N-acetyl-Ser–Asp–Lys–Pro (Ac-SDKP) has emerged as a potent antifibrotic agent and negative regulator of haematopoietic stem cell differentiation which is processed exclusively by ACE. Here we provide a detailed biochemical and structural basis for the domain preference of Ac-SDKP. The high resolution crystal structures of N-domain ACE in complex with the dipeptide products of Ac-SDKP cleavage were obtained and offered a template to model the mechanism of substrate recognition of the enzyme. A comprehensive kinetic study of Ac-SDKP and domain co-operation was performed and indicated domain interactions affecting processing of the tetrapeptide substrate. Our results further illustrate the molecular basis for N-domain selectivity and should help design novel ACE inhibitors and Ac-SDKP analogues that could be used in the treatment of fibrosis disorders.Angiotensin-I converting enzyme (ACE) is a zinc dipeptidylcarboxypeptidase with two active domains and plays a key role in the regulation of blood pressure and electrolyte homeostasis, making it the principal target in the treatment of cardiovascular disease. More recently, the tetrapetide  Angiotensin-1-converting enzyme ACE, EC 3.4.15.1) is a zinc peptidase and a key enzyme in cardio-vascular physiology1.4.15.1 i567N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP) is specifically cleared by ACE11141516171819202123The N- and C-domains of ACE have been shown to display negative co-operativity in substrate hydrolysis2526The X-ray crystal structures of both individual homologous ACE domains have been previously determined28The purpose of this study was to understand the kinetic nature of Ac-SDKP hydrolysis and the structural basis for its interaction with the N-domain of ACE. The high resolution (1.8 Å) X-ray crystal structures of the N-domain in complex with the two products of Ac-SDKP cleavage, the dipeptides Ac-SD and KP, were determined. The extent of domain interaction during substrate hydrolysis and the domain selectivity of these processes were further assessed using a fluorescamine assay. Together the data reported here allowed the construction of a comprehensive model of N-domain selective substrate recognition and peptidase activity.P1. The structure at 1.8 Å showed electron difference density within the catalytic channel , of the Ac-Ser-Asp fragment, presents a different orientation. Its Hydroxyl side chain makes direct electrostatic contacts with His331, His491 and Tyr501, as well as a water-mediated interaction with Zn2+. The AcS amino group interacts with a single water molecule itself connected to Thr358 (main chain) and Asp393. The acetyl moiety may be stabilised in the small hydrophobic area composed of Ala332 and Thr358.The two peptides have their C-terminal end strongly anchored by hydrogen bonds with residues Lys489, Tyr498 and Gln259 forming the S2′ site, amino acids previously shown to be key anchoring residues . The arod Tyr501  and its Km values of 199.6 μM and 138.2 μM, respectively  similar to the individual N-domain. Thus, the combined domain turnover rate of sACE (25.2 s−1) was slightly higher than the sum of the kcat values for the individual domains.Wild-type sACE, with both domains active, displayed a Km values of the two domain (X-sACE) enzymes were approximately 3-fold higher than their single domain counterparts, and twice that of wild-type sACE, again showing an effect of domain interaction on substrate on-off rates. The kcat values were approximately 2-fold higher in C-sACE and N-sACE constructs than their respective single domains, which may be indicative of an inter-domain influence on substrate release. Overall, this resulted in a similar catalytic efficiency between the individual N-domain and the same domain in full length form (N-sACE), whereas C-sACE was approximately half that of its single domain counterpart  were employed . InteresKm compared to wild-type sACE but in a similar range to that of the other full length constructs (−1) showed a two-fold increase compared to C-sACE (8.2 s−1). The resulting improved catalytic efficiency therefore suggests a role of the N-domain in lowering C-domain activity in the full length somatic enzyme towards the substrate.So as to better appreciate the role of the C-domain in the full length molecule, the CC-sACE molecule, comprising two active C-domains connected with the interdomain linker , was chanstructs . InteresThe crystal structure of the N-domain with the KP fragment gave useful insight into the peptide binding mechanism and allowed us to propose a model for the full substrate interaction . The lys35ACE belongs to the gluzincin family of metalloproteases and catalysis is expected to follow a general base-type mechanism. The structures determined here allow us to more accurately predict the substrate hydrolysis mechanism employed by ACE. The cleavage of Ac-SDKP is predicted to happen after displacement of the zinc-bound water molecule which then attacks the scissile carbonyl bond to form an oxyanion . The nucN-acetyl-AngI and substance P4042Ac-SDKP is an important substrate of ACE that, through specific inhibition, could lead to beneficial therapeutic effects in the treatment of fibrosis disorders. Therefore, a thorough enzymatic and structural analysis would be useful in assisting our understanding of the requirements for hydrolysis of tetrapeptide substrates. Previous work involving Ac-SDKP kinetics involved the use of paper chromatography or HPLC, neither of which allow for medium to high throughput kinetic characterisation738Km values determined in this study are generally higher than the original publication presenting Ac-SDKP hydrolysis as a highly N-domain specific processKm values that differ depending on the enzymatic construction and methodology employedet al. also found a decrease in selectivity, with Ac-SDKP having approximately 8-fold N-selectivity6in vivo N-domain inactivation results in a 4–7 fold increase in plasma Ac-SDKP concentrations2245in vivo studies state that the modest increase in serum peptide levels through the use of N- domain selective inhibitors can still possibly provide the desired therapeutic effects.The N-domain selectivity of Ac-SDKP processing was found to be markedly reduced compared to some previous reports. Literature reports differ on the degree of catalytic efficiencies and selectivities of Ac-SDKP hydrolysis7The structural basis of Ac-SDKP’s preferential hydrolysis by the N-domain was investigated by analysing the molecular interactions of the N-domain with the peptide. The two peptides occupied the same area of the S′ catalytic sub-pocket and displayed noticeable differences in their mode of binding within the S1′ site while showing common features in carboxy-terminal ends binding within with the S2′ sites. This common anchoring in the S2′ site is homologous to that of peptide binding in C-domain ACEN-acetyl group is easily accommodated in the cavernous S2 site with the primary contacts in this region being carried out by the P2 Ser of the substrate. Our structural model provides visual insight for observations of other kinetic studies, whereby selectivity of Ac-SDKP analogues could be influenced by changing the unprimed substrate amino acids while having benzoyl derivatives (considerably larger than an acetyl group) on the substrate N-terminus8Using the structural information above, we were able to generate a model for Ac-SDKP binding into the enzyme active site and revealed the importance of the S2 site in providing possible unique interactions for preferential processing. Further, it suggests a minimal set of amino acids that are responsible for enzyme selectivity that, if properly exploited, could result in domain selective inhibitors and/or drugs. Some of these residues have been implicated in selective inhibitor binding35The two active sites within human and bovine somatic ACE exhibit negative co-operativity with certain, but not all, substrates26274927In summary, the detailed molecular interactions between Ac-SDKP and ACE were characterised. We have found that the tetrapeptide is indeed preferentially processed by the N-domain although this preference is less than some previous reports. The observations of the dipeptide fragments resulting from Ac-SDKP cleavage provided the structural basis to offer a complete model of domain preferential substrate hydrolysis, from peptide recognition by specific domain amino acids to a detailed catalytic mechanism. Further, we emphasize important sets of amino acids: conserved anchoring residues that appear to play a role in ACE substrate binding generally and N-domain unique residues that could provide contacts for preferential positioning. Domain interactions were also shown to influence ACE activity with an observed inhibitory effect of the N-domain over C-domain cleavage of Ac-SDKP. Overall, these findings enhance our understanding of peptide hydrolysis generally and should help the development of N-domain selective inhibitors that could be useful in the treatment of tissue injury and fibrosis.For single, soluble enzymatic domains: a modified tACE construct, tACEΔ36NJ, that lacks the transmembrane region and unique 36 amino acid N-terminus  had been generated previously29The full length domain knock outs of sACE were a kind gift from Dr. Vincent DiveZ-FHL)All enzymes were expressed in Chinese Hamster Ovary (CHO) cells using standard tissue culture approaches as formerly describedIn order to determine loss enzymatic activity due to storage, specific activities were calculated immediately after purification assuming that the enzyme which is eluted off the lisinopril-Sepharose column must be active in order to bind the ligand. Specific activities were re-determined prior to kinetic analysis and active protein concentrations adjusted accordingly.4 buffer), ranging in concentration from 0 to 1000 μM, were warmed to 37 °C in a 96-well plate. The assay was commenced by the addition of 10 μl pre-warmed enzyme (0.2 pmols in Hepes buffer) and incubated for 15 minutes for N-domain and sACE enzymes and 30 minutes for the C-domain enzymes. The assay was stopped by the addition of 50 μl 1 M HCl. The solution was neutralised by the addition of 50 μl 1 M NaOH and the pH increased to 8.3 by the addition of 100 μl 500 mM K2HPO4/KH2PO4 buffer pH 8.3. Ten microlitres of fluorescamine  was added and the resulting mixture incubated for 3 minutes at room temperature. Fluorescence intensities were measured at λex = 390 nm and λem = 475 nm using a Cary Eclipse spectrofluorimeter (Varian Inc.). Changes in fluorescence compared to unhydrolysed substrate were converted to reaction velocities by the use of a standard curve obtained by complete AcSDKP hydrolysis and kinetic constants calculated from nonlinear regression analysis . For full length ACE constructs, the presence of 2 moles active sites for every 1 mole of enzyme was taken into account when calculating turnover ratesA plate adapted fluorescamine assay was employed as publishedThe crystals of N-ACE in complex with the peptides were obtained by co-crystallization with a 2.5 mM concentration of Ac-SDKP . Crystals were grown with 1 μl of the N-ACE:Ac-SDKP sample  mixed with an equal volume of reservoir solution consisting of 30% PEG550 MME/PEG20000, 100 mM Tris/Bicine, pH 8.5, and 0.06 M divalent cations (Molecular Dimensions) and suspended above the well as a hanging drop.Fo − Fc electron density peaks exceeded 3σ, and potential hydrogen bonds could be made. Validation was performed with MOLPROBITY+X-ray diffraction data were collected on station IO3 at the Diamond Light Source  equipped with a PILATUS-6M detector . Crystals were kept at constant cryo-temperature (100 K) during data collection. Raw data images were processed and scaled with MOSFLMHow to cite this article: Masuyer, G. et al. Structural basis of Ac-SDKP hydrolysis by Angiotensin-I converting enzyme. Sci. Rep. 5, 13742; doi: 10.1038/srep13742 (2015).


