IntroductionRho guanosine triphosphatases (GTPases), belonging to the Ras superfamily, play key roles in regulating actin organization, membrane trafficking, migration, and cell adhesion. 1 The activity of most Rho GTPases is characterized by reversible binding of guanosine triphosphate (GTP). The cycling of Rho GTPases between GTP-bound, active and guanosine diphosphate-bound, inactive forms is regulated by distinct cellular proteins including guanine nucleotide exchange factors and GTPase activating proteins (GAPs). 2,3 Rac subfamily members, Rac1 and Rac2, are essential for cortical filamentous-actin (F-actin) assembly and chemotaxis in hematopoietic stem/progenitor cells. 4,5 Loss of Rac1 and Rac2 alleles leads to defective engraftment and massive mobilization of hematopoietic progenitor cells (HPCs), which is associated with loss of adhesion, chemotaxis, and cortical F-actin polymerization. 5 Rhoh was first identified as a fusion transcript with LacZ3/Bcl6 in non-Hodgkin lymphoma 6 and encodes a hematopoietic-specific member of the RhoE/Rnd3 subfamily that lacks intrinsic and/or agonist-induced GTPase activity. Therefore, RhoH remains in a GTP-bound, constitutively active state without cycling. The cellular activity of RhoH has been postulated to be regulated by cell-specific transcription that determines its protein level and posttranslational modification. 7-9 Alteration of RhoH expression level disrupts normal proliferation, survival, and engraftment of HPCs in lethally irradiated recipient mice, as well as cortical F-actin assembly and chemokine-induced migration. 10 Overexpression of RhoH also impairs the activation of Rac GTPases in HPCs 10 and inhibits Rac1-mediated downstream signaling pathways in T cells. 7 These findings suggest that RhoH may antagonize Rac function in regulating some cellular processes. However, the molecular mechanisms underlying the crosstalk between Rac and RhoH have not been defined.Rho GTPases associate with effector proteins at the cell plasma membrane, including lipid rafts. 11-15 Detachment of cells induces the internalization of lipid rafts, which inhibits the membrane localization of Rac1. In addition, cell-permeable C-terminal peptides of Rac1 localize to plasma membrane in HL60 cells and inhibit Rac1 membrane localization. This is associated with impaired actin polymerization and cell migration. 12 Thus, translocation of intracellular Rac proteins to the plasma membrane is critical for activating Rac-mediated downstream signaling and function. 16 Rac GTPases contain a conserved C-terminal CAAX motif, which is posttranslationally modified by isoprenylation of the cysteine residue. The isoprenoid moiety is required to anchor Rac proteins to the cell membrane. 17 In this study, we examined the biologic role of RhoH in actin cytoskeleton organization and chemotaxis in response to stromalderived factor-1␣ (SDF-1␣) using freshly isolated Rhoh Ϫ/Ϫ HPCs. Loss of RhoH in these cells causes enhanced Rac-mediated migration and cortical F-actin assembly in the absence or presence...

[structure: see text] Thioxo peptide analogues of the alpha-helical peptide GCN4-p1 were synthesized and evaluated for helicity and oligomeric state. Sedimentation equilibrium and CD measurements indicate that the thioxo peptides fold into parallel alpha-helical coiled coil structures essentially identical to the native structure. This work marks the first incorporation of a thioamide linkage into the backbone of an alpha-helix and demonstrates that a thioamide linkage is compatible with positions within the helix as well as near the C-terminus.

Rho GTPases are recognized as critical mediators of signaling pathways regulating actin assembly, migration, proliferation and survival in hematopoietic cells. Here, we have studied a newly identified hematopoietic-specific Rho GTPase, RhoH. The RhoH gene was first identified as a hypermutable locus in non-Hodgkin’s lymphoma and diffuse large B-cell lymphoma, and alteration of RhoH expression has been implicated in some cases. Unlike most members of the Rho GTPase family, RhoH is GTPase deficient and remains in the active, GTP-bound state suggesting that the level of RhoH expression may be a critical determinant of activity. To elucidate the biological role of RhoH in blood cell development and function, we used two complementary approaches, including overexpression via retrovirus-mediated gene transfer and knock-down via RNA interference, to alter levels of RhoH in hematopoietic progenitor cells (HPCs) and examined the effects of altered RhoH expression on hematopoiesis. In cytokine-stimulated HPCs, knock-down of RhoH expression (to ~ 30% of the endogenous level) significantly induces proliferation, survival and stromal cell derived factor-1 α (SDF-1α)-induced migration and cortical filamentous (F)-actin assembly (Table I). Conversely, RhoH overexpression (>2 folds of the endogenous level) in these cells is associated with reduced proliferation and increased apoptosis in response to stem cell factor, and defective F-actin polymerization at cell peripheral membrane and chemotaxis in response to SDF-1 α (Table I). In the irradiated recipient mice, hematopoietic stem/progenitor cells overexpressing RhoH demonstrate defective reconstitution capability in peripheral blood (Table I), bone marrow, spleen and lymph node as compared with control vector-transduced cells. Also, the RhoH-transduced donor cells displayed an increased level of apoptosis and a reduced proliferative response to the 5-fluorouracil treatment when compared with vector-transduced cells. Interestingly, these defective phenotypes associated with the progenitor cells overexpressing RhoH resemble the phenotypes of Rac1-/- ; Rac2-/- cells. Overexpression of RhoH is also associated with reduced Rac activity when stimulated by a Rac agonist. In addition, overexpression of RhoH disrupts the SDF-α-stimulated subcellular redistribution of Rac1 and Rac2 1 to cell peripheral membrane where Rac proteins are activated and induce F-actin polymerization. These results suggest that aberrant expression of RhoH leads to abnormal growth and actin-based function of HPCs. RhoH functions as a negative regulator of proliferation, survival, migration, actin polymerization and engraftment of HPCs possibly through suppression of Rac activation via preventing localization of Rac proteins to cell peripheral membrane. Table 1. Cellular phenotypes associated with the altered expression levels of RhoH in HPCs. Proliferation (CPM x 1000) Apoptosis (% Annexin V+ cells) Migration (% migrated cells) F-actin (% cells with cortical F-actin staining) engraftment (%EGFP+ cells in PB) * p < 0.01; ** p < 0.05, HA-RhoH- and siRhoH-transduced vs vector-transduced cells. vector control 16.5 +/− 2.0 3.2 +/− 0.8 22.0 +/− 2.0 75 38.0 +/− 6.0 HA-RhoH 7.0 +/− 0.8 * 6.5 +/− 1.1* 10.5 +/− 1.3 * 22 18.0 +/− 3.5 * siRhoH 23.0 +/− 4.5 ** 1.8 +/− 0.4 ** 32.0 +/− 4.8 * 95 NA

