The recent progress of peptide regulators for the Wnt/β-catenin signaling pathway

Wnt signaling plays an important role in many biological processes such as stem cell self-renewal, cell proliferation, migration, and differentiation. The β-catenin-dependent signaling pathway mainly regulates cell proliferation, differentiation, and migration. In the Wnt/β-catenin signaling pathway, the Wnt family ligands transduce signals through LRP5/6 and Frizzled receptors to the Wnt/β-catenin signaling cascades. Wnt-targeted therapy has garnered extensive attention. The most commonly used approach in targeted therapy is small-molecule regulators. However, it is difficult for small-molecule regulators to make great progress due to their inherent defects. Therapeutic peptide regulators targeting the Wnt signaling pathway have become an alternative therapy, promising to fill the gaps in the clinical application of small-molecule regulators. In this review, we describe recent advances in peptide regulators for Wnt/β-catenin signaling.


. Introduction
Wnt was initially discovered ∼40 years ago (1), and the interest in Wnt signaling has increasingly risen. Wnt signaling plays an important role in many biological processes such as stem cell self-renewal, cell proliferation, migration, and differentiation (2). The Wnt signaling pathway is generally divided into canonical and non-canonical signaling pathways, i.e., β-catenin-dependent and β-catenin-independent signaling pathways, respectively (3). The β-catenin-dependent signaling pathway mainly regulates cell proliferation, differentiation, and migration (4). In the canonical signaling pathway, the Wnt family ligands transduce signals through LRP5/6 and Frizzled (FZD) receptors to the Wnt/β-catenin signaling cascades (5). The Porcupine (Porc) enzyme, a multiple-pass transmembrane O-acyltransferase in the endoplasmic reticulum, lipid-modifies the Wnt proteins. Through the Evi/Wntless multiple-pass transmembrane protein, the Wnt proteins are transported and secreted (6). The secreted Wnt ligands that are generally considered to stimulate the Wnt/β-catenin signaling pathway include Wnt1, Wnt2, Wnt3, Wnt8, and Wnt10 (7). Moreover, Wnt signals play a role by binding to the receptors during acute disease (8). The Wnt proteins commonly bind to the FZD receptor family, which are seventransmembrane receptors, including 10 members. FZD has an intracellular C-terminal domain transducing downstream signals and an N-terminal cysteine-rich domain binding to Wnt ligands (7). In addition, LRP5/6 are co-receptors for FZD. LRP5/6 receptors lead prepare and have difficulty entering cells, and can only act on a few types of targets (29). Interestingly, many of the properties of peptide drugs are intermediate between those of small molecular drugs and biologics. Peptide drugs have many advantages such as high specificity, good efficacy, good safety, low immunogenicity, membrane permeability, and low cost. They are widely used in clinical practice and have broad prospects (30,31). Therefore, therapeutic peptide regulators targeting the Wnt signaling pathway have become an alternative therapy, promising to fill the gaps left by small-molecule drugs and biologics. In this review, we describe recent advances in peptide regulators for the Wnt/βcatenin signaling pathway, aiming to use them better for the clinical treatment of diseases ( Figure 1; Table 1).

. Peptide regulators for Wnt ligands
Wnt signaling is important to many physiological, developmental, and disease processes (66). The Wnt signaling pathway is initiated by Wnt ligands. Wnt proteins are secreted proteins. They are synthesized and then lipid-modified by the Porc enzyme. Wnt proteins are further transported and secreted via the Evi/Wntless multiple-pass transmembrane (66,67). There are 19 Wnt genes, which are divided into 20 conserved Wnt subfamilies (68). Wnt1, Wnt2, Wnt3, Wnt8, and Wnt19 ligands are frequently considered to activate the canonical (also named Wnt/β-catenin) signaling pathway (69,70). Wnt4, Wnt5a, Wnt7, and Wnt11 are commonly considered to stimulate the non-canonical signaling pathway (71). In the absence of Wnt, β-catenin is not stabilized, and the Wnt/β-catenin signaling pathway is not activated. Therefore, the peptide regulators can inhibit the Wnt/β-catenin signaling pathway by inhibiting or binding to Wnt ligands (11).
