Search

Background: Synthetic indolyl- pyridinyl- propenones (IPPs) induce methuosis, a form of non-apoptotic cell death, in glioblastoma and other cancer cell lines. Methuosis is characterized by accumulation of cytoplasmic vacuoles derived from macropinosomes and late endosomes, followed by metabolic failure and rupture of the plasma membrane. However, not all IPPs that cause vacuolization are cytotoxic. The main goals of the present study were to identify key signaling pathways that contribute to methuosis induced by cytotoxic IPPs and to evaluate the anti-tumor potential of a prototype IPP in vivo., Methods: We utilized metabolic flux analysis, glucose uptake, immunoblotting, and selective pharmacological inhibitors to compare the effects of closely related cytotoxic and non-cytotoxic IPPs in cultured glioblastoma cells. To determine whether the use of methuosis-inducing IPPs might be feasible in a therapeutic context, we quantified the distribution of our lead IPP compound, MOMIPP, in mouse plasma and brain, and tested its ability to inhibit tumor growth in an intracerebral glioblastoma xenograft model., Results: The cytotoxic IPP compound, MOMIPP, causes early disruptions of glucose uptake and glycolytic metabolism. Coincident with these metabolic changes, MOMIPP selectively activates the JNK1/2 stress kinase pathway, resulting in phosphorylation of c-Jun, Bcl-2 and Bcl-xL. At the same concentration, the non-cytotoxic analog, MOPIPP, does not activate these pathways. Pharmacologic inhibition of JNK activity promotes survival, even when cells are extensively vacuolated, but suppression of c-Jun transcriptional activity offers no protection. MOMIPP readily penetrates the blood-brain barrier and is moderately effective in suppressing progression of intracerebral glioblastoma xenografts., Conclusions: The results suggest that interference with glucose uptake and induction of JNK-mediated phosphorylation of pro-survival members of the Bcl-2 family represent key events in the methuosis death process. In addition to providing new insights into the underlying molecular mechanism of methuosis, the results indicate that compounds of the cytotoxic IPP class may have potential for further development as therapeutic agents for brain tumors.

Chemotherapy, a mainstay modality for cancer, is often hindered by systemic toxicity and side effects. With the emergence of nanomedicine, the development of drug therapy has shifted toward targeted therapy. Hyaluronan (HA) is an ideal molecule as a targeted delivery system because many carcinomas overexpress HA receptors. We have conjugated resveratrol, a natural polyphenol, and 3-(5-methoxy, 2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP), a chalcone, to HA with the goal of enhancing drug bioavailability and targeting triple negative breast cancers. We demonstrate the ability of HA conjugates to accumulate in the tumor interstitium within 6 h after tail vein injections. In vitro, these conjugates interact with their target receptors, which are overexpressed by triple negative breast cancer cells under static and physiological flow. These interactions result in enhanced uptake and efficacy of the therapeutic, as demonstrated by a reduced IC 50 over that of nonconjugated drugs. Thus, HA offers a platform to solubilize, target, and enhance the efficacy of chemotherapeutics.

Methuosis is a form of non-apoptotic cell death involving massive vacuolization of macropinosome-derived endocytic compartments, followed by a decline in metabolic activity and loss of membrane integrity. To explore the induction of methuosis as a potential therapeutic strategy for killing cancer cells, we have developed small molecules (indole-based chalcones) that trigger this form of cell death in glioblastoma and other cancer cell lines. Here, we report that in addition to causing fusion and expansion of macropinosome compartments, the lead compound, 3-(5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP), disrupts vesicular trafficking at the lysosomal nexus, manifested by impaired degradation of EGF and LDL receptors, defective processing of procathepsins, and accumulation of autophagosomes. In contrast, secretion of the ectodomain derived from a prototypical type-I membrane glycoprotein, β-amyloid precursor protein, is increased rather than diminished. A closely related MOMIPP analog, which causes substantial vacuolization without reducing cell viability, also impedes cathepsin processing and autophagic flux, but has more modest effects on receptor degradation. A third analog, which causes neither vacuolization nor loss of viability, has no effect on endolysosomal trafficking. The results suggest that differential cytotoxicity of structurally similar indole-based chalcones is related, at least in part, to the severity of their effects on endolysosomal trafficking pathways.

