A conformational switch driven by phosphorylation regulates the activity of the evolutionarily conserved SNARE Ykt6

Ykt6 is a soluble N-ethylmaleimide sensitive factor activating protein receptor (SNARE) critically involved in diverse vesicular fusion pathways. While most SNAREs rely on transmembrane domains for their activity, Ykt6 dynamically cycles between the cytosol and membrane-bound compartments where it is active. The mechanism that regulates these transitions and allows Ykt6 to achieve specificity toward vesicular pathways is unknown. Using a Parkinson’s disease (PD) model, we found that Ykt6 is phosphorylated at an evolutionarily conserved site which is regulated by Ca2+ signaling. Through a multidisciplinary approach, we show that phosphorylation triggers a conformational change that allows Ykt6 to switch from a closed cytosolic to an open membrane-bound form. In the phosphorylated open form, the spectrum of protein interactions changes, leading to defects in both the secretory and autophagy pathways, enhancing toxicity in PD models. Our studies reveal a mechanism by which Ykt6 conformation and activity are regulated with potential implications for PD.

A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy

ABSTRACTMembrane fusion, an essential process in all eukaryotes, is driven by SNARE proteins. Ykt6 is an essential SNARE that plays critical roles throughout the secretory, endocytic, and autophagy pathways. Ykt6 activity is thought to be regulated by a conformational change from a closed cytosolic form to an open membrane-bound form, yet the mechanism that regulates this transition is unknown. Through genetic, pharmacologic, and structural modeling approaches in mammalian cells, we found that phosphorylation regulates Ykt6 conversion from a closed to an open state. The phosphorylation site we identified is highly conserved in evolution and is regulated by the Ca2+-dependent phosphatase, calcineurin. We found that phosphorylation is a key determinant for intracellular localization of Ykt6 and its function in macroautophagy. Our studies reveal a novel mechanism by which Ykt6 conformation and activity is regulated by Ca2+ signaling with implications in Parkinson’s Disease in which Ykt6 has been shown to play a role.

Alveolar Ridge Preservation Using Allografts and Dense Polytetrafluoroethylene Membranes With Open Membrane Technique in Unhealthy Extraction Socket

We evaluated the effectiveness of the open membrane technique using a high-density polytetrafluoroethylene (dPTFE) membrane with freeze-dried bone allografts in damaged sockets for alveolar ridge preservation (ARP). This retrospective study included 26 sites from 20 patients who had received ARP for the placement of dental implants. ARP was conducted using dPTFE membrane with allografts on the day of extraction without primary closure. When the membrane was removed after 4 weeks, the newly formed reddish tissue at the grafted site was checked (first outcome, clinical evaluation). Four months after membrane removal, a core biopsy was performed from the center of the grafted site before implant placement (second outcome, histomorphometric evaluation). Radiographic measurements of alveolar bone changes between implant prosthesis delivery and the 1-year follow-up were obtained (third outcome, radiographic evaluation). A total of 23 sites from 18 patients had no complications during the follow-up period. Three sites from two patients were excluded because of early membrane removal. Newly formed reddish tissue was found at 15 sites, and partially formed tissue was found at 8 sites. Although we were unable to harvest bone core from all sites, histomorphometric analysis in 11 patients indicated that the mean area of new bone was 28.48% ± 6.60%, that of the remaining graft particle was 27.68% ± 9.18%, and that of fibrous tissue was 43.84% ± 6.98%. The mean loss of marginal bone was 0.13 ± 0.06 mm at the mesial area and 0.15 ± 0.06 mm at the distal area, as assessed using radiographic evaluations. The results of this nonrandomized study suggest that this technique may be an appropriate procedure for ARP. Further studies with a control group and more subjectives can be designed based on this study.