Cystine knot growth factors and their functionally versatile proregions
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Elisabeth Schwarz
Abstract
The cystine knot disulfide pattern has been found to be widespread in nature, since it has been detected in proteins from plants, marine snails, spiders and mammals. Cystine knot proteins are secreted proteins. Their functions range from defense mechanisms as toxins, e.g. ion channel or enzyme inhibitors, to hormones, blood factors and growth factors. Cystine knot proteins can be divided into two superordinate groups. (i) The cystine knot peptides, also referred to – with other non-cystine knot proteins – as knottins, with linear and cyclic polypeptide chains. (ii) The cystine knot growth factor family, which is in the focus of this article. The disulfide ring structure of the cystine knot peptides is made up by the half-cystines 1-4 and 2-5, and the threading disulfide bond is formed by the half-cystines, 3-6. In the growth factor group, the disulfides of half-cystines 1 and 4 pass the ring structure formed by the half-cystines 2-5 and 3-6. In this review, special emphasis will be devoted to the growth factor cystine knot proteins and their proregions. The latter have shifted into the focus of scientific interest as their important biological roles are just to be unravelled.
Introduction: Common features of cystine knot proteins
Several prominent and general characteristics of cystine knot proteins and their proregions are listed below while more detailed information with references will be presented in the chapters thereafter.
Size
Cystine knot proteins are mostly small. Even in large multidomain proteins, the cystine knot domain hardly exceeds 120 residues. In case of the small knottin proteins, often as few as 30 amino acids provide the structure for the cystine knot ring structure, which is remarkable given that the six cysteines make up 20% of the overall amino acid contributions. Cystine knot domains of growth factors consist typically of ca. 120 residues. From those proteins that have been characterised with regard to their intrinsic stability, it has become apparent that the cystine knot stabilises the structure of the protein.
Secondary structure
The smallest denominator for secondary structural contributions of the cystine knot domains is their prevailing β-structure.
Quaternary structure
Growth factor cystine knot proteins associate to homo- or heterodimers with van Willebrand factor (vWF) that forms large multimers being an exception. Many growth factor cystine knot proteins are stabilised by intermolecular disulfide bonds. In contrast, proteins belonging to the knottin group are monomeric. The cyclotides contain two ring structures, the peptide backbone and the cystine knot.
Secretion
Since oxidation to disulfides occurs only in secretory compartments or in the extracellular space, cystine knot proteins exist in their native, i.e. oxidised, form upon being released into a secretory compartment or the extracellular space. Within the cells, the proteins are synthesised as precursor forms that contain secretion signals besides proregions (see below).
Proregions
The presence of proregions (in the literature also referred to as propeptides or prodomains) is a general hallmark of growth factor cystine knot proteins. In contrast to the signal sequences that ensure secretion, the roles of the proregions are versatile and differ depending on the individual protein. Knowledge of their functions, biophysical properties and biochemical or structural characteristics of the proregions will be summarized in this review.
Cystine knot peptides
Within the cystine knot peptide group, the threading disulfide bond is made up of the half-cystines 3 and 6; while in the cystine knot growth factors, the threading bond is based on the half-cystines 1 and 4 (Figure 1) (for review see Isaacs, 1995). Cystine knot peptides or small cysteine-rich proteins display many diverse bioactivities. The peptides have been identified from animal and plant kingdoms (Reinwarth et al., 2012). This group of cystine knot proteins is structurally subdivided into the linear inhibitor cystine knot peptides (ICK) and cyclic cystine knot peptides (CCK) or cyclotides. One of the plant prototype cyclotides, kalata B1, was originally discovered because of its labour-inducing effects in pregnant women (Gran, 1973). NMR studies proved the circular structure and the disulfide connectivities of this small protein (Rosengren et al., 2003). The cyclotides have been demonstrated to be extremely stable. This property, their small size and their distinct bioactivities predestine them as scaffold structures for biotechnological application. The cyclotides are synthesised as precursor proteins with a signal peptide and an N-terminal proregion of ca. 20–25 residues. The cystine knot domain of kalata B1 is flanked by both an N- and a C-terminal segment. The N-terminal segment, ntr, has been found to target the protein to the vacuole and to be intrinsically unstructured (Conlan et al., 2011; Daly et al., 2016).
Scheme of the disulfide connectivities of small cystine knot peptides (A) and cystine knot growth factors (B).
Numbers indicate the half-cystines engaged in the disulfide bonds. Disulfide bonds of the ring are shown in yellow and the disulfide bridge passing the ring is shown in orange.