The Klotho protein is an anti-aging protein abundantly expressed in normal kidneys. Klotho, a single-pass transmembrane protein, includes a short cytoplasmic tail (KL2) and a transmembrane segment (KL1). Based on the KL1 domain sequence of human Klotho, KP6 (sequence: QPVVTLYHWDLPQRLQDAYGGWANRALADH) was designed and synthesized by Chen et al. KP6 is 30 amino acids in length and contains a Wnt-binding motif ranging from Gln186 to His215 within the KL1 domain. KP6 can mimic Klotho's function. KP6 blocks the Wnt/β-catenin signaling pathway by binding to Wnt, which sequesters its ability to interact with LRP6. Furthermore, KP6 promotes survival and ameliorates kidney injury in diabetic mice (32).
A study indicated that the expression level of Wnt3a is high in myocardial hypertrophy mice and is positively related to cardiomyocyte apoptosis (72). The low-molecularmass peptide 2 (LMP2, sequence: TYGPVFMCL) is a major catalytic subunit of the immunoproteasome. Dysregulation of the immunoproteasome has been linked with a variety of diseases (73). LMP2 can activate the Wnt/β-catenin signaling pathway by improving the expression levels of Wnt3a and βcatenin. The expression of tight junction proteins (occludin, claudin-1, and ZO-1) is downregulated to decrease blood-brain barrier (BBB) permeability after activating the pathway (33) ( Figure 2; Table 2).
. Peptide regulators for Frizzled/LRP receptors When binding to target cells, Wnt proteins bind a receptor complex harboring FZD and LRP5/6 proteins (74). FZDs are a subset of seven-transmembrane proteins. They are the principal receptors of the Wnt/β-catenin signaling pathway (75). The Nterminal CRD domain of FZDs binds to Wnt ligands and LRP5/6 co-receptors. The C-terminus of FZDs is located in the cytoplasm, where it recruits and binds to Dsh for triggering subsequent signal cascades (76,77). A previous study indicated that the knockdown of FZD7 decreases cell proliferation, invasion, and viability (78). FZD7 plays an important role in cell development, progression, and stem cell biology (79). Several approaches, such as adenoviral therapy (80) and monoclonal antibodies (81), have been applied to inhibit the Wnt/β-catenin pathway by targeting FZD.
Atrial natriuretic peptide (ANP, sequence: SLRRSSCFGGRMDRIGAQSGL GCNSFRY) was synthesized as an inactive precursor (pro-ANP) that was converted to a mature active peptide after proteolytic cleavage by the membrane-related serine protease Corin, in which the extracellular region included Fzd1 and Fzd2 (receptors for Wnt signaling) (82). ANP may induce β-catenin stabilization and nuclear translation possibly by .

. . Peptide regulators for LRP receptor
ACBP is a polypeptide that was identified from goat spleen extract after immunization with gastric cancer lysates (83). ACBP can inhibit phosphor-LRP6 and stimulate β-catenin, further leading to active β-catenin in the cytoplasm. Subsequently, the Wnt/β-catenin signaling pathway was inhibited to suppress the invasion, migration, and proliferation of colorectal cancer (35).
. Peptide regulators for the cytoplasmic Axin /GSKβ destruction complex The destruction complex may be a dynamic multiprotein assembly. Its core components include the serine-threonine kinase GSK-3, APC protein, scaffolding protein Axin, CK1, and E3ubiquitin ligase β-TrCP (11). Peptide regulators may control the production of stable β-catenin by regulating whether the destruction complex falls apart.

. . Peptide regulators for Axin
Axin is a largely unstructured, flexible protein that contains β-catenin, GSK-3, and CK1-binding sites. Axin is a key control point for catenin destruction because of its ability to promote the phosphorylation of β-catenin (12). A previous study indicated that Axin mutations lead to inappropriate β-catenin-mediated transcription (85). In addition, overexpression of Axin in colon cancer cells can reduce the accumulation of β-catenin (86).
. /fmed. .   Conductin, also named Axin2, is an Axin paralog exhibiting similar domain architecture (39). In contrast to Axin, there are no indications for Axin2 polymerization. Axin2 appears to be diffusely distributed in the cytoplasm. In the lower polymerization of Axin2, it is less active in degrading β-catenin than Axin (87). The regulators of the G protein signaling (RGS) domain of Axin2 may prevent Axin2 polymerization, because of the considerable molecular structure of the RGS domain. A short peptide P 182−195 (sequence: MEENAYQVFLTSDI) is designed and synthesized, which is centered on aggregation preventing polymerization and consists of Axin amino acids 182−185 . The short peptide can induce the polymerization of Axin and further block Wnt signaling and colorectal cancer growth (39) ( Figure 5; Tables 5, 6).

