Article in press - uncorrected proof BioMol Concepts, Vol. 1 (2010), pp. 305–322 • Copyright ᮊ by Walter de Gruyter • Berlin • New York. DOI 10.1515/BMC.2010.025 Review Propeptides as modulators of functional activity of proteases Ilya V. Demidyuk*, Andrey V. Shubin, Eugene protein sorting into specific cellular compartments or extra- V. Gasanov and Sergey V. Kostrov cellular space. Fourth, they can mediate the precursor inter- action with other molecules (such as peptides, proteins, and Institute of Molecular Genetics, Russian Academy of polysaccharides) or supramolecular structures (e.g., cell Sciences, Kurchatov Sq. 2, Moscow 123182, Russia walls). It should be noted that a single propeptide can * Corresponding author perform several or even all these functions. e-mail: [email protected] At the same time, a growing body of data demonstrates that propeptides can modulate protein functional activity irre- spective of their specific role or mechanism of action. They Abstract make it possible to substantially alter biological properties Most proteases are synthesized in the cell as precursor- of proteins without cardinal changes in the major functional containing propeptides. These structural elements can deter- (e.g., catalytic) domains of the molecules. This seems to be mine the folding of the cognate protein, function as an the key property of prosequences that allows propeptides to inhibitor/activator peptide, mediate enzyme sorting, and regulate protein activity at the post-translational level and to mediate the protease interaction with other molecules and function as specific evolutionary modules providing for supramolecular structures. The data presented in this review functional variation of protein molecules. demonstrate modulatory activity of propeptides irrespective The range of proteins synthesized as propeptide-contain- of the specific mechanism of action. Changes in propeptide ing precursors is very wide; it includes structural proteins, structure, sometimes minor, can crucially alter protein func- hormones, cytokines, various enzymes, and their inhibitors. tion in the living organism. Modulatory activity coupled with wA list of examples, although incomplete, can be found in high variation allows us to consider propeptides as specific Ref. (4).x Proteases are prominent among such proteins, as evolutionary modules that can transform biological proper- their synthesis as a proenzyme is typical of most represen- ties of proteases without significant changes in the highly tatives of this vast group (5). Thus, it is not surprising that conserved catalytic domains. As the considered properties of proteolytic enzymes considered in the current review have propeptides are not unique to proteases, propeptide-mediated become one of the main models to study the propeptide evolution seems to be a universal biological mechanism. functions and mechanisms of action. Keywords: folding; inhibition; protein interaction; protein precursor; sorting. Propeptides assisting protein folding Introduction The requirement of the propeptide for the active protein for- mation was originally demonstrated for subtilisin E (SbtE), On numerous occasions, proteins substantially change in the a secretory serine protease of Bacillus subtilis (6). Later, period from their synthesis to degradation. Often they are similar data were obtained for another bacterial secretory synthesized in the cell as precursors; later, these precursors enzyme of the same catalytic type, Lysobacter enzymogenes lose sequence fragments to form new species, each of which a-lytic proteinase (7, 8). To date, the involvement of pro- can have different physicochemical and biological properties. sequences in the folding has been demonstrated for a variety In some cases, the removed fragments direct their proteins of proteases of all major catalytic types and different organ- along a secretory pathway. Such fragments share a typical isms (9–40). At the same time, subtilisin (Sbt) and a-lytic structural organization and are called signal peptides protease (aLP) remain the most thoroughly developed mod- wreviewed in Ref. (1)x. Apart from signal peptides, there are els that contributed most to our understanding of propep- other removed fragments called propeptides, prosequences or tide-assisted folding and the underlying mechanisms. proregions. Prosequence-assisted folding of proteins, largely protea- To date, different functions of propeptides including four ses, has been reviewed previously (3, 4, 41–43), and here major functions are recognized. First, proregions can func- we will briefly consider its main aspects significant for tion as intramolecular chaperones (2) or folding assistants discussion. (3) by determining the three-dimensional structure of their