An updated evolutionary study of the Notch family reveals a new ancient origin and novel invariable motifs as potential pharmacological targets

Dataset collection and filtering

Data was collected from the NCBI database (ncbi.nlm.nih.gov) as previously described in Mitsis et al. (2020), towards to extracting the amino acid sequences that are related to the Notch proteins using the keyword “Notch” (Mitsis et al., 2020). Protein sequences that responded to the query but did not include the Notch family members were eliminated from the primary dataset, by using related keywords and regular expressions techniques in the header information, and local alignments with reference protein sequences. Furthermore, a final dataset for each species class was produced by using internal protein alignments and protein identity score. Duplicated protein sequences in each species that were found share 95% > protein identity within the dataset were removed. In total, 25,761 Notch family related protein sequences were identified from several species, and a dataset containing 603 unique, non-duplicate protein sequences was created (Dataset S1).

Multiple sequence alignment

Multiple sequence alignment (MSA) was executed using the MATLAB Bioinformatics Toolbox, utilizing a guide tree and the progressive MSA method as previously described in several studies (Mitsis et al., 2020; Papageorgiou et al., 2016; Sobie, 2011). Pairwise distances among sequences were estimated based on the pairwise alignment with the “Gonnet” method and followed by calculating the differences between each pair of sequences. The Neighbor-Joining method was used towards to estimating the guide tree by assuming equal variance and independence of evolutionary distance estimates (Dataset S2). Finally, consensus sequence was calculated and visualized through the JalView platform (Waterhouse et al., 2009) using the multiple sequences alignment results and parameters including amino acid conservation. The commentary section of Jalview, which presents the amino-acid conservation using logos and histograms, was further observed to uncover innovative motifs.

Notch family protein clusters

Notch family protein clusters were identified by using phylogenetic analysis. The phylogenetic analysis was performed using the MATLAB Bioinformatics Toolbox (Kufareva & Abagyan, 2012) utilizing the Unweighted Pair-Group Method (UPGMA) (Michener & Sokal, 1957; Pavlopoulos et al., 2010; Sneath & Sokal, 1973) while the matrix of the pairwise distances was calculated using the protein-adapted Jukes-Cantor statistical method (Papageorgiou et al., 2016; Yang & Zhang, 2008). The constructed phylogenetic tree visualized using MEGA radiation option and the final Notch family protein clusters separated in different sub-datasets using a threshold. In total, eight major sub-clusters including Notch Bacteria, Notch_Plants, Notch_Invertebrates, Notch_Protist, Notch1, Notch2, Notch3 and Notch4 were identified (Dataset S3).

Consensus sequences and a specialized phylogenetic analysis

Representative consensus protein sequences of the Notch family sub-clusters were estimated according to the clustering results of the Notch family protein clusters (Mitsis et al., 2020). The representative protein sequences for each sub-cluster were calculated using the MATLAB Bioinformatics Toolbox (Nanni, Lumini & Brahnam, 2014). Two representative protein sequences of the GLP1 and LIN12 homologs of the Caenorhabditis elegans species (Girard et al., 2007; Sorkac et al., 2018) were also included in the final dataset of the consensus protein sequences (Dataset S4). Last but not least, a specialized phylogenetic analysis was performed using the MATLAB Bioinformatics Toolbox (Kufareva & Abagyan, 2012) utilizing the Unweighted Pair-Group Method (UPGMA) (Michener & Sokal, 1957; Pavlopoulos et al., 2010; Sneath & Sokal, 1973) with 100 bootstrap replicates. Finally, the constructed topology of the phylogenetic tree was visualized with MEGA traditional option (Dataset S5).

Dataset

The primary NCBI dataset contained 26,646 entries, related to Notch family members. This dataset consisted of not only Notch protein and its homologs, but also unrelated proteins, such as the example of strawberry notch homolog (SNO). These irrelevant proteins, along with synthetic, hypothetical, partial, low quality, predicted proteins and which were referred as to noisy data and were removed from our dataset. Since the NCBI database provides partial duplicates sequences, the sequences with greater than 95% similarity were also removed, retaining the one with the longer length. Thus, the final dataset involved 603 Notch proteins and specifically, 90% of these corresponded to Notch protein, 4% to Notch1, 3% to Notch2, 2% to Notch3, and 1% to Notch4 (Fig. 1). Herein, representatives of all kingdoms from simpler organisms such as bacteria, to more complex organisms including Homo sapiens were detected. The length of the Notch family proteins ranges between ≈71 aa (bacteria’s Notch-like protein) and ≈4,835 aa (invertebrates Notch). As for the Notch1–4 paralogues, an exponential pattern of length was observed, with N4 being the shortest, followed by Notch3, Notch2 and finally Notch1 being the longest.