Plant ICKs have been shown to represent efficient enzyme blockers. For representative ICKs, the oxidative folding pathway has been studied by a series of biophysical or biochemical techniques such as time-resolved NMR, mutagenesis, RP-HPLC and mass spectrometry (Wentzel et al., 1999; Cemazar et al., 2004; Reinwarth et al., 2013). The plant ICK, EETI, binds to integrins and is discussed as a candidate structure for therapeutic use (Kim et al., 2015). Animal ICKs can act as potent venoms. For example, marine snails of the genus Conus are predators and disable their prey via toxin cocktails, the conotoxins, consisting of several ICKs (for review see Robinson and Norton, 2014). The short peptides belonging to this functional group bind with high affinities to neurotransmitter receptors or ion channels, leading to paralysis of prey animals. Proteins of the group of ω-conotoxins have been extensively characterised with regard to structure and folding properties (Price-Carter et al., 1996a,b). In particular, the roles of the short and conserved proregions have been studied. These segments comprise ca. 25 residues and are more conserved than the mature parts. The original assumption that the proregions would stimulate in vitro folding could not be substantiated (Price-Carter et al., 1996b). Thus, it remains unclear which biological mechanism led to the preservation of the short proregions.
Mammalian blood and growth factor cystine knot proteins
A bioinformatics investigation that searched more than 100 000 open reading frames in five model organisms (human, fly, nematode, slime mold, yeast) for potential cystine knot signatures gave rise to the compilation of phylogenetic trees of known and potential cystine knot proteins (Vitt et al., 2001). Despite the fact that the cystine knot signature cannot be detected in unicellular organisms, the analysis confirmed that the disulfide pattern is a prominent motif of many extracellular proteins. Alone in man, 134 potential cystine knot proteins were identified in silico. Most proteins possess a cystine knot ring composed of eight residues according to the assignments of Sun and Davies (1995). The largest group of eight-membered cystine knot proteins is the TGF-β/BMP structure family (see below). The other eight-membered groups are represented by the glycoprotein hormone (GH)-, PDGF-like-, BMP-antagonist-, slit-like- and the jagged-like families. Several prototypes of the mammalian cystine knot growth factors will be described with a focus on the roles of the proregions and structure formation processes of these proteins.
von Willebrand factor (vWF)
vWF is a multimeric blood glycoprotein that ensures sealing of injured blood vessels by recruiting platelets and stabilising clotting factor VIII (for review see Sadler, 1998). vWF folds in the endoplasmic reticulum as a 2813 residue multidomain pro-protein with a high percentage of cysteines (8.3%). In total, 15 domains make up the pro-protein. The cystine knot domain is the most C-terminal domain. Dimerisation of the cystine knot domain occurs via protein disulfide isomerase-mediated formation of three disulfide bridges (Katsumi et al., 2000; Zhou and Springer, 2014; Lippok et al., 2016). The 741-residue comprising proregion consists of two N-terminal domains that are cleaved by the prohormone convertase, furin. The proregion domains jointly act as an oxido-reductase and ensure multimerisation of mature vWF by enabling interdimer disulfide bond formation (Wise et al., 1988; Voorberg et al., 1990; Brehm, 2017).
Platelet-derived growth factors (PDGFs)
PDGFs and the vascular endothelial growth factors (VEGFs) have been found to be evolutionarily related. The members of the PDGF group (PDGF-A, -B, -C and -D) act as mitogens and play important roles in wound healing. The mature monomers associate in an antiparallel manner, the dimeric form is stabilised by two intermolecular disulfide bridges (Shim et al., 2010). Besides the homodimeric form, PDGF-A and -B have also been detected as the heterodimeric protein, PDGF-AB. PDGF-A and PDGF-B are produced with N-terminal proregions of 66 and 61 residues, respectively. A blast query indicated that the proregions share, upon introduction of a single gap, 38% identical amino acids. It is assumed that the proregions play a role in cellular folding of the growth factors (Shim et al., 2010). The crystal structure of PDGF-A in complex with its non-covalently associated proregions revealed that the proregions flank the mature parts on opposite sites, in particular, at the clamps formed by the two associated monomers (Figure 2). The proregions of PDGF-C and -D have been recognised as CUB domains (Clr/Cls, urchin endothelial growth factor, bone morphogenetic protein) that are separated from the cystine knot domains by so-called ‘hinge regions’ of ca. 70 and 90 residues for PDGF-C and PDGF-D, respectively (Fredriksson et al., 2005; Reigstad et al., 2005). Currently, no functional analysis on the hinge regions is available. The CUB domains may render PDGF-C and -D latent, and in the case of PDGF-D removal of the CUB domain by matriptase led to increased association with the extracellular matrix (Huang and Kim, 2015).
Models of pro-form structures.
