Synergistic effects of systemic trefoil factor family 1 (TFF1) peptide and epidermal growth factor in a rat model of colitis

Novel therapies for the treatment of colitis are required. We therefore examined the potential value of the trefoil factor family 1 (TFF1) peptide and epidermal growth factor (EGF) alone and in combination. Effects of TFF1- Cys58 +/- EGF on an in vitro HT29 cell wounding model of restitution showed synergistic activity when used in combination. In addition, animals had colitis induced by adding 4% dextran sulphate sodium (DSS) to the drinking water for 7 days and they also received twice daily subcutaneous injections of test peptides. Treatment with TFF1-Cys58 alone (100 microg/kg) reduced histological colitis score by 22%, but the TFF1-Ser58 variant was ineffective. In a second study, TFF1-Cys58 reduced histological colitis score by 15%, EGF (600 microg/kg) by 26%, and an additive response (42% reduction) was demonstrated when used together (P < 0.01 versus either peptide given alone). Similar results were found using tissue myeloperoxidase (MPO) activity as a marker of inflammation. Where clinical risk/benefit seems justified, these initial studies suggest that combination therapy of systemic EGF and TFF peptides may prove useful for treatment of colitis in patients with disease extending beyond the reach of topical (enema) therapy.     

Autoinduction of the trefoil factor 2 (TFF2) promoter requires an upstream cis-acting element

Most benign brain tumors are associated with loss of the Nf2 gene tumor suppressor product schwannomin/merlin. Interactions between schwannomin fragments have given rise to hypotheses of in vivo schwannomin folding and dimerization. Previously, we showed that schwannomin with missense mutations L360P, L535P, and Q538P alters interaction with betaII-spectrin and Hrs. Using yeast two-hybrid tests of interaction, we now show the effects of 11 Nf2 missense mutations on schwannomin self-interaction as well as schwannomin interaction with Hrs isoforms 1 and 2, betaII-spectrin, and p110. Missense mutations L46R and K364I significantly decreased affinity of schwannomin for binding all interacting proteins. The schwannomin L46R mutation may result in a complex conformational change that alters folding and denies betaII-spectrin access to an intact binding site in the C-terminal half of schwannomin. We show that unique inter- and intramolecular interactions occur for schwannomin isoform 2, suggesting that this schwannomin isoform has unique functional properties compared to schwannomin isoform 1.     

Mona/Gads SH3C binding to hematopoietic progenitor kinase 1 (HPK1) combines an atypical SH3 binding motif, R/KXXK, with a classical PXXP motif embedded in a polyproline type II (PPII) helix

Hematopoietic progenitor kinase 1 (HPK1) is implicated in signaling downstream of the T cell receptor. Its non-catalytic, C-terminal half contains several prolinerich motifs, which have been shown to interact with different SH3 domain-containing adaptor proteins in vitro. One of these, Mona/Gads, was also shown to bind HPK1 in mouse T cells in vivo. The region of HPK1 that binds to the Mona/Gads C-terminal SH3 domain has been mapped and shows only very limited similarity to a recently identified high affinity binding motif in SLP-76, another T-cell adaptor. Using isothermal titration calorimetry and x-ray crystallography, the binding of the HPK1 motif to Mona/Gads SH3C has now been characterized in molecular detail. The results indicate that although charge interactions through an RXXK motif are essential for complex formation, a PXXP motif in HPK1 strongly complements binding. This unexpected binding mode therefore differs considerably from the previously described interaction of Mona/Gads SH3C with SLP-76. The crystal structure of the complex highlights the great versatility of SH3 domains, which allows interactions with very different proteins. This currently limits our ability to categorize SH3 binding properties by simple rules.