Inhibition of RhoA Translocation and Calcium Sensitization by In Vivo ADP-Ribosylation with the Chimeric Toxin DC3B

Pretreatment of intact rabbit portal vein smooth muscle with the chimeric toxin DC3B (10−6 M, 48 h; Aullo et al., 1993; Boquet et al. 1995) ADP-ribosylated endogenous RhoA, including cytosolic RhoA complexed with rhoGDI, and inhibited the tonic phase of phenylephrine-induced contraction and the Ca2+-sensitization of force by phenylephrine, endothelin and guanosine triphosphate (GTP)γS, but did not inhibit Ca2+-sensitization by phorbol dibutyrate. DC3B also inhibited GTPγS-induced translocation of cytosolic RhoA (Gong et al., 1997a) to the membrane fraction. In DC3B-treated muscles the small fraction of membrane-associated RhoA could be immunoprecipitated, even after exposure to GTPγS, which prevents immunoprecipitation of non-ADP–ribosylated RhoA. Dissociation of cytosolic RhoA–rhoGDI complexes with SDS restored the immunoprecipitability and ADP ribosylatability of RhoA, indicating that both the ADP-ribosylation site (Asn 41) and RhoA insert loop (Wei et al., 1997) are masked by rhoGDI and that the long axes of the two proteins are in parallel in the heterodimer. We conclude that RhoA plays a significant role in G-protein-, but not protein kinase C-mediated, Ca2+ sensitization and that ADP ribosylation inhibits in vivo the Ca2+-sensitizing effect of RhoA by interfering with its binding to a membrane-associated effector.

BAX-induced cell death may not require interleukin 1β-converting enzyme-like proteases

Expression of BAX, without another death stimulus, proved sufficient to induce a common pathway of apoptosis. This included the activation of interleukin 1β-converting enzyme (ICE)-like proteases with cleavage of the endogenous substrates poly(ADP ribose) polymerase and D4-GDI (GDP dissociation inhibitor for the rho family), as well as the fluorogenic peptide acetyl-Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC). The inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) successfully blocked this protease activity and prevented FAS-induced death but not BAX-induced death. Blocking ICE-like protease activity prevented the cleavage of nuclear and cytosolic substrates and the DNA degradation that followed BAX induction. However, the fall in mitochondrial membrane potential, production of reactive oxygen species, cytoplasmic vacuolation, and plasma membrane permeability that are downstream of BAX still occurred. Thus, BAX-induced alterations in mitochondrial function and subsequent cell death do not apparently require the known ICE-like proteases.

The Gfi-1 protooncoprotein represses Bax expression and inhibits T-cell death

The Gfi-1 protooncogene encodes a nuclear zinc-finger protein that carries a novel repressor domain, SNAG, and functions as a position- and orientation-independent active transcriptional repressor. The Gfi-1 repressor allows interleukin 2 (IL-2)-dependent T cells to escape G1 arrest induced by IL-2 withdrawal in culture and collaborates with c-myc and pim-1 for the induction of retrovirus-induced lymphomas in animals. Here we show that overexpression of Gfi-1 also inhibits cell death induced by cultivation of IL-2-dependent T-cell lines in IL-2-deficient media. Similarly, induction of Gfi-1 in primary thymocytes from mice carrying a metal-inducible Gfi-1 transgene inhibits cell death induced by cultivation in vitro. The protein and mRNA levels of the proapoptotic regulator Bax are down-regulated by Gfi-1 in both immortalized T-cell lines and primary transgenic thymocytes. The repression is direct and depends on several Gfi-1-binding sites in the p53-inducible Bax promoter. In addition to Bax, Gfi-1 also represses Bak, another apoptosis-promoting member of the Bcl-2 gene family. Therefore, Gfi-1 may inhibit apoptosis by means of its repression of multiple proapoptotic regulators. The antiapoptotic properties of Gfi-1 provide a potential explanation for its strong collaboration with c-myc during oncogenesis.