. . Peptide regulators for GSKβ
GSK-3β is an evolutionarily conserved serine/threonine kinase functioning in numerous cellular processes such as cell cycle, DNA repair, cell proliferation, and metabolic and signaling pathways (88). GSK-3β can phosphorylate the serine of the PPPSP motif found in many Wnt signaling components (86). When its function is inhibited, the Wnt/β-catenin signaling pathway can be activated (86). Peptides can regulate Wnt/β-catenin by activating or inhibiting GSK-3β.

. . . Activators
The C-peptide (sequence: EVEDPQVPQLELGGGPEAGDL QTLALEVARQ) was initially identified in 1970 (89). It is cleaved from proinsulin during proteolytic processing. It can activate protein-protein interaction protein phosphatase 1 activity using a GSK-3β-dependent mechanism, which dephosphorylates GSK-3β at Ser9. The cross-activation between GSK-3β and protein phosphatase 1 decreases apoptosis and increases cellular senescence, leading to the inhibition of decidualization (46).
Three antioxidant peptides, namely, PIIVYWK (1004.57 Da, P1), TTANIEDRR (1074.54 Da, P2), and FSVVPSPK (860.09 Da, P3), were identified from a peptide fraction of 1-5 kDa obtained from blue mussels by peptic hydrolysis. It was found that the P1, P2, and P3 antioxidant peptides had a higher 2,2diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity than previously reported antioxidant peptides from blue mussels (90,91). In addition, the IC 50 value against DPPH of P1 and P3 (0.71 ± 0.01 and 1.09 ± 0.01 mM, respectively) was higher than that of the P2 antioxidant peptide (2.33 ± 0.56 mM). Therefore, P1 and P3 antioxidant peptides warrant further study (92). P1 and P3 antioxidant peptides may enhance protein expression of Wnt1 and Wnt3a and downregulate protein expression of GSK-3β, ultimately stimulating the nuclear translocation of β-catenin. Subsequently, the Wnt/β-catenin signaling pathway was activated to promote human bone marrow-derived mesenchymal stem cell differentiation into osteoblasts (43).  Pituitary adenylate cyclase-activating polypeptide (PACAP) is a type of peptide hormone superfamily, including glucagonlike peptides, glucagon, and vasoactive intestinal peptides (93), which exists in the form of PACAP38 and PACAP27 in vivo (94). PACAP has poor stability in vivo, and its half-life is less than 10 min. PACAP27-derived mutant peptide (named MPAPO, MHSDGIFTDSYSRYRKQLAVKKYLA AVKK) (95) was designed and synthesized. MPAPO, compared with PACAP27, has the following improvements: first, the binding and specific agonistic effects of MPAPO to the PAC1 receptor are significantly enhanced. Second, MPAPO has a longer half-life, more stable activity, and better stability. MPAPO can phosphorylate GSK-3β, which, once phosphorylated, loses the ability to phosphorylate β-catenin. β-Catenin enters the nucleus and drives the transcription of target genes to accelerate cell cycle progression and promote cell proliferation (42). IAGIP (sequence: EPVPPPPTPRSSRHDSGLDSMKD) was designed by linking the IKK-recognized (inflammation-responsive) motif to the LRP peptide (the GSK-3β inhibitory motif) (95). IAGIP can directly target the N-terminal region of GSK-3β, which includes Ser9, and can block GSK-3β activity. The inhibition of excessive GSK-3β activity provided a possible approach to controlling neuroinflammation in neurodegenerative diseases (44).
Adiponectin (APN) is a 247-amino-acid polypeptide with a molecular weight of 30 kDa. BBB permeability was restricted by APN under physiological conditions. The APN peptide (APNp, sequence: LQVYGDGDHNGLYADNVN) was chemically synthesized based on the functional area in the globular domain of APN (96). APNp could effectively cross the BBB and bind with the intercephalic APN receptors to reliably simulate and maintain the function of endogenous APN. APNp can upregulate the phosphorylation of GSK-3β to improve neurological function and exert antiapoptotic, anti-inflammatory, and antioxidative effects against cerebral ischemia-reperfusion injury (41).
p-GSK-3β can be upregulated by exogenous administration of apolipoprotein E (apoE), thereby promoting neuronal survival. However, apoE has difficulty crossing the BBB because of its 34-kDa molecular weight, thereby limiting its translational study (97). COG1410 (sequence: AS(Aib)LRKL(Aib)KRLL, Aib: 2-Aminoisobutyric acid), a modified apoE mimetic peptide, was designed with a composition of apoE residues 138-149 and the modification of two residues. COG1410 could effectively cross the BBB and have a long-lasting effect (98). COG1410 promoted neuronal autophagy by promoting the phosphorylation of GSK-3β (40).