C. thermophilum have been determined and their arrangement in solution has been studied by SAXS.The crystal structures of the individual domains of the Mex67–Mtr2 complex from  Chaetomium thermophilum are presented together with small-angle X-ray scattering (SAXS) and in vitro RNA-binding data that indicate that, not withstanding the limited sequence conservation between different NXF family members, the molecules retain similar structural and RNA-binding properties. Moreover, the resolution of crystal structures obtained with the C. thermophilum domains was often higher than that obtained previously and, when combined with solution and biochemical studies, provided insight into the structural organization, self-association and RNA-binding properties of Mex67–Mtr2 that facilitate mRNA nuclear export.Members of the Mex67–Mtr2/NXF–NXT1 family are the principal mediators of the nuclear export of mRNA. Mex67/NXF1 has a modular structure based on four domains  that are thought to be present across species, although the level of sequence conservation between organisms, especially in lower eukaryotes, is low. Here, the crystal structures of these domains from the thermophilic fungus  In vitro SAXS and RNA-binding studies have also demonstrated that ctMex67–Mtr2 has similar biochemical properties as H. sapiens NXF1–NXT1 (hsNXF1–NXT1) and, in particular, have shown that the ctMex67 NTF2L domain contributes to mRNA binding.Here, we present the crystal structures of the individual domains of 2.2.1.ctMex67 and ctMtr2 were PCR-amplified from C. thermophilum cDNA using standard procedures. Fragments corresponding to the RRM  and LRR  domains were cloned into the first multiple cloning site (MCS) of pETDuet-1 to generate His6-tagged constructs. Fragments corresponding to the NTF2L domain  and ctMtr2 were cloned into the first and second MCS sites of pETDuet-1, respectively, to generate His6-tagged ctMex67NTF2L–Mtr2. A fragment corresponding to the UBA domain  was cloned into pMCSG10 to generate a TEV-cleavable GST-tagged construct. Longer constructs containing multiple domains of ctMex67  and Mtr2 were also cloned into the first and second MCS sites of pETDuet-1, respectively, to generate TEV-cleavable His6-tagged constructs. All protein constructs were expressed in Escherichia coli BL21-CodonPlus(DE3)-RIL cells by IPTG induction at 291 K over 16 h and all purification steps and manipulations were conducted at 277 K unless stated otherwise. Harvested cell pellets were resuspended at 5 ml per gram of wet cell pellet in 50 mM Tris–HCl pH 7.4, 500 mM NaCl, 20 mM imidazole pH 8.0 (His6-tagged constructs) or 50 mM Tris–HCl pH 7.4, 500 mM NaCl, 5 mM DTT (GST-tagged constructs) and lysed by high-pressure cavitation using two passes through an EmulsiFlex C3 (Avestin). Lysates were clarified by centrifugation and the supernatant was incubated with Ni–NTA agarose beads or glutathione Sepharose 4B beads for 1 h. Nonspecifically bound proteins were removed by washing with the buffer used for lysis, after which the His6-tagged proteins were eluted with 50 mM Tris–HCl pH 7.4, 500 mM NaCl, 250 mM imidazole and GST-tagged proteins were eluted with 50 mM Tris–HCl pH 7.4, 500 mM NaCl, 5 mM DTT, 2 mM reduced glutathione. TEV-cleavable tags were then removed by incubation with TEV protease overnight. Constructs with more than one domain were purified further using heparin affinity chromatography as described in Aibara, Valkov et al. , 20 mM Na HEPES pH 8.0, 750 mM NaCl, 5 mM DTT (for ctMex67UBA) or 20 mM Tris–HCl pH 7.4, 50 mM NaCl . Small aliquots of purified proteins were flash-frozen in liquid nitrogen and stored at 193 K until required.Genes corresponding to  al. 2015. All pro2.2.XDS  and scMtr2  as search models, whereas ctMex67NTF2L–Mtr2 was solved by molecular replacement using Phaser  and ctMtr2 (PDB entry 4x2m) as search models. The structure of ctMex67UBA was solved by SAD phasing in Phaser cysteine that was derived from cysteines modified by the cacodylate buffer  and their maximum sizes Dmax were computed using GNOM  data were collected on beamline BM29 at the European Synchrotron Radiation Facility, Grenoble, France using an online HPLC system (Viscotek) equipped with a Superdex 200 Increase 3.2/300 column  with a sample flow rate of 0.1 ml min2.4.ctMex67 domain-based constructs was assessed using fluorescence anisotropy, in which 10 nM DY-547-labelled homopolymeric RNA  was mixed with serially diluted protein in 20 mM Tris–HCl pH 8.0, 50 mM NaCl as described previously , the LRR domain (1.7 Å resolution), ctMtr2 alone (2.0 Å resolution), NTF2L–Mtr2 (2.9 Å resolution) and the UBA domain (1.7 Å resolution) of ctMex67–Mtr2  and was based on the characteristic RRM βαββαβ fold . Like other NXF family members, ctMex67RRM did not contain a typical RRM consensus sequence that is based on two motifs: RNP1, (K/R)o-G-(F/Y)o-(G/A)i-Fo-Vi-Xo-(F/Y)i, and RNP2, (L/I)i-(Y/F)o-(V/I)i-(G/N)o-(G/N)o-(L/M)i, where the subscript ‘o’ or ‘i’ signifies whether the side chain is surface-exposed or facing the core of the RRM fold  and RNP2 (residues 101–106) sequences were G-Do-Yo-Vi-Wo-Li-Ko-Vi and Ii-Ko-Ii-Lo-G-Li, respectively. The RNP2 motif appeared to be more conserved than RNP1, although the characteristic aromatic group at position 2 that typically forms ring-stacking interactions with RNA was instead lysine in ctMex67RRM. The large deviations from the RNP1 and RNP2 sequences found in different members of the NXF1/Mex67 family may reflect that they bind RNA in a more general, non-base-specific way, as appeared to be the case in the crystal structure of hsNXF1RRM-LRR–CTE-B, in which the hsNXF1 RRM domain formed mainly phosphate–backbone interactions with the CTE-B RNA .The 2.4 Å resolution crystal structure of ctMex67NTF2L and ctMtr2 chains interacted through their highly curved β-sheets in a pseudo-twofold-symmetric manner in the 2.9 Å resolution crystal structure, similar to that observed in the analogous complexes from H. sapiens and S. cerevisiae . Neither ctMex67NTF2L nor ctMtr2 had the larger internal loops between strands β4 and β5 present in S. cerevisiae but which were disordered in previous structures . These loops have been implicated in pre-60S ribosomal export, where a surface flanked by these loops is formed on the NTF2L–Mtr2 region that is separate from that used for bulk mRNA export  of the ctMex67NTF2L chain made contact with the cavity formed between α2′ and β1′ from a symmetry-related ctMex67NTF2L chain resulted in ctMex67NTF2L having an additional β-strand  compared with H. sapiens NXF1NTF2L, although the biological significance of this interaction remains to be established. There was clear density for the N-terminal region of ctMex67NTF2L  that placed this region in a position in which it spanned across the surface of ctMtr2 . In particular, Leu368 was buried in a hydrophobic pocket present in ctMtr2. This arrangement of the Mex67NTF2L pre-α1 loop is also present in the H. sapiens homologue as well as in the multi-domain structure of S. cerevisiae Mex67ΔUBA–Mtr2  and thus the observation that this interaction is conserved between S. cerevisiae, C. thermophilum and H. sapiens indicates that it represents a conserved structural feature within these complexes.The ae Fig. 3a. Neithr2 Fig. 4a. In pans Fig. 4b and thctMex67LRR-NTF2L crystal structure had two copies of the protein in the asymmetric unit that generated a pseudo-homodimer through extensive interactions between the highly curved β-sheets of each Mex67 NTF2L domain  that were analogous to those seen in the S. cerevisiae Mex67–Mtr2 heterodimer or NTF2 homodimer . Comparison of the fold of ctMex67NTF2L in this structure with that observed when it was complexed with Mtr2 indicated that the major secondary-structural elements of the NTF2-like core were not perturbed, although there were several rearrangements in the loop regions . However, the previously ordered pre-α1 loop that gave rise to the asymmetry in the NTF2L–Mtr2 region was now disordered, perhaps thereby allowing the symmetric homodimeric Mex67 to be generated. Although it has been proposed that a motif known as the ‘NXF plug’ formed by residues present in the core of the NTF2L domain would inhibit Mex67 homodimerization . Moreover, ctMex67LRR-NTF2L appeared to form a homodimer in solution, and in size-exclusion chromatography coupled with multi-angle light scattering the protein migrated as a single peak with a molecular mass of 82 g mol−1 , consistent with a dimer .The 2.95 Å resolution in Fig. 5a that w Å Fig. 5b. Compans Fig. 5c. HowevctMex67UBA showed a clear As anomalous signal from a dimethylarsenic group on Cys623 that was derived from the cacodylate buffer . The ctMex67UBA fold was based on three α-helices similar to those seen in H. sapiens and S. cerevisiae . The ctMex67 UBA domain contained a four-residue C-terminal extension compared with the H. sapiens homologue that formed interactions with the end of α1 in seven copies of the eight in the asymmetric unit and which might contribute to thermostability.The 1.7 Å resolution crystal structure of et Fig. 6d. The cC. thermophilum Mex67 and Mtr2 was conserved when compared with the H. sapiens homologue and displayed a low Cα r.m.s.d. values when aligned structurally across species (Table 2α r.m.s.d. < 0.8 Å), whereas the NTF2L domain showed the greatest variation (Cα r.m.s.d. = 1.9 Å). However, despite the conservation of the fold, even after structure-based alignment the number of identical equivalent residues remained remarkably low. The UBA domain showed the greatest conservation (29% sequence identity over 55 residues), whereas the RRM domain had the lowest (15% identity over 73 residues). An unexpected homodimeric form of ctMex67 was also observed in which the two copies of ctMex67NTF2L formed a dimer analogous to that in S. cerevisiae NTF2.In summary, the fold of all four domains of s Table 2. The UBA3.2.et al., 2015ctMex67 complexed with ctMtr2 could not be obtained, and therefore small-angle X-ray scattering (SAXS) was used to investigate the solution state of ctMex67–Mtr2 using systematic truncations of domains , consistent with this domain arrangement probably being conserved. The scMex67ΔUBA–Mtr2 crystal structure  fitted the SAXS profile of ctMex67ΔNΔUBA–Mtr2 well (χFoXS = 1.03), configuration 2  fitted less well (χFoXS = 2.5). Models of ctMex67ΔN–Mtr2 were generated by molecular dynamics in BILBOMD , 2 (green) and 3 (blue), respectively; Fig. 7c]. Aligning the three best models based on the NTF2L–Mtr2 region showed that consistent with the SAXS data obtained with Mex67LRR-NTF2L–Mtr2, the RRM domain showed flexibility whereas the LRR domain position remained constant. Strikingly, the UBA domain in these models was placed in three very different positions, consistent with the spatial positions of the NTF2L and UBA domains not being strongly constrained.Previous studies indicated that there is a defined spatial relationship between the Mex67 RRM, LRR and NTF2L domains  in vitro but with different affinities. Similar to that from S. cerevisiae, ctMex67–Mtr2 bound polyA15 and polyG15  more strongly than polyU15 and polyC15 . Removal of the UBA domain did not reduce the affinity significantly and the observed Kd for ctMex67ΔUBA–Mtr2 (370 nM) was indistinguishable from that for the complete complex (350 nM). However, deletion of either the RRM domain or the NTF2L–Mtr2 region reduced the affinity more than 15-fold. Thus, the Kd for the ctMex67LRR-NTF2L–Mtr2 (RRM domain deletion) was 6.8 µM, whereas that for ctMex67RRM-LRR (NTF2L–Mtr2 region deletion) was 5.5 µM. These data indicated that as in S. cerevisiae  indicated that a continuous RNA-binding interface could be generated from the way in which the LRR and NTF2L domains of Mex67 assumed a defined three-dimensional arrangement through interactions with Mtr2. Although this arrangement has not directly been observed for ctMex67–Mtr2 using X-ray crystallography, binding assays identifying interactions between RNA and the NTF2L–Mtr2 region in addition to the RRM and LRR domains are consistent with ctMex67–Mtr2 adopting a similar conformation to generate an extended positively charged region to mediate RNA binding.Fluorescence anisotropy assays Fig. 8 showed t