Abstract

   Short linear motifs (SLiMs) are evolutionarily conserved functional
   modules of proteins that represent amino acid stretches composed of 3
   to 10 residues. The biological activities of two short peptide segments
   of human alpha-fetoprotein (AFP), a major embryo-specific and
   cancer-related protein, have been confirmed experimentally. This is a
   heptapeptide segment LDSYQCT in domain I designated as AFP[14–20] and a
   nonapeptide segment EMTPVNPGV in domain III designated as GIP-9. In our
   work, we searched the UniprotKB database for human proteins that
   contain SLiMs with sequence similarity to the both segments of human
   AFP and undertook gene ontology (GO)-based functional categorization of
   retrieved proteins. Gene set enrichment analysis included GO terms for
   biological process, molecular function, metabolic pathway, KEGG
   pathway, and protein–protein interaction (PPI) categories. We
   identified the SLiMs of interest in a variety of non-homologous
   proteins involved in multiple cellular processes underlying embryonic
   development, cancer progression, and, unexpectedly, the regulation of
   redox homeostasis. These included transcription factors, cell adhesion
   proteins, ubiquitin-activating and conjugating enzymes, cell signaling
   proteins, and oxidoreductase enzymes. They function by regulating cell
   proliferation and differentiation, cell cycle, DNA
   replication/repair/recombination, metabolism, immune/inflammatory
   response, and apoptosis. In addition to the retrieved genes, new
   interacting genes were identified. Our data support the hypothesis that
   conserved SLiMs are incorporated into non-homologous proteins to serve
   as functional blocks for their orchestrated functioning.

   Keywords: short linear motifs, alpha-fetoprotein, cancer, redox
   regulation, embryonic development, bioinformatics

1. Introduction

   Proteins are key cellular components involved in practically all the
   essential processes in a living organism. The functioning of proteins
   is assured by the presence of functionally important regions and
   modules, which can be organized at different structural levels, from
   primary through secondary to tertiary, three-dimensional (3D)
   structures [[30]1]. These modules include independently folded 3D
   domains, secondary structure elements (SSEs), and short linear motifs
   (SLiMs), which provide multimodular and multifunctional features of
   many proteins [[31]2,[32]3].

   Functional modules of proteins have been attained over a long
   evolutionary time and are implicated in a vast array of biological
   processes, including metabolism, cell division, stress response, signal
   transduction, and cell-to-cell and cell-to extracellular matrix (ECM)
   interactions [[33]4]. SLiMs are short amino acid stretches composed of
   3 to 10 highly conserved residues that can be involved in
   protein–protein interactions (PPIs) underlying various protein
   functions [[34]5]. Such motifs have been implicated in many fundamental
   processes, including Arg-Gly-Asp (RGD) tripeptide, which provides an
   interaction of ECM proteins with their receptors, integrins [[35]6].
   Other examples are the KDEL motif, which marks proteins for their
   retainment in the endoplasmic reticulum (ER) [[36]7], and Src-homology
   3 (SH3)-binding proline-rich motif, PxxP [[37]8].

   SLiMs are stable in a variety of proteins and protein families, but the
   same sequence can code for different native structures that are covered
   by current fragment libraries [[38]9]. They can be intertwined and
   overlapped and are incorporated into proteins to serve as functional
   building blocks. This multiplicity of structural states for a single
   sequence explains why the same structure may be found in a variety of
   unrelated non-homologous proteins. Thus, despite highly differentiated
   amino acid sequences and 3D structures, proteins may share similar
   functions. These protein segments can have a native conformation around
   the local minimum of potential energy function, allowing proteins to
   reuse the same patterns of recurring motifs [[39]10]. This reuse
   enables transition from one conformation to another by sampling
   conformational assemblies of the protein backbone.

   Domain–SLiM interactions mediate many PPI pathways, whereas
   post-translational modifications of a SLiM may provide a switch from
   its interaction with one domain to another [[40]11,[41]12]. Most
   domains maintain unique amino acid conservation patterns, which suggest
   that they can bind SLiMs with high intrinsic specificity, and this
   influences the PPIs [[42]13]. Moreover, SLiMs have evolved to coadapt
   their specificity and affinity to the functional diversity of
   domain–peptide interactions [[43]14], and the interplay between a
   modular domain, such as SH3, and its host protein is important in
   establishing the specificity to wire PPI networks [[44]15]. However,
   their very small size and poorly folded nature make SLiMs difficult to
   detect experimentally. Many computational and bioinformatics tools have
   been developed for PPI analysis on the basis of known SLiM-recognition
   domains [[45]16].

   Here, we used a local sequence alignment algorithm for a computational
   search of SLiMs that have sequence similarity to two biologically
   active peptide segments of human alpha-fetoprotein (AFP), a major
   mammalian embryo-specific and cancer-related protein [[46]17]. One of
   these segments, LDSYQCT, encompasses amino acid residues from 14 to 20
   in mature AFP and is designated as AFP[14–20] [[47]18]. Another one is
   a C-terminal fragment of AFP-derived growth inhibitory peptide (GIP)
   composed of nine residues, EMTPVNPGV, and designated as GIP-9 [[48]19].
   It has been experimentally shown that the GIP peptide displays the
   ability to inhibit mouse uterine cell proliferation and anticancer
   effects in MSF-7 breast cancer cells [[49]20]. AFP[14–20] demonstrated
   an immunomodulatory capability in a phytohemagglutinin (PHA)-activated
   lymphocyte culture [[50]21]. We used gene ontology (GO) functional
   enrichment, Kyoto encyclopedia of genes and genomes (KEGG) pathway,
   metabolic pathway, and PPI network analyses to categorize human
   proteins containing both AFP[14–20]-like and GIP-9-like motifs. We
   found that the retrieved proteins belong to transcription factors, cell
   adhesion proteins, cell signaling proteins, ubiquitin-activating and
   conjugating enzymes, and oxidoreductase enzymes and are involved in
   embryonic development, cancer progression, and redox regulation.

2. Results

2.1. Human Proteins Aligned to Human AFP Segments

   In total, 222 human proteins with sequence similarity to the AFP[14–20]
   segment and 55 proteins with sequence similarity to the GIP-9 segment
   of human AFP were retrieved from UniprotKB knowledgebase. These
   proteins include AFP itself, putative proteins, and uncharacterized
   proteins from both Swiss-Prot and TrEMBL sections.