The mature parts of TGF-β1, PDGF-A and BMP9 are shown in lighter colours than the structures of the proregions. In case of proNGF, the grey and blue shaded areas indicate the position of the proregions in either the crab-like or extended conformation. Except for proNGF, the non-covalent complexes were analysed. Used pdb file codes 1bet (NGF), 3rjr (proTGF-β1), 4yci (proBMP9) and 3mjk (proPDGF-A).
Vascular endothelial growth factors (VEGFs)
VEGFs promote angiogenesis and lymphangiogenesis (Achen and Stacker, 1998). As the growth factors lead to blood vessel formation in tumours, they present important targets for cancer therapy. In addition, the VEGFs are diagnostic tools for tumour prognosis. The four members of the VEGF family, VEGF-A, -B, -C and -D, form anti-parallel homodimers. The structure of VEGF-A bound to the VEGF-receptor extracellular domain has recently been elucidated (Markovic-Mueller et al., 2017). In case of VEGF-C and -D, the central cystine knot domain, also referred to as VHD (VEGF homology domain), is flanked by N- and C-terminal proregions. VEGF-D can be recombinantly expressed without proregions (Harris et al., 2013). This observation indicates that the role of the proregions in the folding of the VEGF proteins may be dispensible. Mutational studies with VEGF-D variants lacking either the N- or C-terminal proregion demonstrated that the proregions determine receptor heterodimerisation (Harris et al., 2013). The mutational analysis also revealed that the C-terminal proregion binds to heparin and that removal of this domain could be rate-limiting for the angiogenic and tumourigenic properties of VEGF. In vitro refolding studies of mutant forms of mature VEGF indicated that the cystine knot is necessary for structure formation rather than for the thermodynamic stability of the protein (Muller et al., 2002).
Glycoprotein hormones (GHs)
Glycoprotein hormones are heterodimeric cystine knot proteins with a constant α-subunit. The associated individual β-subunit determines the physiological identity of the hormones, chorionic gonadotropin, luteinising hormone, follicle stimulating hormone and thyroid-stimulating hormone (for review see Jiang et al. 2014). The monomers of the GHs are associated in a parallel manner as has been found typical for the neurotrophins (NTs) (see below). Yet, in contrast to the NTs, the translation products of α- and β-chains lack proregions. Intracellular folding of the α-subunit has been demonstrated to depend on the sequential formation of disulfide bonds including non-cystine knot disulfide bridges (Darling et al., 2001). A similar architecture with regard to the quaternary scaffold has been detected for the pro-inflammtory cytokines, the interleukin 17s (IL17s). The crystal structures of these proteins revealed a cystine knot-like structure with prevailing β-contributions. However, IL17s lack the cysteines that would build the cystine knot, thus compensatory covalent and non-covalent interactions in these proteins can obviously make up for the absence of the cystine knot (Hymowitz et al., 2001; Gerhardt et al., 2009).
Neurotrophins (NTs)
Biology
NTs are growth factors that elicit trophic responses and differentiation of neuronal tissue besides inducing differentiation into nerve cells and maintaining their vitality. Yet, especially in their pro-forms, the NTs can cause apoptotic processes (for review see Bothwell, 2016). Four mammalian NTs share the cystine knot motif: nerve growth factor (NGF), neurotrophin-3 (NT3), neurotrophin-4/5 (NT4/5) and brain-derived neurotrophic factor (BDNF).
Structure and structure formation
Murine NGF was the first neurotrophin whose structure has been elucidated (McDonald et al., 1991). As the other NTs, NGF has a homo-dimeric quaternary structure with the two monomers in a head to head association. A large hydrophobic interface of 2332 Å2 stabilises the non-covalent dimeric form. This quaternary structure is also present in the other NTs, NT-3, NT 4/5 and BDNF (Butte et al., 1998; Robinson et al., 1999). Structural comparisons revealed that heterodimerisation of the NTs can occur. Equilibrium re/unfolding studies of mouse NGF with denaturants were consistent with a two state model, i.e. a folding process lacking intermediates (Timm and Neet, 1992). These findings and the very high affinity of the two monomers to each other with a KD=10 pm led to the conclusion that a natively folded monomeric species may not exist (Bothwell and Shooter, 1977; Timm and Neet, 1992). Unfolding of NGF has been postulated to proceed via a loop-threading mechanism, this means back-threading of the N-terminal region through the cystine knot (De Young et al., 1996, 1999). The hypothesis has, however, been challenged upon NMR-monitored unfolding studies of the considerably longer pro-form in which no threading could be detected (Kliemannel et al., 2006).