al. 2000 showed tC. thermophilum Mex67–Mtr2 provide atomic resolution information on the Mex67–Mtr2 complex from a single species and indicate that although there is only low sequence identity in some regions within this family of nuclear-export proteins, the folds of the four domains are conserved, demonstrating the highly structurally conserved nature of the NXF family. C. thermophilum Mex67–Mtr2 retained features found in both S. cerevisiae and H. sapiens and conserved the hydrophobic pocket identified as the nucleoporin-binding site found in NTF2L domains from other organisms. The position of the pre-α1 loop, which has been implicated in the spatial arrangement of the Mex67 domains in S. cerevisiae, occupied a similar position in ctMex67NTF2L to that observed in S. cerevisiae and H. sapiens. Notably, Leu368 was positioned in a structurally equivalent position to scMex67Leu263 and hsNXF1Leu370, indicating that the NTF2L pre-α1 loop position may be conserved across species (Fribourg et al., 2001et al., 2015et al., 2015ctMex67LRR-NTF2L crystals was unanticipated because it had been thought that complex formation between Mex67 and Mtr2 was a prerequisite for efficient nuclear export of mRNA (Santos-Rosa et al., 1998ctMex67LRR-NTF2L is homodimeric in solution, whether Mex67 is ever not complexed to Mtr2 in vivo is currently unclear and whether the configuration observed simply represents an inactive form of the Mex67–Mtr2 complex or whether homodimeric Mex67 has another role in the cell has yet to be established.In summary, the crystal structures of all four domains of 10.1107/S2053230X15008766/mn5088sup1.pdfSupplementary figures. DOI: Mex67 RRM, 4wpmPDB reference: Mex67 UBA, 4wp2PDB reference: Mex67 NTF2L, 4wp5PDB reference: Mex67 LRR, 4wp6PDB reference: Mtr2, 4x2mPDB reference: Mex67 LRR+NTF2L, 4xm4PDB reference:


Homologues of calpain, often thought to be an essential, cytoplasmic, calcium-dependent cysteine endopeptidase found exclusively in eukaryotes, have been found in bacterial proteomes. The homologues lack calcium-binding sites, have differing domain architectures, and can be secreted or membrane-associated. Homologues are rare and occur in a minority of bacterial phyla and often in a minority of species in a genus. However, the differences in domain architecture argue against a recent, horizontal gene transfer from a eukaryote. From analysis of a phylogenetic tree and absence of homologues in archaea, calpains in eukaryotes may be derived from genes horizontally transferred from a bacterium.Reviewers: This article was reviewed by L. Aravind and Frank Eisenhaber.The online version of this article (doi:10.1186/s13062-015-0095-0) contains supplementary material, which is available to authorized users. Gallus gallus) calpain, a typical example, is a heterodimer of a large and a small subunit. The peptidase active site is present on the large subunit and consists of a Cys, His, Asn catalytic triad in which the cysteine bears the nucleophilic thiol group and the histidine is the general base, with its ring orientated in the optimal conformation by the asparagine. A glutamine N-terminal to the cysteine is an important residue that helps stabilize the acyl-intermediate formed during substrate hydrolysis by forming a structure known as the “oxyanion hole” [Homo sapiens) and many other mammals, there are calpains sensitive to different concentrations of intracellular calcium. In the human, there are fifteen proteins with domains homologous to the peptidase domain of chicken calpain. Two of these homologues, calpamodulin and androglobin, are not peptidases because the active site cysteines have been replaced [Calpains are cysteine endopeptidases from peptidase family C2 . Chickenon hole” . The pepon hole” . In humareplaced , 6.Of the fifteen human calpains, eight are ubiquitous in cells, whereas six have a restricted distribution. Calpain-3 is found in skeletal muscle, calpains 8 and 9 in the gastrointestinal tract, calpain-11 and androglobin in the testis, calpain-12 in the hair follicle, and calpamodulin in embryonic muscle. Deficiency in a calpain can lead to disease: platelet dysfunction , muscular dystrophy , stress-induced gastric ulcer , and calpain-2 deficiency is embryonically lethal . CalpainRattus norvegicus) calpain-2 was solved and the fold was found to be similar to that of cysteine peptidases of the papain family (peptidase family C1) [The structure of the peptidase domain of rat mily C1) . Because . BecausDrosophila melanogaster (four homologues), Caenorhabditis elegans (fourteen homologues), Schistosoma mansoni (seven homologues), plants , fungi , yeast (Rim13 peptidase), Leishmania and Trypanosoma spp.  and bacteria. The domain architecture of many of these proteins differs considerably from the chordate calpains, with only the peptidase domain being in common [Calpain homologues have been found in a variety of non-chordate species, including n common .Porphyromonas gingivalis [d-Arg (where Pz is Nα-(4-phenylazo)benzyloxycarbonyl), where cleavage occurs between Leu and Gly. Tos-LysCH2Cl (TLCK), Tos-PheCH2Cl (TPCK), ethylenediaminetetraacetic acid (EDTA), and iodoacetate were found to be inhibitory, which is suggestive of a calcium-dependent cysteine peptidase. However phenylmethane sulfonylfluoride (PMSF) was also found to be inhibitory, and given that PMSF is predominantly a serine peptidase inhibitor, it has been argued that the characterized entity may have been a mixture of peptidases [The only bacterial calpain homologue that has been subject to any biochemical characterization is the Tpr protein from ngivalis , 21. Theptidases . If thisThe evolution of the calpain family had been studied by Jékely & Friedrich  when fewMEROPS batch Blast [The chicken calpain sequence was chosen to represent the family, and homologues were found by using the peptidase domain from the known structure to search for homologues in the NCBI non-redundant protein sequence database using BlastP , 24. TBlch Blast . Becausech Blast . Sequencch Blast , which cwww.ebi.ac.uk/Tools/hmmer).Complete protein sequences of all homologues detected by the method above were submitted to PfamScan . The resBranchiostoma floridae, Ciona intestinalis, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, Saccharomyces cerevisiae and Plasmodium falciparum; 3) all homologues from prokaryotes; and 4) a representative sequence from each phylum possessing a homologue. In order to root the phylogenetic tree, the peptidase domain of papain, a member of the structurally-related cysteine endopeptidases from family C1, was included in the selection to provide an outgroup.Because so many homologues were detected, a full sequence alignment of peptidase domains and a phylogenetic tree determined from this alignment would be too large to present here. Instead, sequences were selected from the family. To ensure that the selection was representative, the following criteria were used: 1) sequences corresponding to characterized peptidases; 2) all homologues from the following model organisms: human, mouse, chicken, zebrafish, An alignment of peptidase domains from these selected homologues was made using ClustalW . A neighIn total, 3795 calpain homologues were detected , the sea lamprey (Petromyzon marinus), the sea squirt Ciona savigni, the chlorophytes Chlorella variabilis and Coccomyxa subellipsoidea and the rhodophyte Cyanidioschyzon merolae. The absences from animals are most likely the results of errors in sequencing and assembly rather than because genes have been lost, especially in the case of C. savigni because homologues are known from C. intestinalis. The absences in the algae may result from genuine gene losses. Absences of homologues in fungi and protozoa were more widespread: no homologues were detected in the ascomycetes Eremothecium cymbalariae, Passalora fulva, Schizosaccharomyces japonicas and S. pombe; the basidiomycetes Postia placenta, Rhodotorula glutinis and Tremella mesenterica; the apicomplexan Cryptosporidian hominis; any cercozoan, diplomonad, microsporidian or mycetozoan. Although no homologous protein has been identified in the basidiomycete Dichomitus squalens, a fragment could be detected with a TBlastN search against the genome sequence.All completely sequenced animal and plant genomes possessed a homologue except those of the three-toed sloth (Streptomyces where three species are each known to possess a homologue out of 30 species with completely sequenced genomes). With the exception of Porphyromonas gingivalis, no homologues were found in well-known bacteria such as Escherichia coli, Vibrio cholera, Bacillus subtilis, Staphylococcus aureus, or Streptomyces griseus.Homologues in bacteria were few, with most bacterial genomes lacking a homologue. Sixty-seven homologues were found in species in the following seven phyla: Actinobacteria (1 species of 3 with completely sequenced genomes), Bacteroidetes (8 of 173), Chloroflexi (1 of 17), Cyanobacteria (14 of 53), Firmicutes (19 of 681), Planctomyces  and Proteobacteria (11 of 815). Homologues were found in a minority of species in a phylum, and in some cases examples were found in only a minority of species in a genus .From the structure of the complex of rat calpain 2 with calpastatin, the calcium binding sites in the peptidase domain were found to consist of Ile89, Gly91, Asp96, Glu175 (site 1) and Glu292, Asp299, Gln319, Glu323 (site 2) . All of Caenorhabditis elegans.Many calpain homologues are multidomain proteins. Figure Most eukaryotic calpain homologues possess a CIII domain, the function of which is unknown .Many calpains bind calcium through EF hand structure domains, and a variety of different EF-hand domains are found, with the commonest being EF-hand_8 and EF-hand_6 . However, whether an EF hand structure is a member of one family or another is often debatable because the E values are very similar. All EF hand structure domains are structurally related and members of the same clan . EF handDaphnia pulex) to seven . Zinc fingers from the zf-H2 and zf-H2_2 families are found in calpain-15 from the gnat Culex pipiens quinquefasciatus. Zinc fingers are also found in hypothetical proteins from the cryptophyte Guillardia theta and the chlorophytes Micromonas and Ostreococcus, which are zf-GRF domains.Calpain-15 possesses zinc-finger domains (zf-RanBP domains) which interact with nucleic acids . The numSchistosoma mansoni, and a SNARE  domain is found in a single calpain-3 orthologue.A C2 domain targets a protein to a cell membrane . C2 domaHerpetosiphon aurantiacus and one from Segniliparus rugosus); Brix (a homologue from Ichthyophthirius multifiliis); CARDB (nine domains in a homologue from Cyanothece sp. PCC 7822); Abhydrolase_3 (Emericella nidulans); PPC ; Collagen (Leptosphaeria maculans); TPR_1, TPR_8 and TPR_11 (homologues from Naegleria gruberi); YwqJ-deaminase and Tox-PL ; Pkinase_Tyr (Anopheles darlingi); ZZ (three homologues from Piriformospora indica), DUF1935 (Leishmania and Trypanosoma species); F5_F8_type_C (Bacteroides species); LIM (Entamoeba species); and HemolysinCabind (eight domains in a homologue from Gloeobacter violaceus). It is entirely possible that the association with the peptidase domain for any of these is the result of erroneous gene assembly, where an exon from one gene has been incorrectly assigned to the gene up- or downstream.A variety of other domains are predicted to occur in other, uncharacterized calpain homologues. These include: WXG100 (three homologues from Streptomyces is a good example). For the Tpr peptidase, not all strains of Porphyromonas gingivalis have a homologue: it is known from strains W83 and TDC60, but not ATCC 33277. This distribution is reminiscent of multiple  horizontal gene transfers, and the alternative explanation, multiple gene losses, is unlikely. It would also seem unlikely that horizontal gene transfer occurred from a eukaryote to a bacterium because the eukaryote and bacterial sequences are so divergent. A much more likely scenario would be multiple horizontal gene transfers within the bacteria, then a horizontal gene transfer from a bacterium to a eukaryote, perhaps accompanying the development of the mitochondrion (or chloroplast) from an endosymbiont