Movement of hematopoietic stem/progenitor cells into (engraftment) and out of (mobilization) the bone marrow involves actin cytoskeleton and chemotaxis. Members of the Rho GTPase family have been well known for their critical roles in morphogenesis and cell migration via regulating actin assembly. Loss of Rac1 and Rac2 alleles leads to defective engraftment and massive mobilization of hematopoietic progenitor cells (HPCs), which are associated with impaired chemotaxis and cortical filamentous (F)-actin polymerization (Gu et al., Science 302: 445–449). RhoH, a hematopoietic-specific member of the RhoE subfamily, negatively regulates HPC engraftment, chemotaxis, F-actin polymerization and Rac activities (Gu et al., Blood 105: 1467–1475). These findings suggest that RhoH may antagonize Rac function in regulating these cellular processes. However, molecular mechanism of the cross-talk between these Rho GTPases is not defined. In this study, we examined the role of RhoH in actin cytoskeleton organization, chemotaxis and Rac membrane translocation in response to stromal-derived factor 1α (SDF-1α) using RhoH-deficient HPCs and retrovirus-mediated expression of EGFP-fusion proteins. RhoH−/− HPCs exhibit increased migration in response to SDF-1α, especially at low concentration, as compared with wild-type (WT) cells [10ng/ml SDF-1α: 3.5 +/− 0.9 vs. 12.3 +/− 1.8; 100ng/ml SDF-1α: 21.4 +/− 1.7 vs. 32.3 +/− 3.4, migrated cells (%), WT vs. RhoH−/−, n=3, p< 0.01]. Migration without SDF-1α stimulation of RhoH−/− cells is also enhanced. RhoH−/− HPCs assemble cortical F-actin without SDF-1α stimulation, under conditions in which WT cells do not show F-actin polymerization [cells with F-actin (%): 8.9 +/− 0.9 vs. 72.8 +/− 4, WT vs. RhoH−/−, n=6, p<0.001]. Additionally, RhoH−/− HPCs exhibit increased active, GTP-bound Rac GTPases. PAK, a known downstream effector of Rac in regulating actin cytoskeleton, also shows hyperphosphorylation in RhoH-/− HPCs, suggesting that RhoH may regulate actin assembly and cell migration through Rac-mediated pathway. In support of this, expression of a dominant negative Rac1N17 mutant blocks cortical F-actin assembly in RhoH−/− cells [cells with F-actin (%): 60 +/− 1 vs. 19 +/− 7, EGFP-Rac1 vs. Rac1N17, n=2]. To further address the mechanism by which RhoH cross-talks to affect Rac signaling, we examine the role of RhoH in subcellular localization of EGFP-Rac proteins. SDF-1α induces activation of Rac, leading to translocation to the cell membrane where it co-localizes with lipid rafts and mediates cortical F-actin assembly in HPCs. In contrast, the dominant negative Rac1N17 does not localize to the cell membrane after SDF-1α stimulation. In RhoH−/− HPCs, EGFP-Rac protein presents at the cell membrane in the absence of SDF-1α [cells with membrane-localized EGFP-Rac1 (%): 7.5 +/− 3.9 vs. 44.5 +/− 6.4, WT vs. RhoH−/−, n=2]. In contrast, overexpression of RhoH in HPCs blocks translocation to the cell membrane after SDF-1α stimulation of Rac1, Rac2 and active Rac1V12. Finally, we found that RhoH, a constitutively active, GTP-bound protein, preferentially localizes to the cell membrane even in the absence of SDF-1α. This localization is dependent upon the prenylation site and the c-terminal domains of RhoH. Lack of membrane localization is associated with defective biological function. Together, our data suggest that RhoH is essential for proper cortical F-actin assembly and chemotaxis of HPCs via regulating Rac activation and membrane localization, and implicates a functional cross-talk between RhoH and Rac.