mM sodium conditions (22, 23, 29). WPI is a mixture of these proteins phosphate buffer of pH 7.0. Elution was carried out at a flow rate of 1.0 (about 20% of total milk proteins), which remain in the milk ml/min. Samples were monitored by their absorbance at 280 nm. serum after removal of casein (29). When 0.5 mg/ml WPI solu- RESULTS AND DISCUSSION tion in phosphate buffer, pH 6.6, is heated to 70 °C, the solution Prevention of thermal aggregation of substrate proteins by develops visible turbidity with time because of aggregation molecular chaperones is a commonly used method for in vitro (Fig. 2A, trace 1). The presence of 0.4 mg/ml a -casein com- assay of their activity. When solutions of substrate proteins pletely prevents this aggregation (Fig. 2A, trace 2). Similarly, such as alcohol dehydrogenase, carbonic anhydrase, and when 0.5 mg/ml BSA solution at pH 6.6 was heated at 70 °C, a b-crystallin are heated at 40, 60, and 60 °C, respectively, the low level of scattering was visible because of aggregated pro- solutions get turbid because of the formation of large aggre- teins (Fig. 2B, trace 1). The presence of 0.5 mg/ml completely gates. Fig. 1 shows the kinetic traces of the apparent absorb- prevents the formation of scattering BSA particles (Fig. 2B, ance at 400 nm of these proteins in the presence and absence of trace 2). Our results show that a -casein not only prevents the a -casein. In the absence of a -casein, the substrates at the aggregation of unrelated proteins but also protects its natural s s respective temperatures undergo denaturation followed by ag- substrates in vivo against thermal aggregation. Early work by gregation. However in the presence of a -casein, aggregation Morr and co-workers (30, 31) also clearly demonstrated that was suppressed. Approximately 92% protection was found at a whole casein prevented gross heat-induced aggregation of whey 1:1 (w/w) ratio of alcohol dehydrogenase:a -casein (Fig. 1A). proteins through nonspecific interaction, even in calcium-con- Complete protection occurred at a 1:1.5 (w/w) ratio of alcohol taining systems. dehydrogenase:a -casein, corresponding to a mole ratio of 1:4. Many investigators (32–38) have extensively studied the In the case of b-crystallin (Fig. 1B), complete suppression of thermal aggregation properties of whey proteins. It is well aggregation required a b-crystallin:casein weight ratio 1:0.3 known that the major whey proteins b-lactoglobulin, a-lactal- Chaperone-like Properties of a -casein 15507 FIG.4. Percentage protection of DTT-induced aggregation of insulin B-chain with a -casein. Insulin concentration was 0.25 FIG.3. Effect of a -casein on the aggregate size of heated mix- s mg/ml and a -casein concentration was 0.01 mg/ml at each tempera- tures of a-lactalbumin (2 mg/ml) and b-lactoglobulin (2 mg/ml) ture. The inset shows the aggregation of insulin and its complete pro- in 10 mM phosphate buffer, pH 7.0. Trace 1, only a-lactalbumin and tection by 0.1 mg/ml a -casein at 27C. b-lactoglobulin heated at 70 °C for 5 min; trace 2, plus a -casein, (4 mg/ml); trace 3, plus a -casein (6 mg/ml). Arrows on top show elution ence of sufficient quantity of a -casein formation of soluble peaks of the standards. aggregates of any appreciable size is prevented. bumin, and BSA have disulfide bridges, and the former two For nonthermal aggregation of substrate proteins, insulin have free thiol groups as well (21–23, 29). It was shown that became a popular choice for as the assay system, because thermal aggregation of whey protein was caused by a combi- reduction of disulfide bond by DTT leads to aggregation of its nation of hydrophobic as well as thiol-disulfide interchange B-chain at room temperature (16, 26, 40). Like other chaper- reactions (34 –38). a -Casein, being a highly hydrophobic pro- ones, a -casein also can prevent disulfide cleavage-induced ag- s s tein, interacts instantly with the exposed hydrophobic groups gregation of insulin at 27 °C, requiring a 1:0.35 weight ratio of denaturing proteins, preventing aggregation. However we between insulin and a -casein for complete prevention of ag- feel that the lack of disulfide bridges and free thiol groups in gregation (Fig. 4, inset). Using this assay system, we investi- a -casein sequence (39) is another very important and unique gated the effect of temperature on its chaperoning efficiency. feature that plays a significant role in inhibiting thiol-disulfide Fig. 4 shows the bar diagram of the percentage protection of interchange reactions. These covalent reactions require close insulin aggregation with a -casein (1:0.035 w/w ratio) at 37, 27, contact of appropriate residues, and a -casein creates a nonre- 22, and 18 °C. At 37, 27, and 22 °C, the suppression of aggre- active barrier by placing itself between the whey proteins. gation is 39, 52, and 90%, respectively, whereas there was Whey proteins under various conditions are also known to complete protection against insulin aggregation at 18 °C at the form soluble aggregates, which apparently cannot be detected same ratio. This finding was in sharp contrast to the behavior by the light-scattering technique we have employed as the of other known chaperones such as a-crystallin, tubulin, etc., aggregation assay method. To check if a -casein can prevent whose activity were generally found to increase with the in- formation of soluble aggregates of