Certain indolyl-pyridinyl-propenone analogues kill glioblastoma cells that have become resistant to conventional therapeutic drugs. Some of these analogues induce a novel form of non-apoptotic cell death called methuosis, while others primarily cause microtubule disruption. Ready access to 5-indole substitution has allowed characterization of this position to be important for both types of mechanisms when a simple methoxy group is present. We now report the syntheses and biological effects of isomeric methoxy substitutions on the indole ring. Additionally, analogues containing a trimethoxyphenyl group in place of the pyridinyl moiety were evaluated for anticancer activity. The results demonstrate that the location of the methoxy group can alter both the potency and the mechanism of cell death. Remarkably, changing the methoxy from the 5-position to the 6-position switched the biological activity from induction of methuosis to disruption of microtubules. The latter may represent a prototype for a new class of mitotic inhibitors with potential therapeutic utility., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Methuosis is a form of nonapoptotic cell death characterized by an accumulation of macropinosome-derived vacuoles with eventual loss of membrane integrity. Small molecules inducing methuosis could offer significant advantages compared to more traditional anticancer drug therapies that typically rely on apoptosis. Herein we further define the effects of chemical substitutions at the 2- and 5-indolyl positions on our lead compound 3-(5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one (MOMIPP). We have identified a number of compounds that induce methuosis at similar potencies, including an interesting analogue having a hydroxypropyl substituent at the 2-position. In addition, we have discovered that certain substitutions on the 2-indolyl position redirect the mode of cytotoxicity from methuosis to microtubule disruption. This switch in activity is associated with an increase in potency as large as 2 orders of magnitude. These compounds appear to represent a new class of potent microtubule-active anticancer agents.

Although macropinocytosis is widely recognized as a distinct form of fluid-phase endocytosis in antigen-presenting dendritic cells, it also occurs constitutively in many other normal and transformed cell types. Recent studies have established that various genetic or pharmacological manipulations can hyperstimulate macropinocytosis or disrupt normal macropinosome trafficking pathways, leading to accumulation of greatly enlarged cytoplasmic vacuoles. In some cases, this extreme vacuolization is associated with a unique form of non-apoptotic cell death termed "methuosis," from the Greek methuo (to drink to intoxication). It remains unclear whether cell death related to dysfunctional macropinocytosis occurs in normal physiological contexts. However, the finding that some types of cancer cells are particularly vulnerable to this unusual form of cell death has raised the possibility that small molecules capable of altering macropinosome trafficking or function might be useful as therapeutic agents against cancers that are resistant to drugs that work by inducing apoptosis. Herein we review examples of cell death associated with dysfunctional macropinocytosis and summarize what is known about the underlying mechanisms.