A novel peptide purified from the hydrolysates of tuna backbone protein (APTBP, VKAGFAWTANQQLS) was identified to have strong antioxidant activity (99). APTBP had high stability and a significant amphipathic characteristic. APTBP with a cellpenetrating peptide (CPP: GRKKRRQRRRPPQ) was chemically synthesized and derived from HIV-1 Tat (34-39, 80-86) attached to the N-terminus. The expression of APC and Axin2 was decreased by treatment with CPP-APTBP, which indicated that the CPP-APTBP function could be related to the disruption of the destruction complex. Subsequently, the activity of Wnt/βcatenin could be restored by CPP-APTBP. The accumulation of βcatenin can ameliorate hypoxia-induced cardiomyocyte apoptosis (47) (Figure 6; Table 7).
. Peptide regulators for β-catenin and its target genes β-Catenin is a type of plasma membrane-associated protein, and it plays a dual role in cellular signaling. On the one hand, βcatenin can stabilize cell-cell contact. On the other hand, it plays a role in activating gene transcription after β-catenin translocation to the nucleus (100). β-Catenin plays a crucial role in a multitude .

. . . Activators
Apelin is the endogenous ligand for APJ, which is a seventransmembrane G protein-coupled receptor (102). Apelin consists of numerous isoforms, including Apelin-12, 13, 17, and 36, which are all derived from the C-terminal fragment of the Apelin pre-proprotein with 77 amino acids (103). Among them, Apelin-13 needs further study because it exhibits the most active isoforms binding to the APJ receptor (104). Apelin-13 (sequence: QRPRLSHKGPMPF) could activate the Wnt/β-catenin signaling cascade by increasing the expression levels of total β-catenin and active β-catenin, thereby promoting osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (54).
Gastrin-releasing peptide is a neuropeptide that acts through G protein-coupled receptors, which stimulate the proliferation of mesenchymal cells in the fetal monkey lung (105). GRP increased active β-catenin and total β-catenin levels and β-catenin mRNA expression. Myofibroblast differentiation was promoted after the activation of the pathway (56).
The peptide vaccine for melanoma includes four peptides: Melan-A (sequence: AAGI GILTV), gp100 (sequence: KTWGQYWQV), MAGE-3 (sequence: FLWGPRALV), and NA-17 (sequence: VLPDVFIRCV). Melan-A has a "bulged" and "stretched" conformation. To identify the MART-1 epitopes for tumor-infiltrating lymphocytes, 23 peptides were selected based on the known peptide-binding motif to HLA-A2.1 and synthesized (>90% purity). Among them, Malan-A could most effectively sensitize T2 cells for lysis. Gp100 is a melanocyte-derived lineage-restricted intracellular glycoprotein that can be presented on the cell surface in association with major histocompatibility complex molecules in T lymphocytes (106). Several peptides generated from gp100 have been isolated and characterized. Among them, gp154-162 (sequence: KTWGQYWQV) necessitated further study because it was the most abundant at the cell surface (107). Six genes of the MAGE family, namely, MAGE-1, 2, 3, 4, 6, and 12, were found to be expressed at a high level in many tumor types of various histological origins (108). A MAGE-3 .
/fmed. .   antigen presented by HLA-A2, which is the most common HLA allele, might therefore be widely used in the immunotherapy of melanoma. Based on the initial screening for binding to HLA-A2, the peptide (MAGE-3, sequence: FLWGPRALV) was synthesized (109). NA17 was designed and synthesized based on N-acetylglucosaminyltransferase-V from the GnT-V gene intron, nt 38-67, which appeared to induce peptide-specific recognition of target cells (110). After the vaccine is injected into patients with metastatic melanoma, the β-catenin protein stabilization is enhanced. In addition, the high-level expression of β-catenin is found in recurrent tumors after developing new treatmentresistant metastases. Therefore, the peptides in the vaccine are considered to be closely related to β-catenin (58).