appreciable size, we have crease of temperature (16, 26, 27). Also unlike other chaperones employed a gel filtration assay using fast protein liquid chro- (16, 26, 27), preheating of a -casein solution to 50 °C for 30 min matography. Although a-lactalbumin does not aggregate on its and cooling back to 27 °C did not alter its chaperone efficiency. own on heating, it is known to form soluble aggregates in the a -Casein can also prevent other nonthermal aggregation of presence of b-lactoglobulin in the early stages of heat treat- proteins as well such as those induced by UV light. The eye lens ment (36, 37). A mixture of a-lactalbumin (2 mg/ml) and b-lac- protein g-crystallin in solution on being exposed to UV light toglobulin (2 mg/ml) at pH 7.0 was heated to 70 °C for 5 min (295 nm) becomes turbid because of aggregation (27, 28). Like and rapidly cooled to room temperature. The sample on gel the chaperone-like a-crystallin, a -casein also can prevent this filtration showed the presence of aggregated species of molec- aggregation (Fig. 5). Complete prevention requires a 1:2 weight ular mass in excess of 300 kDa eluting at the void volume, ratio between a-crystallin and a -casein, corresponding to a unreacted proteins corresponding to dimeric b-lactoglobulin molar ratio of ;1:0.7. (36.5 kDa), monomeric a-lactalbumin (14.4 kDa), and some It is known that molecular chaperones bind only aggrega- intermediate aggregates centered around molecular masses of tion-prone conformers of the substrate protein but do not in- approximately 100 –120 kDa (Fig. 3, trace 1). In the presence of teract with native proteins or proteins that have already ag- a -casein (4 mg/ml), a considerable reduction in the high mo- gregated (41). To test if a -casein acted similarly, we started an s s lecular mass species was observed, and most of the proteins insulin aggregation reaction by adding DTT to it, and when was eluted in a relatively single peak centered around 60 kDa. nearly 50% aggregation occurred, we added a -casein to the In presence of 6 mg/ml casein, no trace of any species of more reaction mixture. Our results show that a -casein not only than ;80 kDa was observed. This clearly shows that in pres- prevented further aggregation of insulin, but unlike other 15508 Chaperone-like Properties of a -casein FIG.5. Photo-aggregation of 0.2 mg/ml g-crystallin in absence FIG.6. Effect of addition of casein on partially aggregated (curve A) and presence (curve B) of 0.4 mg/ml a -casein. 295 nm s insulin in 10 mM phosphate buffer, pH 7.0. The kinetics of DTT- wavelength light from a spectrofluorometer was used to irradiate the induced aggregation of 0.35 mg/ml insulin was followed at 400 nm. 0.7 sample, and emission due light scattering at the same 295-nm wave- mg/ml a -casein was added at 2900 s, and kinetics were continued for length was measured to monitor aggregation. 12,000 s. known chaperones, it also slowly solubilized the already-aggre- cyclophilin was able to refold properly from the unfolded state gated insulin (Fig. 6). It has also been observed that GroEL can in the presence of a-casein (43). The discovery of RNA chaper- prevent aggregation of substrate proteins both on the unfolding ones is considered to be of vital importance because their early and refolding pathway (42, 43) (e.g. on dilution from 6 M gua- appearance in the evolution is believed to have made the tran- nidine hydrochloride solution). However, unlike GroEL but like sition from RNA to a protein/RNA world by rescuing the RNA a-crystallin (42), a -casein can effectively prevent aggregation from possible kinetic trap (49). It has recently been hypothe- in the unfolding pathway but fails to prevent aggregation com- sized that proteins in the early stages of evolution of life were pletely on the refolding pathway (data not shown). unfolded proteins, which through long evolutionary process We have thus identified a -casein, which exits in nature as s ultimately became folded (50). Our observation for the first an unfolded random coil protein, as having chaperone-like time that a commonly occurring unfolded protein a -casein can functions. Like all other known chaperones, it can prevent function as a molecular chaperone may be significant in under- irreversible aggregation of proteins induced by thermal as well standing this aspect. as nonthermal stress by providing hydrophobic surfaces to REFERENCES unfolding proteins. The important features responsible for its 1. Sigler, P. B., Xu, Z., Rye, H. S., Burston, S. G., Fenton, W. A., and Horwich, chaperone-like activity are (i) its high hydrophobicity charac- A. L. (1998) Annu. Rev. Biochem. 67, 581– 608 terized by Bigelow’s parameter of 1170 (44) along with high 2. Netzer, W. J., and Hartl, F. U. (1998) Trends Biochem. Sci. 23, 68 –73 3. Gething, M. J. (ed.) (1997) Guidebook to Molecular Chaperones and Protein- estimated net negative charge of about 22 at pH 6.5 (39), (ii) its Folding Catalysts, Sambrook and Tooze at Oxford University Press, Oxford highly flexible nature because of the relatively high amount 4. Das, K. P., Petrash, J. M., and Surewicz, W. K. (1996) J. Biol. Chem. 271, (8.5%) of proline residues distributed uniformly throughout the 10449 –10452 5. Frydman, J., and Hohfeld, J. (1997) Trends Biochem. Sci. 22, 87–92 chain (29, 39), (iii) its lack of a cystine residue in the sequence 6. Ruddon, R. W., and Bedows, E. (1997) J. Biol. Chem. 272, 3125–3128 (39). Interestingly a -casein possesses a few features similar to s 7. Hartl, F. U. (1996) Nature 381, 571–580 8. Ellis, R. J. (1996) Science 272, 1448 –1449 that of the CSP. Generally CSPs in bacteria have higher levels 9. Braig, K., Otwinowski, Z., Hedge, R., Biosvert, D. C., Joachimak, A., Horwich, of expression following a cold shock (45) and play a major A. L., and Sigler, P. B. (1994) Nature 371, 578 –585 regulatory role in the activation (46) and physiology of adapta- 10. Rommelaere, H., Van Troys, M., Gao, Y., Melki, R., Cowan, N. J., van der Kerckhove, J., and Ampe, C. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, tion to low temperature (45). Similarly greater amounts of 11975–11979 a -casein are found in mammals of the low temperature zones s 11. Smulders, R. H. P. H., van Boekel, M. A. M., and de Jong, W. W. (1998) Int. J. Biol. Macromol. 22, 187–196 such as reindeer (47), moose, yak, etc. compared with mammals 12. Walsh, M. T., Sen, A. C., and Chakrabarti, B. (1991) J. Biol. Chem. 266, living in other temperature zones (21). CSPs are known to be 20079 –20084 devoid of disulfide bonds in their sequence (48) and so is a - 13. Sharma, K. K., Kaur, H., Kumar, S. G., and Kester, K. (1998) J. Biol. Chem. 273, 8965– 8970 casein (39). These similarities between a -casein and CSPs of 14. Sharma, K. K., Kumar, S. G., Murphy, A. S., and Kester, K. (1998) J. Biol. prokaryotes might suggest that a -casein act as a CSP in s Chem. 273, 15474 –15478 15. Martin, J., Langer, T., Boteva, R., Schramel, A., Horwich, A. L., and Hartl, eukaryotes. F. U. (1991) Nature 352, 36–42 It is now recognized that molecular chaperones, all of which 16. Guha, S., Manna, T. K., Das, K. P., and Bhattacharyya, B. (1998) J. Biol. are known to date, are folded protein molecules and provide Chem. 273, 30077–30080 17. Merck, K. B., Groenen, P. J. T. A., Voorter, C. E. M., de Haard-Hoekman, guidance to newly synthesized peptides through the folding W. A., Horwitz, J., Bloemendal, H., and de Jong, W. W. (1993) J. Biol. Chem. process, and their presence is essential for cell survival (1–3, 268, 1046 –1052 18. Das, B., Chattopadhyay, Bera, A. K., and Dasgupta, C. (1996) Eur. J. Biochem. 5– 8). How the chaperones themselves get folded, however, 235, 613– 621 remains a mystery. It is too early to say if a -casein in vivo may 19. Kudlicki, W., Coffman, A., Kramer, G., and Hardesty, B. (1997) Folding Design help fold other proteins. It has very recently been shown that 2, 101–108 Chaperone-like Properties of a -casein 15509 20. Bogdanov, M., Sun, J., Kaback, H. R., and Dowhan, W. (1996) J. Biol. Chem. 36. Dalgleish, D. G., van Mourik L., and Corredig, M. (1997) J. Agric. Food Chem. 271, 11615–11618 45, 4806 – 4813 21. Waugh, D. F. (1971) Milk Proteins: Chemistry and Molecular Biology 37. Dalgleish, D. G., Senaratne, V., and Francois, S. (1997) J. Agric. Food Chem. (McKenzie, H. A., ed) Vol. 2, pp. 3–79, Academic Press, New York 45, 3459 –3464 22. Jenness, R. (1970) Milk Proteins: Chemistry and Molecular Biology (McKenzie, 38. Laligant, A., Dumay, E, Valencia, C. C., Cuq, J. L., and Cheftel, J. C. (1991) J. H. A., ed) Vol. 1, pp. 17– 40, Academic Press, New York Agric. Food Chem. 39, 2147–2155 23. Kinsella, J. E. (1984) CRC Crit. Rev. Food Sci. Nutr. 21, 197–262 39. Mercier, J. C., Grosclaude, F., and Dumas, B. R. (1971) Eur. J. Biochem. 23, 24. Susi, H., Timasheff, S. N., and Stevens, L. (1967) J. Biol. Chem. 242, 41–51 5460 –5466 40. Farahbakhsh, Z. T. Huang, Q. L., Ding, L. L., Altenbach, C., Steinhoff, H. J., 25. Creamer, L. K., Richardson, T., and Parry, D. A. D. (1981) Arch. Biochem. Horwitz, J., and Hubbell, W. L. (1995) Biochemistry 34, 509 –516 Biophys. 211, 689 – 696 41. Wang, K., and Spector, A. (1994) J. Biol. Chem. 269, 13601–13608 26. Das, K. P., and Surewicz, W. K. (1995) FEBS Lett. 369, 321–325 42. Das, K. P., and Surewicz, W. K. (1995) Biochem. J. 311, 367–370 27. Bhattacharyya, J., and Das, K. P. (1998) Biochem. Mol. Biol. Intl. 46, 249 –258 43. Wang, J. D., Michelitsch, M. D., and Weissman, J. S. (1998) Proc. Natl. Acad 28. Raman, B., and Rao, Ch. M. (1994) J. Biol. Chem. 269, 27264 –27268 Sci. U. S. A. 95, 12163–12168 29. Morr, C. V., and Ha, E. Y. W. (1993) Crit. Rev. Food Sci. Nutr. 33, 431– 476 44. Bigelow, C. C. (1967) J. Theor. Biol. 16, 187–211 30. Morr, C. V., and Josephson, R. V. (1968) J. Dairy Sci. 51, 1349 –1355 45. Graumann, P., and Marahiel, M. A. (1996) Arch. Microbiol. 166, 293–300 31. Kenkare, D. B., Morr, C. V., and Gould, I. A. (1964) J. Dairy Sci. 47, 947–952 46. Jones, P. G., and Inouye, M. (1994) Mol. Microbiol. 11, 811– 818 32. Ruegg, M., Moor, U., and Blanc, B. (1977) J Dairy Res. 44, 509 –515 47. McMeekin T. L., and Polis, B. D. (1949) Adv. Protein Chem. 5, 201–228 33. De Wit, J. N., Klarenbeck, G., and Hontelez-Backx, E. (1983) Neth. Milk Dairy 48. Reid, K. L., Rodriguez, H. M., Hiller, B. J., and Gregoret, L. M. (1998) Protein J. 37, 37– 43 Sci. 7, 470 – 479 34. Xiang, Y. L. (1992) J. Agric. Food Chem. 40, 380 –384 35. Gezimati, J. Singh, H., and Creamer, L. K. (1996) ACS Symp. Ser. 650, 49. Herschlag, D. (1995) J. Biol. Chem. 270, 20871–20874 113–123 50. Doi, N., and Yanagawa, H. (1998) FEBS Lett. 430, 150 –153 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Molecular Chaperone-like Properties of an Unfolded Protein, αs-Casein

Bhattacharyya, Jaya; Das, Kali P.