Apoptosis is the most widely recognized form of physiological programmed cell death. During the past three decades, various nonapoptotic forms of cell death have gained increasing attention, largely because of their potential importance in pathological processes, toxicology, and cancer therapy. A recent addition to the panoply of cell death phenotypes is methuosis. The neologism is derived from the Greek methuo (to drink to intoxication) because the hallmark of this form of cell death is displacement of the cytoplasm by large fluid-filled vacuoles derived from macropinosomes. The demise of the cell resembles many forms of necrosis, insofar as there is a loss of metabolic capacity and plasma membrane integrity, without the cell shrinkage and nuclear fragmentation associated with apoptosis. Methuosis was initially defined in glioblastoma cells after ectopic expression of activated Ras, but recent reports have described small molecules that can induce the features of methuosis in a broad spectrum of cancer cells, including those that are resistant to conventional apoptosis-inducing drugs. This review summarizes the available information about the distinguishing morphological characteristics and underlying mechanisms of methuosis. We compare and contrast methuosis with other cytopathological conditions in which accumulation of clear cytoplasmic vacuoles is a prominent feature. Finally, we highlight key questions that need to be answered to determine whether methuosis truly represents a unique form of regulated cell death., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Because many cancers harbor mutations that confer resistance to apoptosis, there is a need for therapeutic agents that can trigger alternative forms of cell death. Methuosis is a novel form of non-apoptotic cell death characterized by accumulation of vacuoles derived from macropinosomes and endosomes. Previous studies identified an indole-based chalcone, 3-(5-methoxy-2-methylindol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP), that induces methuosis in human cancer cells. Herein, we describe the synthesis of related 2-indolyl substituted pyridinylpropenones and their effects on U251 glioblastoma cells. Increasing the size of the 2-indolyl substituent substantially reduces growth inhibitory activity and cytotoxicity, but does not prevent cell vacuolization. Computational models suggest that the results are not due to steric-driven conformational effects. The unexpected uncoupling of vacuolization and cell death implies that the relationship between endosomal perturbations and methuotic cell death is more complex than previously realized. The new series of compounds will be useful in further defining the molecular and cellular mechanisms underlying methuosis.

Methuosis is a novel caspase-independent form of cell death in which massive accumulation of vacuoles derived from macropinosomes ultimately causes cells to detach from the substratum and rupture. We recently described a chalcone-like compound, 3-(2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (i.e., MIPP), which can induce methuosis in glioblastoma and other types of cancer cells. Herein, we describe the synthesis and structure-activity relationships of a directed library of related compounds, providing insights into the contributions of the two aryl ring systems and highlighting a potent derivative, 3-(5-methoxy, 2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (i.e., MOMIPP) that can induce methuosis at low micromolar concentrations. We have also generated biologically active azide derivatives that may be useful for future studies aimed at identifying the protein targets of MOMIPP by photoaffinity labeling techniques. The potential significance of these studies is underscored by the finding that MOMIPP effectively reduces the growth and viability of Temozolomide-resistant glioblastoma and doxorubicin-resistant breast cancer cells. Thus, it may serve as a prototype for drugs that could be used to trigger death by methuosis in cancers that are resistant to conventional forms of cell death (e.g., apoptosis).

Background: Methuosis is a unique form of non-apoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell., Results: Here we describe a novel chalcone-like molecule, 3-(2-methyl-1H- indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MIPP) that induces cell death with the hallmarks of methuosis. MIPP causes rapid accumulation of vacuoles derived from macropinosomes, based on time-lapse microscopy and labeling with extracellular fluid phase tracers. Vacuolization can be blocked by the cholesterol-interacting compound, filipin, consistent with the origin of the vacuoles from non-clathrin endocytic compartments. Although the vacuoles rapidly acquire some characteristics of late endosomes (Rab7, LAMP1), they remain distinct from lysosomal and autophagosomal compartments, suggestive of a block at the late endosome/lysosome boundary. MIPP appears to target steps in the endosomal trafficking pathway involving Rab5 and Rab7, as evidenced by changes in the activation states of these GTPases. These effects are specific, as other GTPases (Rac1, Arf6) are unaffected by the compound. Cells treated with MIPP lose viability within 2-3 days, but their nuclei show no evidence of apoptotic changes. Inhibition of caspase activity does not protect the cells, consistent with a non-apoptotic death mechanism. U251 glioblastoma cells selected for temozolomide resistance showed sensitivity to MIPP-induced methuosis that was comparable to the parental cell line., Conclusions: MIPP might serve as a prototype for new drugs that could be used to induce non-apoptotic death in cancers that have become refractory to agents that work through DNA damage and apoptotic mechanisms.