Role and characteristics of the proregions
The proregions of NT3, BDNF and NGF nearly match in length: the mature growth factors with ca. 120 residues. The proregion of NGF, when covalently attached to mature NGF, has been shown to enhance refolding yields of NGF from reduced unfolded species (Rattenholl et al., 2001a). Via mass spectrometry, partially oxidised folding intermediates of proNGF were identified (Rattenholl et al., 2001b). It has been concluded that the proregion acts as a scaffold during oxidative folding, allowing the cystine knot domain to adopt a conformation in which the cysteines can arrange in such a way that the correct disulfide bonds can form. The proregion of NT-3 can functionally complement the proregion of NGF during oxidative refolding (Hauburger et al., 2007). Early investigations into the role of the NGF proregion during recombinant production of NGF in COS-7 cells showed that two conserved segments of the proregion are required for the secretion of active NGF (Suter et al., 1991). Thus, it is likely that the proregions guide the folding pathway of NTs within cells.
Modulation of neurotrophin function by their proregions
The presence of the proregions alters receptor preference of the NTs (Lee et al., 2001; Nykjaer et al., 2004). While the mature growth factors bind with low nanomolar dissociation constants to the tyrosine receptor kinases, TrkA, TrkB and TrkC with different neurotrophin ligand-defined specificities (reviewed in Bothwell, 2016), the pro-forms bind with high affinities to a complex consisting of p75NTR (75 kDa neurotrophin receptor) and sortilin. P75NTR belongs to the death domain receptors that initiate, upon ligand binding, apoptotic cascades (Dechant and Barde, 2002). Sortilin is a member of the Vps10p-domain receptor family (Hermey, 2009). Both receptors build a complex even in the absence of pro-neurtrophins (Nykjaer et al., 2004). Notably, the proregion in proNGF reduces the affinity of mature NGF to TrkA by a factor of 10. Binding of proNGF and NGF to p75NTR leads to pro-apoptotic signalling via a death domain in p75NTR and a conserved cysteine in the transmembrane helix (Tanaka et al., 2016). The proregion-mediated altered receptor preference has also been demonstrated for BDNF and NT-3 (Lee et al., 2001; Teng et al., 2005; Tauris et al., 2011).
Numerous reports have shown that a delicate balance of the levels of secreted pro-forms and mature NTs on one hand and cell surface presence of neurotrophin receptors on the other hand, decides over life and death. Especially, in pathological scenarios and, in general, age-related processes, the pro-apoptotic processes predominate (Fahnestock et al., 2001; Al-Shawi et al., 2007; Jansen et al., 2007; Nakamura et al., 2007; Yune et al., 2007; Volosin et al., 2008; Tauris et al., 2011). A mouse model, in which the balance was shifted towards elevated levels of proNGF through the expression of an non-cleavable pro-form, revealed learning and memory defects due to enhanced neurodegeneration (Tiveron et al., 2013).
Structure of the NGF proregion
The proregion of NGF can be separately expressed in Escherichia coli cells. In its isolated form, however, the proregion does not appear to be stabilised by tertiary interactions (Kliemannel et al., 2007). However, in covalent association with the native part, the proregion became stabilised via tertiary contacts (Kliemannel et al., 2004). The flexible conformation of the neurotrophin proregions has hampered structural studies. Yet, crystallisation of the proNGF- and proNT3-p75NTR complexes at least allowed for speculation about the location of the proregion within the complex (Feng et al., 2010). Due to the absence of significant electron densities, ‘empty spaces’ were observed for the pro-form receptor complexes. These unoccupied areas very likely represent the position of the proregions. The authors concluded that the crystal structures represent the fluid states of the proregions as even association of the pro-forms with p75NTR did not induce tertiary contacts to confine the proregions into a stable conformation. An earlier study employing small angle X-ray scattering (SAXS) of proNGF provided low resolution data that enabled modelling of the overall shape of proNGF by ab initio reconstructions (Paoletti et al., 2009). Two conformations were predicted for proNGF: a crab-like and an extended conformation (Figure 2). Circumstantial evidence for the association of the proregion on the ‘back’ of the two monomers has been obtained by tryptophan spectroscopy of folded and unfolded species, which revealed involvement of tryptophan 21 in association with the proregion moiety (Kliemannel et al., 2007). Early studies on the role of tryptophan 21 had already indicated that this residue is surface-exposed and involved in receptor binding (Cohen et al., 1980).
