Rabbit Anti-Human NGEF Polyclonal antibody

Dbl-related oncoproteins are guanine nucleotide exchange factors (GEFs) specific for Rho-family GTPases and typically possess tandem Dbl (DH) and pleckstrin homology (PH) domains that act in concert to catalyze exchange. Although the exchange. potential of many Dbl-family proteins is constitutively activated by truncation, the precise mechanisms of regulation for many Dbl-family proteins are unknown. Tim and Vav are distantly related Dbl-family proteins that are similarly regulated; their Dbl homology (DH) domains interact with N-terminal helices to exclude and prevent activation of Rho GTPases. Phosphorylation, substitution, or deletion of the blocking helices relieves this autoinhibition. Here we show that two other Dbl-family proteins, Ngef and Wgef, which like Tim contain a C-terminal SH3 domain, are also activated by tyrosine phosphorylation of a blocking helix. Consequently, basal autoinhibition of DH domains by direct steric exclusion using short N-terminal helices likely represents a conserved mechanism of regulation for the large family of Dbl-related proteins. N-Terminal truncation or phosphorylation of many other Dbl-family GEFs leads to their activation; similar autoinhibition mechanisms could explain some of these events. In addition, we show that the C-terminal SH3 domain binding to a polyproline region N-terminal to the DH domain of the Tim subgroup of Dbl-family proteins provides a unique mechanism of regulated autoinhibition of exchange activity that is functionally linked to the interactions between the autoinhibitory helix and the DH domain.

The formation of isoaspartate (isoAsp) from asparaginyl or aspartyl residues is a spontaneous post-translational modification of peptides and proteins. Due to isopeptide bond formation, the structure and possibly function of peptides and proteins is altered. IsoAsp modifications within the peptide chain have been reported for many cytosolic proteins. Amyloid peptides (A beta) deposited in Alzheimer's disease may carry an N-terminal isoAsp-modification. Here, we describe a quantitative investigation of isoAsp-formation from N-terminal Asn and Asp using model peptides similar to the A beta N-terminus. The study is based on a newly developed separation of peptides using capillary electrophoresis (CE). H-1 NMR was employed to validate the basic finding of N-terminal isoAsp-formation from Asp and Asn. Thereby, the isomerization of Asn at neutral pH (0.6 day(-1), peptide NGEF) is approximately six times faster than that within the peptide chain (AANGEF). The difference in velocity between Asn and Asp isomerization is approximately 50-fold. In contrast to N-terminal Asn, Asp isomerization is significantly accelerated at acidic pH. The kinetic solvent isotope (k (D2O)/k (H2O)) effect of 2.46 suggests a rate-limiting proton transfer in isoAsp-formation. The proton inventory is consistent with transfer of one proton in the transition state, supporting the previous notion of rate-limiting deprotonation of the peptide backbone amide during succinimide-intermediate formation. The study provides evidence for a spontaneous N-terminal isoAsp-formation within peptides and might explain the accumulation of N-terminal isoAsp in amyloid deposits.