. . . Inhibitors
The first Buthus martensii Karsch (BmK) analgesic peptide was identified from a venom (111). Since then, more BmK analgesic peptides, including BmK AGAP, have been purified (112). BmK AGAP (sequence: VRDGYIADDKNCA YFCGRNAYCDDECEKNGAESGYC-QWAGVYGNACWCYKLPDKVPIRVPGKCNGG) belongs to a group of long-chain scorpion peptides, and it has a molecular mass of 7,142 Da (113). The rBmK has a molecular scaffold consisting of an α-helix and three-stranded antiparallel β-sheets, resulting in a compact core and several loops and c-turns extending outside (114). It can target PTX3 and further decrease the expression of β-catenin to influence the Wnt/β-catenin signaling pathway. BmK AGAP can effectively inhibit breast cancer cells (IC 50 for MCF-7 = 40 µM; IC 50 for MDA-MB-231 cells = 50 µM). In addition, the expression of the target gene of β-catenin, E-cadherin, is upregulated, thereby reducing breast cancer cell stemness and epithelial-mesenchymal transition (48).
/fmed. . Enhancing β-catenin protein stabilization a S5: (S)-α-(4-pentenyl) alanine, a pair of S5 was coupled through the ring-closing metathesis reaction. b Ahx: 6-aminocaproic acid; S5: (S)-α-(4-pentenyl) alanine, a pair of S5 was coupled through the ring-closing metathesis reaction. enhanced affinity for β-catenin (K d = 60 ± 2 nM). The StAx-3 sequence was thus selected for further optimization. In a parallel series of experiments, affinity optimization of the Axin peptide sequence was examined. In addition, 32 new sequences were obtained. Among them, peptide fStAx-35R (sequence: RRWPRSILDSHVRRVWR) with high binding affinity for βcatenin (K d = 53 ± 9 nM) and positive charge was obtained upon the addition of Arg and Trp at positions 467 and 468 (fStAx-34) and addition of arginine residues. Proteolysis-targeting chimera is a useful technology to degrade proteins in vivo.
PTHrP is synthesized and expressed by various cells and tissues. After posttranslational processing, PTPrp could generate a variety of fragments, including PTHrP (1-34, sequence: AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTA), PTHrP , and PTHrP (107-139) peptides. Among them, PTHrP  can bind to the PTHrP receptor (PTH1R), a common G proteincoupled receptor. PTHrP (1-34) warrants further study because of its important role in tooth and bone development (115). PTHrP  can inactivate the Wnt/β-catenin pathway and further inhibit the expression of osteogenic-related genes (ALP, RUNX2, BSP, and OPN) in dental follicle cells. Ultimately, osteogenesis of dental follicle cells is inhibited (51). SOX9, the most extensively studied SOX family member, was both a downstream target and an inhibitor of the Wnt/β-catenin signaling pathway (116).
S9pep (sequence: ADSPHSSSGMSEVHSPGEHSG) was designed and synthesized based on the amino acid sequence Frontiers in Medicine frontiersin.org . /fmed. . located in the SOX9 central region. The peptide has S9pep, which could inhibit the expression of c-Myc (its oncogenic target gene) by interacting with and relocating β-catenin from the chromatin to the cytosol, like SOX9 (52). A deca-peptide library of the DXMXXPQQTE sequence with 6,859 members was produced in MATLAB, where X denotes one of the 19 standard amino acids, except for cysteine. PEP8 (sequence: DEMEEPQQTE) was selected and synthesized after a series of screenings, for example, the calculation of affinity energy values for each amino acid and the root mean square deviation from the reference conformation. Peptide PEP8 expanded the size of the repository of druggable target proteins. PEP8 can inhibit the formation of the β-catenin/LRH-1 complex, thereby inhibiting the Wnt/β-catenin signaling pathway. In addition, PEP8 can inhibit the growth of AsPC-1 cells (IC 50 value = 288 µM). To promote entry into the cells, TAT-PEP8 (sequence: GRKKRRQRRRPPQDEMEEPQQTE) was designed and synthesized by attaching the cell-penetrating peptide TAT to PEP8. TAT-PEP8 can inhibit the growth of AsPC-1 cells (IC 50 value = 111 µM) (53).