Ras GTPases are best known for their ability to serve as molecular switches regulating cell growth, differentiation and survival. Gene mutations that result in expression of constitutively active forms of Ras have been linked to oncogenesis in animal models and humans. However, over the past two decades, evidence has gradually accumulated to support a paradoxical role for Ras proteins in the initiation of cell death pathways. In this review we survey the literature pointing to the ability of activated Ras to promote cell death under conditions where cancer cells encounter apoptotic stimuli or Ras is ectopically expressed. In some of these cases Ras acts through known effectors and well defined apoptotic death pathways. However, in other cases it appears that Ras operates by triggering novel non-apoptotic death mechanisms that are just beginning to be characterized. Understanding these mechanisms and the factors that go into changing the nature of Ras signaling from pro-survival to pro-death could set the stage for development of novel therapeutic approaches aimed at manipulating pro-death Ras signaling pathways in cancer.

Methuosis is a unique form of nonapoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Methuosis can be induced in glioblastoma cells by expression of constitutively active Ras. This study identifies the small GTPases, Rac1 and Arf6, and the Arf6 GTPase-activating protein, GIT1, as key downstream components of the signaling pathway underlying Ras-induced methuosis. The extent to which graded expression of active H-Ras(G12V) triggers cytoplasmic vacuolization correlates with the amount of endogenous Rac1 in the active GTP state. Blocking Rac1 activation with the specific Rac inhibitor, EHT 1864, or coexpression of dominant-negative Rac1(T17N), prevents the accumulation of vacuoles induced by H-Ras(G12V). Coincident with Rac1 activation, H-Ras(G12V) causes a decrease in the amount of active Arf6, a GTPase that functions in the recycling of clathrin-independent endosomes. The effect of H-Ras(G12V) on Arf6 is blocked by EHT 1864, indicating that the decrease in Arf6-GTP is directly linked to the activation of Rac1. Constitutively active Rac1(G12V) interacts with GIT1 in immunoprecipitation assays. Ablation of GIT1 by short hairpin RNA prevents the decrease in active Arf6, inhibits vacuolization, and prevents loss of cell viability in cells expressing Rac1(G12V). Together, the results suggest that perturbations of endosome morphology associated with Ras-induced methuosis are due to downstream activation of Rac1 combined with reciprocal inactivation of Arf6. The latter seems to be mediated through Rac1 stimulation of GIT1. Further insights into this pathway could suggest opportunities for the induction of methuosis in cancers that are resistant to apoptotic cell death.

Expression of activated Ras in glioblastoma cells induces accumulation of large phase-lucent cytoplasmic vacuoles, followed by cell death. This was previously described as autophagic cell death. However, unlike autophagosomes, the Ras-induced vacuoles are not bounded by a double membrane and do not sequester organelles or cytoplasm. Moreover, they are not acidic and do not contain the autophagosomal membrane protein LC3-II. Here we show that the vacuoles are enlarged macropinosomes. They rapidly incorporate extracellular fluid-phase tracers but do not sequester transferrin or the endosomal protein EEA1. Ultimately, the cells expressing activated Ras detach from the substratum and rupture, coincident with the displacement of cytoplasm with huge macropinosome-derived vacuoles. These changes are accompanied by caspase activation, but the broad-spectrum caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethylketone does not prevent cell death. Moreover, the majority of degenerating cells do not exhibit chromatin condensation typical of apoptosis. These observations provide evidence for a necrosis-like form of cell death initiated by dysregulation of macropinocytosis, which we have dubbed "methuosis." An activated form of the Rac1 GTPase induces a similar form of cell death, suggesting that Ras acts through Rac-dependent signaling pathways to hyperstimulate macropinocytosis in glioblastoma. Further study of these signaling pathways may lead to the identification of other chemical and physiologic triggers for this unusual form of cell death.