Multiple sequence alignment and motifs

Multiple sequence alignment (MSA) of protein sequences from the Notch family was performed to identify highly conservative regions within all organisms, from monera to invertebrates. As known, Notch receptors consist of specific conservative domains including EGF-like domain, NRR, TMD, RAM, NLS, ANK, NOD/NODP and PEST, that could be identified in the MSA (Aster, Pear & Blacklow, 2017; Sander, Krysinska & Powell, 2006) (Fig. 2). Monitoring and analyzing the MSA using Jalview resulted in identifying EGF, LNR and NOD/NODP domains appearance in most kingdoms, though a variation of the number of repeats and length is noticeable (Gordon, Arnett & Blacklow, 2008). Even bacteria Notch-like proteins share some of the characteristic motifs of those regions in their receptors; therefore, we can speculate that lateral gene transfer might have occurred via bacterial transfers (Gazave et al., 2009; Ponting et al., 1999). Two other members of the Notch pathway, including Fringe (Irvine & Wieschaus, 1994) and Strawberry Notch (Sno) (Gazave et al., 2009) show the same scenario, in which the eventuality of lateral gene transfer cannot be excluded.

Epidermal growth factors repeats are crucial components of the Notch signaling. Thus, it was expected to identify them as the most conserved site. EGF’s 6 Cysteine residues, being the key element of the domain, are responsible for the formation of disulfide bonds and influence the native 3D structure of the Notch members (Fig. 3). The importance of these key residues is also apparent in the pathological phenotype. In the case of a Notch3 mutation, if any of the 6 Cys is mutated into another amino acid, it leads to a rare neurodegenerative syndrome, called CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) (Papakonstantinou et al., 2019; Vlachakis et al., 2014). It is worth mentioning that the EGF-domain is also characterized by the conserved Glycine residues, as shown by the current multiple alignments (Fig. 3). Furthermore, the highly conserved EGFs, led us to suggest that two new motifs A and B are crucial for the Notch family evolution. The CXNGGXC motif (Fig. 3/Motif A) consisting of two highly conserved Gly residues and a second motif (Fig. 3/Motif B), CXCXXG[FY]XG, characterized by a conserved Cys and Gly and a non-polar aromatic amino acid (F/Y) among two conserved Gly (Fig. 3). These motifs could possibly be εGF-domain’s precursors, since they are repeated with a different number of repetitions depending on the respective species, where the number of repetitions is directly related to the complexity of the organism (Kovall et al., 2017).

Additionally, a segment of the LNR-domain appears to be highly conserved in organisms from all kingdoms and the characteristic motif of the LNR-repeat was also found in bacteria. Motif C could be considered as the LNRs’ single unit of short peptide that has been repeated via internal tandem duplications through evolution in eukaryotes (Bjorklund, Ekman & Elofsson, 2006) (Fig. 4/Motif C). NOD/NODP domain seems to have maintained throughout the evolutionary history of all eukaryotes and it contains two conserved motifs (Motifs D and E). Remarkably, the conserved motif D has been identified also in bacteria’s Notch-like proteins, which are significantly smaller than the homolog proteins of the other species.

Structural characterization of Notch Receptor

Despite the progress achieved recently, in the structure determination of limited fragments of the Notch receptor, its quaternary 3D structure remains undetermined (Fig. 5). Notch receptor 3D models are based on the structural features of the long NECD region, since it contains many calcium binding EGF-like domains (Fig. 5). The NECD differs between species. Drosophila and mammalian Notch receptors are much larger than their counterparts from other invertabrate species like Caenorhabditis elegans, although each invariably maintains the same molecular architecture (Kopan & Ilagan, 2009). On the other hand, in monera like bacteria, and in protists, the NECD size is much shorter and compact (Fig. 2). Based on previous studies it has been observed that the NECD region of the Notch receptor is expected to have a rigid near-linear 3D structure, however potential sites of flexibility may occur at the 3D structure of the EGF domain which is less conserved between species/kingdoms (Figs. 2 and 3) (Kopan & Ilagan, 2009; Morgan et al., 1999).