Journal of Biological Chemistry – May 1, 1999
Download PDF
5 pages

Loading next page...
 
/lp/unpaywall/molecular-chaperone-like-properties-of-an-unfolded-protein-s-casein-TWJUvepgR0

References

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
Unpaywall
ISSN
0021-9258
DOI
10.1074/jbc.274.22.15505
Publisher site
See Article on Publisher Site

Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 22, Issue of May 28, pp. 15505–15509, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Molecular Chaperone-like Properties of an a -Casein* Unfolded Protein, (Received for publication, March 5, 1999) Jaya Bhattacharyya‡ and Kali P. Das§ From the Protein Chemistry Laboratory, Department of Chemistry, Bose Institute, Calcutta-700 009, India All molecular chaperones known to date are well or- chaperone function (9 –12). Although no common sequence has ganized, folded protein molecules whose three-dimen- been identified among chaperones of different family of pro- sional structure are believed to play a key role in the teins, some common features emerges. Chaperones have dis- mechanism of substrate recognition and subsequent as- tinct hydrophilic and hydrophobic domains to enhance solubil- sistance to folding. A common feature of all protein and ity and to bind lipophilic molecules (13–15). Many of them have nonprotein molecular chaperones is the propensity to a characteristic micelle-like-associated structure. GroEL exits form aggregates very similar to the micellar aggregates. as an associated 14-mer (7, 9). TriC has a ring-like structure of a -casein, abundant in mam- In this paper we show that 8 to 9 subunits (10). Tubulin, reported recently by us (16) to act malian milk, which has no well defined secondary and like a chaperone, associates to form microtubules. a-Crystallin, tertiary structure but exits in nature as a micellar ag- which belongs to small heat shock protein (sHSP) family (17), gregate, can prevent a variety of unrelated proteins/ has been proposed to have a micellar architecture (11, 12). enzymes against thermal-, chemical-, or light-induced Even nonprotein biological molecules such as ribosomal RNA aggregation. It also prevents aggregation of its natural (18, 19) and phospholipid (20), which can form micelle-type a -Casein interacts with substrates, the whey proteins. aggregates, were shown to function as molecular chaperones. partially unfolded proteins through its solvent-exposed It is long known that casein in bovine skim milk remains as hydrophobic surfaces. The absence of disulfide bridge large (40 –300 nm) stable micelles (21). Bovine milk contains or free thiol groups in its sequence plays important role about 78% casein, of which 65% is a-casein. More than 75% of in preventing thermal aggregation of whey proteins a-casein is Ca -sensitive and called a -casein (22). The rest is caused by thiol-disulfide interchange reactions. Our re- Ca -insensitive and is mainly k-casein. a -Casein has a mo- a -casein not only prevents the for- sults indicate that s lecular mass of 23.6 kDa (23). It is present in the milk of all mation of huge insoluble aggregates but it can also in- mammals as a random coil protein (24, 25) and is the major hibit accumulation of soluble aggregates of appreciable protein constituent of casein micelle (21). In the absence of size. Unlike other molecular chaperones, this protein can solubilize hydrophobically aggregated proteins. Ca ions, it is a highly soluble protein. Despite its unorga- This protein seems to have some characteristics of cold nized secondary and tertiary structure, there are similarities shock protein, and its chaperone-like activity increases between a -casein and other known chaperones in their tend- with decrease of temperature. ency to self-associate into micelle-like aggregate. This prompted us to test if a -casein possessed any chaperone-like behavior. In this paper we report for the first time that a From the time of synthesis through their entire lifetime in random coil protein a -casein can prevent in vitro the thermal the cell, proteins are under constant threat to structural desta- aggregation of whey proteins from milk as well as the aggre- bilization because of misfolding, stress, or other unfavorable gation of a variety of unrelated proteins/enzymes caused by interactions. Molecular chaperones recognize these unstable thermal-, chemical-, and light-induced stress. We also show nonnative conformers of proteins and bind to them instantly, that unlike other chaperones, a -casein can solubilize hydro- preventing their aggregation in the cell (1– 8). Molecular chap- phobically aggregated proteins and possesses some features erones comprise several structurally unrelated protein families similar to the cold shock proteins (CSP). and assist not only in folding of other proteins but also in EXPERIMENTAL PROCEDURES subcellular transport, oligomeric assembly, and degradation of Materials—a -Casein, bovine serum albumin (BSA), rhodanese, in- undesirable proteins (1–2, 4, 7– 8). It is not understood at sulin, carbonic anhydrase, alcohol dehydrogenase from equine lever, present whether the chaperone function of a protein is because and citrate synthase were purchased from Sigma. Whey protein isolate of the presence of a particular sequence or three-dimensional (WPI) was obtained from Le Sueur Isolates (Le Sueur, MN). Dithiothre- structure, although the role of higher levels of organization of itol (DTT) was purchased from Sisco Research Laboratory, India. Re- GroEL, TriC, a-crystallin, etc. have been emphasized for their agents used for SDS-polyacrylamide gel electrophoresis were from Fisher. All reagents used for making buffer solutions were of analytical grade. Preparation of b- and g-Crystallin—Freshly excised bovine eyes were * This work was supported by the Council of Scientific and Industrial obtained from a local slaughterhouse. The lenses were surgically re- Research CSIR grant No. 37 (0943)/97/EMR-II (to K. P. D.) and Depart- moved and homogenized in 10 mM Tris-HCl buffer, pH 8.0, containing ment of Biotechnology (DBT), Government of India. The costs of publi- 0.1 M NaCl and 0.02% (w/v) NaN . The homogenate was centrifuged at cation of this article were defrayed in part by the payment of page 3 15,000 3 g for 20 min at 4 °C. The supernatant was then loaded to a charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Sephacryl S-300 column (1.5 cm 3 90 cm). Five distinct peaks corre- ‡ Research associate under the Umbrella Program of the Department sponding to high molecular weight a (a -), low molecular weight a (a -), H L of Biotechnology. high molecular weight b (b -), low molecular weight b (b -), and g-crys- H L § To whom correspondence should be addressed: Dept. of Chemistry, Bose Institute, Main campus, 93/1 A.P.C. Rd. Calcutta-700 009, India. Tel.: 91 33 350 6619; Fax: 91 33 350 6790; E-mail: kalipada@boseinst. The abbreviations used are: CSP, cold shock proteins; BSA, bovine ernet.in. serum albumin; DTT, dithiothreitol; WPI, whey protein isolate. This paper is available on line at http://www.jbc.org 15505 This is an Open Access article under the CC BY license. 15506 Chaperone-like Properties of a -casein FIG.1. Prevention of thermal aggregation of proteins by a - casein. A, aggregation assay of alcohol dehydrogenase (0.4 mg/ml, 10 mM phosphate buffer, pH 7.0, 40 °C). Curve a, only alcohol dehydrogen- ase; curve b, plus 0.1 mg/ml a -casein. B, aggregation assay of b - s L crystallin (0.2 mg/ml, 10 mM phosphate, pH 7.0, 60 °C). Curve a, only b -crystallin; curve b, plus 0.03 mg/ml a -casein; and curve c, plus 0.06 L s mg/ml a -casein. C, aggregation assay of carbonic anhydrase (0.1 mg/ ml, 10 mM phosphate buffer, pH 7.0, 60 °C). Curve a, only carbonic anhydrase; curve b, plus 0.35 mg/ml a -casein. tallin were obtained. Pooled fractions were stored at 220 °C until use. Only b - and g-crystallin were used in the present study. Assay of Aggregation of Protein—Thermally induced aggregation of proteins was measured in a Shimadzu UV-2401PC spectrophotometer fitted with thermostatic cell holder assembly with electronic tempera- ture control. Protein solution and buffer with or without a -casein were FIG.2. Prevention of thermal aggregation of whey proteins by a -casein. A, aggregation assay of whey protein isolate (0.5 mg/ml, 10 mixed in the cuvette at room temperature and then placed in the mM phosphate buffer, pH 6.6, 70 °C). Curve 1, only whey protein isolate; thermostatic cell holder, and the apparent absorbance at 400 nm was curve 2, plus 0.4 mg/ml a -casein. B, aggregation assay of bovine serum monitored as a function of time. Aggregation of insulin was assayed as s albumin (0.5 mg/ml, 10 mM phosphate buffer, pH 6.6, temperature described by us earlier (26). Briefly, insulin was dissolved in a minimum 70 °C). Curve 1, only bovine serum albumin; curve 2, plus 0.5 mg/ml volume of 0.02 M NaOH and immediately diluted in 10 mM phosphate a -casein. buffer, pH 7.0. The aggregation of insulin B-chain was initiated by adding to insulin solution (0.35 mg/ml) DTT (from a 500 mM stock solution) to a final concentration of 20 mM. The extent of aggregation was followed by measuring apparent absorbance because of light scat- (1:0.6 molar ratio). At 1:3.5 weight ratio between carbonic tering at 400 nm. The ultraviolet light-induced aggregation of g-crys- anhydrase and a -casein, 90% protection was obtained (Fig. tallin (27, 28) was followed in a Hitachi F-4500 spectrofluorometer by 1C), and complete protection required approximately a 1:5 mo- setting both excitation and emission wavelengths at 295 nm with exci- lar ratio (data not shown). a -Casein was also effective in tation and emission slits of 10 and 5 mm, respectively. preventing thermal aggregation of a number of other proteins, Gel Filtration—The gel filtration of the samples was performed using including citrate synthase, g-crystallin, and rhodanese (data a prepacked column (1 3 30 cm) of Suparose 12 attached to a fast protein liquid chromatography system of Amersham Pharmacia Bio- not shown). tech. Protein samples with or without a -casein were first heated at the Milk contains a number of globular proteins such as b-lacto- given temperature and immediately put on ice to bring the temperature globulin, a-lactalbumin, BSA, etc., collectively called whey pro- to 25 °C. The solution was filtered through a 0.22-mm filter before 100 teins, which are highly sensitive to temperature, pH, and other ml of it was loaded onto the column equilibrated with 20 mM sodium conditions (22, 23, 29). WPI is a mixture of these proteins phosphate buffer of pH 7.0. Elution was carried out at a flow rate of 1.0 (about 