B-cell lymphoma 9 (BCL9) is a type of co-activator of βcatenin, which was first discovered in some B-cell malignant tumors (117). Peptide regulators targeted in the β-catenin/BCL9 interaction have garnered extensive attention, such as SAH-BCL9. Hydrocarbon stapling was applied to generate cellpermeable α-helical peptides of the BCL9 HD2 domain for in vivo and in vitro studies (118, 119). Non-natural amino acids with olefinic side chains were substituted at (i, i+4) positions, followed by ruthenium-catalyzed olefin metathesis to yield SAH-BCL9 peptides A through C. Among SAH-BCL9 A−C , SAH-BCL9 B (sequence: LSQEQLEHRERSLXTRXIQRMLF) was identified as the most effective β-catenin-targeting peptide. In addition, circular dichroism analysis confirmed that hydrocarbon stapling consistently enhanced peptide α-helicity compared with the corresponding unmodified peptide (BCL9 HD2 ). SAH-BCL9 could target β-catenin in cells with a high affinity (IC 50 = 135 nM) and selectively disrupt the BCL9/β-catenin complex in a dose-dependent manner. Blockade of these interactions inhibited β-catenin-dependent transcriptional activity and target gene expression and suppressed tumor cell growth, metastasis, and angiogenesis (49) (Figure 7; Table 8).
. . Peptide regulators for c-Myc c-Myc (a type of target gene of β-catenin) is a type of proto-oncogene. It encodes a phosphoprotein that acts in cellular transformation, apoptosis, and cell cycle progression (120). When Kaposi sarcoma herpesvirus (KSHV) reactivation begins, RNA polymerase II molecules are effectively recruited to viral episomes and form a complex with a viral protein, KSHV replication, and transactivator (K-Rta), for vital gene expression (121). Moreover, KSHV reactivation induced the interaction of coactivators with both RNA polymerase II and K-Rta. A small peptide was designed based on the binding interface of K-Rta and the cellular coactivator complex. The homologous protein sequences were extracted from other gamma-herpesvirus homologs and a bacterial transcription factor. Based on the conserved protein sequence, a series of K-Rta peptides (P1-P5) were synthesized. Among them, P1 (renamed VGN50, sequence: LSSILQGLYQLDT) needs further study because of its major effect on cell viability and viral replication. VGN50 could recruit the SWI/SNF complex by engaging the MYC promoter, resulting in the downregulation of both MYC and MYCtarget gene transcription. Ultimately, cell proliferation in leukemia and lymphoma is inhibited (65) (Figure 8; Table 9).

. Peptide regulators for TCF/LEF
Although it is challenging to directly target β-catenin, it is possible to indirectly activate it through transcriptional cofactors and by controlling its stabilization. TCF and LEF are transcription factors mainly involved in the Wnt/β-catenin signaling pathway (122). The mammalian TCF/LEF consists of four nuclear factors, namely, TCF7L1, TCF7L2, LEF1, and TCF7 (also known as TCF3,   TCF4, LEF1, and TCF1, respectively) (123). The activation of target genes by the β-catenin/TCF complex has been considered the main mode of the Wnt/β-catenin signaling pathway (124). LEF1 is widely expressed during mouse embryonic development. Moreover, its expression is tissue-specific in adults (125). A full-length LEF isoform that interacts with β-catenin is regulated by β-catenin-TCF complexes (122). Cementum markers, such as cementum protein 1 (CEMP1), were shown to promote the proliferation and differentiation of periodontal ligament cells toward a "mineralizing-like" phenotype (126). The secondary structure analysis of CEMP1 demonstrated that it had a random coil structure. A short peptide (named CEMP1-p4, sequence: QGQGDTEDGRMTLMG) was designed and synthesized based on the C-terminus of CEMP1, activating β-catenin signaling in human oral mucosa stem cells. CEMP1-p4 also had a random coil structure and could mimic the biological capabilities of CEMP1. CEMP1-p4 may activate the Wnt/β-catenin pathway by enhancing the expression of β-catenin, decreasing the expression of GSK-3β, and activating downstream transcriptional factors (TCF1/7 and LEF1). Subsequently, the expression of mineralization-related markers (OSX, RUNX2, IBSP, and OCN) was upregulated in human oral mucosal stem cells at both the mRNA and protein levels, driving the differentiation of HOMSCs to a mineralizing-like phenotype (64) (Figure 9; Table 10).
OA-GL17d (sequence: GLFKWHPRCGEEQSMWT), a new natural peptide homodimer, was identified from the skin secretions of Oreochromis andersonii. OA-GL17d was degraded after 10 h, and its half-life was ∼1.86 h, much longer than that of some peptides (130). It may act on TGF-β1 through miR-663a to activate the SMAD signaling pathway, thereby promoting skin wound regeneration (59).