   [51]Table 1 contains representative proteins aligned to the AFP[14–20]
   segment; the lower the E-value, the more significant the sequence
   alignment. As shown in [52]Table 1, proteins significantly aligned with
   the AFP[14–20] segment are involved in a diversity of functions,
   including cell proliferation and differentiation, development,
   metabolism, immune/inflammatory response, redox homeostasis, and
   apoptosis. These proteins include transcription factors, such as
   tripartite motif (TRIM)-containing protein 3,
   haematopoietically-expressed homeobox protein HHEX, and zinc finger
   protein 714. The AFP[14–20] segment was also found in proteins that are
   involved in DNA replication, cell cycle regulation, and cell division,
   including DNA polymerases, nucleotide transferases, and growth factors.
   Multiple epidermal growth factor (EGF)-like repeat-containing,
   calcium-binding, and membrane-bound extracellular matrix proteins, such
   as members of neurogenic locus notch, von Willebrand factor A
   domain-containing protein and fibulin-4, which are crucial for cellular
   homeostasis and functioning, were also identified. Additionally,
   ubiquitin-activating enzyme (E1) and ubiquitin-protein ligase
   (transferase) enzyme (E3), which are involved in protein modification
   and protein quality control, were also aligned to the AFP[14–20]
   segment. Interestingly, there were oxidoreductases involved in
   oxidative stress response among the retrieved proteins, including
   prostaglandin G/H synthase 1, glutathione S-transferase LANCL1, and
   prolyl hydroxylase.

Table 1.

   Selected human proteins retrieved from UniprotKB knowledgebase
   containing AFP[14–20]-like motifs.
   Protein Name Entry Code Gene Symbol Alignment Aa Positions Identity
   E-Value GO Molecular Functions GO Biological Processes Reference
   Tripartite motif-containing protein 3 (RING finger protein HAC1)
   TR:[53]Q1KXY7 TRIM3 LDSYQCT
   : :  . : : :
   LDRYQCP 26–32 71.4% 2.2 × 10^−4 Metal ion binding, ubiquitin-protein
   ligase/transferase activity Transcriptional regulation, UPS-mediated
   protein degradation [[54]22]
   Zinc finger protein 714 TR:A0A087WV13 ZNF714 LDSYQCT
   . : :  : : .
   ENSYQCE 15–21 57.1% 3.0 × 10^−2 Transcription factor Transcriptional
   regulation [[55]22]
   Hematopoietically-expressed homeobox protein HHEX TR:F8VU08 HHEX
   LDSYQCT
   : :  :  : : .
   LDSSQCS 59–65 71.4% 9.4 × 10^−4 DNA binding, transcription activator
   activity Transcriptional regulation, anterior–posterior pattern
   specification, B- cell differentiation [[56]23]
   Neurogenic locus notchhomolog protein 2 TR:A0A494C1F1 NOTCH2 LDSYQCT
   : . :  . : :
   RDTYECT 87–93 57.1% 7.9 × 10^−2 Calcium ion binding, signaling receptor
   activity Tissue morphogenesis, cell fate determination [[57]24]
   von Willebrand factor A domain-containing protein 2 SP: Q5GFL6-2 VWA2
   LDSYQCT
   : :  : :  :
   LDGYQCL 315–321 71.4% 0.36 Calcium binding activity Cell–matrix
   adhesin, insulin-receptor signaling [[58]25]
   EGF-containing fibulin-like extracellular matrix protein 2 TR: E9PRQ8
   EFEMP2 LDSYQCT
   .  : : : : :
   PGSYQCT 144–150 71.4% 4.0 Calcium ion binding Extracellular matrix
   assembly, developmental processes [[59]26]
   Slit-Robo RhoGTPase-activating protein 2B TR:[60]A0A087 × 1G6 SRGAP2B
   LDSYQCT
   :  : . :  : .
   LYSHQCS 22–28 57.1% 0.17 GTPase activator activity Neuronal
   morphogenesis developmental process [[61]27]
   Calcium and integrin-binding family member 2 TR:H0YND4 CIB2 LDSYQ-CT
   : : .  : :  : :
   LDNYQDCT 13–20 75.0% 3.3 × 10^−2 Calcium ion binding, integrin binding
   Calcium ion homeostasis, response to ATP [[62]28]
   F-box protein Fbx3 TR: [63]Q9UKC5 FBX3 LDSYQCT
   : : .  : . : .
   LDDYRCS 137–143 57.1% 4.0 × 10^−2 Ubiquitin-protein transferase
   activity Protein ubiquitination and degradation [[64]29]
   Ubiquitin-like modifier-activating enzyme 6 TR:Q2MD40_ UBA6 LDSYQCT
   : : .    : : :
   LDKYQCV 151–157 71.4% 5.0 × 10^−3 ATP binding, ubiquitin-activating
   enzyme activity Response to DNA damage, protein ubiquitination,
   embryonic development [[65]30,[66]31]
   Epidermal growth factor TR:[67]Q6QBS2 EGF LDSYQCT
   : : .  :  : .
   LDKYACN 26–32 57.1% 3.8 × 10^−2 Growth factor activity Cell
   proliferation and survival [[68]32]
   Proliferating cell nuclear antigen TR:[69]Q7Z6A3 PCNA LDSYQCT
   . :  . : . :
   FDTYRCD 57–63 42.9% 0.42 DNA binding Cell cycle regulation, DNA
   replication and repair [[70]33]
   Ethanolamine-phosphate cytidylyltransferase TR:I3L1F9 PCYT2  LDSYQCT
   : : .  : .  : .
   LDKYNCD 24–30 57.1% 5.7 × 10^−3 Catalytic activity Biosynthetic
   process, cell division, cell fusion, and apoptosis [[71]34]
   Cysteine protease ATG4D SP: Q86TL0-2 ATG4D  LDSYQCT
   : . : .  . : :
   LESFHCT 40–46 57.1% 0.18 Peptidase activity
   Apoptosis/autophagy/mitophagy/proteolysis [[72]35]
   CTP:phosphoethanolamine cytidylyltransferase TR:I3L1C4 PCYT2  LDSYQCT
   : : .  : .  :
   LDKYNCD 24–30 57.1% 0.21 Transferase activity Biosynthetic process
   [[73]36]
   B-cell linker protein TR: [74]Q2MD40 BLNK LDSYQCT
   . : :  : . :
   MDSYSCL 1–7 57.1% 9.7 × 10^−4 SH2-domain binding, signaling adaptor
   activity B-cell differentiation, immune and inflammatory response
   [[75]37]
   3-alpha hydroxysteroid dehydrogenase III TR:[76]Q1KXY7 AKR1C2  LDS –
   YQCT
   . : :  : : :
   MDSKYQCV 1–7 62.5% 4.4 × 10^−5 Oxidoreductase, metabolic activity
   Steroid hormone metabolism [[77]38]
   Prostaglandin G/H synthase 1 SP: P23219-3 PTGS1  LDSYQCT
   : :  . : : :
   LDRYQCD 26–32 71.4% 0.35 Cyclooxygenase/peroxidase activity, heme
   binding, metal ion binding Response to oxidative stress, inflammatory
   process [[78]39]
   Glutathione S-transferase LANCL1 TR:H7C2E3 LANCL1 LDSYQCT
    :  : :  :
   CDAYQCA 59–65 57.1% 0.22 Glutathione binding, zinc ion binding
   Oxidative stress response [[79]40]
   HSPB1-associated protein 1 SP: Q96EW2-2 HSPBAP1 LDSYQCT
   : : :  :  : :
   LDSYGCN 176–182 71.4% 0.12 Oxidoreductase, dioxygenase activity Brain
   development [[80]41]
   Prolyl hydroxylase EGLN2 TR:M0R2X9 EGLN2  LDSYQCT
   :  : : . :
   LPSYHCP 45–51 57.1% 2.5 Dioxygenase activity, oxygen sensor activity
   Cell redox homeostasis, response to hypoxia [[81]42]
   [82]Open in a new tab

