title: Precursor of brain-derived neurotrophic factor (proBDNF) forms a complex with Huntingtin-associated protein-1 (HAP1) and sortilin that modulates proBDNF trafficking, degradation, and processing.
abstract: proBDNF, a precursor of brain-derived neurotrophic factor (BDNF), is anterogradely transported and released from nerve terminals, but the mechanism underlying this process remains unclear. In this study, we report that proBDNF forms a complex with Huntingtin associated protein-1 (HAP1) and sortilin, which plays an important role in proBDNF intracellular trafficking and stabilization. The interaction of proBDNF with both HAP1A and sortilin in co-transfected HEK293 cells is confirmed by both fluorescence resonance energy transfer and co-immunoprecipitation. The frequent co-localization (>90%) of endogenous HAP1, sortilin, and proBDNF is also found in cultured cortical neurons. Mapping studies using GST pulldown and competition assays has defined the interacting region of HAP1 with proBDNF within amino acids 371-445 and the binding sequences of proBDNF to HAP1 between amino acids 65 and 90. Fluorescence recovery after photobleaching confirms the defective movement of proBDNF-containing vesicles in neurites of HAP1(-/-) neurons, which can be partially restored by reintroducing HAP1 cDNA into the neurons. However, the effect is significantly increased by simultaneously reintroducing both HAP1 and sortilin. proBDNF and HAP1 are highly co-localized with organelle markers for the Golgi network, microtubules, molecular motor, or endosomes in normal neurons, but this co-localization is reduced in HAP1(-/-) neurons. Co-immunoprecipitation and Western blot showed that sortilin stabilizes the proBDNF·HAP1 complex in co-transfected HEK293 cells, helping to prevent proBDNF degradation. Furthermore, the complex facilitates furin cleavage to release mature BDNF.
authors.affiliation: Department of Human Physiology and Centre for Neuroscience, Flinders University, Adelaide, South Australia, Australia.
authors.forename: Miao
authors.initials: M
authors.lastname: Yang
authors.forename: Yoon
authors.initials: Y
authors.lastname: Lim
authors.forename: Xiaojiang
authors.initials: X
authors.lastname: Li
authors.forename: Jin-Hua
authors.initials: JH
authors.lastname: Zhong
authors.forename: Xin-Fu
authors.initials: XF
authors.lastname: Zhou
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics / Q000378:metabolism*; D002540:Cerebral Cortex / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D015388:Organelles / Q000235:genetics / Q000378:metabolism; D016716:PC12 Cells; D010449:Peptide Mapping / Q000379:methods; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D055550:Protein Stability; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D000099183:Sortilin
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D019208:Brain-Derived Neurotrophic Factor; D046912:Multiprotein Complexes; D009419:Nerve Tissue Proteins; D011498:Protein Precursors; D000099183:Sortilin; C096896:HAP1 protein, human; C490758:Hap1 protein, mouse; C490757:Hap1 protein, rat