Neurotrophin-related proteins
Spätzle
A close relative to NGF is the cytokine Spätzle from Drosophila melanogaster. As the NTs, Spätzle is a homodimer in a parallel arrangement, however, the two monomers are covalently connected by an intermolecular disulfide bond. Spätzle mediates dorsoventral differentiation and innate immunity by binding to the transmembrane receptor Toll. The crystal structure of receptor bound Spätzle has been elucidated (Parthier et al., 2014). Spätzle is produced in several pro-forms that result from alternative splicing (DeLotto et al., 2001). The proregion is required for biogenesis and secretion of Spätzle (Weber et al., 2007). Processing to the mature form is mediated by the protease Easter and a related protease (for review see Moussian and Roth, 2005). After cleavage, the proregion stays non-covalently associated with the mature domain. Upon binding to Toll, the prodomain becomes displaced (Weber et al., 2007). It is noteworthy that similar to those of the NTs, the proregion of Spätzle has been described as ‘loopy’ or unstructured (Weber et al., 2003). A clear difference to the NTs is, however, that the Spätzle-induced signalling system via the Toll receptor lacks resemblence to the neurotrophin receptor cascades (Bothwell, 2016).
Coagulogen
Curiously, from horseshoe crabs – being considered as living fossils – coagulogen, a clotting protein has been characterised and shown to exhibit an NGF-fold including cystine knot connectivities (Bergner et al., 1996). Obviously, this protein fold has made a long journey through evolution.
Growth factors of the TGF-β/BMP family
TGF-β structure superfamily
In contrast to the NTs, monomers of the TGF-β/BMP family form dimers in an anti-parallel arrangement. With the exception of Lefty 1/2 and three BMPs, an intermolecular disulfide bond stabilises the dimers. Phylogenetic trees of the proteins have been deduced on the basis of sequence alignments (Shi et al., 2011; Hinck, 2012).
The very large TGF-β/BMP superfamily can be subdivided into several subfamilies: the transforming growth factors-β (TGF-βs), the bone morphogenetic proteins (BMPs) together with the growth and differentiation factors (GDFs), the activins (Act) and inhibins (Inh), and a group of proteins that appear phylogenetically and functionally more distant like the BMP antagonist, nodal, GDF 15, a sex determination factor, Müllerian inhibiting substance (MIS) and Lefty1,2 that determine vertebrate body asymmetry. As the proteins’ names disclose, members of the superfamily engage in many versatile roles: from regulation of differentiation during embryogenesis via coordination of housekeeping processes in the adult organism towards, finally, the regeneration of injured tissue. Individual proteins of the superfamily have specific receptor preferences. Yet, all receptors are single membrane-spanning proteins that transfer signals upon ligand binding by phosphorylation cascades. Two basic modes of receptor activation have been described: the ligand or BMP-induced signalling complex (BISC) and binding of the ligands to preformed receptor complexes (PFC). The intricate oligomerisation processes differ depending on the individual growth factor ligand. There exists a wealth of data on ligand-receptor interactions and subsequent signalling pathways, which are summarised in reviews by (Wu and Hill, 2009; Hinck, 2012; Macias et al., 2015; Yadin et al., 2016).
TGF-β and its proregion-mediated latent form
TGFs have been originally retrieved from neoplastic mouse tissue and bladder carcinoma (Roberts et al., 1981). The important role of TGF-β1 has been recognised upon gene-knockout that resulted in early post-natal death. The study implied a role of TGF-β1 in the control of immune cell proliferation (Kulkarni et al., 1993). One of the earliest biochemically and structurally characterised member of the superfamily was TGF-β2 (Daopin et al., 1992; Schlunegger and Grutter, 1992). The proregions of the TGF-βs were shown to exert inhibitory functions (Bottinger et al., 1996; McMahon et al., 1996; Shi et al., 2011). After proteolytic cleavage, the proregion of TGF-β1 was found in a non-covalent association with the mature domain (Gentry and Nash, 1990). Even when independently expressed in CHO cells, the proregions became secreted as a disulfide-linked dimer that in complex with the mature part led to its inactivation (Pircher et al., 1984; Bottinger et al., 1996). Denaturation of the proregion with acid or heat caused the release of active growth factor (Gentry and Nash, 1990). The fact that the association of the proregion with the mature part resulted in a transient inactivation coined the name latency-associated peptide (LAP) for the proregion. The complex consisting of mature TGF-β and its proregion is referred to as small latent complex.