20% of total milk proteins), which remain in the milk ml/min. Samples were monitored by their absorbance at 280 nm. serum after removal of casein (29). When 0.5 mg/ml WPI solu- RESULTS AND DISCUSSION tion in phosphate buffer, pH 6.6, is heated to 70 °C, the solution Prevention of thermal aggregation of substrate proteins by develops visible turbidity with time because of aggregation molecular chaperones is a commonly used method for in vitro (Fig. 2A, trace 1). The presence of 0.4 mg/ml a -casein com- assay of their activity. When solutions of substrate proteins pletely prevents this aggregation (Fig. 2A, trace 2). Similarly, such as alcohol dehydrogenase, carbonic anhydrase, and when 0.5 mg/ml BSA solution at pH 6.6 was heated at 70 °C, a b-crystallin are heated at 40, 60, and 60 °C, respectively, the low level of scattering was visible because of aggregated pro- solutions get turbid because of the formation of large aggre- teins (Fig. 2B, trace 1). The presence of 0.5 mg/ml completely gates. Fig. 1 shows the kinetic traces of the apparent absorb- prevents the formation of scattering BSA particles (Fig. 2B, ance at 400 nm of these proteins in the presence and absence of trace 2). Our results show that a -casein not only prevents the a -casein. In the absence of a -casein, the substrates at the aggregation of unrelated proteins but also protects its natural s s respective temperatures undergo denaturation followed by ag- substrates in vivo against thermal aggregation. Early work by gregation. However in the presence of a -casein, aggregation Morr and co-workers (30, 31) also clearly demonstrated that was suppressed. Approximately 92% protection was found at a whole casein prevented gross heat-induced aggregation of whey 1:1 (w/w) ratio of alcohol dehydrogenase:a -casein (Fig. 1A). proteins through nonspecific interaction, even in calcium-con- Complete protection occurred at a 1:1.5 (w/w) ratio of alcohol taining systems. dehydrogenase:a -casein, corresponding to a mole ratio of 1:4. Many investigators (32–38) have extensively studied the In the case of b-crystallin (Fig. 1B), complete suppression of thermal aggregation properties of whey proteins. It is well aggregation required a b-crystallin:casein weight ratio 1:0.3 known that the major whey proteins b-lactoglobulin, a-lactal- Chaperone-like Properties of a -casein 15507 FIG.4. Percentage protection of DTT-induced aggregation of insulin B-chain with a -casein. Insulin concentration was 0.25 FIG.3. Effect of a -casein on the aggregate size of heated mix- s mg/ml and a -casein concentration was 0.01 mg/ml at each tempera- tures of a-lactalbumin (2 mg/ml) and b-lactoglobulin (2 mg/ml) ture. The inset shows the aggregation of insulin and its complete pro- in 10 mM phosphate buffer, pH 7.0. Trace 1, only a-lactalbumin and tection by 0.1 mg/ml a -casein at 27C. b-lactoglobulin heated at 70 °C for 5 min; trace 2, plus a -casein, (4 mg/ml); trace 3, plus a -casein (6 mg/ml). Arrows on top show elution ence of sufficient quantity of a -casein formation of soluble peaks of the standards. aggregates of any appreciable size is prevented. bumin, and BSA have disulfide bridges, and the former two For nonthermal aggregation of substrate proteins, insulin have free thiol groups as well (21–23, 29). It was shown that became a popular choice for as the assay system, because thermal aggregation of whey protein was caused by a combi- reduction of disulfide bond by DTT leads to aggregation of its nation of hydrophobic as well as thiol-disulfide interchange B-chain at room temperature (16, 26, 40). Like other chaper- reactions (34 –38). a -Casein, being a highly hydrophobic pro- ones, a -casein also can prevent disulfide cleavage-induced ag- s s tein, interacts instantly with the exposed hydrophobic groups gregation of insulin at 27 °C, requiring a 1:0.35 weight ratio of denaturing proteins, preventing aggregation. However we between insulin and a -casein for complete prevention of ag- feel that the lack of disulfide bridges and free thiol groups in gregation (Fig. 4, inset). Using this assay system, we investi- a -casein sequence (39) is another very important and unique gated the effect of temperature on its chaperoning efficiency. feature that plays a significant role in inhibiting thiol-disulfide Fig. 4 shows the bar diagram of the percentage protection of interchange reactions. These covalent reactions require close insulin aggregation with a -casein (1:0.035 w/w ratio) at 37, 27, contact of appropriate residues, and a -casein creates a nonre- 22, and 18 °C. At 37, 27, and 22 °C, the suppression of aggre- active barrier by placing itself between the whey proteins. gation is 39, 52, and 90%, respectively, whereas there was Whey proteins under various conditions are also known to complete protection against insulin aggregation at 18 °C at the form soluble aggregates, which apparently cannot be detected same ratio. This finding was in sharp contrast to the behavior by the light-scattering technique we have employed as the of other known chaperones such as a-crystallin, tubulin, etc., aggregation assay method. To check if a -casein can prevent whose activity were generally found to increase with the in- formation of soluble aggregates of appreciable size, we have crease of temperature (16, 26, 27). Also unlike other chaperones