RESUMEN
BACKGROUND: Adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1) plays a crucial role in regulating insulin signaling and glucose metabolism. Mutations in the APPL1 gene have been associated with the development of maturity-onset diabetes of the young type 14 (MODY14). Currently, only two mutations [c.1655T>A (p.Leu552*) and c.281G>A p.(Asp94Asn)] have been identified in association with this disease. Given the limited understanding of MODY14, it is imperative to identify additional cases and carry out comprehensive research on MODY14 and APPL1 mutations. AIM: To assess the pathogenicity of APPL1 gene mutations in diabetic patients and to characterize the functional role of the APPL1 domain. METHODS: Patients exhibiting clinical signs and a medical history suggestive of MODY were screened for the study. Whole exome sequencing was performed on the patients as well as their family members. The pathogenicity of the identified APPL1 variants was predicted on the basis of bioinformatics analysis. In addition, the pathogenicity of the novel APPL1 variant was preliminarily evaluated through in vitro functional experiments. Finally, the impact of these variants on APPL1 protein expression and the insulin pathway were assessed, and the potential mechanism underlying the interaction between the APPL1 protein and the insulin receptor was further explored. RESULTS: A total of five novel mutations were identified, including four missense mutations (Asp632Tyr, Arg633His, Arg532Gln, and Ile642Met) and one intronic mutation (1153-16A>T). Pathogenicity prediction analysis revealed that the Arg532Gln was pathogenic across all predictions. The Asp632Tyr and Arg633His variants also had pathogenicity based on MutationTaster. In addition, multiple alignment of amino acid sequences showed that the Arg532Gln, Asp632Tyr, and Arg633His variants were conserved across different species. Moreover, in in vitro functional experiments, both the c.1894G>T (at Asp632Tyr) and c.1595G>A (at Arg532Gln) mutations were found to downregulate the expression of APPL1 on both protein and mRNA levels, indicating their pathogenic nature. Therefore, based on the patient's clinical and family history, combined with the results from bioinformatics analysis and functional experiment, the c.1894G>T (at Asp632Tyr) and c.1595G>A (at Arg532Gln) mutations were classified as pathogenic mutations. Importantly, all these mutations were located within the phosphotyrosine-binding domain of APPL1, which plays a critical role in the insulin sensitization effect. CONCLUSION: This study provided new insights into the pathogenicity of APPL1 gene mutations in diabetes and revealed a potential target for the diagnosis and treatment of the disease.
RESUMEN
OBJECTIVE: To study the effects of TrkB-BDNF signal pathway on the synthesis and secretion of vascular endothelial growth factor (VEGF) in human neuroblastoma cells (NB). METHODS: TrkB protein expression in SY5Y cells before and after all-trans-retinoicacid (ATRA) treatment was detected by Western blot. P-TrkB protein expression in SY5Y cells before and after the treatment of ATRA along with BDNF was also detected by Western blot. VEGF concentrations in the SY5Y cell culture supernatants were measured using ELISA after the treatment with ATRA, BDNF, tyrosine kinase inhibitor K252a and PI3k inhibitor LY294002. RESULTS: TrkB protein was undetectable in SY5Y cells before ATRA treatment. After the treatment of 1, 10 and 100 nM/L ATRA for five days, TrkB protein was expressed in SY5Y cells and the TrkB protein level increased with the increasing ATRA concentration. P-TrkB protein was not expressed in SY5Y cells treated only with 10 nM/L ATRA, but it was detectable after the treatment of ATRA along with BDNF. VEGF concentrations in the group treated with ATRA+BDNF were significantly higher than those in the untreated control and the ATRA alone treatment groups (P<0.01). VEGF concentrations in the K252a pretreated ATRA+BDNF group were significantly lower than those in the group treated with ATRA+BDNF (P<0.05). VEGF concentrations in the LY294002 treatment group (ATRA+LY294002+BDNF group) were also significantly lower than those in the group treated with ATRA+BDNF (P<0.01). CONCLUSIONS: Activation of TrkB-BDNF signal pathway may increase the synthesis and secretion of VEGF in human NB cells. The synthesis and secretion of VEGF can be inhibited by blocking TrkB-BDNF signal pathway with K252a or blocking the TrkB-BDNF downstream signal pathway PI3K/Akt with LY294002.