Propeptide-assisted folding means that an unfolded protein protein. Second, they can function as inhibitors or activation without prosequence cannot form the proper biologically peptides by maintaining the proteins (commonly enzymes) active three-dimensional structure. This applies to both in that contain them inactive. Third, prosequences can direct vitro denatured mature proteins and proteins synthesized 2010/030 Article in press - uncorrected proof 306 I.V. Demidyuk et al. without propeptides in artificial expression systems. An proteolytic degradation (55, 60, 61), high temperature (62), active protein can be produced after the propeptide is added or low pH (63). to the unfolded protein in trans, i.e., they are not covalently The transition of protein folding from the thermodynam- bound (7, 14, 15, 31, 44–47). For the purpose of complete- ically controlled propeptide-independent pathway (Figure ness, even when the prosequence is a folding assistant, the 1C) to the kinetically controlled propeptide-mediated path- protein can fold under specific conditions without the pro- way is probably an important evolutionary mechanism. This peptide, although it is usually much less efficient (45, can be exemplified by bacterial subtilisin-like proteases (sub- 48–52). tilases) including proteins both with and without the propep- Direct transition of an unfolded protein (U) into the native tide. Intracellular and extracellular bacterial subtilases have catalytically active form (N) in the absence of the propeptide highly conserved primary structure (more than 50% identity), is thermodynamically forbidden as demonstrated for Sbt, similar three-dimensional organization of the catalytic aLP, and protease B of Streptomyces griseus. This is due to domains and closely resembling catalytic activities; however, a higher stability (lower free energy) of the unfolded the intracellular enzymes have no propeptides. A study of conformation than the native conformation in such proteins folding of two homologous proteins of B. subtilis, secretory (Figure 1) (53–55). In the absence of the propeptide, they SbtE and intracellular serine protease 1 (IPS1), has demon- transform into a partially folded stable intermediate (I) with strated substantially different folding pathways and kinetics. the conformation similar to that of a molten globule and SbtE folding requires the propeptide to form a kinetically lower free energy than N. In addition, the I is separated from stable molecule at a local energy minimum. IPS1 folding is the kinetically trapped N by a high-energy barrier (Figure more than a million times faster, does not depend on the 1A). After the propeptide (P) is added in trans, the I•P com- propeptide, and gives rise to a thermodynamically stable pro- plex is formed and the energy barrier is lowered, which tein (54). Thus, the propeptide makes possible cardinal allows the fast formation of the thermodynamically stable changes in the energy state of the active enzyme. This N•P complex. The metastable native state, protected from the requires minimum modifications in the catalytic domain that transition into the unfolded conformation by the same energy are largely limited to substitutions of individual surface barrier, is formed after the propeptide degradation, which is amino acids without affecting the hydrophobic core so that usually autocatalytic in active proteases (53, 55, 56). Thus, the catalytic activity is retained. Essentially, the propeptide the propeptide actually catalyzes the protein folding similar allows a single protein structure to form two principally dif- to an enzyme w‘foldase’ (57)x. ferent molecules: a high-stability molecule persists in aggres- The folding energy profile of the full-length precursor with sive extracellular environments (42, 61), whereas the other covalently bound mature and propeptide parts is similar to molecule is probably optimal for the intracellular protein the in trans folding described above (Figure 1B). In the case turnover (54). of Sbt and aLP, the unfolded precursor (Up) was shown to In addition, the kinetic stability make possible the mech- transform into the intermediate (Ip) analogous to the non- anisms of adaptation to harsh environmental conditions covalent I•P complex in the molten globule state. Then, the unavailable for thermodynamically stable proteins, as dem- Ip is folded into the thermodynamically stable propeptide- onstrated by comparative analysis of the structure and mature part complex (P-N), which enters the native state unfolding behavior of two homologous kinetically stable pro- after the propeptide is removed (54, 58). teins, acid-resistant Nocardiopsis alba protease A (NAPase) Thus, in all studied cases, the native state of proteases with and neutrophilic aLP. As the unfolded