Alanine-Scanning Mutagenesis of α-Conotoxin GI Reveals the Residues Crucial for Activity at the Muscle Acetylcholine Receptor.

Recently, the muscle-type nicotinic acetylcholine receptors (nAChRs) have been pursued as a potential target of several diseases, including myogenic disorders, muscle dystrophies and myasthenia gravis, etc. α-conotoxin GI isolated from Conus geographus selectively and potently inhibited the muscle-type nAChRs which can be developed as a tool to study them. Herein, alanine scanning mutagenesis was used to reveal the structure⁻activity relationship (SAR) between GI and mouse α1ß1δε nAChRs. The Pro5, Gly8, Arg8, and Tyr11 were proved to be the critical residues for receptor inhibiting as the alanine (Ala) replacement led to a significant potency loss on mouse α1ß1δε nAChR. On the contrary, substituting Asn4, His10 and Ser12 with Ala respectively did not affect its activity. Interestingly, the [E1A] GI analogue exhibited a three-fold potency for mouse α1ß1δε nAChR, whereas it obviously decreased potency at rat α9α10 nAChR compared to wildtype GI. Molecular dynamic simulations also suggest that loop2 of GI significantly affects the interaction with α1ß1δε nAChR, and Tyr11 of GI is a critical residue binding with three hydrophobic amino acids of the δ subunit, including Leu93, Tyr95 and Leu103. Our research elucidates the interaction of GI and mouse α1ß1δε nAChR in detail that will help to develop the novel analogues of GI.

Effect of Methionine Oxidation and Substitution of α-Conotoxin TxID on α3ß4 Nicotinic Acetylcholine Receptor.

α-Conotoxin TxID was discovered from Conus textile by gene cloning, which has 4/6 inter-cysteine loop spacing and selectively inhibits α3β4 nicotinic acetylcholine receptor (nAChR) subtype. However, TxID is susceptible to modification due to it containing a methionine (Met) residue that easily forms methionine sulfoxide (MetO) in oxidative environment. In this study, we investigated how Met-11 and its derivatives affect the activity of TxID using a combination of electrophysiological recordings and molecular modelling. The results showed most TxID analogues had substantially decreased activities on α3β4 nAChR with more than 10-fold potency loss and 5 of them demonstrated no inhibition on α3β4 nAChR. However, one mutant, [M11I]TxID, displayed potent inhibition at α3β4 nAChR with an IC50 of 69 nM, which only exhibited 3.8-fold less compared with TxID. Molecular dynamics simulations were performed to expound the decrease in the affinity for α3β4 nAChR. The results indicate replacement of Met with a hydrophobic moderate-sized Ile in TxID is an alternative strategy to reduce the impact of Met oxidation, which may help to redesign conotoxins containing methionine residue.

CK1δ/ε inhibition induces ULK1-mediated autophagy in tumorigenesis.

INTRODUCTION: Autophagy is an important mechanism of cell homeostasis maintenance. As essential serine/threonine-protein kinases, casein kinase I family members affect tumorigenesis by regulating a variety of cellular progression. However, the mechanism by which they regulate autophagy remains unclear. MATERIALS AND METHODS: We silenced CK1δ/ε in cancer cells and observed cell morphology, the expression of autophagy-related genes, and its impact on cancer cell growth and viability. By inhibiting CK1δ/ε-induced upregulation of autophagy genes, we profiled the regulatory mechanism of CK1δ/ε on autophagy and cancer cell growth. The impact of CK1δ/ε inhibition on tumor cell growth was also assessed in vivo. RESULTS: Here, we found that CK1δ/ε played an important role in ULK1-mediated autophagy regulation in both lung cancer and melanoma cells. Mechanically, silencing CK1δ/ε increased ULK1 expression with enhanced autophagic flux and suppressed cancer cell proliferation, while ULK1 knockdown blocked the activation of autophagy caused by CK1δ/ε inhibition. By silencing CK1δ/ε in syngeneic mouse model bearing LLC1 murine lung cancer cells in vivo, we observed tumor growth suppression mediated by CK1δ/ε inhibition. CONCLUSION: Our results provide evidence for the role of CK1δ/ε in the regulation of tumorigenesis via the ULK1-mediated autophagy, and also suggest the impact of CK1δ/ε inhibition on tumor growth and its significance as a potential therapeutic target.

The CK1ε/SIAH1 axis regulates AXIN1 stability in colorectal cancer cells.

Casein kinase 1ε (CK1ε) and axis inhibitor 1 (AXIN1) are crucial components of the ß-catenin destruction complex in canonical Wnt signaling. CK1ε has been shown to interact with AXIN1, but its physiological function and role in tumorigenesis remain unknown. In this study, we found that CK1δ/ε inhibitors significantly enhanced AXIN1 protein level in colorectal cancer (CRC) cells through targeting CK1ε. Mechanistically, CK1ε promoted AXIN1 degradation by the ubiquitin-proteasome pathway by promoting the interaction of E3 ubiquitin-protein ligase SIAH1 with AXIN1. Genetic or pharmacological inhibition of CK1ε and knockdown of SIAH1 downregulated the expression of Wnt/ß-catenin-dependent genes, suppressed the viability of CRC cells, and restrained tumorigenesis and progression of CRC in vitro and in vivo. In summary, our results demonstrate that CK1ε exerted its oncogenic role in CRC occurrence and progression by regulating the stability of AXIN1. These findings reveal a novel mechanism by which CK1ε regulates the Wnt/ß-catenin signaling pathway and highlight the therapeutic potential of targeting the CK1ε/SIAH1 axis in CRC.