Insulin-stimulated translocation of GLUT4 glucose transporters requires SNARE-complex proteins

A major physiological role of insulin is the regulation of glucose uptake into skeletal and cardiac muscle and adipose tissue, mediated by an insulin-stimulated translocation of GLUT4 glucose transporters from an intracellular vesicular pool to the plasma membrane. This process is similar to the regulated docking and fusion of vesicles in neuroendocrine cells, a process that involves SNARE-complex proteins. Recently, several SNARE proteins were found in adipocytes: vesicle-associated membrane protein (VAMP-2), its related homologue cellubrevin, and syntaxin-4. In this report we show that treatment of permeabilized 3T3-L1 adipocytes with botulinum neurotoxin D, which selectively cleaves VAMP-2 and cellubrevin, inhibited the ability of insulin to stimulate translocation of GLUT4 vesicles to the plasma membrane. Furthermore, treatment of the permeabilized adipocytes with glutathione S-transferase fusion proteins encoding soluble forms of VAMP-2 or syntaxin-4 also effectively blocked insulin-regulated GLUT4 translocation. These results provide evidence of a functional role for SNARE-complex proteins in insulin-stimulated glucose uptake and suggest that adipocytes utilize a mechanism of regulating vesicle docking and fusion analogous to that found in neuroendocrine tissues.

The IgG Fc receptor, FcγRIIB, is a target for deregulation by chromosomal translocation in malignant lymphoma

Rearrangement of chromosomal bands 1q21–23 is one of the most frequent chromosomal aberrations observed in hematological malignancy. The genes affected by these rearrangements remain poorly characterized. Typically, 1q21–23 rearrangements arise during tumor evolution and accompany disease-specific chromosomal rearrangements such as t(14;18) (BCL2) and t(8;14) (MYC), where they are thus thought to play an important role in tumor progression. The pathogenetic basis of this 1q21–23-associated disease progression is currently unknown. In this setting, we surveyed our series of follicular lymphoma for evidence of recurring 1q21–23 breaks and identified three cases in which a t(14;18)(q32;q21) was accompanied by a novel balanced t(1;22)(q22;q11). Molecular cloning of the t(1;22) in a cell line (B593) derived from one of these cases and detailed fluorescent in situ hybridization mapping in the two remaining cases identified the FCGR2B gene, which encodes the immunoreceptor tyrosine-based inhibition motif-bearing IgG Fc receptor, FcγRIIB, as the target gene of the t(1;22)(q22;q11). We demonstrate deregulation of FCGR2B leading to hyperexpression of FcγRIIb2 as the principal consequence of the t(1;22). This is evidence that IgG Fc receptors can be targets for deregulation through chromosomal translocation in lymphoma. It suggests that dysregulation of FCGR2B may play a role in tumor progression in follicular lymphoma.

Nucleocytoplasmic translocation of Stat1 is regulated by a leucine-rich export signal in the coiled-coil domain

Signal transducer and activator of transcription (Stat) proteins are latent transcription factors that reside in the cytoplasm before activation. On cytokine-induced tyrosine phosphorylation, these molecules dimerize and accumulate transiently in the nucleus. No specific signals mediating these processes have been identified to date. In this report, we examine the nuclear export of Stat1. We find that treatment of cells with the export inhibitor leptomycin B does not affect steady-state localization of Stat1 but impedes nuclear export after IFNγ-induced nuclear accumulation. We identify a conserved leucine-rich helical segment in the coiled-coil domain of Stat1, which is responsible for the efficient nuclear export of this protein. Mutation of two hallmark leucines within this segment greatly attenuate the back transport of Stat1 in the cytoplasm. When fused to a carrier protein, the Stat1 export sequence can mediate nuclear export after intranuclear microinjection. We show that prolonging the nuclear presence of Stat1 by inhibiting nuclear export reduces the transcriptional response to stimulation with IFNγ. These data suggest that Stats are actively exported from the nucleus via several separate pathways and link this activity to transcriptional activation.