bacterium.The phylogenetic tree Fig.  appears E. coli and Salmonella typhimurium diverged between 100 and 160 million years ago. A study of 16S RNA sequences has shown that in some species of bacteria from the same genus, the sequence divergence can be as great  or greater  than that between Escherichia and Salmonella [It has been estimated that lmonella . For theSaccharomyces Rim13 peptidase and the androglobins are much longer than any of the other branches, indicating a faster rate of mutation for these sequences. However, it is also clear that the divergences between sequences from closely related bacteria are frequently much greater than between calpain orthologues of human and chicken . It couPhotorhabdus and Diplorickettsia, that cluster with the eukaryote sequences: these are presumably derived from horizontal gene transfers from eukaryote to prokaryote.In support of the hypothesis that horizontal gene transfer occurred between bacteria, the phylogenetic tree Fig.  shows muArguments against the hypothesis of horizontal gene transfers are the degree of dissimilarity, the different domain architectures and length between the sequences. This diversity in sequences argues against a recent origin. However, the bacterial species that have retained (or gained) a calpain homologue are from a variety of environments, being free-living or symbiotic, aerobic, anaerobic or autotrophic. Without knowing the function of a calpain homologue in a bacterium it is perhaps impossible to understand the distribution.It might be expected that the original calpain was a simple protein containing just a peptidase domain, and that the source organism was free-living, perhaps in an unusual, and poorly researched, environment. A reconstructed tree of life based on 31 orthologous domains from 191 organisms whose genomes had been completely sequenced shows a fundamental divergence amongst the bacteria into Gram-positive (Firmicutes and Actinobacteria) and Gram-negative, with the Firmicutes as the most ancient of the bacterial phyla . So it iBrachybacterium, Streptomyces and Frankia) and Actinobacteria (Leucobacter). It is possible that any of these sequences might be representative of the ancestral calpain. The most divergent sequences are from Brachybacterium faecium and B. paraconglomeratum. Both sequences are short, containing only the peptidase domain. However, neither Brachybacterium species is free-living: both were isolated from poultry litter [Frankia alni is a nitrogen-fixing symbiont associated with alder and myrtle [Frankia spp are uncharacterized bacteria, but these may also not be free-living. The calpain homologue from Leucobacter is a C-terminal fragment bearing only the peptidase domain, so it is impossible to tell what the complete domain architecture is. Leucobacter is a soil organism tolerant to high levels of chromium [Leucobacter species with completely-sequenced genomes.The rooted tree shows that thirteen sequences are derived from nodes that predate the bacterial-eukaryote split Fig. , node A.y litter , implyind myrtle . The othchromium . HomologFrankia sp. EUN1f. All genes for bacterial calpain homologues are found on the chromosome: none are on plasmids.All bacterial homologues of calpain are predicted to be peptidases, with the single exception of the homologue from Brachybacterium faecium) to 3,754 residues . A homologue from Nocardia brasiliensis is predicted to be 15,203 residues, but only the N-terminal half of the peptidase domain can be detected. In most bacterial homologues the peptidase domain is the C-terminal domain, but the homologue from Granulibacter bethesdensis is an exception where the peptidase domain is N-terminal (residues 49–247 in a sequence of 681 residues).The sequence length of bacterial homologues varies immensely from 258 residues (Geobacter lovleyi (UniProt:B3E5A8), Segniliparus rotundus (D6ZC85) and Bacteroides thetaiotaomicron (Q8A0R1). These may be periplasmic proteins. However, other mechanisms for protein secretion exist in bacteria. The Gloeobacter violaceus calpain homologue possesses HemolysinCaBind domains, which are known to be important in metallopeptidases known as serralysins for secretion outside of the cell wall and which bind calcium [Gloeobacter calpain homologue the eight HemolysinCaBind domains are N-terminal to the peptidase domain. If these domains are used for secretion, then it is possible that the calpain homologue functions extracellularly.Most eukaryote calpain homologues are known to be intracellular, but this may not be the case for the bacterial homologues. Of the 66 bacterial calpain homologues identified, only three are predicted to have signal peptides: homologues from  calcium . Unlike Bacteroides sp. D2, Salinispora tropica and S. pacifica, are predicted to have transmembrane helices and therefore to be membrane-associated.Three bacterial calpains, from Salinispora). The deaminase domain is also found in a putative ribonuclease YwqJ from Bacillus subtilis (Uniprot: P96722), but no protein with this domain has been characterized biochemically. The Salinispora calpain homologue is also the only one to have Tox-PL domains. As mentioned above, the calpain homologue from Gloeobacter is the only one to possess HemolysinCaBind domains. One homologue from Cyanothece has a CARDB domain, which is predicted to be a cell-adhesion domain also found in glycoyl hydrolases and peptidases, and may indicate that the protein is attached to the cell membrane. Homologues from Herpetosiphon and Segniliparus have WXG100 domains. This is a domain of approximately 100 residues containing a Trp-Xaa-Gly motif. Proteins with a WXG100 domain may be toxins and be exported by the poorly characterized TS77/ESX/ESAT-6 secretion system [Segniliparus is a member of the phylum Chloroflexi. All of the homologues from Bacteroides have a discoidin (or F5_F8_type_C) domain, originally identified in human blood coagulation proteins. Proteins with a discoidin domain are predominantly extracellular. Homologues from cyanobacteria may have PPC domains, which are C-terminal propeptides associated predominantly with secreted peptidases such as vibriolysin [Most bacterial calpain homologues are simple proteins with only the peptidase domain. However, some sequences are multidomain. Domains that occur just in just one homologue are YwqJ-deaminase  are ancestral. Calpain-5 and calpamodulin arose via a gene duplication at node H. This occurred early in vertebrate evolution, because both calpains are present in zebrafish. A further gene duplication at node I in early metazoan evolution, gave rise to calpain-10 , a single fusion event and two genome duplications - the first in an ancestral chordate approximately 550–450 MYA and the second 450–400 MYA. The genome duplications are based on the ‘2R hypothesis’ of Ohno [Comparing this evolution scheme to that of Jékely & Friedrich , who use of Ohno . The ordHydra confirms that the gene duplications that gave rise to the CAPN15 and CAPN5/6 genes occurred before the divergence of the protostomes and deuterostomes, and dates the event even older to before the divergence of the Radiata and Bilatera. The duplication that gave rise to the genes ancestral to CAPN14/14 and CAPN1/2/3/8/9/11/12 also occurred before the divergence of Protostomia and Deuterostomia. There is no evidence from Fig. Branchiostoma floridae) genome concluded, from comparison of the synteny between the amphioxus and human genomes, that two rounds of genome duplication occurred in the vertebrate lineage after the divergence of cephalochordates, and identified 17 ancient chordate linkage groups. Table In the present study, the presence of paralogues of calpains 5, 6, 7, 10 and 15 in Capitella and the Pacific oyster (Crassostrea gigas); and another (XP_002590876) is most closely related to Drosophila calpains A and B. The others are orthologues and paralogues of vertebrate calpains: one (XP_002611732) is an orthologue of calpain 15; one (EEN46888) an orthologue of calpain 10; one (XP_002603439) is paralogous to calpains 5 and 6; and the other two (XP_002607996 and XP_002607981) are closely related to each other and calpains 1, 2, 3, 8, 9, 11 and 12. The latter three lend support to the vertebrate genome duplication hypothesis described above.Amphioxus was not included in the set of model organisms from which calpain sequences were selected because there are questions about the gene assembly. Of the eleven sequences showing homology to the calpain peptidase domain, four were partial and did not overlap all of the active site residues. One sequence (XP_002586006) matched a region between the active site Cys and His and thus did not overlap any of the active site residues; the other three overlapped the N-terminal two residues only. Of the seven sequences with complete calpain peptidase domains, two are more closely related to protostome sequences: one (EEN62485) is most closely related to sequences from in the annelid A variety of additional domains have been appended to the peptidase domain of eukaryote calpains and the nodes where these were acquired (or lost) are indicated by letters on the phylogenetic tree Fig. .Naegleria possess TPR (tetratricopeptide repeat) domains, which are involved in protein-protein interactions [Emericella has a fragment resembling the region around the catalytic serine of α/β hydrolase domain, but with the serine replaced by histidine.Homologues from the sarcomastigophoran ractions . Calpainractions . Other dPlasmodium but not the heterokontophyte Ectocarpus. Calpain-III domains were lost in calpain-15 (node E) and other calpains with zinc fingers.CIII domains are found in almost all eukaryote calpains, and were acquired early in eukaryote evolution (node B): they are found in calpains from the apicomplexan Drosophila [Piriformospora have ZZ zinc finger domains and chlorophytes such as Ostreococcus have zf_GRF zinc-finger domains. In addition, the chlorophyte calpains possess a UBA (ubiquitin-associated) domain. Other protozoa possess domains that interact with nucleotides. A homologue from Entamoeba has a LIM domain, which consists of two zinc finger-like domains. A homologue from Ichthyophthirius has a Brix domain which may be important for binding single-stranded RNA [Zinc fingers are associated with binding nucleotides, and are associated with calpain-15 (node E). Deficiency of this enzyme leads to degeneration of the cells forming the small optic lobes in osophila . Surprisnded RNA .C2 domains are found in calpain-5, calpamodulin and tra-3. These were acquired at node D. Calcium-binding EF hand domains are present in “classical” calpains and were acquired at node I.Calpains had been thought to be exclusively eukaryotic. However, proteins with calpain-like cysteine endopeptidase domains occur in both bacteria and eukaryotes.Homologues have not been found in archaea. The presence of sequences in bacteria apparently derived from a node that predates the bacteria-eukaryota divergence and absence in archaea raises the possibility that the original eukaryote calpain gene was horizontally transferred from a bacterium.Schizosaccharomyces, and some basidiomycetes.Calpains were thought to be essential proteins in eukaryotes, however homologues are absent in a number of single-celled eukaryotes, including algae and Calpains were also thought to be exclusively cytoplasmic, however some bacterial homologues may be secreted because they are predicted to possess signal peptides. Some bacterial and eukaryote calpain homologues are predicted to possess transmembrane regions, suggesting that activity occurs in close proximity to plasma membranes.Calpains were thought to be exclusively calcium-dependent, but although some bacterial homologues retain one of the two calcium-binding sites found in the peptidase domain, none possess the EF-hand domain, and most are predicted not to be calcium-dependent.Streptomyces, only a small minority of species within a genus possess homologues. Such a limited distribution could be taken as evidence of recent, multiple horizontal gene transfers, however, the diversity in sequence, differences in length and domain architecture are arguments against this. Bacteria with homologues do not occur in similar