At the N-terminal end, for most species, the Notch receptor contains 36 EGF-like domains, a region of containing calcium-binding sites. Next to the EGF region there are three LNR and a hydrophobic region which has been shown to mediate heterodimerization (HD) (Fig. 5). Together, the LNR repeats and the HD form the NRR, adjacent to the cell membrane (Fig. 5). This region prevents ligand-independent activation of the Notch receptor by concealing and protecting from metalloproteases. When Notch activation is achieved via ligand binding to repeats within the EGF-like domain, then two sequential proteolytic events termed S2 and S3 cleavages are induced (Sanchez-Irizarry et al., 2004). The S3 cleavage site lies within the transmembrane segment and is cleaved by the γ-secretase complex to liberate NICD. NICD comprises one RAM domain, seven ANK, one TAD and one PEST domain (Fig. 5). Both the RAM domain and ANK repeats have been identified as regions involved in the interaction with CSL transcription factors (Chillakuri et al., 2012). The TAD region is found in Notch-1 and -2 but not in -3 and -4 in mammals (Chillakuri et al., 2012). The C-terminal PEST domain is involved in NICD degradation by proteolysis.

Recently, they have been determined the complete 3D structures of the extracellular domain (ECD) of the Drosophila Notch receptor and the human Notch1 receptor, by using single particle electron microscopy and antibody labeling (Chillakuri et al., 2012; Kelly et al., 2010). Since the inherent resolution of the method is poorly corresponding to the small size of the domains involved, these are significant challenging experiments.

Ca⁺² signaling activation and digital emulation

Notch family proteins, being capable of recognizing and binding to the neighbor cell’s ligand, achieve selective cell-cell adhesion initiated by protein-protein interaction. They comprise an extracellular region, composed of EGF-repeats, LNR repeats and NOD/NODP. EGF-repeats act as transmitters that promote the signal to the LNRs and along those extracellular regions, intracellularly to the ANK repeats. In this process, calcium plays a crucial role and is required for the biological activation of transmembrane proteins that contain EGF and LNR repeats. More precisely, Ca⁺² binds to cbEGFs, a large subgroup of EGFs. CbEGFs have essentially similar structure as EGFs, but they are so conserved, that they cluster together in EGF dendrograms (Stenflo, Stenberg & Muranyi, 2000). The central role of Ca⁺² has been demonstrated through crystallization experiments, where Ca⁺² seems to stabilize the N-terminal of tandem EGFs, establishing a stable surface for the protein-protein interaction (Downing et al., 1996). The Ca⁺² binding coordination is related to cbEGFs’ amino acid modifications, including hydroxylation and glycosylation of an asparagine or aspartic acid residue. Moreover, when a Ca⁺² is binding, the extracellular structure is locally changing, making it versatile and difficult to model. Ca⁺² role is also, observed in the LNR repeats, as each cysteine-rich Lin-12/Notch repeat binds one calcium ion to achieve the correct folding and maintenance of its 3-D structure. Mutations in calcium binding sites lead to dysregulated pathways, introducings pathogenic phenotypes. A substantial case of defective Ca⁺² binding is the Marfan syndrome and Hemophilia B.

The extracellular region of a Notch family precursor is a mix of calcium-loaded and calcium-free sites, with each EGF repeat forming a beta-sheet and the LNR repeats forming loops. Simulating each beta-sheet as a hoop, the whole EGF region could be presented as a strongly-connected chain. Furthermore, emulating the chain into a digital format, each hoop, depending on its Ca⁺² binding or Ca⁺² binding disability, is emulated by a sequence of 1 and 0, respectively. In that way, simulating each Lin12/Notch repeat as a gear (are more specialized forms than EGF repeats), the complex of LNRs could be emulated by a compact gear mechanism, which produces a more specialized signal based on the calcium interactions (Fig. 6). Combining all this mechanism together we can easily understand its specialization in producing different unique codes (signals) to the cell in order to start different biological tasks. This proposed emulation could represent a way to present if the signal is going to be promoted from the EGF repeats to the LNRs etc. and finally to the nucleus, completing a the signaling pathway. In case of mutation in these residues due to defective Ca⁺² binding, the corresponding digit changes and an error in the code occurs, indicating signal transduction failure associated with the possibility of a functional abnormality (Fig. 6).