   Note: colons between the aligned sequences indicate identity of the
   residues, whereas dots indicate similarity between residues.

   [83]Table 2 shows the most representative proteins that were aligned
   with high significance to the GIP-9 segment of human AFP. They include
   developmental proteins, such as isoforms of C-C motif chemokine 4-like;
   and Wnt-signaling regulators, such as AXIN2 and L1 cell adhesion
   molecule (L1CAM or CD171). Like AFP[14–20], the GIP-9 segment was
   aligned to various proteins with transcription factor activity, such as
   homeobox protein Hox-C5 (HOXC5), forkhead box protein O1 (FOXO1), and
   zinc finger proteins 547 and 213. Among the aligned proteins, there
   were cell-cycle regulators, such as antiproliferation factor 3
   (BTG3)-associated nuclear protein and cyclin-dependent kinase inhibitor
   1B. There were also proteins involved in DNA replication, repair, and
   recombination. Additionally, various proteins with receptor activity,
   including IGF-like family receptor 1, brain-specific angiogenesis
   inhibitor (BAI) family proteins, and E3 ubiquitin-protein ligase
   TRIM35, were aligned to GIP-9. Growth hormone receptor (GHR) was also
   involved in metabolic regulation. Importantly, various oxidoreductase
   enzymes, such as ceruloplasmin and pyridoxine 5’-phosphate oxidase
   (PNPO), were also aligned to the GIP-9 segment.

Table 2.

   Selected human proteins retrieved from UniprotKB knowledgebase
   containing GIP-9-like motifs.
   Protein Name Entry Code Gene Symbol Alignment Aa Positions Identity
   E-Value Go Molecular Functions Go Biological Processes Reference
   Zinc finger protein 547 TR: M0QYW2 ZNF547 EMTPVNPGV
   :  : .  . : : :
   EEAPLEPGV 60–68 55.6% 3.4 DNA binding, metal ion binding, transcription
   factor activity Transcriptional regulation [[84]22]
   C-C motif chemokine 4-like SP: Q8NHW4-7 CCL4L1 EMTPVNPGV
    . : :  : . : :
   ALTPVSPGS 31–39 66.7% 1.7 × 10^−2 Chemokine activity Response to INF-γ,
   IL-1, and TNF-α; cell signaling [[85]43]
   Axin 2 TR: A0A024R8M3 AXIN2 EMTPVNPGV
   : :  : : : . : .
   EMTPVEPAT 361–369 66.7% 1.7 Beta-catenin binding, ubiquitin protein
   ligase binding Regulation of Wnt signaling, cell death, bone
   mineralization [[86]44]
   L1 cell adhesion molecule TR: [87]Q7Z2J6 L1CAM EMTPVNPGV
    . : .  : : . :
   ATSP I NPAV 54–62 44.4%1 1.0 Cell adhesion molecule activity Nervous
   system development [[88]45]
   Paired-like homeodomain transcription factor LEUTX SP: A8MZ59-1 LEUTX
   EMTPVNPGV
   . .  . : : .  : : .
   N I RPVSPG I 69–77 44.4% 2.3 DNA binding activity Transcriptional
   regulation, embryogenesis [[89]46]
   Homeobox protein Hox-C5 SP: [90]Q00444 HOXC5 EMTPVNPGV
   : .  : .  : :  : .
   EAAPLNPGM 90–98 55.6% 0.48 DNA-binding activity, transcription factor
   Anterior/posterior specification, embryonic development [[91]47]
   Forkhead box protein O1 SP: [92]Q12778 FOXO1 EMTPVNPGV
   : : : :  . : : :
   IMTPVDPGV 476–484 77.8% 0.12 DNA-binding activity, transcription factor
   Transcriptional regulation, metabolic response to oxidative stress
   [[93]48,[94]49]
   RUNX1/CBFA2T2 fusion protein type 1 TR:D1LYX4 RUNX1/CBFA2T2 EMTPVNPGV
   .  :  . : :  : .
   PLP P I NPGG 50–58 44.4% 8.5 Transcription corepressor activity
   Transcriptional regulation [[95]50]
   Cyclin-dependent kinase inhibitor 1B TR: H7C2T1 CDKN1B EMTPVNPGV
   :  : :  . : : .
   EQTPKKPGL 91–99 55.6% 2.3 Cyclin binding, chaperone binding Cell-cycle
   regulation, autophagy, response to chemicals [[96]51]
   DNA replication complex GINS protein PSF2 SP: [97]Q9Y248 GINS2
   EMTPVNPGV
   .  .  : . : : : .
   DLGPFNPGL 32–40 44.4% 7.2 DNA binding DNA replication, DNA repair
   [[98]52]
   IGF-like family receptor 1 TR: K7ESC2 IGFLR1 EMTPVNPGV
   . : : .  : : : .
   PLTPGNPGA 124–132 55.6% 2.7 Receptor activity IGF-mediated signaling,
   inflammation process [[99]53,[100]54]
   Brain-specific angiogenesis inhibitor 1-associated protein 2-like
   protein 2 TR: B0QYF0 BAIAP2L2  EMTPVNPGV
   : : : .  : : :
   PMTPMNPGN 98–106 66.7% 4.5 × 10^−2 Cadherin-binding and
   cytoskeletal-binding activities Actin cytoskeleton organization, brain
   development [[101]55,[102]56]
   E3 ubiquitin-protein ligase TRIM35 TR: H0YBF3 TRIM35   EMTPVNPGV
   : . . :  : . :  : .
   EPEPVQPGM 33–41 55.6% 4.6 Zinc ion binding, ubiquitin-protein ligase
   activity Protein ubiquitination, innate immune response, apoptotic
   process [[103]57]
   Ceruloplasmin TR: H7C5N5 CP    EMTPVNPGV
   : :  :  .  : .
   EMFPRTGG I 162–170 55.6% 2.8 Oxidoreductase activity, copper binding
   Redox homeostasis [[104]58]
   Pyridoxine-5’-phosphate oxidase TR: A0A286YF38 PNPO    EMTPVNPGV
   : .   : .   : :
   EVPPLGPGL 46–54 44.4% 1.5 Oxidoreductase activity Biosynthetic process
   [[105]59]
   Growth hormone receptor TR: [106]Q9NRZ8 GHR EMTPVNPGV
   . . .  : : :  : .
   SLQSVNPGL 11–19 44.4% 1.3 Cytokine receptor activity Response to
   stimulus, cell signaling [[107]60]
   [108]Open in a new tab

