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The VHS domain of the Stam2 protein is a ubiquitin binding domain involved in the recognition of ubiquitinated proteins committed to lysosomal degradation. Among all VHS domains, the VHS domain of Stam proteins is the strongest binder to monoubiqiuitin and exhibits preferences for K63-linked chains. In the present paper, we report the solution NMR structure of the Stam2-VHS domain in complex with monoubiquitin by means of chemical shift perturbations, spin relaxation, and paramagnetic relaxation enhancements. We also characterize the interaction of Stam2-VHS with K48- and K63-linked diubiquitin chains and report the first evidence that VHS binds differently to these two chains. Our data reveal that VHS enters the hydrophobic pocket of K48-linked diubiquitin and binds the two ubiquitin subunits with different affinities. In contrast, VHS interacts with K63-linked diubiquitin in a mode similar to its interaction with monoubiquitin, We also suggest possible structural models for both K48- and K63-linked diubiquitin in interaction with VHS. Our results, which demonstrate a different mode of binding of VHS K48. and K63-linked diubiquitin, may explain the preference of VHS for K63- over K48-linked diubiquitin chains and monoubiquitin. [ABSTRACT FROM AUTHOR]

We have previously reported that endogenous vascular endothelial growth factor (VEGF) concentration in older pig wounds peaked later and at one-fourth the level of young pigs. These data suggested that VEGF might play a major role in the healing of full-thickness wounds in the aged pig. By in vivo gene transfer using the microseeding technique, we treated full-thickness wounds with different doses of VEGF-expressing adenoviral vector (Ad-VEGF) varying from 1× 107 to 2.7× 1011 particles per wound (ppw). We found that the VEGF expression in wound fluid followed a dose–response pattern. However, when wounds were microseeded with the highest concentration of Ad-VEGF (2.7× 1011 ppw), diminished healing rates were found. We then determined the minimal functional concentrations of Ad-VEGF. We used five aged Yucatan minipigs, all retired breeders, to analyze the role of over-expression of 1× 108 and 1× 109 ppw of Ad-VEGF (n= 78) in terms of healing of full-thickness wounds, all 2.5× 2.5× 1 cm in size (n= 158). The Ad-VEGF solutions were delivered to the wound floor and borders by in vivo microseeding. Control wounds (n= 80) were microseeded with Ad-Lac-Z (n= 25), treated with saline (n= 49) or treated dry (n= 6). All wounds except for the dry-treated ones were covered with a wound chamber and a wet environment was created by injecting 2.5 ml saline into the chamber. Peak VEGF expression (2300–4000 pg/ml) was detected on days 2 or 3 post gene delivery. This level of VEGF expression was not seen in the saline (n= 49) or Ad-null (n= 25) control groups. The VEGF expression in wounds treated with 1× 108 and 3× 108 ppw (n= 39) exhibited a slower onset with a peak concentration of 400–920 pg/ml on days 5–7. Although high levels of VEGF expression were achieved in the local wound environment, we could not show a significant increase in neovascularization as compared to saline-treated wounds. No significant differences were observed in the rate of reepithelialization and wound contraction among groups of full-thickness wounds treated with Ad-VEGF, Ad-null mutant, or saline in the aged“wet wound healing” pig model. These results indicate that increased levels of VEGF in wounds produced by in vivo gene transfer have little effect on the healing of full-thickness wounds in the aged pig model. Moreover, significantly higher levels of VEGF expression by Ad-VEGF could lead to impaired wound healing. [ABSTRACT FROM AUTHOR]

As the upstream component of the ESCRT (endosomal sorting complexes required for transport) machinery, the ESCRT-0 complex is responsible for directing ubiquitinated membrane proteins to the multivesicular body pathway. ESCRT-0 is formed by two subunits known as Hrs (hepatocyte growth factor-regulated substrate) and STAM (signal transducing adaptor molecule), both of which harbor multiple ubiquitin-binding domains (UBDs). In particular, STAM2 possesses two UBDs, the VHS (Vps27/Hrs/Stam) and UIM (ubiquitin interacting motif) domains, connected by a 20-amino acid flexible linker. In the present study, we report the interactions of the UIM domain and VHS-UIM construct of STAM2 with monoubiquitin (Ub), Lys48- and Lys63-linked diubiquitins. Our results demonstrate that the UIM domain alone binds monoubiquitin,Lys48-andLys63-linkeddiubiquitinswiththesame affinity and in the same binding mode. Interestingly, binding of VHS-UIM to Lys63-linked diubiquitin is not only avid, but also cooperative. We also show that the distal domain of Lys63-linked diubiquitin stabilizes the helical structure of the UIM domain and that the corresponding complex adopts a specific structural organization responsible for its greater affinity. In contrast, binding of VHS-UIM to Lys48-linked diubiquitin and monoubiquitin is not cooperative and does not show any avidity. These results may explain the better sorting efficiency of some cargoes polyubiquitinated with Lys63-linked chains over monoubiquitinated cargoes or those tagged with Lys48-linked chains. [ABSTRACT FROM AUTHOR]