FEBS Letters 425 (1998) 382^384 FEBS 20039 Minireview Enzymes as chaperones and chaperones as enzymes Chih-Chen Wang, Chen-Lu Tsou* National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, 15 Datun Road, Beijing 100101, People's Republic of China Received 9 February 1998 for a protein to be a molecular chaperone. In addition, for Abstract Chaperones and foldases are two groups of accessory proteins which assist maturation of nascent peptides into PDI to promote the joining of thiol groups distantly situated functional proteins in cells. Protein disulfide isomerase, a in the peptide sequence to form correct disul¢des the peptide foldase, and ATP-dependent proteases, responsible for degrada- chain has to be folded at least to some extent to bring the tion of misfolded proteins in cells, both have intrinsic chaperone thiol groups together. The spontaneous folding of the peptide activities. Trigger factor and DnaJ, well known Escherichia coli chain and the oxidation of thiol groups in vitro are often slow chaperones, show peptidyl prolyl isomerase and protein disulfide processes and the PDI-catalyzed folding does not need the isomerase activities respectively. It is suggested that the presence of other chaperones. We therefore put forward the combination of chaperone and enzyme activities in one molecule hypothesis that PDI is both an enzyme and a molecular chap- is the result of evolution to increase molecular efficiency. erone [5]. Many in vitro and in vivo data have now accumu- z 1998 Federation of European Biochemical Societies. lated to support the hypothesis that in addition to its isomer- Key words: Foldase; Chaperone; Protein disul¢de isomerase; ase activity in the catalysis of native disul¢de formation PDI Trigger factor; ATP-dependent protease; Protein folding does have intrinsic activity as a chaperone [6^13]. The folding of a peptide chain and the formation of its native disul¢de(s) are two intimately connected processes and work in cooperation. In order to distinguish explicitly 1. From spontaneous to assisted folding the possible chaperone activity of PDI from its isomerase activity in assisting protein folding, proteins with no disul¢de, It is now widely accepted that a large number of proteins D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [7] do not fold and assemble spontaneously in vivo to form the and rhodanese [8], were employed as target proteins. The biologically active molecules but require the help of other presence of PDI in the folding system at near stoichiometric proteins. This results in a conceptional transition for protein instead of catalytic amounts indeed increases greatly the re- folding: from the classical `self-assembly' to the new `assisted activation yield of both guanidine hydrochloride denatured assembly' principle [1]. Most in vitro experiments employed enzymes upon dilution, and suppresses their aggregation dur- conditions considerably di¡erent from the in vivo situation of ing refolding without being a part of the ¢nal functional struc- high protein concentrations with a high risk of aggregation [2] ture. In addition, PDI suppresses aggregation of rhodanese and relatively high temperature for warm-blooded animals. during thermal denaturation. The above properties are en- The evolution of higher organisms must create a mechanism tirely consistent with the de¢nition by Ellis of chaperones [1] to overcome problems resulting from the crowded protein and fully meet the four criteria proposed by Jakob and Buch- solution and the elevated temperature. ner [14] for characterization of a protein as a molecular chap- Two groups of proteins have been found to function as erone. accessory proteins for folding: molecular chaperones and The two thioredoxin-like -CGHC- sequences and the pep- foldases which catalyze the necessary covalent reactions tide segment 451^476 in the C-terminal region of PDI have directly involved in protein folding [3]. Only two foldases, been characterized to be the active sites for its isomerase ac- protein disul¢de isomerase (PDI) and peptidyl prolyl cis-trans tivity [15] and the peptide binding site [16] respectively. PDI isomerase (PPI), have so far been characterized as foldases. alkylated at the active site thiols shows little isomerase activity Based on the de¢nition of molecular chaperones as proteins but retains nearly full ability for increasing reactivation and assisting correct folding without covalent changes the foldases decreasing aggregation during refolding of denatured were therefore excluded as chaperones [1]. GAPDH as that of native PDI [17], suggesting that the chap- erone activity of PDI is independent of its -CGHC- active 2. Chaperone activity of protein disul¢de isomerase sites. It has also been found that mutant PDI with no isomer- ase activity is still in many in vivo cases functional for cell PDI, a multifunctional protein present in the endoplasmic viability [18], assisting folding of other proteins [11], and as- reticulum at high concentration [4], is remarkable in its ca- sembly of prolyl-4-hydroxylase [12] and microsomal