   Note: colons between the aligned sequences indicate identity of the
   residues, whereas dots indicate similarity between residues.

2.2. Biological Process Categories

   We performed GO term categorization to assess the involvement of genes
   from our gene list in various biological processes. This enabled the
   identification of unique human genes and total gene amounts associated
   with a given GO biological process term. In total, we identified 120
   and 39 unique genes encoding proteins that contain AFP[14–20]-like and
   GIP-9-like motifs, respectively. The results of biological process
   enrichment of the genes encoding AFP[14–20]-like motif-containing
   proteins are shown in [109]Figure 1A. With the PANTHER17.0
   classification system, 116 of 120 unique genes were mapped to the whole
   human genome, and a total of 224 biological process hits associated
   with our gene list were found. The most statistically significant GO
   categories (p-value ˂ 0.05) belonged to developmental processes, such
   as “multicellular organism development”, “tissue development”,
   “biological adhesion”, and “positive regulation of cell
   differentiation”. At a p-value cutoff of 0.2, more biological process
   terms were identified: 36 genes were implicated in biological
   regulation, 32 genes were implicated in metabolic processes, 26 genes
   were implicated in response to various stress stimuli, and 17 genes
   were implicated in cell signaling. These GO categories can overlap with
   one another. For example, 62 genes were involved in variety of cellular
   processes, including protein biosynthesis, protein transportation,
   protein quality control, metabolism, cellular component organization,
   cell communication, signal transduction, and cellular response to
   chemical stimulus.

Figure 1.

   [110]Figure 1

   [111]Figure 1
   [112]Open in a new tab

   Representation of gene-ontology-based biological process enrichment
   categorization of genes encoding (A) AFP[14–20]-like motif-containing
   proteins and (B) GIP-9-like motif-containing proteins. The gene list
   enrichment analysis tool of PANTHER17.0 was applied. All query genes
   were retrieved from UniprotKB knowledgebase and then converted to
   ENSEMBL gene IDs.

   Categorization of genes encoding GIP-9-like motif-containing proteins
   on the basis of GO biological process terms is shown in [113]Figure 1B.
   Of the 39 unique genes, 37 were mapped to the whole human genome and
   were involved in 95 biological processes. The overrepresented genes
   were involved in biological regulation (20), metabolic processes (17),
   localization (9), response to stimulus (6), cell signaling (4), and
   immune response (4). Due to overlapping of various biological process
   categories, a general term, “cellular process”, included 25 genes
   subcategorized as transcriptional regulation, cytokine signaling,
   ubiquitin-mediated protein degradation, DNA replication/repair,
   cytoskeletal organization, ion channel regulation, protein transport,
   and localization.

2.3. Molecular Function Categories

   For a more detailed gene set enrichment analysis, molecular function
   categorization was performed with the use of the ShinyGO v0.75 suite.
   [114]Figure 2A depicts genes encoding AFP[14–20]-like motif-containing
   proteins ranked by the number of genes in each category at a p-value
   cutoff of 0.2. As many as 48 genes belonged to the “metal ion binding”,
   “cation binding”, and “enzyme binding” categories. These molecular
   function categories are inherent to transcription factors and
   oxidoreductase enzymes. The additional “calcium ion binding” category
   is mostly inherent to cell signaling and developmental proteins.
   Additionally, the “extracellular matrix structural constituent”
   category evidences the implication of 6 AFP[14–20]-like
   motif-containing proteins in cell adhesion processes. [115]Figure 2B
   depicts the ranking of genes of interest by fold enrichment. This shows
   that oxidoreductase enzymes with prostaglandin-endoperoxide synthase
   (cyclooxygenase) activity, which are involved in ROS generation and
   redox regulation, were aligned with the most significance to the
   AFP[14–20] segment.

Figure 2.

   [116]Figure 2

   [117]Figure 2
   [118]Open in a new tab

   Gene ontology term-based molecular function categorization of genes
   encoding (A,B) AFP[14–20]-like motif-containing proteins and (C,D)
   GIP-9-like motif-containing proteins with the use of the ShinyGO v075
   suite. Categories are ranked by (A,C) number of genes and (C,D) fold
   enrichment. Lolipop chats with an aspect ratio of 1.5 and −log10 (FDR)
   heat maps for each category are shown.