environments, or share similar unusual metabolic pathways, so what the evolutionary pressure is to maintain a homologue is unknown.The distribution of calpain homologues amongst bacteria is unusual. Although homologues are found in several phyla, most phyla do not possess homologues. Within a phylum, only a small minority of species with completely sequenced genomes possess a homologue, and in some cases, e.g., A bacterium possesses only one calpain homologue, but a eukaryote frequently possesses more than one paralogue. Gene duplication in eukaryotes is frequently associated with acquisition of an additional domain, such as a CIII domain, zinc-finger, C2 domain or EF-hand structure. Most vertebrate calpain genes are derived via gene duplication, but there is some limited evidence that some calpain genes result from genome duplication.Neil Rawlings describes the presence of calpains in bacteria and makes the case for their ultimate origin in the bacterial superkingdom. I am convinced by both the calpain affinities of the bacterial proteins and the essence of the proposed evolutionary scenario. Hence, I recommend publication of this work.The author might want to consider the following points in the revision of the manuscript: the versions fused to the WXG domains are likely to be secreted toxins of the T7SS. Such toxins including those with several types of papain-like fold peptidase domains have been described before here: PMID 22731697. The author lists several domains fused to the peptidase catalytic domain in different types of calpains. However, description of the C-termini of several eukaryotic calpains is unclear: two widespread conserved domains might have bearing on the membrane interactions of calpains (see figure 7 in PMID: 24582806).The singular of archaea is archaeon rather archaean which could convey a different sense.I thank the reviewer for pointing out these two references, and I have incorporated both into the text. However, I would prefer the paper to be more focused on the evolution of the calpain domain and I have chosen not to go into depth concerning additional domains.Author’s response: The singular of archaea has been corrected.The proposed review about the calpain family is timely and it is very carefully and well done.I suggest to add files containing the lists of calpains together with annotations  as well as the reasons for inclusions  for future reference. The problem is that any repeated search will result in at least slightly changed outcomes. Further, I suggest an additional figure showing representative domain architecture of calpains.To be able to repeat the search exactly, not only are a list of hits required, but also a copy of the seed alignment used for the HMMER3 search and a copy of the UniProtKB sequence library that was searched against. I have provided an additional figure  which shows the seed alignment used for the search, and an additional table  which lists all the hits returned from the HMMER3 search, with their E-values. Note that the table includes more hits than mentioned in the paper, because the UniProtKB database is not non-redundant and in my list only one of a pair of sequences from the same organism was selected if the sequences were identical or more than 94 % identical. I am unable to provide a copy of the UniProtKB library for obvious reasons. Note also that the existing Supplementary Table and Figure have been renumbered.Author’s response: A figure Fig.  to show


Reconstructing evolution provides valuable insights into the processes of gene evolution and function. However, while there have been great advances in algorithms and software to reconstruct the history of gene families, these tools do not model the domain shuffling events  that drive the evolution of multidomain protein families. Protein evolution through domain shuffling events allows for rapid exploration of functions by introducing new combinations of existing folds. This powerful mechanism was key to some significant evolutionary innovations, such as multicellularity and the vertebrate immune system. A method for reconstructing this important evolutionary process is urgently needed.Here, we introduce a novel, event-based framework for studying multidomain evolution by reconciling a domain tree with a gene tree, with additional information provided by the species tree. In the context of this framework, we present the first reconciliation algorithms to infer domain shuffling events, while addressing the challenges inherent in the inference of evolution across three levels of organization.http://www.cs.cmu.edu/˜durand/Notung.We apply these methods to the evolution of domains in the Membrane associated Guanylate Kinase family. These case studies reveal a more vivid and detailed evolutionary history than previously provided. Our algorithms have been implemented in software, freely available at  Reconstruction of the history of change in a protein family provides valuable insight into processes of mutation and selection. Evolutionary reconstruction can reveal the context and order in which changes occurred, distinguish between shared history and convergent evolution, and identify interacting mutations that together result in a functional shift. Further, considering protein evolution in the context of species evolution makes it possible to correlate mutations with metabolic, physiological, and morphological changes, indicating which mutations are likely to be functionally important.multidomain families are lacking. Genes that encode this large and important class of proteins are characterized by a mosaic of sequence fragments that encode structural or functional modules, called domains. Multidomain families are central to the two-component histidine kinase signaling systems that are the backbone of cellular communication in prokaryotes. In metazoans, the expansion of multidomain families drove the evolution of cell signaling and cell adhesion. In human health, multidomain families are implicated in tissue repair, apoptosis, inflammation response, antigen recognition, and innate immunity.Despite tremendous advances in molecular evolution and phylogenetics, methods for reconstructing the evolutionary history of domain shuffling  models have been used to achieve the computational efficiency necessary for genome-scale analyses -4, and wIn a phylogenetic context, patterns of domain gain and loss have been investigated by treating domain architectures as binary character data; a domain is either present or absent in a given architecture. Given a tree with architectures on the leaves, Wagner and Dollo parsimony have been used to infer ancestral domain architectures and the history of domain gains and losses ,21-24. IThe benefits of the DA model include computational and conceptual tractability. However, it relies on several unrealistic simplifying assumptions: First, the DA model ignores sequence variation within domain superfamilies, treating all instances of a domain family as indistinguishable. Second, the DA model captures change in the form of domain gain and loss, but is not sufficiently powerful to infer the events that caused the change. For example, a domain gain could result from a domain insertion or an ancestral duplication followed by losses. Without an explicit event model, it is not possible to distinguish between these two scenarios. Third, the DA model will make incorrect inferences and underestimate the degree of domain shuffling activity in the presence of parallel gains or losses, as illustrated in Figure Here, we introduce a reconciliation-based framework for multidomain event inference. Reconciliation is the process of inferring evolutionary events by comparing the phylogenies of entities at two levels of biological organization. Given a rooted, binary gene tree, a rooted, binary species tree, and a mapping from extant genes to extant species, reconciliation seeks to infer the association between ancestral genes and ancestral species and the optimal history of gene duplications, gene losses, and horizontal gene transfers (HGTs) that explains this association.DL) model was first proposed by Goodman et al. [DT ) model is NP-complete. The field has expanded with further algorithmic development for both the DL and DT models [temporally feasible; that is, if there exists a partial ordering of the nodes in the species tree that satisfies the temporal constraints imposed by the transfers in the inferred event history. There is no known constructive algorithm for generating minimum cost, temporally feasible DTL-reconciliations. Instead, dynamic programming is used to construct candidate minimum-cost reconciliations, which are then tested for temporal feasibility. A restricted model that only considers transfers between contemporaneous species, reviewed in [Reconciliation for the duplication-loss  family.Here we present an event inference algorithm for reconstructing domain evolution. We define a set of domain shuffling events that includes domain duplication, domain loss, domain insertion within the same genome, and horizontal domain transfer between different genomes. We also consider a restricted model that allows domain insertion, but not horizontal transfer of genes or domains. In the context of this model, the events in the history of a multidomain family are inferred by reconciling a domain tree with a gene tree that has been previously reconciled with a species tree. This procedure also yields the timing of those events relative to gene and species divergences, as well as ancestral domain content. Consideration of the co-evolution of domains with both genes and species enables our algorithm to distinguish between domain losses and gene losses, and between domain insertions within the same genome and domain transfers across genomes. Further, our algorithm can determine whether a domain tree co-divergence is due to a species divergence  or a gene divergence . In order to ensure the biological validity of the inferred evolutionary histories, we present criteria for temporal feasibility that capture the complex temporal constraints associated with reconciliation involving domains, genes, and species. The implementation of these algorithms is freely available at GS T= , a rooted, binary domain tree, D T= , and a mapping, L(DT), the leaves in the domain tree, to L(GST), the leaves in gene tree, the goal of domain shuffling event inference is to infer the association between ancestral taxa in the species, gene, and domain trees and the set of events that best explains this association. In our model, we define the following set of events:Given a rooted, binary gene tree, Co-divergence (C ) A bifurcation in the domain tree that arose through a bifurcation in the gene tree. The gene tree bifurcation may have arisen via speciation (SC), gene duplication (DC), or gene transfer (TC).Duplication A single domain is copied resulting in two separate copies of the domain within the same gene.Loss A domain is deleted from the gene (and genome).Domain insertion (I) A new copy of the domain is inserted into a different gene within the same genome.Horizontal domain transfer A new copy of a domain is inserted into a gene in a different genome.DG M: D V→ GV, the association between ancestral domains and ancestral genes. The result is a DG reconciled domain tree, T= , in which every node, d ∈ DV, is annotated with DGM(d) = g, where g is the ancestral gene that contained domain d; d; and E(d), the event that caused the divergence at d. Co-divergences and domain duplications correspond to internal nodes in DGT. Each insertion and transfer corresponds to an edge,  ∈ DE, where d2 is the inserted (or transferred) domain and d1 is the donor domain. In a parsimony framework, the cost of a reconciliation is ε κis the cost of event ε and ε nis the number of occurrences of ε in the reconciliation.Reconciliation is the process of inferring Formally, we define the problem of inferring a domain event history as follows:Domain events: .Input: A rooted, binary domain tree, D T= ; a rooted, binary, DTL-reconciled gene tree, GST; and a leaf mapping, Output: The set of all temporally feasible, domain shuffling histories DG Tthat minimizeSolving DE-DTL entails challenges that do