Vesicle budding, membrane trafficking, and lipid metabolism depend on the switching of Arf and Rab GTPases from the inactive GDP bound state to the active GTP bound state. However, Arf and Rab GTPases have intrinsic rates of GDP to GTP exchange that are much slower (hours to days) than the time scale of the relevant trafficking processes (seconds or less). In cells, the activation of Arf and Rab GTPases is tightly regulated by guanine nucleotide exchange factors (GEFs) with Sec7 or Vps9 domains, respectively. Full length Cytohesins, which have a domain architecture consisting of heptad repeats, a Sec7 domain, a pleckstrin homology (PH) domain, and a polybasic motif, have 100-fold lower exchange activity than the isolated Sec7 domain. Insights into the low exchange activity were obtained by structural, biochemical and kinetic analyses. It was found that the Sec7-PH domain linker and a C-terminal amphipathic helix physically block the docking sites for the switch regions of Arf GTPases. Mutations within either element result in partial or complete relief of autoinhibition. Autohibition is also strongly relieved by phosphorylation of protein kinase C (PKC) sites in the polybasic motif of Cytohesin-1 or by phosphoinositide head group-dependent binding of active Arf6. Despite unrelated folds, Sec7 and Vps9 domains engage cognate GTPases in a strikingly similar manner and supply a critical acidic residue that interacts with an invariant lysine residues from phosphate binding (P) loop of the GTPase in the nucleotide free complex. The key acidic residues have also been proposed to disrupt the Mg2+ binding site; however, it is not known whether disruption of Mg2+ binding contributes to the rate limiting step for nucleotide release. To investigate the kinetic mechanism for catalysis of nucleotide exchange in the absence of autoinhibitory interactions, a detailed stopped flow kinetic analysis of the intrinsic and GEF mediated exchange reactions was conducted for the isolated catalytic cores. Using three different fluorescence methods to monitor Mg2+ dissociation, formation of the nucleotide free intermediate, and subsequent nucleotide binding, the catalytic cores of Cytohesin-1 and Rabex-5 were found to robustly accelerate nucleotide exchange on Arf1 and Rab5, respectively, by at least 105- fold at physiological concentrations of Mg2+. The acceleration of nucleotide exchange was reduced by roughly an order of magnitude at sub-micromolar concentrations of Mg2+. In addition, the Cytohesin-1 and Rabex-5 catalytic cores have similarly high catalytic efficiencies (kcat/KM) as well as high lower limits on both the rate (kcat) and steady state (KM) constants for GDP release at physiological as well as low Mg2+ concentration. The limits on kcat and KM are comparable to the highest values reported for other well characterized GEFs and likely reflect dual requirements of membrane targeting and autoregulatory mechanisms for tight control of catalytic output. These results provide a solid structural and mechanistic foundation for future experiments to investigate the spatial-temporal dynamics of Cytohesin and Rabex-5 activation in cellular contexts.

Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) is the ninth member of the Ca+2-dependent mammalian proprotein convertase super family of serine endoproteases that is structurally related to the bacterial subtilisin and yeast kexin enzymes. It plays a critical role in the regulation of lipid metabolism and cholesterol homeostasis by binding to and degrading low-density lipoprotein-receptor (LDL-R) which is responsible for the clearance of circulatory LDL-cholesterol from the blood. Owing to this functional property, there is plenty of research interest in the development of functional inhibitors of PCSK9 which may find important biochemical applications as therapeutic agents for lowering plasma LDL-cholesterol. The catalytic domain of PCSK9 binds to the EGF-A domain of LDL-R on the cell surface to form a stable complex and re-routes the receptor from its normal endosomal recycling pathway to the lysosomal compartments leading to its degradation. Owing to these findings, we propose that selected peptides from PCSK9 catalytic domain, particularly its disulphide (S-S) bridged loop1 323-358 and loop2 365-385, are likely to exhibit strong affinity towards the EGF-A domain of LDL-R. Several regular peptides along with corresponding all- dextro and retro-inverse peptides as well as the gain-of-function mutant variants were designed and tested for their regulatory effects towards LDL-R expression and PCSK9-binding in human hepatic HepG2 and mouse hepatic Hepa1c1c7 cells. Our data indicated that disulfide bridged loop1-hPCSK9323-358 and its H357 mutant as well as two short loop2-hPCSK9372-380 and its Y374 mutant peptides modestly promote the LDL-R protein levels. Our study concludes that specific peptides from the PCSK9 catalytic domain can regulate LDL-R and may be useful for development of novel class of therapeutic agents for cholesterol regulation.