   [119]Figure 2C depicts the categorization of genes encoding GIP-9-like
   motif-containing proteins at a p-value cut-off 0.2 by molecular
   function terms that are ranked by the number of identified genes.
   Nucleic acid (DNA)-binding activity that is inherent to transcriptional
   regulators was the most represented term. This was followed by
   phospholipid and phosphatase binding, sterol transporter, beta-catenin
   binding, and chemokine and transcription coregulator activities
   ([120]Figure 2C). However, ranking of these genes by fold enrichment
   revealed the highest significance of proteins with oxysterol binding
   and ferroxidase activities that are specific to redox regulation. Axon
   guidance receptor activity, ubiquitin-activating enzyme activity, CCR1
   chemokine receptor binding, and cyclin-dependent serine/threonine
   kinase inhibitor activity were also inherent to GIP-9-like
   motif-containing proteins, although with less significance ([121]Figure
   2D).

2.4. KEGG Pathways

   To elucidate molecular mechanisms underlying the functioning of the
   retrieved proteins, we undertook KEGG pathway enrichment analysis.
   [122]Figure 3A,C shows that 10 and 4 genes encoding AFP[14–20]-like and
   GIP-9-like motif-containing proteins, respectively, are involved in
   cancer-associated pathways. The AFP[4–20]-like motif group includes
   genes associated with cAMP-signaling, Ras-signaling, MAPK-signaling,
   FOXO-signaling, JAK-STAT-signaling, notch-signaling, and ErbB-signaling
   pathways ([123]Figure 3A). Among these pathways are those involved in
   metabolic processes and drug resistance. The GIP-9-like group includes
   cytokine signaling, NF-kB-signaling, Toll-like-receptor-signaling,
   AGE-RAGE-signaling, FoxO-signaling, and PI3K-Akt-signaling pathways
   ([124]Figure 3C). Therefore, proteins containing the SLiMs of interest
   mediate the abovementioned signal transduction pathways implicated in
   cancer initiation and progression.

Figure 3.

   [125]Figure 3

   [126]Figure 3

   [127]Figure 3
   [128]Open in a new tab

   KEGG pathway enrichment analysis of genes encoding (A,C)
   AFP[14–20]-like motif-containing proteins and (B,D) GIP-9-like
   motif-containing proteins. Rankings by both number of genes (A,B) and
   fold enrichment value (C,D) are shown. Bar plot representation with an
   aspect ratio of 1.5 and −log10 (FDR) heat maps for each category are
   shown.

   KEGG pathway ranking by fold enrichment showed the involvement of genes
   encoding AFP[14–20]-like motif-containing proteins in phosphonate and
   phosphinate metabolism associated with glycolysis and phosphorylation
   of proteins, lipids, and carbohydrates ([129]Figure 3B). Ranking of
   genes encoding GIP-9-like motif-containing proteins by fold enrichment
   showed the overrepresentation of KEGG pathways involved in metabolism
   of vitamin B6, which, in turn, is associated with metabolism of amino
   acids and their derivatives essential for cell growth ([130]Figure 3D).
   Therefore, these metabolic pathways are essential for the functioning
   of the retrieved proteins.

2.5. Metabolic Pathways

   Further, we used the Reactome resource to obtain more detailed
   information on the involvement of the retrieved proteins in cell
   metabolism and signaling. [131]Figure 4A shows that terms associated
   with elastic fiber formation are overrepresented among pathways that
   involve AFP[14–20]-like motif containing proteins. The elastic fiber
   proteins, such as fibulin family members, including finulin-4
   (identified here), play key roles in the assembly of elastic fibers, as
   well as sequestering and binding of growth factors to ECM, and contain
   the RGD tripeptide to interact with integrins. Additionally, pathways
   implicated in pre-NOTCH transcription, translation, and processing were
   overrepresented in our work. Remarkably, nascent NOTCH peptides are
   cotranslationally targeted to the endoplasmic reticulum for processing
   and further modification in the Golgi apparatus, as well as trafficking
   to the plasma membrane. In addition, we found that biosynthesis of
   prostaglandins (PGs) and thromboxanes (TXs), synthesis of
   phosphatidylethanolamine (PE), activation of RAC1, diseases associated
   with O-glycosylation of proteins, and NOTCH1 signaling in cancer were
   among the significant pathways.

Figure 4.

   [132]Figure 4

   [133]Figure 4
   [134]Open in a new tab

   Hierarchical tree representation of the reactome metabolic pathway
   categories of genes encoding (A) AFP[14–20]-like motif-containing
   proteins and (B) GIP-9-like motif-containing proteins. The tree
   summarizes the correlation among significant pathways in the gene
   enrichment list. Pathways with many shared genes are clustered
   together. Larger dots indicate more significant p-values.

   The most significant pathways that involve GIP-9-like motif-containing
   proteins included constitutive signaling mediated by Akt1 carrying the
   E17K mutation, which is implicated in cancer ([135]Figure 4B). A
   low-frequency point mutation, E17K, in Akt1 enables binding to
   phosphatidylinositol-2-phosphate (PIP2) for phosphorylation by TORC2
   complex, as well as activation. FOXO transcription-factor-mediated
   transcription of cell-cycle genes, including cyclin-dependent kinase
   inhibitor CDKN1A (p21Cip1), were also found among significant pathways.
   High significance of the runt-related transcription factor family
   (RUNXs), including RUNX1, RUNX2, and RUNX3, which are involved in
   developmental processes, immune response, and cancer, was also
   identified. Another pathway involved in cell-cycle regulation involves
   protein tyrosine kinase 6 (PTK6), which promotes cell-cycle progression
   by phosphorylation/inactivation of CDKN1A. Additionally, genes
   regulated by beta-catenin and TCF/LEF that participate in cell
   proliferation, differentiation, embryonic development, and tissue
   homeostasis were identified with high significance. Regulation of tumor
   suppressor gene TP53 through the association with cofactors was also
   identified among metabolic pathways, which involve GIP-9-like
   motif-containing proteins. Additionally, significant metabolic pathways
   included acyl chain remodeling of phosphatidylserine (PS), pregnenolone
   biosynthesis, and pathways that involve organic cation and metal ion
   solute carrier (SLC) transporters.