Mutational inactivation of the proapoptotic gene BAX confers selective advantage during tumor clonal evolution

A remarkable instability at simple repeated sequences characterizes gastrointestinal cancer of the microsatellite mutator phenotype (MMP). Mutations in the DNA mismatch repair gene family underlie the MMP, a landmark for hereditary nonpolyposis colorectal cancer. These tumors define a distinctive pathway for carcinogenesis because they display a particular spectrum of mutated cancer genes containing target repeats for mismatch repair deficiency. One such gene is BAX, a proapoptotic member of the Bcl-2 family of proteins, which plays a key role in programmed cell death. More than half of colon and gastric cancers of the MMP contain BAX frameshifts in a (G)8 mononucleotide tract. However, the functional significance of these mutations in tumor progression has not been established. Here we show that inactivation of the wild-type BAX allele by de novo frameshift mutations confers a strong advantage during tumor clonal evolution. Tumor subclones with only mutant alleles frequently appeared after inoculation into nude mice of single-cell clones of colon tumor cell lines with normal alleles. In contrast, no clones of BAX-expressing cells were found after inoculation of homozygous cell clones without wild-type BAX. These results support the interpretation that BAX inactivation contributes to tumor progression by providing a survival advantage. In this context, survival analyses show that BAX mutations are indicators of poor prognosis for both colon and gastric cancer of the MMP.

Molecular signals required for type III secretion and translocation of the Xanthomonas campestris AvrBs2 protein to pepper plants

Strains of Xanthomonas campestris pv. vesicatoria (Xcv) carrying avrBs2 are specifically recognized by Bs2 pepper plants, resulting in localized cell death and plant resistance. Agrobacterium-mediated transient expression of the Xcv avrBs2 gene in plant cells results in Bs2-dependent cell death, indicating that the AvrBs2 protein alone is sufficient for the activation of disease resistance-mediated cell death in planta. We now provide evidence that AvrBs2 is secreted from Xcv and that secretion is type III (hrp) dependent. N- and C-terminal deletion analysis of AvrBs2 has identified the effector domain of AvrBs2 recognized by Bs2 pepper plants. By using a truncated Pseudomonas syringae AvrRpt2 effector reporter devoid of type III signal sequences, we have localized the minimal region of AvrBs2 required for type III secretion in Xcv. Furthermore, we have identified the region of AvrBs2 required for both type III secretion and translocation to host plants. The mapping of AvrBs2 sequences sufficient for type III delivery also revealed the presence of a potential mRNA secretion signal.

Visualization of elongation factor G on the Escherichia coli 70S ribosome: The mechanism of translocation

During protein synthesis, elongation factor G (EF-G) binds to the ribosome and promotes the step of translocation, a process in which tRNA moves from the A to the P site of the ribosome and the mRNA is advanced by one codon. By using three-dimensional cryo-electron microscopy, we have visualized EF-G in a ribosome–EF-G–GDP–fusidic acid complex. Fitting the crystal structure of EF-G–GDP into the cryo density map reveals a large conformational change mainly associated with domain IV, the domain that mimics the shape of the anticodon arm of the tRNA in the structurally homologous ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog. The tip portion of this domain is found in a position that overlaps the anticodon arm of the A-site tRNA, whose position in the ribosome is known from a study of the pretranslocational complex, implying that EF-G displaces the A-site tRNA to the P site by physical interaction with the anticodon arm.

The vav exchange factor is an essential regulator in actin-dependent receptor translocation to the lymphocyte–antigen-presenting cell interface

During the interaction of a T cell with an antigen-presenting cell (APC), several receptor ligand pairs, including the T cell receptor (TCR)/major histocompatibility complex (MHC), accumulate at the T cell/APC interface in defined geometrical patterns. This accumulation depends on a movement of the T cell cortical actin cytoskeleton toward the interface. Here we study the involvement of the guanine nucleotide exchange factor vav in this process. We crossed 129 vav−/− mice with B10/BR 5C.C7 TCR transgenic mice and used peptide-loaded APCs to stimulate T cells from the offspring. We found that the accumulation of TCR/MHC at the T cell/APC interface and the T cell actin cytoskeleton rearrangement were clearly defective in these vav+/− mice. A comparable defect in superantigen-mediated T cell activation of T cells from non-TCR transgenic 129 mice was also observed, although in this case it was more apparent in vav−/− mice. These data indicate that vav is an essential regulator of cytoskeletal rearrangements during T cell activation.

