Functions of Prolyl Isomerases

After their discovery in 1984 (Fischer et al, 1984), a huge number of prolyl isomerases have been found in all organisms and all subcellular compartments. It is clear that not all these proteins are involved in de novo protein folding. Rather, they are assumed to participate in many cellular functions. The following section discusses several of these functions. 

The periplasm of E. coli contains prolyl isomerases of all three families. PPIA is a member of the cyclophilin family (Liu and Walsh, 1990 Hayano et al., 1991), FkpA or FKBP26 (Horne and Young, 1995) contains a FKBP12-like domain, and SurA (Rouviere and Gross, 1996) and PpiD (Dartigalongue and Raina, 1998) show parvulin domains. SurA is a soluble protein, whereas PpiD is attached to the inner membrane with its catalytic domain exposed to the periplasmic space. 

Pliickthun and co-workers searched for proteins that improve folding of recombinant proteins in the periplasm by coexpressing a poorly folding single-chain Fv antibody fragment in a phagemid together with a library of/. , coli proteins. In this selection they identified Skp (Bothmann and Pliickthun, 1998) and FkpA (Bothmann and Pliickthun, 2000  

Ramm and Pliickthun, 2000). Overexpression of FkpA strongly improved the functional expression of several Fv fragments, even of ones without cis prolines. Although FkpA shows a high prolyl isomerase activity toward proteins in vitro, this suggests that the improvement of protein folding in the periplasm by FkpA does not necessarily require its prolyl isomerase activity (Bothmann and Pliickthun, 2000 Ramm and Pliickthun, 2000). 

Behrens and colleagues (2001) investigated SurA both in vivo and in vitro. All regions of this large protein contribute to its function in vivo. Prolyl isomerase activity could be demonstrated for one of the two parvulin domains, but, as in the case of FkpA, this activity seems to be unimportant for SurA function in vivo. SurA has a chaperone function in vitro, which requires the N-terminal part of the protein to be present. 

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The amino acid sequences of the cyclophilins remained highly conserved during evolution. This holds in particular for the proteins from eukaryotes. The cyclophilins from bovine thymus and from porcine kidney are identical in sequence (Takahashi et al., 1989), and the human and the bovine cyclophilins share 98% identical amino acids (Haendler et al., 1987). The homology between the mammalian cyclophilins and the cytosolic PPl from E. coli is about 25% (Hayano et al., 1991). The PPIs from porcine kidney and E. coli cytoplasm were used in most of the work on the function of prolyl isomerases as catalysts of protein folding that will be discussed herein. 

Most of the small proteins that were used initially as substrates to test the function of prolyl isomerases contained disulfide bonds, which were left intact during unfolding and refolding. These proteins were used because their unfolding is reversible under a wide variety of conditions and because good evidence existed for a number of them that prolyl isomerizations were involved as rate-limiting steps in their slow-folding reactions. A protein chain without disulfides should be a better model... 

In this section RNase A and RNase T1 are used as examples to illustrate the role of prolyl isomerizations for the unfolding and refolding of small single-domain proteins. Bovine pancreatic RNase A is selected because the history of the proline hypothesis and its experimental verification are closely related with this protein. The mechanism of RNase T1 folding is described because it is one of the major in vitro systems for investigating the function of prolyl isomerases as catalysts of proline-limited protein folding. 

FKBP12 is a member of immunophilin family that has prolyl isomerase activity and is related to the cyclophi-lins in function. FKBP12 binds immunosuppressant molecule FK506 (tacrolimus). The FBKP-FK506 complex inhibits calcineurin, a protein phosphatase, thus blocking signal transduction in the T-lymphocyte... 

Lu PJ, Wulf G, Zhou XZ, Davies P, Lu KP. The prolyl isomerase Pinl restores the functions of Alzheimer-associated phosphorylated tau protein [see comments]. Nature 1999 399 784-788. 