2.6. PPI Networks

   Because SLiMs are involved in the interactions underlying protein
   functioning, we further undertook STRING network analysis. As shown on
   [136]Figure 5A, proteins containing AFP[14–20]-like motifs constructed
   a PPI network with 112 nodes and 449 edges (interactions) at a
   confidence score of 0.150, an average node degree of 8.02, and a
   p-value of 1.6 × 10^-2. This approach allowed for the identification of
   hub genes with the most interaction partners, which include NOTCH1 and
   NOTCH2 isoforms, as well as EGF, FBN3, SLIT2, LTBP1, LAMA2, SFRP2, EMR1
   (ADGRE1), MIB1, POLR1B, LOXL2, GLI3, PCNA, CRB1, and PTGS2. However,
   there were genes with no interactions, including TCP11L2, OR4M1,
   SLC39A14, FAM10A, and ART4. Interestingly, novel genes that were not
   retrieved by local alignment algorithms were identified in our PPI
   network. They included NAT10-encoding N-acetyltransferase 10,
   CBX3-encoding DNA binding chromobox protein homolog 3, GALNT12-encoding
   N-acetylgalactosaminyltransferase 12, MT-ND6-encoding electron
   transportation chain protein NADH-ubiquinone oxidoreductase chain 6,
   and DPH7-encoding diphthamide biosynthesis 7, which is essential for
   posttranslational modification of elongation factor 2.

Figure 5.

   [137]Figure 5

   [138]Figure 5
   [139]Open in a new tab

   Protein–protein interaction networks constructed by STRING resource for
   genes encoding (A) AFP[14–20]-like motif-containing proteins and (B)
   GIP-9-like motif-containing proteins. ENSEMBL gene IDs or STRING-db
   protein IDs were used. Colored nodes—query proteins and first shell of
   interactions; white nodes—second shell of interactions; filled
   nodes—proteins of known or predicted 3D structure; empty nodes—proteins
   of unknown 3D structure. Known interactions: blue—from curated
   databases and violet (experimentally determined). Predicted
   interactions: red—gene fusions; green—gene neighborhood; purple—gene
   co-occurrence. Other interactions: lilac—protein homology; black—gene
   coexpression; light green—text mining.

   The proteins that contain GIP-9-like motifs were identified to create a
   PPI network with 48 nodes and 133 interactions at a confidence score of
   0.150 and an enrichment p-value of 4.26 × 10^−4 ([140]Figure 5B). POGZ,
   GINS1, GINS2, MCM4, MCM5, CDK4, CCND1, SIRT1, and RAD548 demonstrated
   the hub gene properties. Additionally, there were novel genes, such as
   SIRT1-encoding NAD-dependent deacetylase sirtuin-1; APC-encoding
   adenomatous polyposis coli protein, which is a negative regulator of
   beta-catenin involved in Wnt signaling; and RAB35-encoding Ras
   GTPase-related protein Rab-35, which is involved in endosomal
   trafficking. Furthermore, two novel genes encoding DNA replication
   licensing factors MCM4 and MCM5, which interact with GINS2 and its
   isoform, GINS1 complexes, were identified as hub genes. Cell-cycle
   regulator genes that interact with the retrieved CDKN1B, such as
   CCND1-encoding cyclin 1, as well as CDK1- and CDK4-encoding
   cyclin-dependent kinases 2 and 4, were among the novel genes.
   Additionally, the UBA2 isoform of UBA6 aligned to the AFP[14–20]
   segment but not the GIP-9 segment, was among new genes not retrieved by
   local alignment.

3. Discussion

   AFP is a major mammalian embryo-specific and cancer-related protein
   [[141]17]. We previously constructed a 3D structure of human AFP
   [[142]61] and performed mapping of its short linear sequences with
   putative and experimentally confirmed biological activities [[143]62].
   Two human AFP-derived peptides, AFP[14–20] and GIP-9, have been
   chemically synthesized and experimentally studied [[144]20,[145]21].
   Here, we undertook a search for AFP[14–20]-like and GIP-9-like SLiMs in
   human proteins, as well as GO-term-based comprehensive analysis of the
   retrieved proteins that contain both types of SLiMs of interest. The
   analyses were performed by categorization of the identified proteins in
   biological process, molecular functions, metabolic pathways, KEGG
   pathways, and PPI network terms. We identified both types of SLiMs in a
   variety of unrelated and non-homologous proteins that are involved in
   embryonic development and cancer progression. Surprisingly, we found
   that both SLiM types in multiple oxidoreductase enzymes were implicated
   in the regulation of redox homeostasis. Below, we discuss the
   implication of the most representative proteins retrieved in our work
   in the abovementioned cellular processes.

3.1. AFP[14–20]-like Motif-Containing Proteins

   The majority of proteins aligned to the AFP[14–20] segment belonged to
   transcription factors ([146]Table 1). Among them was TRIM proteins,
   which have three types of domains at their N-terminus: RING finger
   domain, B-box zinc finger domain, and coiled-coil region. These domains
   provide the involvement of TRIM proteins transcriptional regulation,
   cytoskeletal organization, epithelial development, cell adhesion, and
   immune response [[147]22]. Another retrieved transcription factor,
   HHEX, is involved in cell growth and differentiation, hepatic and
   pancreatic development, and anterior–posterior pattern specification
   via the Wnt signaling pathway [[148]23]. Additionally, HHEX has been
   associated with type 2 diabetes, whereas zinc finger proteins are
   linked to the progression of various cancers [[149]63,[150]64,[151]65].

   NOTCH family proteins function as receptors for membrane-bound ligands
   Jagged-1, Jagged-2, and Delta-1 to regulate cell fate and development
   through the formation of transcriptional regulator complexes
   [[152]24,[153]25]. Aberrant NOTCH expression has been linked to the
   progression of various types of cancer [[154]66]. Additionally, various
   EGF-like repeat-containing proteins, such as fibulin-2, implicated in
   embryonic development and tissue homeostasis [[155]26,[156]67] were
   aligned to the AFP[14–20] segment. SRGAP2 protein, which is implicated
   in spatially and temporally balanced development of excitatory and
   inhibitory synapses [[157]27], was also aligned to the AFP[14–20]
   segment. Calcium and integrin-binding family member 2 (CIB2), which
   blocks translocation of sphingosine kinase 1 (SK1) to the plasma
   membrane, was also identified. This protein inhibits cell signaling for
   sensitization to TNFα-induced apoptosis and inhibition of Ras-induced
   neoplastic transformation [[158]28].