Expression of activated H-Ras induces a unique form of non-apoptotic cell death in human glioblastoma cells and other specific tumor cell lines. The major cytopathological features of this form of death are the accumulation of large phase-lucent, LAMP1-positive, cytoplasmic vacuoles. In this study we sought to determine if induction of cytoplasmic vacuolation a) depends on Ras farnesylation, b) is specific to H-Ras, and c) is mediated by signaling through the major known Ras effector pathways. We find that the unusual effects of activated H-Ras depend on farnesylation and membrane association of the GTPase. Both H-Ras(G12V) and K-Ras4B(G12V) stimulate vacuolation, but activated forms of Cdc42 and RhoA do not. Amino acid substitutions in the Ras effector domain, which are known to selectively impair its interactions with Raf kinase, class-I phosphatidylinositide 3-kinase (PI3K), or Ral nucleotide exchange factors, initially pointed to Raf as a possible mediator of cell vacuolation. However, the MEK inhibitor, PD98059, did not block the induction of vacuoles, and constitutively active Raf-Caax did not mimic the effects of Ras(G12V). Introduction of normal PTEN together with H-Ras(G12V) into U251 glioblastoma cells reduced the PI3K-dependent activation of Akt, but had no effect on vacuolation. Finally, co-expression of H-Ras(G12V) with a dominant-negative form of RalA did not suppress vacuolation. Taken together, the observations indicate that Ras activates non-conventional and perhaps unique effector pathways to induce cytoplasmic vacuolation in glioblastoma cells. Identification of the relevant signaling pathways may uncover specific molecular targets that can be manipulated to activate non-apoptotic cell death in this type of cancer.

The human type III phosphatidylinositol 3-kinase, hVps34, converts phosphatidylinositol (PtdIns) to phosphatidylinositol 3-phosphate [PtdIns(3)P]. Studies using inhibitors of phosphatidylinositide 3-kinases have indicated that production of PtdIns(3)P is important for a variety of vesicle-mediated trafficking events, including endocytosis, sorting of receptors in multivesicular endosomes, and transport of lysosomal enzymes from the trans-Golgi network (TGN) to the endosomes and lysosomes. This study utilizes small interfering (si)RNA-mediated gene silencing to define the specific trafficking pathways in which hVps34 functions in human U-251 glioblastoma cells. Suppression of hVps34 expression reduced the cellular growth rate and caused a striking accumulation of large acidic phase-lucent vacuoles that contain lysosomal membrane proteins LAMP1 and LGP85. Analysis of these structures by electron microscopy suggests that they represent swollen late endosomes that have lost the capacity for inward vesiculation but retain the capacity to fuse with lysosomes. Morphological perturbation of the late endosome compartment was accompanied by a reduced rate of processing of the endosomal intermediate form of cathepsin D to the mature lysosomal form. There was also a reduction in the rate of epidermal growth factor receptor (EGFR) dephosphorylation and degradation following ligand stimulation, consistent with the retention of the EGFR on the limiting membranes of the enlarged late endosomes. By contrast, the suppression of hVps34 expression did not block trafficking of cathepsin D between the TGN and late endosomes, or endocytic uptake of fluid-phase markers, or association of a PtdIns(3)P-binding protein, EEA1, with early endosomes. LAMP1-positive vacuoles were depleted of PtdIns(3)P in the hVps34-knockdown cells, as judged by their inability to bind the PtdIns(3)P probe GFP-2xFYVE. By contrast, LAMP1-negative vesicles continued to bind GFP-2xFYVE in the knockdown cells. Overall, these findings indicate that hVps34 plays a major role in generating PtdIns(3)P for internal vesicle formation in multivesicular/late endosomes. The findings also unexpectedly suggest that other wortmannin-sensitive kinases and/or polyphosphoinositide phosphatases may be able to compensate for the loss of hVps34 and maintain PtdIns(3)P levels required for vesicular trafficking in the early endocytic pathway or the TGN.