triglycer- pacity of non-speci¢c peptide binding, an important feature ide transfer protein complex [13] as essential subunits. The mutant PDI, with its C-terminal 51 amino acid residues *Corresponding author. Fax: (86) (10) 6202-2026. responsible for peptide binding deleted, shows neither peptide E-mail: [email protected] binding ability nor chaperone activity in assisting the refold- ing of denatured GAPDH, but keeps most of its catalytic Abbreviations: PDI, protein disulfide isomerase; PPI, peptidyl prolyl activities [19]. The above provides a straightforward demon- cis-trans isomerase; GAPDH, D-glyceraldehyde-3-phosphate dehydro- genase stration that the peptide binding site is directly responsible for 0014-5793/98/$19.00 ß 1998 Federation of European Biochemical Societies. All rights reserved. PII S0014-5793(98)00272-5 FEBS 20039 3-4-98 C. Wang, C. Tsou/FEBS Letters 425 (1998) 382^384 383 its chaperone activity. In vitro, non-speci¢c peptides compete mutation in the protease active site of the overproduced Lon with the target proteins for binding to PDI to prevent the also promotes membrane protein assembly. The chaperone PDI-assisted folding and the suppression of the aggregation activity of proteases is important for proteolysis, as the pep- of target proteins, indicating the role of the peptide binding tide binding domains can interact with substrates and pro- site on its chaperone activity [17]. mote chaperone-like remodeling/refolding of proteins. For Both the chaperone and isomerase activities of PDI are Lon, unfolding is a necessary part of the proteolysis with necessary for its function as a foldase. For the maximal re- high e¤ciency. As a matter of fact, classical chaperones do folding and reactivation of denatured and reduced acidic participate in proteolysis [23]. Thus in vivo, the combined phospholipase A2, a protein composed of 124 amino acid action of chaperone and protease, situated either in di¡erent residues and seven disul¢de bonds, 90% of the native PDI chains or on the same chain, functions for quality control of in the refolding bu¡er can be replaced by the alkylated PDI proteins in removing misfolded chains by degradation. Struc- with only chaperone but no isomerase activity [17], indicating tural di¡erences of the complex formed might be responsible that the in vitro action of PDI as a foldase consists of both its for recognition either by proteases for degradation or by isomerase and chaperone activities [10]. The simultaneous chaperones for folding to its native form. presence of the C-terminal truncated mutant PDI with no peptide binding site [19] and the alkylated PDI with a peptide 4. Chaperones with enzymatic activities binding site but no isomerase activity [17] also shows a col- laborative action to promote the reactivation of acidic phos- The Escherichia coli chaperone, trigger factor, has recently pholipase A2 [19]. been shown to be a member of FK506-binding proteins of the It has been proposed that PDI, like other chaperones, rec- PPI family [24]. The trigger factor has a peptide binding site ognizes and binds with the non-native structure of unfolded well separated from the isomerase active site and responsible or partially folded intermediates of nascent chains or dena- for its high e¤ciency to promote protein folding with peptid- tured proteins during folding through its peptide binding yl-proline isomerization as the rate limiting step [25]. The site(s), thus prevents incorrect interactions and aggregation small PPIs catalyze peptidyl-proline isomerization in short of its substrate. Unlike most of the ATP-dependent chaper- unstructured peptides much better than in the folding of pro- ones, such as GroEL [20] and Hsp70 [21], but like some other teins, while the trigger factor is much more e¡ective in cata- chaperones, such as Hsp90 [22], the binding of PDI with its lyzing the folding of large proteins than all known small PPIs, substrates could be transient and the dissociation of the com- undoubtedly because in addition to the prolyl isomerization plex is ATP-independent. In such a way PDI prevents non- reaction, protein folding involves other complicated folding productive interactions of peptide chains leading to aggrega- events, which can be handled by the chaperone part of the tion and thus promotes their correct folding to a native-like trigger factor molecule. An excised central fragment from the conformation so as to bring the corresponding thiol groups trigger factor molecule corresponding to the FK506 binding into proximity to be joined up by oxidative formation of protein remains fully active as a prolyl isomerase with a short native disul¢des. This latter reaction is the generally recog- peptide as the substrate, but its activity is reduced about 1000- nized function of PDI. The two intrinsic activities of PDI, fold in assisting protein folding [25]. the chaperone and isomerase activities, are, in a way, inde- DnaJ in E. coli is well known as a chaperone involved in pendent of each other but function in collaboration, possibly protein folding and renaturation after stress.