not arise in gene tree-species tree reconciliation. First, when a domain co-divergence is inferred, additional information is required to determine whether the co-divergence is the result of a speciation, a gene duplication, or a gene transfer. Second, domain insertions are horizontal events between genes in the same species. In contrast, domain transfers are horizontal events between different species. An extra test is needed to ensure that the correct event is inferred. Third, a missing taxon in the domain tree may be due to a domain loss or the loss of a gene. If the latter, then the loss should not be included in the event cost of the domain-gene reconciliation. Finally, when testing for temporal feasibility, temporal constraints arising from gene transfers, domain transfers, and domain insertions must all be considered.Notation: Given a tree i T= , ρi designates the root of the tree. For nodes u, v ∈ i, pV(v) denotes the parent of v and l(v) and r(v) denote the left child and right child of v, respectively. If u is on the path from v to ρi, then u is an ancestor of v, designated u ≥i v, and v is a descendant of u, designated v ≤i u. If u and v are incomparable, designated GS Tand a reconciled domain tree DG, s T= GS M(g) is the species s ∈ S Vthat contained gene g ∈ G Vand g = DG M(d) is the gene g that contained domain d ∈ DV. The species containing d is s = DS M(d), where DS M(d) = GS M(DGM(d)).locus model [Here, we present an algorithm for the domain event inference problem for multidomain families evolving according to a us model , in whicus model ,3,43,44 us model ,45-48. MIn the context of this model, we introduce an algorithm (Alg. 1) for inferring domain events by reconciling a domain tree with a gene tree that has been previously reconciled with a species tree.Algorithm 1 DE-DTLInput: TS; TGS; TD; Output:1     2     costCalc3     4     d ∈ D Vin post-order and determines the cost of the subtree rooted at d for each possible event at d. This information is stored in the cost and event tables, Kd and Hd. In the second pass, traceback traverses D Ttop-down to generate candidate reconciliations from the information stored in the cost and event tables. In general, there may be more than one optimal set of domain events that reconcile D Twith GST. The second pass generates all candidate event histories of minimum cost, m is the number of candidate histories. In the final step, each candidate history is tested for conflicting temporal constraints. The output is the set of all temporally feasible histories, f is the number of feasible, optimal reconciliations.The algorithm proceeds in four steps. First, an additional data structure, Our domain shuffling event inference algorithm is based on the existing framework for gene-species tree reconciliation with a DTL model ,49, but addLoss: We construct GS Tby placing pseudonodes on each edge (p(g), g) ∈ GE, on which losses occurred. Each pseudonode represents an ancestral gene that was present in the species lineage from GS M(p(g)) to GS M(g), but cannot be observed because of a gene loss in one child of each of the intervening species. Let GS M(g) and GS M(p(g)) that are absent from G Tdue to gene losses. We insert pseudonodes ϕs1⋯ ϕsl between g and p(g) such that ϕs1becomes the new parent of g, ϕsi is the parent of ϕsi−1for i = 2 ⋯ l, and p(g) becomes the new parent of ϕsl. For each pseudonode, ϕs, we attached a pseudoleaf, sλ, where S' is the child of s that is not on the path from GS M(g) to GS M(p(g)) and ST, because if a gene is missing from an entire clade of species, then the most parsimonious explanation is a single gene loss in the root of the clade.u in D Twas in a genome on the Eudicot-Rosid lineage because of the position of the pseudonode ϕR, between u and d1_g1_A. Without the pseudonode, it would not be possible to determine whether or not u predates the divergence between Apple and Berry.The addition of pseudonodes allow for more precise estimation of the association between a node in the domain tree and a lineage in the gene tree. Consider, for example, the evolutionary history of the hypothetical family shown in Figure B and ℓC), indicating that only one of the missing domains is due to a domain loss (d2_g2_B).Pseudoleaves are also used to distinguish between gene and domain losses. The domain tree in Figure costCalc: Once the extended reconciled tree has been constructed, the first pass takes D Tand d) for each d ∈ D Vin post-order (Alg. 2). The gene association and event at d depend on l gand rg, the genes associated with the children of d. The outer loop in cost Calc enumerates all possible  pairs, and for each pair determines the gene associations and events implied by DGM(l(d)) = l gand DG M(r(d)) = r g. The cost of each configuration is stored in Kd. A tuple consisting of the event and the mappings of the children of d are stored in Hd. The logic for these assignments is as follows.l gand r gare comparable, then d is g = lca. Horizontal events and co-divergences both result in l gand r gare incomparable, both possibilities are considered. For the co-divergence case, the associated gene is again g = lca. The type of co-divergence is determined by inspecting the gene event associated with gene node g in l gor r gmay be associated with d; i.e., either l gor r gcould be the donor of the domain.Domain duplication is the only event that results in children mapped to comparable genes. Thus, if d to p(d) is the number of non-pseudonodes between g = DGM(d) and p g= DGM(p(d)). If g and p gare not pseudonodes, then this quantity is Δ(g) − Δ(pg) − 1, where Δ (g) is the depth of g in the original gene tree, GT. However, if g is a pseudonode, ϕ, then Δ (g) is undefined. We define Δ (ϕ) to be the depth of the first non-pseudonode ancestor of ϕ in u), where u ≥G ϕ and there exists no v ∈ GV, such that u ≥G v ≥G ϕ. This effectively jumps over all pseudonodes between ϕ and u. If g is a pseudonode and its first non-pseudonode ancestor is a loss, then directly using the depth of the first non-pseudonode ancestor will fail to count this loss. In this case, the number of losses is incremented by one (lines 15 and 16). No such correction is needed when p gis a pseudonode.For each scenario, the cost of domain losses must also be determined, excluding cases where domain loss is due to gene loss. Recall that pseudonodes correspond to gene losses. Therefore, the number of losses, d) is a co-divergence, then the number of losses is (Δ (l g) − Δ (g) − 1) + (Δ (rg) − Δ (g) − 1). If E(d) is a duplication, l, grg, and g should all be at the same depth, and the number of losses is (Δ (l g) − Δ (g)) + (Δ (rg) − Δ (g)).This formulation allows for efficient calculation of the number of losses under each scenario considered in costCalc. If E and determine the labels of the genes associated with the children of d. The losses that occurred between d and p(d) are inferred in a climbing procedure [g that is missing between DG M(d) and DGM(p(d)), a loss is inferred in g', the child of g that is incomparable to DGM(d). If g is a pseudonode in GST, then g' is a gene loss, sλ. Otherwise, a domain loss in g' is inferred.rocedure ,50. For Algorithm 2 DE-DTL: CostCalcInput:D; Output: Kd, costCalc(d) {if d 1 ∈ L(DT) {for each 2    if 3        table }else 4            {5                6                table7            }8        }9    return10 }11 costCalc(l(d)), costCalc(r(d))12 for each 13 g = lca14 15 if(gl is pseudonode) { 16 if(gr is pseudonode) { 17 if NOT 18     // Duplication case;19     20     21     table22 }23 else {24     // Co-divergence case;25     if            // Duplication26     else if    // Transfer27     else { ε ← S C}                     // Speciation28     table29     // Domain insertion or transfer case;30     if MGS (l g) = GSM(rg) {          // Same genome31         // Domain insertion case;32         table      l to gr// g33         table     r to gl// g34     } else if {35         // Domain transfer case;36         table l to gr// g37         table r to gl// g38     }39   }40 }table {41 42 if43     44     45 } else iflist ) }46 }D Tis traversed repeatedly until all minimum cost histories have been generated. The traceback requires no modification to address reconciliation on three levels and is essentially the same as the traceback algorithm for gene tree-species tree reconciliation, as described in [If there is more than one optimal candidate reconciliation, ribed in .checkFeasibility: Once all candidate reconciliations have been returned, their temporal feasibility is checked to ensure that each solution is valid. This requires criteria for temporal feasibility that accommodate the interplay of domain evolution with gene and species evolution. To be temporally feasible, an inferred domain event history must satisfy two criteria. First, it must be possible to assign a timestamp to every species tree node, such that the timestamps are consistent with the temporal constraints imposed by the combined set of gene and domain transfers. Second, it must be possible to assign a timestamp to every gene tree node, such that the timestamps are consistent with the temporal constraints imposed by the combined set of domain transfers and domain insertions.species timing graph, g is horizontally transferred from species s1 to s2, then every domain in g is also implicitly transferred from s1 to s2. Thus, every gene transfer, , corresponds to one or more edges in the domain tree. Let D ⊂ D Ebe gene and domain transfer edges in DGT, respectively, and let d1, d2), such that ∃ ∈ ΛG, where g1 = DGM(d1), g2 = DGM(d2). Then, For the first criterion, we introduce a The temporal constraints on species are encoded in i s= p(js), then i smust have predated js. ∀ ∈ SE, add  to 1 If species i sto species j soccurred, then i sand j smust have co-existed. Further, the parent of j smust have predated is, and vice versa. p(is), js) and (p(js), is) to i s= DSM(id) and j s= DSM(jd).2 If a transfer from species d1, d2) and (s1 = DSM(d1) and s2 = DSM(d2) must have occurred no later than species p(s1), p(s1), p(s2), p(s2), 3 If two transfers occur in the same lineage in the reconciled domain tree , then the species corresponding to the donor and recipient of the more ancestral event must have occurred no later than the donor and recipient species of the more recent event. Let , then i gmust have predated jg. i, gjg) to 1 If gene i gto jg, then i gand j gmust have co-existed. Further, the parent of i gmust have predated j gand vice versa. ∀ ∈ {(DGM(d1), DGM(d2)) |  ∈ ΛD ∪ ○D}, add (p(ig), jg) and (p(jg), ig) to 2 If a domain was transferred or inserted from gene d1, d2) and  and g2 = DGM(d2) must have existed no later than p(g1), p(g1), p(g2), p(g2), 3 Let  [tG.A domain event history is temporally feasible  + |Et|)  is used GST. The number of pseudonodes that can be added to the edge above a node in GS Tis at most Sh, where S his the height of ST. Therefore, the complexity of addLoss is d ∈ D Vand loops through all pairs of gene nodes ld, g, and r grequires only constant time, the total complexity is Our algorithm infers domain shuffling events in polynomial time. addLoss constructs d ∈ DV, the losses between d and p(d) are calculated by climbing from DGM(d) to DGM(p(d)). This requires d, where GS Tis bounded by S hand there are at most G hnodes that are not pseudonodes contributing to O(ShGh|DV|).traceback constructs a single candidate solution in a preorder traversal of the domain tree. Looking up the event, the number of losses, and mapping of each node requires constant time. For each node O(|tV| + |tE|).To determine whether a candidate solution is temporally feasible, checkFeasibility tests both the gene and species timing graphs for cycles, using a topological sorting algorithm that runs in For the species timing graph, SE| edges are added to 1 |DE| edges because 2 Two edges are added to DE|2, the number of added edges is bounded by 4|DE|2.3 Four edges are added to O(|SV| + |SE| + |DE| + |DE|2). Because |SV| = O(|S E|) and |DE| ≥ SE|, |DE|2 is the dominant term. Therefore, the complexity can be written as O(|DE|2).Combining all three constraints, the complexity of cycle checking in the species timing graph is O(|DE|) and O(|DE|2). Thus, the