   F-box motif proteins, which constitute the SCF-E3 ubiquitin ligase
   complex of the ubiquitin-proteasome (UPS) protein degradation pathway,
   were also retrieved. The proteins that are degraded with this complex
   include translational regulatory and cell-cycle proteins during
   embryogenesis [[159]29]. Ubiquitin-like modifier-activating enzyme 6
   (UBA6), which activates ubiquitin and uses ubiquitin-conjugating enzyme
   (E2) to target proteins to proteasomal degradation, was also aligned
   [[160]30]. UBA6 activates human leukocyte antigen F-adjacent transcript
   10 (FAT10), which serves as 26S proteasome-targeting signal, to be
   involved in epithelial-mesenchymal transition (EMT), invasion, and
   apoptosis in hepatocellular carcinoma [[161]31]. Dysregulation of a
   protein ubiquitination cascade is implicated in various human diseases,
   including neurodegenerative disorders and cancer [[162]68].

   Multiple proteins involved in the regulation of cell cycle, cell
   fusion, and apoptosis were also aligned to the AFP[14–20] segment
   [[163]32,[164]33]. An example is ethanolamine-phosphate
   cytidylyltransferase, which is involved in the biosynthesis of membrane
   phospholipid, PE [[165]34]. The Atg 4 cysteine proteases that are
   required for conjugation of Atg 8 to PE on autophagosomal membranes, a
   key step in autophagosome biogenesis during the macroautophagic
   process, were also retrieved [[166]35]. Interestingly, AFP[14–20]-like
   motifs were found in CTP:PE cytidylyltransferase, which is involved in
   phospho-ethanolamine biosynthesis from ethanolamine [[167]36].
   Upregulated phosphoethanolamine biosynthesis is required to meet
   increased demands in energy and metabolites for T-cell activation,
   cellular proliferation, and cancer cell adaptation [[168]69]. Immune
   response regulators aligned to AFP[14–20] include B-cell linker
   protein, which is crucial for B-cell differentiation.
   Downregulation/mutation in the BLNK gene has been shown to induce acute
   lymphoblastic leukemia through JAK3 signaling [[169]37].

   Living organisms have adapted to oxidative stress conditions via
   reversible post-translational chemical modifications of redox-sensitive
   amino acid residues in intracellular effectors of signal transduction
   pathways (protein kinases and protein phosphatases), transcription
   factors, etc. [[170]70]. Dysregulation of these mechanisms has been
   associated with various human diseases, including cancer. Proteins
   involved in redox regulation include 3-alpha hydroxysteroid
   dehydrogenase III, which belongs to steroidogenic oxidoreductase
   enzymes and uses NADPH or NADH, cofactors involved in ROS generation
   [[171]38]. The retrieved proteins include dual cyclooxygenase and
   peroxidase, as well as prostaglandin G/H synthase 1, which is involved
   in the biosynthesis of prostanoids and ROS generation [[172]39].
   Additionally, glutathione S-transferase LANCL1 is involved in oxidative
   stress response and is overexpressed in prostate cancer cells
   [[173]40]. LANCL1 causes the expression of glucose transporters, as
   well as mitochondrial uncoupling and respiration via the
   AMPK/PGC-1α/Sirt1 pathway [[174]71]. HSPB-associated protein 1
   (HSPBAP1) exhibits oxidoreductase activity, and its overexpression has
   been observed in prostate cancer samples [[175]41]. Another
   oxidoreductase, prolyl hydroxylase, catalyzes hydroxylation of proline
   residues in hypoxia-inducible factor-1α (HIF-1α) and other target
   proteins, such as ATF4, IKBKB, and CEP192 under hypoxia conditions
   [[176]42]. This leads to pVHL (von Hippel–Lindau protein)-dependent
   ubiquitination and rapid proteasomal degradation of HIF-1α, which is
   implicated in cancer progression [[177]72].

3.2. GIP-9-like Motif-Containing Proteins

   Proteins aligned with high significance to the GIP-9 segment of human
   AFP include various isoforms of C-C motif chemokine 4-like ([178]Table
   2), which has been shown to promote human trophoblast migration at the
   fetoplacental interface during embryonic development [[179]43].
   Additionally, other development-associated proteins, including
   Wnt-signaling regulators, such as AXIN2, were retrieved from the
   UniprotKB database. Wnt signaling is involved in embryonic pattern
   formation and tissue morphogenesis, whereas dysregulation of Wnt
   signaling has been implicated in various cancer types, including
   colorectal and hepatocellular carcinoma [[180]44]. Additionally, cell
   adhesion proteins were identified, such as L1 cell adhesion molecule
   (L1CAM or CD171), a transmembrane protein and member of the
   immunoglobulin superfamily, which plays a major role in nervous system
   development, as well as cancer cell migration and invasion [[181]45].

   Transcription factor, the paired (PRD)-like leucine twenty homeobox
   (LEUTX) domain protein, is expressed almost exclusively in human
   embryos during preimplantation development [[182]46]. HOXC5
   transcription factor is also involved embryonic development; however,
   the deregulation of HOXC5 has been shown to contribute to activation of
   the TERT gene in human cancers [[183]47]. Another transcription factor,
   FoxO1, is overexpressed and became acetylated due to the dissociation
   from histone deacetylase sirtuin-2 (SIRT2) in response to oxidative
   stress. This causes its binding to Atg7, the E1-like protein, leading
   to cancer cell death via autophagy and tumor suppression
   [[184]48,[185]49]. Additionally, RUNX family transcriptional
   regulators, key regulators of normal embryonic development
   overexpressed in cancer, were among the retrieved proteins [[186]50].

   Among cell-cycle regulators, BTG3-associated nuclear protein and
   cyclin-dependent kinase inhibitor 1B are involved in the cell-cycle
   G[1]/S transition, playing tumor-suppressor roles [[187]51].
   Additionally, various proteins involved in DNA replication, repair, and
   recombination were retrieved from the UniprotKB knowledgebase,
   identified as containing GIP-9-like motifs. An example is DNA
   replication complex GINS protein, a part of the human replisome, a
   molecular machine responsible for accurate chromosome replication
   [[188]52].

   Proteins with receptor activity include IGF-like family receptor 1,
   which is implicated in T-cell-mediated inflammation and associated with
   the prognosis of various cancers correlating with immune cell
   infiltration [[189]53,[190]54]. Another example is brain-specific
   angiogenesis inhibitor (BAI)-associated protein 2-like 1 (BAIAP2L1),
   known as insulin receptor tyrosine kinase (RTK) substrate. It belongs
   to putative G-protein-coupled receptors, with a wide spectrum of
   cellular activities, including inflammation and tumorigenesis
   [[191]55,[192]56]. E3 ubiquitin-protein ligase TRIM35, which
   participates in multiple biological processes, including cell death,
   glucose metabolism, and innate immune response to viral infection, was
   also found to contain the GIP-9-like motif [[193]57].