Still, regulation of TGF-β activity does not only involve the proregion but additional proteins, namely, the latent complex binding proteins (LTBPs) that build intermolecular disulfide bonds with proTGF-β already during ER passage (Miyazono et al., 1988, 1991; Wakefield et al., 1988; Annes et al., 2004). The proregion of TGF-β1 with 249 residues is a dimer, stabilised by an intermolecular disulfide bridge. Via an N-terminally positioned cysteine, the proregion can additionally engage in an intermolecular disulfide bond with the LTBPs (Rifkin, 2005). The crystal structure of TGF-β1 in complex with the proregion enabled a mechanistic understanding of the latency mechanism (Shi et al., 2011). Both proregions embrace the dimeric mature part and mask binding epitopes for both type I and type II receptors. The N-terminal extended α1 helices of the proregions are positioned at the interface of the two mature parts. Association of the α1 helices with the mature parts causes a severe reduction of the monomer contact area. The firm association of these helices with the mature parts has been compared to a ‘life jacket’ (Figure 2). The so-called ‘latency lasso’ lies C-terminal to the α1-helix and connects the α-helix to the core structure of the proregion, which is formed by the ‘arm domain’. The arm domains, while having minor interactions with the mature parts, position the dimerisation area or ‘bow tie’ of the two proregions. The bow tie stabilises the dimeric proregion via two disulfide bridges. The essential residues for activation of TGF-β1 are RGD motifs in the proregions that enable binding to integrins and thus the cytoskeleton.
Activation of TGF-β1 from the latent complex
How can activation of TGF-β1 by dissociation from the proregion take place? It had been speculated that tensile force originating from cellular contraction and actin movements could eventually lead to the dissociation of the small latent complex (Shi et al., 2011). This hypothesis has been substantiated by a multi-method approach to gain insight into the structure of proTGF-β1 in complex with the head domain of integrin αvβ6 (Dong et al., 2017). The crystal structure, a H/D exchange-mass spectrometry approach together with SAXS experiments revealed that integrin binding leads to the reshaping of an extended segment close to the RGD motif within the proregion. The interface between the proregion and the integrin binding site has been described as interdigitated. The authors concluded that the constrained conformation within the proregion imposed by integrin binding may constitute a pivotal process in the activation of TGF-β1. Thus, the proregion within the proTGF-β1 complex, which is sandwiched between the integrin αvβ6 domain and the LTBPs, probably undergoes an unfolding process which is induced by a pulling force from the integrin interaction site and finally leads to the release of active TGF-β1. The deduced mechanistic model is supported by mutational studies of the proregion (Walton et al., 2010). The importance of the correct proregion structure is reflected by natural mutations that lead to Camurati-Engelmann disease, a rare bone disorder (Saito et al., 2001) (Table 1). Yet, the role of the proregion is not limited to controlling availability of TGF-β. The proregions of TGF-β1 and Act A are required for the biogenesis and secretion of the mature growth factors (Gray and Mason, 1990). Springer and co-workers speculated about the specific proregion segments that could be involved in TGF-β folding (Shi et al., 2011).
Phenotype-associated mutations in proregions of selected growth factors within the TGF-β/BMP family.
| Growth factor (uniprot accession number) | Proregion length | Mutation | Clinical, biochemical or biological phenotype (not found in all mutations) |
|---|---|---|---|
| TGF-β (P01137) | 249 aa | Y81H, R218C/H, H222D, C223G/R, C225R | Camurati-Engelmann disease, Altered levels of growth factors, intracellular accumulation |
| GDF8 (O14793) | 243 aa | A55T, K153R | Increased life expectance, Altered pro-form processing |
| GDF5 (P43026) | 354 aa | M173V, T201P, T203N, L263P, G319V, R380Q | Brachydactyly A/C, Reduced biological activity, decreased thermodynamic stability of proregion |
| BMP9 (Q9UK05) | 297 aa | R68L, P85L | Hereditary haemorrhagic telangiectasia, Impaired protein processing and/or function |
aa, amino acid. For references see either text or uniprot with the accession numbers indicated.
Myostatin and its latency-conferring proregion
Myostatin, also referred to as GDF8, is a potent antagonist of muscle growth. Knock-out of the myostatin gene in mice and a deletion in the myostatin coding sequence in certain cattle breeds resulted in strongly enhanced muscle mass (Grobet et al., 1997; McPherron et al., 1997). Transgenic mice that overproduced the proregion exhibited a similar phenotype with regard to muscle mass as the myostatin knock-out mice (Lee and McPherron, 2001). It has been concluded that the myostatin proregion acts as a latenty factor, similar to the situation in the TGF-βs. The authors also demonstrated that the proregion effectively inhibited the binding of myostatin to receptor ActRIIB. Myostatin has been recognised as a candidate growth factor that may determine life span control, because several single nucleotide polymorphisms in the coding region of myostatin correlate with longevity in humans (Saunders et al., 2006; Garatachea et al., 2013). Notably, some exchanges lie within the proregion (Table 1). The conservative exchange K153R in the proregion has been shown to increase pro-form processing by furin (Szlama et al., 2015). As an extracellular pool of pro-myostatin has been identified, post-secretion activation of the pro-form by furin may control availability of the mature growth factor (Anderson et al., 2008).
