There is now considerable experimental evidence that aberrant activation of Rho family small GTPases promotes the uncontrolled proliferation, invasion, and metastatic properties of human cancer cells. Therefore, there is considerable interest in the development of small molecule inhibitors of Rho GTPase function. However, to date, most efforts have focused on inhibitors that indirectly block Rho GTPase function, by targeting either enzymes involved in post-translational processing or downstream protein kinase effectors. We recently determined that the EHT 1864 small molecule can inhibit Rac function in vivo. In this study, we evaluated the biological and biochemical specificities and biochemical mechanism of action of EHT 1864. We determined that EHT 1864 specifically inhibited Rac1-dependent platelet-derived growth factor-induced lamellipodia formation. Furthermore, our biochemical analyses with recombinant Rac proteins found that EHT 1864 possesses high affinity binding to Rac1, as well as the related Rac1b, Rac2, and Rac3 isoforms, and this association promoted the loss of bound nucleotide, inhibiting both guanine nucleotide association and Tiam1 Rac guanine nucleotide exchange factor-stimulated exchange factor activity in vitro. EHT 1864 therefore places Rac in an inert and inactive state, preventing its engagement with downstream effectors. Finally, we evaluated the ability of EHT 1864 to block Rac-dependent growth transformation, and we determined that EHT 1864 potently blocked transformation caused by constitutively activated Rac1, as well as Rac-dependent transformation caused by Tiam1 or Ras. Taken together, our results suggest that EHT 1864 selectively inhibits Rac downstream signaling and transformation by a novel mechanism involving guanine nucleotide displacement.Rho family proteins (20 human members) comprise a major branch of the Ras superfamily of small GTPases (1, 2). Like Ras, Rho GTPases function as GDP/GTP-regulated binary on-off switches. In resting, quiescent cells, Rho GTPases exist predominately in their inactive, GDP-bound state. Upon growth factor stimulation, Rho-specific guanine nucleotide exchange factors (RhoGEFs) 4 are activated, and they stimulate the intrinsic guanine nucleotide exchange activity to promote formation of the active GTP-bound protein. Rho-GTP binds preferentially to downstream effector proteins. Rho-specific GTPaseactivating proteins then stimulate the intrinsic GTP hydrolysis activity of Rho GTPases, returning the protein to its inactive state and terminating effector interaction.RhoA, Rac1, and Cdc42 are the best studied and understood members of the Rho GTPase family (1, 2). Rho GTPases are regulators of a diverse variety of cellular processes that include regulation of cell proliferation, actin cytoskeleton reorganization, and gene expression (3). Like Ras and other members of the Ras superfamily (4, 5), the aberrant activity of Rho GTPases has been implicated in cancer and other human diseases (6 -10). In particular, the Rac subfamily has also been linked to c...
Nramp2, also known as DMT1 and DCT1, is a 12-transmembrane (TM) domain protein responsible for dietary iron uptake in the duodenum and iron acquisition from transferrin in peripheral tissues. Nramp2/DMT1 produces by alternative splicing two isoforms differing at their C terminus (isoforms I and II). The subcellular localization, mechanism of action, and destination of divalent cations transported by the two Nramp2 isoforms are not completely understood. Stable CHO transfectants expressing Nramp2 isoform II modified by addition of a hemaglutinin epitope in the loop defined by the TM7-TM8 interval were generated. Immunofluorescence with permeabilized and intact cells established that Nramp2 isoform II is expressed at the plasma membrane and demonstrated the predicted extracytoplasmic location of the TM7-TM8 loop. Using the fluorescent, metal-sensitive dye calcein, and a combination of membrane-permeant and -impermeant iron chelators, Nramp2 transport was measured and quantitated with respect to kinetic parameters and at steady state. Iron transport at the plasma membrane was time-and pH-dependent, saturable, and proportional to the amount of Nramp2 expression. Iron uptake by Nramp2 at the plasma membrane was into the nonferritin-bound, calcein-accessible so-called "labile iron pool." Ion selectivity experiments show that Nramp2 isoform II can also transport Co 2؉ and Cd 2؉ but not Mg 2؉ into the calceinaccessible pool. Parallel experiments with transfectants expressing the lysosomal Nramp1 homolog do not show any divalent cation transport activity, establishing major functional differences between Nramp1 and Nramp2. Monitoring the effect of Nramp2 on the calcein-sensisitve labile iron pool allows a simple, rapid, and nonisotopic approach to the functional study of this protein.
Mutations at the natural resistance–associated macrophage protein 1 (Nramp1) locus cause susceptibility to infection with antigenically unrelated intracellular pathogens. Nramp1 codes for an integral membrane protein expressed in the lysosomal compartment of macrophages, and is recruited to the membrane of phagosomes soon after the completion of phagocytosis. To define whether Nramp1 functions as a transporter at the phagosomal membrane, a divalent cation-sensitive fluorescent probe was designed and covalently attached to a porous particle. The resulting conjugate, zymosan–FF6, was ingested by macrophages and its fluorescence emission was recorded in situ after phagocytosis, using digital imaging. Quenching of the probe by Mn2+ was used to monitor the flux of divalent cations across the phagosomal membrane in peritoneal macrophages obtained from Nramp1-expressing (+/+) and Nramp1-deficient (−/−) macrophages. Phagosomes from Nramp1+/+ mice extrude Mn2+ faster than their Nramp−/− counterparts. The difference in the rate of transport is eliminated when acidification of the phagosomal lumen is dissipated, suggesting that divalent metal transport through Nramp1 is H+ dependent. These studies suggest that Nramp1 contributes to defense against infection by extrusion of divalent cations from the phagosomal space. Such cations are likely essential for microbial function and their removal from the phagosomal microenvironment impairs pathogenesis, resulting in enhanced bacteriostasis or bactericidal activity.
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