Longins and the longin domain: pivotal elements in subcellular trafficking and neuronal differentiation
Year: 2012
Type: dissertation
Abstract: Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are the most investigated trafficking proteins due to their role in directing the fusion complex. Among these, long VAMPs, or longins are characterized by N-terminal longin domain (LD), controlling both SNARE complex formation and subcellular localization (SCL) and are prototyped by VAMP7, Sec22b and Ykt6. The LD adopts a stable, closed conformation, but the contribution of this state and each different domain to sorting is still unclear. Human VAMP7, encoded by gene SYBL1, is involved in multiple cell pathways, including control of neurite outgrowth. Furthermore, alternative splicing (AS) of SYBL1 results in the production of two isoform subfamilies that retain an intriguingly domain architecture. Non-SNARE longin variants share the inhibitory LD, whereas non-longin SNARE variants share the SNARE motif. Since previous evidence suggests inhibitory functions for the LD construct and growth promoting activity for the Δ-longin construct, these subfamilies are likely to play opposite functions. Therefore, mechanisms mediating neurotogenesis are not clear, in particular the contribution of extracellular stimuli and different SNAREs. This work focused on the characterization of VAMP7 LD and its isoforms in SCL and neuronal development. Expression analysis in different tissues and cell lines, real time RT-PCR and confocal microscopy analyses demonstrated that VAMP7 variants have different tissue specificities and SCL; furthermore, the LD-only isoform VAMP7i displays also a nuclear localization. Considering their variant domain combinations, these physiological splice variants were used as tools for studying targeting determinants in SCL. Moreover, recombinant fragments of the VAMP7a cytoplasmic region confirmed that individual domains are unable to determine sorting by alone, and open/closed conformational switch is not relevant to SCL in the absence of transmembrane region. Gain-of-function experiments on both neuroblastoma cells and primary neurons revealed that VAMP7 AS is able to regulate neurite outgrowth by balanced production of stimulatory (VAMP7dh) and inhibitory (VAMP7i) isoforms. These effects are also subjected to the substrate (Poly-D-Lysine or Laminin) in which neurons are cultured and to the co-expression of other VAMP7 isoforms or SNAREs (VAMP2), suggesting a fine regulatory mechanism mediated by VAMP7 AS. Additional investigation will be helpful in order to manipulate neuritogenesis in cell therapy and clarify the role of tissue-specific variants in some neurological diseases. Further characterization of VAMP7 LD and isoforms can unravel novel molecular partners and mechanisms, helpful in some biotechnological applications.
Author: Albania, Lara
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W189832800