Beclin 1 was originally identified as a novel Bcl-2-interacting protein, but co-immunoprecipitation studies suggest that the major physiological partner for Beclin 1 is the mammalian class III phosphatidylinositol 3-kinase (PI 3-kinase) Vps34. Beclin 1 has been proposed to function as a tumor suppressor by promoting cellular macroautophagy, a process that is known to depend on Vps34. However, an alternative role for Beclin 1 in modulating normal Vps34-dependent protein trafficking pathways has not been ruled out. This possibility was examined in U-251 glioblastoma cells. Immunoprecipitates of endogenous Beclin 1 contained human Vps34 (hVps34), but not Bcl-2. Suppression of Beclin 1 expression by short interfering (si)RNA-mediated gene silencing blunted the autophagic response of the cells to nutrient deprivation or C2-ceramide. However, other PI 3-kinase-dependent trafficking pathways, such as the post-endocytic sorting of the epidermal growth factor receptor (EGFR) or the proteolytic processing of procathepsin D en route from the trans-Golgi network (TGN) to lysosomes, were not affected. Depletion of Beclin 1 did not reduce endocytic internalization of a fluid phase marker (horseradish peroxidase, HRP) or cause swelling of late endosomal compartments typically seen in cells where the function of hVps34 is impaired. These findings argue against a role for Beclin 1 as an essential chaperone or adaptor for hVps34 in normal vesicular trafficking, and they support the hypothesis that Beclin 1 functions mainly to engage hVps34 in the autophagic pathway.

Tyrosine phosphorylation is a fundamental mechanism for regulating the functions of numerous proteins in eukaryotic cells. It has been known for some time that several members of the Rab GTPase family can undergo phosphorylation on serine or threonine residues, but the potential for tyrosine phosphorylation has been appreciated only recently, based on a single example-Rab24. Herein we describe a series of straightforward methods to facilitate an initial assessment of the potential for tyrosine phosphorylation of epitope-tagged Rab proteins transiently expressed in mammalian cells. The approach takes advantage of the availability of highly specific monoclonal antibodies against phosphotyrosine and specific chemical inhibitors for tyrosine kinases. We also describe the use of site-directed mutagenesis to identify tyrosine residues that may be targets for phosphorylation, and we discuss the possible relevance of this modification for regulating Rab function.

Several members of the large family of Rab GTPases have been shown to function in vesicular trafficking in mammalian cells. However, the exact role of Rab24 remains poorly defined. Rab24 differs from other Rab proteins in that it has a low intrinsic GTPase activity and is not efficiently prenylated. Here we report an additional unique property of Rab24; i.e., the protein can undergo tyrosine phosphorylation when overexpressed in cultured cells. Immunoblot analyses with specific anti-phosphotyrosine monoclonal antibodies revealed the presence of phosphotyrosine (pTyr) on myc-Rab24 in whole cell lysates and immunoprecipitated samples. No pTyr was detected on other overexpressed myc-tagged GTPases (H-Ras, Rab1b, Rab6, Rab11 or Rab13). Comparisons of myc-Rab24 in the soluble and particulate fractions from HEK293 and HEp-2 cells indicated that the cytosolic pool of Rab24 was more heavily phosphorylated than the membrane pool. Treatment of transfected cells with the broad-spectrum tyrosine kinase inhibitor, genistein, as well as the specific Src-family kinase inhibitor, PP2, eliminated the pTyr signal from Rab24. In contrast the receptor tyrosine kinase inhibitor, tyrphostin A25, had no effect. Tyrosine phosphorylation of Rab24 was reduced by alanine substitution of two unique tyrosines, one found in a strong consensus phosphorylation motif (Y [Formula: see text] ) in the hypervariable domain (Y172) and the other falling within the GXXXGK(S/T) motif known as the P-loop (Y17). The latter region is known to influence GTP hydrolysis in Rab proteins, so the phosphorylation of Y17 could contribute to the low intrinsic GTPase activity of Rab24. This is the first report of tyrosine phosphorylation in any member of the Ras superfamily and it raises the possibility that this type of modification could influence Rab24 targeting and interactions with effector protein complexes.