Myostatin and GDF11 share 90% sequence identity within the mature domains and have been assigned to the myostatin/inhibin group close to the TGF-β branch (Shi et al., 2011). However, their proregions exhibit only 52% identity (for review see Walker et al., 2016). An interesting hypothesis has been raised of whether the dissimilarities of the proregions may reflect differing shielding of the mature parts by proregions from antagonists (Mi et al., 2015).
BMPs: multi-functional factors in developing and adult organisms
BMPs have been originally discovered in demineralised bone matrix (Urist, 1965). Decades later, the proteinaceous agents were identified (Wang et al., 1990). Recombinant BMP2 and BMP7, derived from mammalian expression systems, are administered as protein therapeutics for problematic osseous defects. In particular, tibial fractures show accelerated healing in the presence of recombinant BMPs (Nauth et al., 2011).
The BMP subgroup is by numbers the largest family belonging to the TGF-β/BMP superfamily. At least 20 BMPs have been identified, including the GDFs of which individual proteins have been phylogenetically grouped closely to specific BMPs (Shi et al., 2011; Hinck, 2012). As the members of the TGF-β family, the monomers are arranged in an anti-parallel fashion. With the exception of GDF3, GDF9 and BMP15, the dimers are stabilised by intermolecular disulfide bonds (Rider and Mulloy, 2010). Dimer formation leads to the masking of a large hydrophobic area from solvent exposure (for representative structures, see Griffith et al., 1996; Scheufler et al., 1999).
The functionally versatile BMP proregions
The BMP proregions are similar in size to those of the TGF-β subgroup, with GDF5 having the most extended proregion (354 residues). It has been demonstrated for some BMPs that the proregions are involved in the biogenesis of the growth factors and required for the secretion. In case of BMP4, the proregion of BMP2 improved the yield of recombinant BMP4 in the cell culture supernatant (Hammonds et al., 1991). Likewise, deletion of the proregion of BMP2 resulted in a secretion defect of recombinant BMP2 (Kuhfahl and Schwarz, 2015). In contrast to the results obtained for TGF-β1 or Act A, secretion of BMP2 required covalent linkage with the proregion and no trans complementation was observed (Gray and Mason, 1990; Kuhfahl and Schwarz, 2015).
Studies with the purified pro-form of BMP2 showed that the presence of the proregion inhibits binding to BMP receptor II, while not affecting binding to BMP receptor type I (Hauburger et al., 2009). Limited proteolysis of the proregions and/or pro-forms of BMP2, GDF5 and the Drosophila orthologue of BMP2, decapentaplegic (dpp), led to the identification of protease-resistant subdomains within the proregions (Kuhfahl et al., 2011). These subdomains or core fragments can be independently expressed and show tertiary contacts. The presence of the core fragment of the BMP2 proregion restored to a certain extent secretion of BMP2.
The proregion of BMP4 has been shown to undergo sequential processing by pro-hormone convertases (Cui et al., 2001; Sopory et al., 2006). Correct two-step processing appears to be required for secretion of active growth factor as inactivation of the second processing site led to a severe reduction of BMP4 activity in transgenic mice (Goldman et al., 2006). Although a role in cellular folding may apply to all proregions of the TGF-β/BMP family, in vitro folding of the mature parts from inclusion body material upon recombinant expression in E. coli does not depend on the presence of the proregions (for selected examples see Ruppert et al., 1996; Honda et al., 2000; Vallejo et al., 2002; Vallejo and Rinas, 2004, 2013; Hillger et al., 2005; von Einem et al., 2010; Gieseler et al., 2017).
Similar to several other members of the TGF-β/BMP family, recombinant BMP7 can be isolated from mammalian host cells with the proregion in a non-covalent association (Jones et al., 1994). The complex has been reported to resemble the overall shape displayed by proBMP9 (Wohl et al., 2016). Interestingly, the authors observed that proregion-mediated binding to fibrillin induced a conformational switch that could be observed by electron microscopy. The interaction with fibrillin probably reflects proregion-caused binding of BMP7 to the extracellular matrix (Gregory et al., 2005; Ono et al., 2009). Furthermore, the purified proregion of BMP7 competes for binding to the ligand binding domain of receptor BRII, however, not that of BRI (Sengle et al., 2008).