Notch family protein clusters

Notch family protein clusters were estimated using a phylogenetic analysis (Fig. 7). A phylogenetic tree could provide clustering information for the Notch family members, so that representative consensus sequences could be properly defined. Having such a large dataset with proteins of variant sequence length, we initiated the construction of an unrooted tree. Phylogenetic analysis revealed a distinct separation of Notch family members into eight monophyletic branches, the Notch bacteria cluster, the Notch plants cluster, the Notch protist cluster, the Notch invertebrates cluster, as well as the Notch1, Notch2, Notch3 and Notch4 clusters. Each cluster’s content was carefully examined and reviewed (proteins, classes and kingdoms it contained), eventually leading to clustering the data into the eight groups (Table 2). Moreover, it was clear that the resulting clusters of the phylogenetic tree were formed by related protein members and not by classes/organisms, even if we included the same species in different clusters for the Notch1 to 4 homologs. Furthermore, considering the conservation and similarity of the characteristic domains presented in each Notch cluster, it was verified that the Notch family members classification was accurate because they belong in the same phylum/kingdom (Fig. 7). In this study, using protein sequences from representatives of all kingdoms, it was necessary to observe the agreement of the emerging phylogenetic tree with the tree of life. Thus, a bibliographic examination was carried out, which confirmed the correct and proper positioning and clustering of organisms according to evolution, by examining the position of the invertebrates, such as cnidarians, flatworms, nematodes, arthropods, chordates, as well as the phylogenetic position of the protists and plants, in the tree of life (Keeling, 2019) (Table 2).

Notch family evolution

In this study, we identified novel Notch protein clusters from various kingdoms extending from bacteria to chordates and conducted a comprehensive phylogenetic analysis in order to confirm and expand the evolutionary history of the Notch family (Fig. 8). Notch family evolution was examined in a more specialized phylogenetic analysis with 100 bootstrap replicates using representative consensus protein sequences of each identified cluster from the previous phylogenetic analysis. Each consensus sequence, with its amino acids corresponding to the most frequently encountered, fully represented the organisms and the members of the NOTCH family, accordingly. Hence, the specialized phylogenetic tree contained meaningful information, as needed for further evolutionary research (Papageorgiou et al., 2018). Based on results, both constructed phylogenetic trees of the Notch family members were found to share similar topology.

Due to the small number of sequences which were available the past decade, all previously performed evolutionary studies have a significantly smaller dataset. In this study, a more comprehensive phylogenetic analysis was achieved and it was important to compare the evolutionary scenarios presented here with the previous ones. The evolutionary distances between Notch2 and Notch3 groups have been found shorter than Notch, Notch1 and Notch4. This finding is correspondingly consistent with the evolutionary analysis performed by Gazave et al. (2009). On the other hand, Notch4 group found to be evolutionary older than Notch1, Notch2 and Notch 3, and with this result being consistent with the evolutionary study performed by Theodosiou et al. (2009). However, in this study we have introduced and present for the first time, the plant, protist and bacteria groups which are in the early Notch evolution (Fig. 8). Another remarkable observation is that Notch protein groups are positioned before Notch 1–4 groups, indicating the origin of the Notch family. In consideration of the tree of life, bacteria, protists and plants, appeared before the rest of the eukaryotes and therefore their position in the constructed tree is validated. Bacteria belongs to prokaryotes, while protists and plants are eukaryotic organisms and thus, much more complex than prokaryotes. The fact that the latter two are eukaryotes, combined with the absence of evidence for the existence of Notch proteins in Archaea, partially modifies the obtained results, compared to the current evolutionary theories. In addition, bearing in mind the limited number of representatives given in the kingdom of plants, this slight divergence in the present evolutionary study of the Notch family is justified. However, future enrichment of the dataset, with additional Notch family protein sequences from other species, could provide an improved and even more distinct phylogenetic analysis. Lastly, in the phylogenetic tree, Notch paralogs of Caenorhabditis elegans appear distinctly before Notch1–4, as expected, given the fact of an independent genomic duplication, leading to two Notch genes (Theodosiou et al., 2009).