The CK1δ/ϵ-Tip60 Axis Enhances Wnt/ß-Catenin Signaling via Regulating ß-Catenin Acetylation in Colon Cancer.

Casein kinase 1δ/ϵ (CK1δ/ϵ) are well-established positive modulators of the Wnt/ß-catenin signaling pathway. However, the molecular mechanisms involved in the regulation of ß-catenin transcriptional activity by CK1δ/ϵ remain unclear. In this study, we found that CK1δ/ϵ could enhance ß-catenin-mediated transcription through regulating ß-catenin acetylation. CK1δ/ϵ interacted with Tip60 and facilitated the recruitment of Tip60 to ß-catenin complex, resulting in increasing ß-catenin acetylation at K49. Importantly, Tip60 significantly enhanced the SuperTopFlash reporter activity induced by CK1δ/ϵ or/and ß-catenin. Furthermore, a CK1δ/CK1ϵ/ß-catenin/Tip60 complex was detected in colon cancer cells. Simultaneous knockdown of CK1δ and CK1ϵ significantly attenuated the interaction between ß-catenin and Tip60. Notably, inhibition of CK1δ/ϵ or Tip60, with shRNA or small molecular inhibitors downregulated the level of ß-catenin acetylation at K49 in colon cancer cells. Finally, combined treatment with CK1 inhibitor SR3029 and Tip60 inhibitor MG149 had more potent inhibitory effect on ß-catenin acetylation, the transcription of Wnt target genes and the viability and proliferation in colon cancer cells. Taken together, our results revealed that the transcriptional activity of ß-catenin could be modulated by the CK1δ/ϵ-ß-catenin-Tip60 axis, which may be a potential therapeutic target for colon cancer.

Transmembrane protein 97 exhibits oncogenic properties via enhancing LRP6-mediated Wnt signaling in breast cancer.

Upregulation of transmembrane protein 97 (TMEM97) has been associated with progression and poor outcome in multiple human cancers, including breast cancer. Recent studies suggest that TMEM97 may be involved in the activation of the Wnt/ß-catenin pathway. However, the molecular mechanism of TMEM97 action on Wnt/ß-catenin signaling is completely unclear. In the current study, TMEM97 was identified as an LRP6-interacting protein. TMEM97 could interact with LRP6 intracellular domain and enhance LRP6-mediated Wnt signaling in a CK1δ/ε-dependent manner. The binding of TMEM97 to LRP6 facilitated the recruitment of CK1δ/ε to LRP6 complex, resulting in LRP6 phosphorylation at Ser 1490 and the stabilization of ß-catenin. In breast cancer cells, knockout of TMEM97 attenuated the Wnt/ß-catenin signaling cascade via regulating LRP6 phosphorylation, leading to a decrease in the expression of Wnt target genes AXIN2, LEF1, and survivin. TMEM97 deficiency also suppressed cell viability, proliferation, colony formation, migration, invasion, and stemness properties in breast cancer cells. Importantly, TMEM97 knockout suppressed tumor growth through downregulating the Wnt/ß-catenin signaling pathway in a breast cancer xenograft model. Taken together, our results revealed that TMEM97 is a positive modulator of canonical Wnt signaling. TMEM97-mediated Wnt signaling is implicated in the tumorigenesis of breast cancer, and its targeted inhibition may be a promising therapeutic strategy for breast cancer.

Identification of Crucial Residues in α-Conotoxin EI Inhibiting Muscle Nicotinic Acetylcholine Receptor.

α-Conotoxins (α-CTxs) are small disulfide-rich peptides from venom of Conus species that target nicotinic acetylcholine receptors (nAChRs). The muscle-type nAChRs have been recognized as a potential target for several diseases, such as myogenic disorders, muscle dystrophies, and myasthenia gravis. EI, an α4/7-CTx, mainly blocks α1ß1δε nAChRs and has an extra N-terminal extension of three amino acids. In this study, the alanine scanning (Ala-scan) mutagenesis was applied in order to identify key residues of EI for binding with mouse α1ß1δε nAChR. The Ala-substituted analogues were tested for their abilities of modulating muscle and neuronal nAChRs in Xenopus laevis oocytes using two-electrode voltage clamp (TEVC) recordings. Electrophysiological results indicated that the vital residues for functional activity of EI were His-7, Pro-8, Met-12, and Pro-15. These changes exhibited a significant decrease in potency of EI against mouse α1ß1δε nAChR. Interestingly, replacing the critical serine (Ser) at position 13 with an alanine (Ala) residue resulted in a 2-fold increase in potency at the α1ß1δε nAChR, and showed loss of activity on α3ß2 and α3ß4 nAChRs. Selectivity and potency of [S13A] EI was improved compared with wild-type EI (WT EI). In addition, the structure-activity relationship (SAR) of EI revealed that the "Arg1-Asn2-Hyp3" residues at the N-terminus conferred potency at the muscle-type nAChRs, and the deletion analogue △1-3 EI caused a total loss of activity at the α1ß1δε nAChR. Circular dichroism (CD) spectroscopy studies demonstrated that activity loss of truncated analogue △1-3 EI for α1ß1δε nAChR is attributed to disturbance of the secondary structure. In this report, an Ala-scan mutagenesis strategy is presented to identify crucial residues that are significantly affecting potency of E1 for mouse α1ß1δε nAChR. It may also be important in remodeling of some novel ligands for inhibiting muscle-type nAChRs.