employed a gel filtration assay using fast protein liquid chro- (16, 26, 27), preheating of a -casein solution to 50 °C for 30 min matography. Although a-lactalbumin does not aggregate on its and cooling back to 27 °C did not alter its chaperone efficiency. own on heating, it is known to form soluble aggregates in the a -Casein can also prevent other nonthermal aggregation of presence of b-lactoglobulin in the early stages of heat treat- proteins as well such as those induced by UV light. The eye lens ment (36, 37). A mixture of a-lactalbumin (2 mg/ml) and b-lac- protein g-crystallin in solution on being exposed to UV light toglobulin (2 mg/ml) at pH 7.0 was heated to 70 °C for 5 min (295 nm) becomes turbid because of aggregation (27, 28). Like and rapidly cooled to room temperature. The sample on gel the chaperone-like a-crystallin, a -casein also can prevent this filtration showed the presence of aggregated species of molec- aggregation (Fig. 5). Complete prevention requires a 1:2 weight ular mass in excess of 300 kDa eluting at the void volume, ratio between a-crystallin and a -casein, corresponding to a unreacted proteins corresponding to dimeric b-lactoglobulin molar ratio of ;1:0.7. (36.5 kDa), monomeric a-lactalbumin (14.4 kDa), and some It is known that molecular chaperones bind only aggrega- intermediate aggregates centered around molecular masses of tion-prone conformers of the substrate protein but do not in- approximately 100 –120 kDa (Fig. 3, trace 1). In the presence of teract with native proteins or proteins that have already ag- a -casein (4 mg/ml), a considerable reduction in the high mo- gregated (41). To test if a -casein acted similarly, we started an s s lecular mass species was observed, and most of the proteins insulin aggregation reaction by adding DTT to it, and when was eluted in a relatively single peak centered around 60 kDa. nearly 50% aggregation occurred, we added a -casein to the In presence of 6 mg/ml casein, no trace of any species of more reaction mixture. Our results show that a -casein not only than ;80 kDa was observed. This clearly shows that in pres- prevented further aggregation of insulin, but unlike other 15508 Chaperone-like Properties of a -casein FIG.5. Photo-aggregation of 0.2 mg/ml g-crystallin in absence FIG.6. Effect of addition of casein on partially aggregated (curve A) and presence (curve B) of 0.4 mg/ml a -casein. 295 nm s insulin in 10 mM phosphate buffer, pH 7.0. The kinetics of DTT- wavelength light from a spectrofluorometer was used to irradiate the induced aggregation of 0.35 mg/ml insulin was followed at 400 nm. 0.7 sample, and emission due light scattering at the same 295-nm wave- mg/ml a -casein was added at 2900 s, and kinetics were continued for length was measured to monitor aggregation. 12,000 s. known chaperones, it also slowly solubilized the already-aggre- cyclophilin was able to refold properly from the unfolded state gated insulin (Fig. 6). It has also been observed that GroEL can in the presence of a-casein (43). The discovery of RNA chaper- prevent aggregation of substrate proteins both on the unfolding ones is considered to be of vital importance because their early and refolding pathway (42, 43) (e.g. on dilution from 6 M gua- appearance in the evolution is believed to have made the tran- nidine hydrochloride solution). However, unlike GroEL but like sition from RNA to a protein/RNA world by rescuing the RNA a-crystallin (42), a -casein can effectively prevent aggregation from possible kinetic trap (49). It has recently been hypothe- in the unfolding pathway but fails to prevent aggregation com- sized that proteins in the early stages of evolution of life were pletely on the refolding pathway (data not shown). unfolded proteins, which through long evolutionary process We have thus identified a -casein, which exits in nature as s ultimately became folded (50). Our observation for the first an unfolded random coil protein, as having chaperone-like time that a commonly occurring unfolded protein a -casein can functions. Like all other known chaperones, it can prevent function as a molecular chaperone may be significant in under- irreversible aggregation of proteins induced by thermal as well standing this aspect. as nonthermal stress by providing hydrophobic surfaces to REFERENCES unfolding proteins. The important features responsible for its 1. Sigler, P. B., Xu, Z., Rye, H. S., Burston, S. G., Fenton, W. A., and Horwich, chaperone-like activity are (i) its high hydrophobicity charac- A. L. (1998) Annu. Rev. Biochem. 67, 581– 608 terized by Bigelow’s parameter of 1170 (44) along with high 2. Netzer, W. J., and Hartl, F. U. (1998) Trends Biochem. Sci. 23, 68 –73 3. Gething, M. J. (ed.) (1997) Guidebook to Molecular Chaperones and Protein- estimated net negative charge of about 22 at pH 6.5 (39), (ii) its Folding Catalysts, Sambrook and Tooze at Oxford University Press, Oxford highly flexible nature because of the relatively high amount 4. Das, K. P., Petrash, J. M., and Surewicz, W. K. (1996) J. Biol. Chem. 271, (8.5%) of proline residues distributed uniformly throughout the 10449 –10452 5. Frydman, J., and Hohfeld, J. (1997) Trends Biochem. Sci. 22, 87–92 chain (29, 39), (iii) its lack of a cystine residue in the sequence 6. Ruddon, R. W., and Bedows, E. (1997) J. Biol. Chem. 272, 3125–3128 (39). Interestingly a -casein possesses a few features similar to s 7. Hartl, F. U. (1996) Nature 381, 571–580 8. Ellis, R. J. (1996) Science 272, 1448 –1449 that of the CSP. Generally CSPs in bacteria have higher levels 9. Braig, K., Otwinowski, Z., Hedge, R., Biosvert, D. C., Joachimak, A., Horwich, of expression following a cold shock (45) and play a major A. L., and Sigler, P. B. (1994) Nature 371, 578 –585 regulatory role in the activation (46) and physiology of adapta- 10. Rommelaere, H., Van Troys, M., Gao, Y., Melki, R., Cowan, N. J., van der Kerckhove, J., and Ampe, C. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, tion to low temperature (45). Similarly greater amounts of 11975–11979 a -casein are found in mammals of the low temperature zones s 11. Smulders, R. H. P. H., van Boekel, M. A. M., and de Jong, W. W. (1998) Int. J. Biol. Macromol. 22, 187–196 such as reindeer (47), moose, yak, etc. compared with mammals 12. Walsh, M. T., Sen, A. C., and Chakrabarti, B. (1991) J. Biol. Chem. 266, living in other temperature zones (21). CSPs are known to be 20079 –20084 devoid of disulfide bonds in their sequence (48) and so is a - 13. Sharma, K. K., Kaur, H., Kumar, S. G., and Kester, K. (1998) J. Biol. Chem. 273, 8965– 8970 casein (39). These similarities between a -casein and CSPs of 14. Sharma, K. K., Kumar, S. G., Murphy, A. S., and Kester, K. (1998) J. Biol. prokaryotes might suggest that a -casein act as a CSP in s Chem. 273, 15474 –15478 15. Martin, J., Langer, T., Boteva, R., Schramel, A., Horwich, A. L., and Hartl, eukaryotes. F. U. (1991) Nature 352, 36–42 It is now recognized that molecular chaperones, all of which 16. Guha, S., Manna, T. K., Das, K. P., and Bhattacharyya, B. (1998) J. Biol. are known to date, are folded protein molecules and provide Chem. 273, 30077–30080 17. Merck, K. B., Groenen, P. J. T. A., Voorter, C. E. M., de Haard-Hoekman, guidance to newly synthesized peptides through the folding W. A., Horwitz, J., Bloemendal, H., and de Jong, W. W. (1993) J. Biol. Chem. process, and their presence is essential for cell survival (1–3, 268, 1046 –1052 18. Das, B., Chattopadhyay, Bera, A. K., and Dasgupta, C. (1996) Eur. J. Biochem. 5– 8). How the chaperones themselves get folded, however, 235, 613– 621 remains a mystery. It is too early to say if a -casein in vivo may 19. Kudlicki, W., Coffman, A., Kramer, G., and Hardesty, B. (1997) Folding Design help fold other proteins. It has very recently been shown that 2, 101–108 Chaperone-like Properties of a -casein 15509 20. Bogdanov, M., Sun, J., Kaback, H. R., and Dowhan, W. (1996) J. Biol. Chem. 36. Dalgleish, D. G., van Mourik L., and Corredig, M. (1997) J. Agric. Food Chem. 271, 11615–11618 45, 4806 – 4813 21. Waugh, D. F. (1971) Milk Proteins: Chemistry and Molecular Biology 37. Dalgleish, D. G., Senaratne, V., and Francois, S. (1997) J. Agric. Food Chem. (McKenzie, H. A., ed) Vol. 2, pp. 3–79, Academic Press, New York 45, 3459 –3464 22. Jenness, R. (1970) Milk Proteins: Chemistry and Molecular Biology (McKenzie, 38. Laligant, A., Dumay, E, Valencia, C. C., Cuq, J. L., and Cheftel, J. C. (1991) J. H. A., ed) Vol. 1, pp. 17– 40, Academic Press, New York Agric. Food Chem. 39, 2147–2155 23. Kinsella, J. E. (1984) CRC Crit. Rev. Food Sci. Nutr. 21, 197–262 39. Mercier, J. C., Grosclaude, F., and Dumas, B. R. (1971) Eur. J. Biochem. 23, 24. Susi, H., Timasheff, S. N., and Stevens, L. (1967) J. Biol. Chem. 242, 41–51 5460 –5466 40. Farahbakhsh, Z. T. Huang, Q. L., Ding, L. L., Altenbach, C., Steinhoff, H. J., 25. Creamer, L. K., Richardson, T., and Parry, D. A. D. (1981) Arch. Biochem. Horwitz, J., and Hubbell, W. L. (1995) Biochemistry 34, 509 –516 Biophys. 211, 689 – 696 41. Wang, K., and Spector, A. (1994) J. Biol. Chem. 269, 13601–13608 26. Das, K. P., and Surewicz, W. K. (1995) FEBS Lett. 369, 321–325 42. Das, K. P., and Surewicz, W. K. (1995) Biochem. J. 311, 367–370 27. Bhattacharyya, J., and Das, K. P. (1998) Biochem. Mol. Biol. Intl. 46, 249 –258 43. Wang, J. D., Michelitsch, M. D., and Weissman, J. S. (1998) Proc. Natl. Acad 28. Raman, B., and Rao, Ch. M. (1994) J. Biol. Chem. 269, 27264 –27268 Sci. U. S. A. 95, 12163–12168 29. Morr, C. V., and Ha, E. Y. W. (1993) Crit. Rev. Food Sci. Nutr. 33, 431– 476 44. Bigelow, C. C. (1967) J. Theor. Biol. 16, 187–211 30. Morr, C. V., and Josephson, R. V. (1968) J. Dairy Sci. 51, 1349 –1355 45. Graumann, P., and Marahiel, M. A. (1996) Arch. Microbiol. 166, 293–300 31. Kenkare, D. B., Morr, C. V., and Gould, I. A. (1964) J. Dairy Sci. 47, 947–952 46. Jones, P. G., and Inouye, M. (1994) Mol. Microbiol. 11, 811– 818 32. Ruegg, M., Moor, U., and Blanc, B. (1977) J Dairy Res. 44, 509 –515 47. McMeekin T. L., and Polis, B. D. (1949) Adv. Protein Chem. 5, 201–228 33. De Wit, J. N., Klarenbeck, G., and Hontelez-Backx, E. (1983) Neth. Milk Dairy 48. Reid, K. L., Rodriguez, H. M., Hiller, B. J., and Gregoret, L. M. (1998) Protein J. 37, 37– 43 Sci. 7, 470 – 479 34. Xiang, Y. L. (1992) J. Agric. Food Chem. 40, 380 –384 35. Gezimati, J. Singh, H., and Creamer, L. K. (1996) ACS Symp. Ser. 650, 49. Herschlag, D. (1995) J. Biol. Chem. 270, 20871–20874 113–123 50. Doi, N., and Yanagawa, H. (1998) FEBS Lett. 430, 150 –153

Journal

Journal of Biological Chemistry – Unpaywall

Published: May 1, 1999

There are no references for this article.