Accelerated apoptosis of lymphocytes by augmented induction of Bax in SSI-1 (STAT-induced STAT inhibitor-1) deficient mice

Growth, differentiation, and programmed cell death (apoptosis) are mainly controlled by cytokines. The Janus kinase–signal transducers and activators of transcription (JAK-STAT) signal pathway is an important component of cytokine signaling. We have previously shown that STAT3 induces a molecule designated as SSI-1, which inhibits STAT3 functions. To clarify the physiological roles of SSI-1 in vivo, we generated, here, mice lacking SSI-1. These SSI-1−/− mice displayed growth retardation and died within 3 weeks after birth. Lymphocytes in the thymus and spleen of the SSI-1−/− mice exhibited accelerated apoptosis with aging, and their number was 20–25% of that in SSI-1+/+ mice at 10 days of age. However, the differentiation of lymphocytes lacking SSI-1 appeared to be normal. Among various pro- and anti-apoptotic molecules examined, an up-regulation of Bax was found in lymphocytes of the spleen and thymus of SSI-1−/− mice. These findings suggest that SSI-1 prevents apoptosis by inhibiting the expression of Bax.

Bax ablation prevents dopaminergic neurodegeneration in the 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) damages dopaminergic neurons in the substantia nigra pars compacta (SNpc) as seen in Parkinson's disease. Here, we show that the pro-apoptotic protein Bax is highly expressed in the SNpc and that its ablation attenuates SNpc developmental neuronal apoptosis. In adult mice, there is an up-regulation of Bax in the SNpc after MPTP administration and a decrease in Bcl-2. These changes parallel MPTP-induced dopaminergic neurodegeneration. We also show that mutant mice lacking Bax are significantly more resistant to MPTP than their wild-type littermates. This study demonstrates that Bax plays a critical role in the MPTP neurotoxic process and suggests that targeting Bax may provide protective benefit in the treatment of Parkinson's disease.

ClpA mediates directional translocation of substrate proteins into the ClpP protease

The intracellular degradation of many proteins is mediated in an ATP-dependent manner by large assemblies comprising a chaperone ring complex associated coaxially with a proteolytic cylinder, e.g., ClpAP, ClpXP, and HslUV in prokaryotes, and the 26S proteasome in eukaryotes. Recent studies of the chaperone ClpA indicate that it mediates ATP-dependent unfolding of substrate proteins and directs their ATP-dependent translocation into the ClpP protease. Because the axial passageway into the proteolytic chamber is narrow, it seems likely that unfolded substrate proteins are threaded from the chaperone into the protease, suggesting that translocation could be directional. We have investigated directionality in the ClpA/ClpP-mediated reaction by using two substrate proteins bearing the COOH-terminal ssrA recognition element, each labeled near the NH2 or COOH terminus with fluorescent probes. Time-dependent changes in both fluorescence anisotropy and fluorescence resonance energy transfer between donor fluorophores in the ClpP cavity and the substrate probes as acceptors were measured to monitor translocation of the substrates from ClpA into ClpP. We observed for both substrates that energy transfer occurs 2–4 s sooner with the COOH-terminally labeled molecules than with the NH2-terminally labeled ones, indicating that translocation is indeed directional, with the COOH terminus of the substrate protein entering ClpP first.

Translocation pathway of protein substrates in ClpAP protease

Intracellular protein degradation, which must be tightly controlled to protect normal proteins, is carried out by ATP-dependent proteases. These multicomponent enzymes have chaperone-like ATPases that recognize and unfold protein substrates and deliver them to the proteinase components for digestion. In ClpAP, hexameric rings of the ClpA ATPase stack axially on either face of the ClpP proteinase, which consists of two apposed heptameric rings. We have used cryoelectron microscopy to characterize interactions of ClpAP with the model substrate, bacteriophage P1 protein, RepA. In complexes stabilized by ATPγS, which bind but do not process substrate, RepA dimers are seen at near-axial sites on the distal surface of ClpA. On ATP addition, RepA is translocated through ≈150 Å into the digestion chamber inside ClpP. Little change is observed in ClpAP, implying that translocation proceeds without major reorganization of the ClpA hexamer. When translocation is observed in complexes containing a ClpP mutant whose digestion chamber is already occupied by unprocessed propeptides, a small increase in density is observed within ClpP, and RepA-associated density is also seen at other axial sites. These sites appear to represent intermediate points on the translocation pathway, at which segments of unfolded RepA subunits transiently accumulate en route to the digestion chamber.