In addition to its role as the P-subunit of PHY, PDI acts independently by catalysing thiol/protein disulphide interchange. The role of PDI as the P-subunit in prolyl 4-hydroxylase is not related to its disulphide isomerase activity and experiments where the vertebrate PDI was mutated in both thioredoxin-like active domains had no effect on tetramer assembly (Vuori et al., 1992). PDI appears to function as a molecular chaperone, retaining the a-subunits in the correct catalytically active, non-aggregated form in the ER-lumen (John et al, 1993). Dissociation of the P-subunits results in insoluble aggregates of the a-subunits, analogous to a-subunits expressed in the absence of PDI. An additional function of PDI in the complex is to maintain the ER luminal location of the a-subunits, since deletion of the ER retention signal from PDI results in the secretion of the complex (Vuori et al., 1992). 

Loss of function of the prolyl isomerase Pinl by homologous recombination has been reported to lead to the formation of abundant filaments made of hyperphosphorylated mouse tau and neurodegeneration [39]. 

The potential utility of peptides as therapeutics with clinical applications is limited by its metabolic instability or poor transmembrane mobility. Consequently, the preparation of metabolically stable peptide analogs that can either mimic or block the function of natural peptides or enzymes is an important area of medicinal chemistry research. Synthesis of fluoroolefin amide isosteres, its incorporation in peptidomimetics, and the influence of that isosteric substitution on the inhibition of several enzymes such as peptidyl prolyl isomerases, dipeptidyl peptidase IV, and thermolysin is described. Moreover, protein folding and activity... 

What does tau do normally Although it has been studied for many years, its exact functions are elusive. However, the role of the microtubules in axonal transport is well established. The tau isoforms may play a functional role in this process. The hyperphosphorylated tau of Alzheimer disease doesn t promote proper assembly of microtubules and may interfere with axonal transport of materials along the microtubules (see p. 1119).1214 1215 Alzheimer disease may reflect an imbalance between the phosphorylation and dephosphorylation processes. Another possible problem with tau may be slow isomerization of prolyl linkages because of a deficiency of a prolyl cis-trans isomerase (Box 9-F).1216... 

The above definition of molecular chaperone is entirely fnnctional and contains no constraints on the mechanisms by which different chaperones may act. The term noncovalent is nsed to exclude those proteins that carry out posttranslational covalent modifications. Protein disulfide isomerise may seem to be an exception, bnt it is both a covalent modification enzyme and a molecular chaperone. It is helpful to think of a molecnlar chaperone as a fnnction rather than as a molecnle. Thns, no reason exists why a chaperone function shonld not be a property of the same molecnle that has other fnnctions. Other examples include peptidyl-prolyl isomerase, which possesses both enzymatic and chaperone activities in different regions of the molecnle, and the alpha-crystallins, which combine two essential fnnctions in the same molecnle in the lens of the eye-contribnting to the transparency and the refractive index reqnired for vision as well... 

Beyond protein folding, the discovery of peptidyl prolyl isomerases (PPIases) and related proteins has opened the way to novel concepts in biology the notion of chaperone-assisted receptor binding is an emerging field of research which sheds light on receptor function and protein-protein interactions. The recent discovery of a secondary amide peptide bond cis-trans isomerase (APIase) heralds new advances in this field. 

In 1995, Fischer and co-workers (Stoller et al., 1995) discovered that trigger factor is a ribosome-associated prolyl isomerase. The enzymatic activity originates from a central FKBP domain, which encompasses residues 142—251 (Callebaut and Momon, 1995 Hesterkamp and Bukau, 1996 Stoller et al., 1996). When protein translation is arrested, nascent protein chains can be crosslinked to the trigger factor at the ribosome (Valent etal., 1995 Hesterkamp et al., 1996). The N-terminal domain of the trigger factor (residues 1—118) mediates the interaction with the ribosome (Hesterkamp et al., 1997) the function of the C-terminal domain is unknown. 

In the future we need to elucidate which of the many interactions between prolyl isomerases and target proteins are functionally important in the cell and how prolyl isomerase domains of large proteins contribute to the overall functions of these proteins. At the molecular level the kinetic and structural consequences of native-state prolyl isomerization must be elucidated at high resolution to understand how these isomerizations can contribute to cellular regulation, and how they are modulated by prolyl isomerases and by other processes, such as phosphorylation. 

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