   Proteins involved in redox regulation include ceruloplasmin, an enzyme
   with ferroxidase activity and a major copper-binding protein in the
   blood, which plays a key role in redox homeostasis and metabolic
   regulation [[194]58]. Additionally, PNPO, which converts pyridoxine
   5’-phosphate into pyridoxal 5’-phosphate (PLP), an active form of
   vitamin B6, is implicated in several types of cancer and was aligned to
   GIP-9 [[195]59]. Another example is a growth hormone receptor (GHR)
   involved in metabolic regulation; its deficiency causes upregulation of
   enzymes involved in amino acid catabolism, urea cycle, and
   tricarboxylic acid cycle, as well as reduced mitochondrial import of
   fatty acids for beta-oxidation [[196]60].

4. Materials and Methods

4.1. Search for Short Linear Motifs

   The FastA suite of the European Molecular Biology Laboratory-European
   Bioinformatics Institute (EMBL-EBI) was applied
   ([197]https://www.ebi.ac.uk/Tools/sss/fasta/ (accessed on 22 November
   2021)) [[198]73] for local sequence alignment. Two human AFP-derived
   short sequences, LDSYQCT and EMTPVNPGV, were used as query sequences.
   The search was performed against the UniprotKB human taxonomic subset
   [[199]74]. GLSEARCH (version 36.3.8 h) algorithm provided the most
   optimal search to match the query sequences. The BLOSUM50 matrix and
   the following parameters were used to obtain as many as 500 alignments:
   gap open: 10; gap extension: 2; KTUP: 2; expectation value (E-value)
   upper unit: 10 and lower unit: 0.

4.2. Gene Ontology Analysis

   Lists of genes encoding the retrieved human proteins were composed for
   further analysis. Gene ontology resource ([200]http://geneontology.org/
   (accessed on 15 December 2021)) was utilized for gene enrichment
   analysis in biological process categories. The gene list analysis
   option of PANTHER17.0 classification system ([201]http://pantherdb.org/
   (accessed on 23 December 2021)) was used for this purpose [[202]75].
   GO-Slim annotation and a statistical overrepresentation test were
   applied. Additionally, the GeneCards human gene database
   ([203]https://www.genecards.org/ (accessed on 20 January 2022))
   annotations were applied for gene categorization [[204]76]. All query
   genes were retrieved from the UniprotKB knowledgebase and then
   converted to ENSEMBL [[205]77] gene IDs.

4.3. Gene Set Enrichment Analysis

   The ShinyGO v0.75 suite ([206]http://bioinformatics.sdstate.edu/go/
   (accessed on 8 February 2022)) was utilized [[207]78] for further
   detailed gene set enrichment analysis on the basis of molecular
   functions and metabolic pathway categories. Both fold enrichment and
   gene enrichment options were applied with a color heatmap of
   −log10(FDR). Fold enrichment is calculated by the percentage of genes
   in the list belonging to a pathway divided by the corresponding
   percentage of genes in the background, i.e., the whole human genome.
   Characteristics of genes in our lists were compared to those of genes
   of the whole human genome, and Student’s t-test was applied. Lolipop
   chats with an aspect ratio of 1.5 were utilized for visualization. FDR
   was calculated based on nominal p-value from the hypergeometric test in
   order to determine the likelihood of enrichment by chance.

4.4. KEGG Pathway Enrichment Analysis

   The KEGG pathway database [[208]79] analysis option of the ShinyGO
   v0.75 suite was used with an FDR cutoff of 0.4 for both gene enrichment
   and fold enrichment versions. Bar plot charts with an aspect ratio of
   1.5 were used for visualization to generate log10 (FDR) heat maps for
   each category.

4.5. Metabolic Pathway Analysis

   The reactome pathway database ([209]https://reactome.org/ (10 March
   2022)) was applied with the gene list analysis option and functional
   annotation report [[210]80]. Additionally, the Curated.Reactome option
   of the ShinyGO v0.75 suite was used to assess metabolic pathway
   enrichment analysis. A p-value (FDR) of 0.4 was used to identify as
   many as 40 pathways. The minimum pathway size was 5, and the maximum
   pathway size was 2000.

4.6. PPI Network Analysis

   The STRING ([211]https://string-db.org/ (accessed on 17 March 2022))
   suite was used for PPI network enrichment analysis [[212]81]. Full
   STRING network type, a confidence score of 0.150, and an FDR stringency
   of 1.0 percent were applied.

5. Conclusions

   Short linear motifs with sequence similarity to two biologically active
   sites of human AFP were identified in multiple non-homologous and
   unrelated proteins with the use of a local alignment algorithm. Gene
   ontology term-based categorization was performed on the proteins
   retrieved from the UniprotKB database. Gene set enrichment analysis in
   biological process, molecular functions, metabolic pathways, KEGG
   pathways, and PPI network categories allowed for identification of
   functional classes of the retrieved proteins. Transcription factors,
   proteins involved in DNA replication/repair, cell-cycle progression,
   signal transduction, ubiquitin-mediated protein degradation, immune
   response, and oxidoreductase enzymes were aligned to both types of
   SLiMs. The majority of proteins were involved in embryonic development,
   cancer, and redox regulation. Our data support the concept that
   proteins are composed of evolutionarily conserved short linear segments
   that are incorporated into their primary structure as functional
   building blocks to be reused in a variety of non-homologous proteins.

Author Contributions

   S.S.S., methodology, investigation, and original draft preparation;
   S.P.Z., software, investigation, and original draft preparation;
   I.M.M., formal analysis and visualization; N.T.M., conceptualization,
   methodology, data curation, and review and editing. All authors have
   read and agreed to the published version of the manuscript.

Institutional Review Board Statement

   Not applicable because this study did not involve humans and animals.

Informed Consent Statement

   Not applicable because this study did not involve humans.

Data Availability Statement

   Data are available in a publicly accessible repository. The data
   presented in this study are openly available in FigShare at
   doi:10.6084/m9.figshare.19806037.

Conflicts of Interest

   The authors declare no conflict of interest.

Funding Statement

   This research received no external funding.

Footnotes

   Publisher’s Note: MDPI stays neutral with regard to jurisdictional
   claims in published maps and institutional affiliations.

References