Disease-associated mutations in the TGF-β/BMP proregions
The molecular mechanism of how the proregions support folding and secretion of the mature parts is currently not clear. The important roles of the proregions during biogenesis of the mature growth factors is highlighted by several reports that describe natural mutations in the proregion of human GDF5 (Table 1). Interestingly, three point mutations were identified that lie within the DNA encoding the core fragment of the GDF5 proregion and led to skeletal malformations in fingers and toes, classified as Brachydactyly C (Stange et al., 2014, 2015). The biochemical and structural effects of the exchanges have been analysed in the recombinant purified mutant proteins (Thieme et al., 2014). The study revealed that the mutations affected the thermodynamic stabilities of the protein variants. All three exchanges caused a loss of function with regard to chondrogenesis (Stange et al., 2014, 2015). Another natural amino acid exchange (R380Q) within the proregion of GDF5 had been detected earlier in a patient family diagnosed with Brachydactyly A2, a clinically different malformation of the digits (Ploger et al., 2008). The trait showed a dominant inheritance. The mutation impaired furin cleavage as it changed the protease processing site between the proregion and the mature part. The study demonstrated that a lack in GDF5 pro-form processing can result in the severely reduced activity of the growth factor.
BMP9 was found to be expressed predominantly in the liver (Miller et al., 2000). During recombinant expression for structural investigations, the complex of BMP9 with the proregion was obtained (Brown et al., 2005). Two disease-associated point mutations were identified in the proregion of BMP9 (Table 1) (Wooderchak-Donahue et al., 2013). The amino acid exchanges were linked to the congenital disorder hereditary haemorrhagic telangiectasia (HHT), with vascular abnormalities as the most central phenotypic symptoms. Analysis of the BMP9 proregion complex by electron microscopy and X-ray crystallography showed an open arm conformation that diverges from the orientation of the arm domain in proTGF-β1 (Figure 2) (Mi et al., 2015). Despite differing interfaces of the BMP9 and TGF-β proregions with the mature parts, the secondary structural elements that contribute to the interaction with the mature parts were found to be similar. The observations that the BMP9-proregion complex circulates in detectable concentrations in the blood and that the BMP9 proregion changes type II receptor affinities implicate the physiological role of the proregion. An inhibitory role of the BMP9 proregion has, however, not been demonstrated (Brown et al., 2005; Mi et al., 2015).
Conclusions
The presence of proregions represents a hallmark of cystine knot proteins. Cystine knot growth factors depend on their proregions in several aspects. Very likely, a role during biogenesis of the growth factors is common to all proregions. Even in case of cystine knot peptides, N-terminal peptides may guide intracellular routing. Several more functions apparently depend on the individual pro-form or proregion. Some proregions confer latency or at least alter receptor binding of the mature parts. For certain growth factors, the proregions mediate storage in the extracellular matrix. The importance of the proregions is reflected by several disease-associated mutations that can lead to reduced bioavailability of the mature parts. If the hypothesis that some proregions mediate interactions with regulatory proteins (Mi et al., 2015) turns out to be true, another level of complexity of the proregion-growth factor interplay could be lurking.
Acknoweldgments
I thank Milton T. Stubbs for providing the improved Figure 2. Valuable comments by Andrea Sinz and Hauke Lilie are gratefully acknowledged.
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©2017 Walter de Gruyter GmbH, Berlin/Boston
Articles in the same Issue
- Frontmatter
- Reviews
- Regulation of protein function by S-nitrosation and S-glutathionylation: processes and targets in cardiovascular pathophysiology
- Cystine knot growth factors and their functionally versatile proregions
- Kallistatin: double-edged role in angiogenesis, apoptosis and oxidative stress
- Minireview
- The sphingomyelin synthase family: proteins, diseases, and inhibitors
- Research Articles/Short Communications
- Genes and Nucleic Acids
- Comparison of cytochrome P450 expression in four different human osteoblast models
- Cell Biology and Signaling
- The molecular mechanisms involved in lectin-induced human platelet aggregation
- HDAC1 triggers the proliferation and migration of breast cancer cells via upregulation of interleukin-8
- Heat shock protein 47 effects on hepatic stellate cell-associated receptors in hepatic fibrosis of Schistosoma japonicum-infected mice
Articles in the same Issue
- Frontmatter
- Reviews
- Regulation of protein function by S-nitrosation and S-glutathionylation: processes and targets in cardiovascular pathophysiology
- Cystine knot growth factors and their functionally versatile proregions
- Kallistatin: double-edged role in angiogenesis, apoptosis and oxidative stress
- Minireview
- The sphingomyelin synthase family: proteins, diseases, and inhibitors
- Research Articles/Short Communications
- Genes and Nucleic Acids
- Comparison of cytochrome P450 expression in four different human osteoblast models
- Cell Biology and Signaling
- The molecular mechanisms involved in lectin-induced human platelet aggregation
- HDAC1 triggers the proliferation and migration of breast cancer cells via upregulation of interleukin-8
- Heat shock protein 47 effects on hepatic stellate cell-associated receptors in hepatic fibrosis of Schistosoma japonicum-infected mice