The pilose antler polypeptide (PAP-3.2KD, sequence: VLSATDKTNVLAAW GKVGGNAPAFGAEALERM) is one of the main components of pilose antler. PAP-3.2KD may decrease the expression of TGF-β1 and further reduce SMADs, which are the target proteins of TGF-β1 signal transduction. Ultimately, the TGF-β/SMAD signaling pathway is inhibited, which thereby ameliorates histopathological damage, myocardial fibrosis, and apoptosis in adriamycin-induced myocardial tissues (61).
A total of 1,697 endogenous peptides in hypertrophic scar tissues and matched normal skin were identified using liquid chromatography-mass spectrometry/mass spectrometry. Among these, 78 peptides were highly expressed in normal tissues compared with the corresponding hypertrophic scar tissues. The peptides with fat solubility (aliphatic index > 100) and high stability (instability index < 0) were further screened. Considering that endogenous peptides were derived through hemoglobin reduction in hypertrophic scar tissues, LYENRL and ASGVAVSDGVIKV (ASGVA) were selected for further assessment of biological function. In addition, LYENPL could inhibit the proliferation of human skin fibroblast cells at a lower dose compared with . /fmed. .
ASGVA. LYENRL could reduce SMAD2/3 phosphorylation and the SMAD2/3/4 complexes to the SMAD Binding element, thereby inhibiting TGF-β1/SMAD signaling. Additionally, LYENRL can inhibit the proliferation, migration, and extracellular matrix production of scar fibroblasts in a concentration-dependent manner (62). A region termed the SMAD4-binding domain (SBD) was identified, which was an amino-terminal region of transactivation 2 of p65 (a main subunit of NF-κB), associated with the mad homology 1 domain of SMAD4 to inhibit bone morphogenetic protein signaling. Based on the SBD, a cell-permeable SBD peptide (sequence: RRRRRRRGGGQAGEGTLSEALLHLQF) was designed and synthesized, which could block the association of p65 with SMAD4. In addition, the SBD peptide enhances BMP2-induced osteoblast differentiation and mineralization (63).
APNp could suppress mitochondrial and ATF4-CHOP apoptosis pathways in a SMAD3-dependent manner by targeting GSK-3β. Thus, neural survival was promoted following intracerebral hemorrhage injury in the diabetic setting (60) (Figure 10; Table 11).

. Conclusion and outlook
In summary, we have briefly explored the importance of the Wnt/β-catenin signaling pathway in human diseases. This signaling pathway is mainly involved in regulating biological processes such as cell proliferation, differentiation, and migration. In the Wnt/βcatenin signaling pathway, Wnt family ligands transduce signals to the Wnt/β-catenin signaling cascade through LRP5/6 and FZD receptors. Therefore, targeting molecules in the Wnt/β-catenin signaling pathway may modulate human diseases by activating or inhibiting this pathway, thereby affecting biological processes. Given recent research advancements, we summarize the peptide modulators in the Wnt/β-catenin signaling pathway. Studies have indicated that peptide regulators regulate the Wnt/β-catenin signaling pathway by targeting and antagonizing or agonizing molecules in the pathway.
Peptide regulators of the Wnt/β-catenin signaling pathway have shown promising results in preclinical studies. They have the potential to treat several diseases such as cancer, osteoporosis, and Alzheimer's disease. A recent study has shown that the interaction of the protein FAM83A with catenin is very important for the regulation of the development process of pancreatic cancer. Multiple blocking short peptide drug precursors targeting FAM83A catenin were screened and obtained. Their effectiveness was verified in animal models such as zebrafish and mice (131). In the future, peptide regulators could be developed into therapeutics that target specific diseases with great efficacy and fewer side effects than current treatments. Additionally, the development of peptide drug delivery systems could improve the bioavailability and pharmacokinetic properties of these agents. Furthermore, combining peptide regulators with other signaling pathway inhibitors could offer synergistic effects in treating diseases such as cancer. More research on the safety and efficacy of these peptide regulators is needed to fully understand their potential therapeutic benefits. However, polypeptide drugs also have some potential challenges, such as unstable physical and chemical properties, a short half-life, a fast clearance rate, and a lack of an effective drug delivery system, which are worthy of further exploration in future studies. Overall, peptide regulators of the Wnt/β-catenin signaling pathway have promising future perspectives as potential therapeutics for a range of diseases.