complexity of cycle checking in the gene timing graph is DE| ≥ |GV| and |DE| ≥ Sh, this complexity can be written as For the gene timing graph, DE-DTL model includes horizontal transfer of both genes and domains and is suitable for reconstructing the history of domain shuffling events in species that accept foreign DNA. However, this model is not appropriate for analysis of multidomain families in species that do not participate in genetic exchange with other species. For such families, we also consider domain shuffling inference for the restricted model without transfer events. This model is particularly well-suited to the large and complex multidomain families in vertebrates [The tebrates , in whicDomain events: .Input: A rooted, binary domain tree, D T= ; a rooted, binary, DL-reconciled gene tree, GST; and a leaf mapping, Output: The set of all temporally feasible, domain shuffling histories DG Tthat minimizeDL model, not the DTL model. An extended gene tree, The overall structure of the algorithm for DE-DL is the same as for DE-DTL, i.e., Alg. 1. However, since transfers are not allowed, the input gene tree must be reconciled with a species tree under the The worst-case time complexity for costCalc is the same as in DE-DTL, but because domain transfers are not considered and domain insertions are only allowed between genes in the same species, the number of mappings that can be considered is greatly restricted. This suggests a faster run-time in practice.O(|DE|2).Determining temporal feasibility is a simpler procedure, since only the gene timing graph must be constructed and tested for cycles. The complexity for gene tree timing graph is Here we demonstrate the practical application of our approach using several examples from the Membrane-associated guanylate kinase (Maguk) family ,53.The Maguks are multidomain scaffolding proteins Figure  that plaGuK) domain, in combination with various adapter domains that anchor downstream pathway proteins to the scaffold. There are six Maguk subfamilies, each with a characteristic domain architecture  for all members of the Maguk family in mouse, human, and chicken  subfamily, and the PDZ domain, which is found in all Maguk subfamilies, except the calcium channel β (CACNB) proteins.With this in mind, we constructed a n Figure . Using tThe L27 domain. The MPPs mediate protein complex formation at cell junctions and play a role in establishing cell polarity during development [MPPs contain a core GuK-SH3-PDZ domain architecture and, except MPP1, two N-terminal L27 domains  in human, mouse, and chicken MPPs (see Methods). Outside of the MPP subfamily, only a few proteins encoded in the human genome possess L27 domains . These proteins possess only a single L27 domain, rather than a tandem pair. Structural studies partition single-copy and tandem L27 domains into two distinct subtypes, with distant sequence homology [MPP L27 domains are more closely related to each other than to other L27 domains and, thus, satisfy the assumption of our algorithm, which only considers domains within the current gene family.To determine whether the homology . This suL27 phylogeny was reconciled with the GuK reference tree  is consistent the hypothesis that the L27-2 and GuK domains co-evolved without shuffling. However, the reconciled L27-1 tree  suggestn Figure ).L27-1 domain insertion, we generated the 95% confidence set of trees with high likelihood scores [MPP2/6/3/7 form a clade, which is consistent with the hypothesis that the L27-1 domain was replaced in the MPP2/6 ancestor.Phylogenetic error is of particular concern in domain tree reconstruction, because domain sequences tend to be short and weakly conserved . To inved scores ,63, as dd scores  in FigurMPP evolution [L27-1 domains in MPP2/6 and CASK share a unique, common ancestor. In contrast, our analysis predicts that the L27-1 domains in MPP2/6 and MPP3/7 are more closely related. This has functional, as well as evolutionary, implications. A vertical descent model implies that MPP2/6 are likely to be most functionally similar to CASK, their closest MPP relative. Our analysis predicts that MPP2/6 are more likely to be similar to MPP3/7, with respect to L27 mediated interactions. This is consistent with recent experimental results that show that MPP2, MPP3, and MPP7 share a common L27-mediated complex formation mechanism, while CASK uses a different mechanism [Previous models of volution  predicteechanism .The PDZ domain. All members of the Maguk family, except for the CACNBs, have at least one PDZ domain. Using our newly developed methods, we sought to understand how the varied number of PDZ domains arose in this family. Was there a single PDZ in the earliest Maguk that expanded independently in different subfamilies? Or was there a round of ancestral domain duplication followed reciprocal, independent losses?PDZ domains . The inferred events were scored and ranked based on this support score  tree for the PDZ domain shuffling shows several interesting trends that disagree with prior analyses based on Wagner parsimony. This is especially true for the ZO and Carma subfamilies.Our reconstructed history of ZO/DLG5 subfamily have a cassette of at least three tandemly arranged PDZ domains (and a fourth in DLG5). It has been previously been proposed [ZO/DLG ancestor gave rise to this cassette, which was subsequently inherited by all ZOs and DLG5, with an additional duplication resulting in the fourth PDZ in DLG5.All members of the proposed  that twoZO ancestor had a single PDZ domain that was vertically inherited in ZO2 and ZO3, but lost in ZO1. Second, the cassette expansion was the result of a domain insertion from MAGI into the common ancestor of ZO2 and ZO3. While these insertions are weakly supported, the fact that both the donor domains (PDZ4 and PDZ5 in MAGI) and the recipient domains (PDZ1 and PDZ2 in ZO2 and ZO3) are adjacent is suggestive. Third, the PDZ cassette in ZO1 was the result of three, highly supported domain insertion events from ZO2 in amniotes. Considering the fact that these three PDZs are tandemly located in the domain architecture, it is possible that this was the result of a single insertion event involving all three PDZ domains simultaneously. Interestingly, the C-terminal PDZ domain in DLG5 and the C-terminal PDZ in the ZO proteins were both vertically inherited from the same ancestral copy. The other DLG5 PDZ domains were the result of insertions from the DLG and Carma subfamilies.Our reconstruction, based on domain tree reconciliation, tells a very different story Figure . First, Carma subfamily. All Carma genes have a single PDZ domain that is predicted to be the result of vertical inheritance in Wagner parsimony analysis. According to our domain tree reconciliation analysis, however, this is not the case , parallel losses (PDZ in Carma3), and replacement of a domain with another domain from the same family (L27 in the MPP subfamily). Further, our results suggest that some orthologous proteins with identical architectures nevertheless contain paralogous domains. This has intriguing implications for orthology-based function prediction.As a demonstration of the power of a reconciliation-based approach, we presented an analysis of the domain shuffling in the multidomain Maguk family. This analysis uncovered multiple evolutionary scenarios that could not be reconstructed with Wagner parsimony, including parallel gains  domain, which is unique to this gene family. Domain trees were constructed for the two constituent domains that were analyzed in this study: the L27 domains of the MPP subfamily and the PDZ domains, which are present in all Maguks except for the CACNBs. Sequence acquisition: An initial set of amino acid sequences of the GuK, PDZ, and L27 domains from Gallus gallus, Homo sapiens, and Mus musculus was obtained from te Velthuis et al. [A reference gene tree and two domain trees were constructed for various components of the Maguk gene family. The gene tree was inferred using the non-functional guanylate kinase  estimation. Model selection for all reconstructions was first performed on the trimmed alignment using Modelgenerator [PDZ was LG+G, as supported by AIC1, AIC2, and BIC; the best for GuK was JTT+G, as supported by AIC2 and BIC (AIC1 supports JTT+I+G); and the best for L27 was JTT.enerator . The besGuK, PDZ, and L27 domains were generated in PhyML [GuK tree was rooted using the sequences of three functional guanylate kinase domains, from which the GuK domain is derived [GuK tree with these outgroups, one nearest neighbor interchange operation was performed on the least supported branch (bootstrap = 33), so that the MPP5 GuKs in all species form a monophyletic clade, instead of grouping some MPP5 with MAGI. The L27 tree was rooted based on the duplication that gave rise to L27-1 and L27-2.ML trees for the in PhyML  with 100 derived . After rTreePuzzling: Support for the L27 and PDZ domains was further evaluated by generating a 95% confidence set of trees based on the Expected Likelihood Weight method as implemented in CONSEL [L27 confidence set using SplitsTree [n CONSEL . Tree-Pun CONSEL  was usedlitsTree  with an Event support calculation: For the PDZs, the confidence set consists of 121 trees. To avoid spurious event inference, each tree was manually rooted, so as to separate MPP and MAGI from the other Maguk subfamilies, consistent with the GuK tree. Using the DE-DL model, each tree was then reconciled with the reconciled GuK tree, as described in the Results and Discussion section, using event costs D, the support value for insertion ε isFor each insertion observed in at least one reconciliation, we calculate its support as follows: To ensure that each of the 121 trees has equal voting power, the multiple optimal solutions from each tree were averaged. Given a confidence set d1, d2) and (DG Tand DT(d) is the subtree rooted at d.where The authors declare that they have no competing interests.The project was conceived and directed by MS and DD. MS, HL, and MX designed and implemented the algorithms. Empirical case studies carried out by KS, MX, and MS. MS, DD, MX, and HL wrote the manuscript. All authors read and approved the manuscript.Additional file containing supplementary figures: stolzer32 supplemental.pdf Format: PDFClick here for file


Small GTPases participate in a broad range of cellular processes such as proliferation, differentiation, and migration. The exchange of GDP for GTP resulting in the activation of these GTPases is catalyzed by a group of enzymes called guanine nucleotide exchange factors (GEFs), of which two classes: Dbl-related exchange factors and the more recently described dedicator of cytokinesis proteins family exchange factors. Increasingly, deregulation of normal GEF activity or function has been associated with a broad range of disease states, including neurodegeneration and neurodevelopmental disorders. In this review, we examine this evidence with special emphasis on the novel role of Rho guanine nucleotide exchange factor (RGNEF/p190RhoGEF) in the pathogenesis of amyotrophic lateral sclerosis. RGNEF is the first neurodegeneration-linked GEF that regulates not only RhoA GTPase activation but also functions as an RNA binding protein that directly acts with low molecular weight neurofilament mRNA 3′ untranslated region to regulate its stability. This dual role for RGNEF, coupled with the increasing understanding of the key role for GEFs in modulating the GTPase function in cell survival suggests a prominent role for GEFs in mediating a critical balance between cytotoxicity and neuroprotection which, when disturbed, contributes to neuronal loss. Figure 1). The five subfamilies of small GTPases  have highly diverse cellular roles. For example, GTPases of the Rho family  guanine nucleotide-binding proteins that function as binary molecular switches by alternating between an active GTP-bound state and an inactive GDP-bound state and in doing so, regulate the activation of several effectors -homology family in which the catalytic function is exerted by a tandem of two domains [a Dbl-homology domain (DH) and an adjacent pleckstrin-homology domain (PH) that together bind other proteins and phospholipids; Figure 1).The activation of small GTPases such as Rho or Rab proteins requires the participation of specialized enzymes called guanine nucleotide exchange factors (GEFs) which catalyze the exchange of GTP for GDP in the GTPase with the requirement of MgDbl GEFs are a highly complex family of about 80 proteins containing a variety of differing functional domains, including: (1) domains that modulate protein–protein interactions such as Proline rich domains , PDZ binFigure 2). This module functions similarly to the DH-PH module in that DHR1 mediates phospholipid binding and membrane targeting of Docks while DHR2 catalyzes nucleotide exchange through a mechanism distinct from the Dbl domain. The Dock proteins are further differentiated from the classical GEFs in that they exhibit specificity for activation of Rac and/or Cdc42, but not RhoA or other members of the Rho family. Eleven Dock proteins have been described in mammals . They are divided into four subfamilies based on sequence homology: Dock-A ; Dock-B ; Dock-C , and Dock-D . Dock-A and -B members contain both a Src homology 3 (SH3) domain in the N-terminal region that can bind to the adaptor proteins ELMO (Engulfment and Motility) 1, 2, and 3 in addition to a proline-rich domain in C-terminus portion that binds Crk proteins. Dock-D members contain an N-terminal PH domain. Conversely, Dock-C members lack other recognizable domains outside of the DHR1–DHR2 module 1-DHR2 module (Table 1). In Alzheimer’s disease, there is an increase in the number of Dock2-expressing microglia, a finding of pathogenic significance given that Dock2 deficiency reduces the size of β-amyloid (Aβ) plaque in cerebral cortex and hippocampus of a mouse model of AD  family of proteins play a key role in multiple aspects of neuronal development, including both axonal and dendritic differentiation, in addition to involvement in both neuroinflammation and the differentiation of Schwann cells . Not surel of AD , 2013. Iel of AD . Dock4, el of AD . It is aTable 1; . Less isTable 1; . AlthougTable 1; , the autIt is however Dock3 that has the greatest biological and therapeutic implications for neurodegenerative disorders, in part due to the fact that it is the only Dock protein that demonstrates high tissue specificity for the CNS. Initially discovered through a yeast two-hybrid assay as a binding partner to presenilin 1 that shares 40% homology with Dock1/180 [and termed presenilin binding protein (PBP); There is thus significant evidence to implicate the Dock family of GEFs in both neurodegeneration (largely restricted to Alzheimer’s disease) and a range of both developmental and neuropsychiatric disorders. As suggested earlier, this is not entirely unexpected given the integral role of GEFs in neural development.While over 80 GEFs containing a Dbl-homology domain have been described, to date only a finite number have been associated with neurodegenerative disorders. The most lethal of these, presenting most often as a childhood disorder, is associated with mutations in any of the subunits of GEF eIF2B (eukaryotic initiation factor 2B). Conversely, alterations in the metabolism of the recently discovered Rho guanine nucleotide exchange factor (RGNEF or p190RhoGEF) are associated with the adult onset fatal disorder ALS. As is also typical of these proteins, the mechanisms by which these GEFs mediate neuronal degeneration is complicated by their pluripotential functions.The eukaryotic initiation factors (eIFs) play an essential role in the initiation of translation in eukaryotes by mediating the formation of a complex between Met-tRNA (initiator methionyl-tRNA), the AUG start codon of the mRNA and the 80S ribosome. Key to this process is eIF2, a classical G-protein which recruits Met-tRNA to the 40S ribosome when in its GTP-bound (active) form. Inactive GDP-bound eIF2 is regenerated by the GEF eIF2B in a process that is the major point of regulation of protein synthesis in all eukaryote cells . eIF2B cMutations in the eIF2B ε-subunit were first described in patients with clinical and MRI characteristics of childhood ataxia with CNS hypomyelination (CACH)/leukoencephalopathy with vanishing white matter (VWM) neurodegenerative syndrome . SubsequFigure 2). In fact, puratrophin-1 is a bona fide GEF that facilitates activation of the small GTPases Rac1, Cdc42, and RhoA. The overexpression of this protein induces rearrangements of the actin cytoskeleton, specifically enhancing the formation of lamellipodia and filopodia (Puratrophin-1 (Purkinje cell atrophy associated protein-1) is associated with autosomal dominant spinocerebellar ataxia (ADCA), a cerebellar pathology that manifests primarily as lack of motor coordination. This protein contains spectrin repeats, a GEF for Rho GTPases containing the classical Dbl homologous domain  and a CRilopodia . Mutatioilopodia .Figure 2). ARFGEF2 has been postulated as a new potential biomarker of Huntington’s disease (HD) because of its significant up-regulation in blood samples of HD patients (The regulation of protein and lipid transfer within eukaryotic cells is largely mediated by vesicles that are formed in response to the need to transport between cellular compartments. The ADP-ribosylation factor (ARF) GEF family of proteins are important regulators of the initiation of vesicle formation in that they regulate the ARF GTPases through a GDP/GTP exchange . The ARFpatients .Figure 2). CalDAG-GEFI is highly enriched in striatum and targets the small G proteins, Rap1 and Rap2 (Another guanine nucleotide exchange factor (GEF) that has been described as being involved in HD is a calcium and diacylglycerol-regulated GEFI , also called RasGRP2  which manifests with a progressive retinopathy leading to blindness, dementia, epilepsy, and motor dysfunction. Microarray analyses have identified a GEF protein for small GTPase of the Ras family, Ras-Related Protein GEF1 , which is dysregulated regardless of the clinical course of CLN3 disease, suggesting that this protein could be a potential biomarker  are the most common inherited neurodegenerative disorders mainly affecting children. That associated with mutations in the iomarker . This isiomarker .Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the progressive loss of motor neurons leading to death within three to five years of symptom onset. While currently 1:350 men and 1:450 women are affected, increased incidence rates are predicted as the population ages . Most caNEFL, NEFM, NEFH, respectively). The traditional view of NF function is that they are integral to the formation and maintenance of the cytoskeleton, including axon diameter and integrity. The contemporary view includes a critical role for NF as a protective sink for reactive nitrating species  including RNA binding proteins such as mutant superoxide dismutase 1 (mtSOD1), TAR DNA binding protein (TDP-43), fused in sarcoma/translocated in liposarcoma (FUS/TLS), TATA-binding protein-associated factor 15 (TAF15), Ewing sarcoma breakpoint region 1 protein (EWS), and RNA binding motif protein 45 RBM45;  in addit species , in cell species , and in  species .NEFL mRNA in spinal motor neurons while the levels of NEFM and NEFH mRNA are unchanged .A comparison of alsin with other proteins reveals the presence of several interesting motifs. In its N-terminus domain, alsin contains a GEF domain for Ran GTPase, called RCC1. Alsin has been reported to have 5 RCC1 repeats that form a β-propeller similar to RCC1 protein in which this domain was first described . The midALS2 is a causative gene for the juvenile autosomal recessive form of multiple motor neuron diseases, including type 2 ALS  is a Rho-specific nucleotide exchange factor (GEF) that is part of the Dbl family of GEFs encoded by the 90RhoGEF . Mouse Rotubules . mRGNEF otubules , the 14-otubules  and the otubules . The intotubules .Figure 2). It is this latter aspect that renders RGNEF as being unique amongst the remaining members of the Dbl-homology GEF family of proteins in that it is the only member that combines RNA binding activity with GEF activity.Structurally, RGNEF has five important domains: an L-rich region and a cysteine-rich Zn binding domain in the amino terminus half of the protein; a Dbl homology domain (DH), a Pleckstrin homology domain (PH), and an RNA binding domain in the carboxyl terminus half of the protein  of the murine anscript , 2001. Tanscript , an untranscript . Interesanscript  in whichtability .NEFL mRNA (NEFL mRNA in whole cell lysates (including proteins and RNA) from spinal cord in ALS but not control cases  would play a key role in the pathogenesis of ALS. Our group determined that hRGNEF, similar to mRGNEF, is an RNA binding protein that regulates the stability of EFL mRNA . We obseol cases . This obin vitro .Figure 3) co-localized with other RNA binding proteins, suggesting a common pathway of NCI formation in which multiple RNA binding proteins co-aggregate . The observation that RGNEF is able to immunoprecipitate NEFL mRNA from ALS spinal cord homogenates and not from controls suggests a preferential interaction in ALS that would be predicted to suppress NEFL mRNA levels, consistent with the loss of NEFL mRNA detected by in situ hybridization in ALS motor neurons (NEFL and other mRNAs stability. In a similar vein, the sequestration of RGNEF within NCIs in ALS would also be predicted to render it unavailable to participate in the modulation of RhoA GTPase activation and potentially lead to neuronal death. Understanding this intricate relationship is currently the focus of ongoing studies.In this context, the example of RGNEF and its association with ALS is notable as this is the only GEF that combines RNA binding activity with the capacity to activate RhoA GTPase ( neurons . This maNEFL mRNA stability is critical to the formation of pathological NCIs in spinal motor neurons, the concomitant loss of GEF activity due to RGNEF sequestration may be sufficient to render the cell incapable of responding to the subsequent cellular stressors. Such a conceptualization is consistent with the increasing evidence for oligogenic inheritance in ALS where a second gene might participate as a critical modulator of the pathological phenotype of the disease (It is also interesting to postulate that alterations in GEF activity, while not directly inducing a disease state, could function as a critical “second insult” through the loss of one or more effector pathways. For instance, while the loss of  disease . In this disease .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The hydrocarbon-stapled BH3 peptide not only induces apoptosis but also possesses potent antitumor activity in vivo.The decision phase of apoptosis is mainly regulated by the Bcl-2 family, which renders these proteins potential targets for cancer therapy through apoptotic mechanisms. Bcl2 family members have homology clustered within four conserved Bcl2 homology (BH) domains . Only the antiapoptotic proteins, such as Bcl2, Bcl-XL, Bcl-w and A1, bear the NHin vivo [Recently, an approach called “structure-activity relationship by nuclear magnetic resonance (SAR by NMR)” has been used for the discovery of novel Bcl2/Bcl-XL inhibitors . To genein vivo , this su2-terminal BH4 domain (aa-6-31) of Bcl2 is also required for its antiapoptotic function [in vivo, which provides direct evidence that the BH4 domain contributes to the survival function of the prosurvival Bcl2 family members. Since the BH4 domain is critical for the antiapoptotic function of Bcl2, this amphipathic helix should also be an ideal structural target for the screening of small molecules that may bind to this domain and interfere with Bcl2's survival activity. Solution structure of the BH4 domain exhibits multiple potential binding pockets for small molecule docking [In addition to the hydrophobic surface binding pocket, the NHfunction . The BH4omain aa--31 of Bc docking . Recentl docking .In summary, BH3 mimetics (ABT-263 and ABT-199) and the BH4 antagonist (BDA-366) are two different classes of Bcl2 inhibitors that target Bcl2 at the hydrophobic binding pocket or BH4 domain, respectively. Disruption of Bcl2's antiapoptotic function via BH3 mimetics or the BH4 antagonist may represent attractive strategies for cancer treatment.