The targeting of various Rab proteins to different subcellular compartments appears to be determined by variable amino acid sequences located upstream from geranylgeranylated cysteine residues in the C-terminal tail. All nascent Rab proteins are prenylated by geranylgeranyltransferase II, which recognizes the Rab substrate only when it is bound to Rab escort protein (REP). After prenylation, REP remains associated with the modified Rab until it is delivered to the appropriate subcellular membrane. It remains unclear whether docking of the Rab with the correct membrane is solely a function of features contained within the prenylated Rab itself (with REP serving as a "passive" carrier) or whether REP actively participates in the targeting process. To address this issue, we took advantage of a mutation in the alpha2 helix of Rab1B (i.e. Y78D) that abolishes REP and GDI interaction without disrupting nucleotide binding or hydrolysis. These studies demonstrate that replacing the C-terminal GGCC residues of Rab1B(Y78D) with a CLLL motif permits this protein to be prenylated by geranylgeranyltransferase I but not II both in cell-free enzyme assays and in transfected cells. Subcellular fractionation and immunofluorescence studies reveal that the prenylated Rab1B(Y78D)CLLL, which remains deficient in REP and GDI association is, nonetheless, delivered to the Golgi and endoplasmic reticulum (ER) membranes. When the dominant-negative S22N mutation was inserted into Rab1B-CLLL, the resulting monoprenylated construct suppressed ER --> Golgi protein transport. However, when the Y78D mutation was added to the latter construct, its inhibitory effect on protein trafficking was lost despite the fact that it was localized to the ER/Golgi membrane. Therefore, protein interactions mediated by the alpha2 helical domain of Rab1B(S22N) appear to be essential for its functional interaction with components of the ER --> Golgi transport machinery.

The function of Rab24 is currently unknown, but other members of the Rab GTPase family are known to participate in various protein trafficking pathways. Rab proteins are thought to cycle on and off vesicle membranes in conjunction with changes in their guanine nucleotide state. The present studies indicate that Rab24 possesses several unusual characteristics that distinguish it from other Rab proteins. 1) Based on [(32)P]orthophosphate labeling of protein-bound nucleotide, Rab24 exists predominantly in the GTP state when expressed in cultured cells. The low GTPase activity is related to the presence of serine instead of glutamine at the position cognate to Ras Gln-61. 2) Posttranslational geranylgeranylation of Rab24, determined by metabolic labeling or detergent partitioning assays, is inefficient when compared with other Rabs ending with the common CXC and CC carboxyl-terminal motifs. This is partly due to the presence of two histidines distal to the target cysteines, but also involves other unidentified features. 3) Most of the Rab24 in the cytoplasmic compartment of cultured cells is not associated with Rab GDP dissociation inhibitors. These findings indicate that, if Rab24 functions in vesicular transport processes, it may operate through a novel mechanism that does not depend on GTP hydrolysis or GDP dissociation inhibitor-mediated recycling.

Posttranslational modification of Rab proteins by geranylgeranyltransferase type II requires that they first bind to Rab escort protein (REP). Following prenylation, REP is postulated to accompany the modified GTPase to its specific target membrane. REP binds preferentially to Rab proteins that are in the GDP state, but the specific structural domains involved in this interaction have not been defined. In p21 Ras, the alpha2 helix of the Switch 2 domain undergoes a major conformational change upon GTP hydrolysis. Therefore, we hypothesized that the corresponding region in Rab1B might play a key role in the interaction with REP. Introduction of amino acid substitutions (I73N, Y78D, and A81D) into the putative alpha2 helix of Myc-tagged Rab1B prevented prenylation of the recombinant protein in cell-free assays, whereas mutations in the alpha3 and alpha4 helices did not. Additionally, upon transient expression in transfected HEK-293 cells, the Myc-Rab1B alpha2 helix mutants were not efficiently prenylated as determined by incorporation of [3H]mevalonate. Metabolic labeling studies using [32P]orthophosphate indicated that the poor prenylation of the Rab1B alpha2 helix mutants was not directly correlated with major disruptions in guanine nucleotide binding or intrinsic GTPase activity. Finally, gel filtration analysis of cytosolic fractions from 293 cells that were coexpressing T7 epitope-tagged REP with various Myc-Rab1B constructs revealed that mutations in the alpha2 helix of Rab1B prevented the association of nascent (i.e., nonprenylated) Rab1B with REP. These data indicate that the Switch 2 domain of Rab1B is a key structural determinant for REP interaction and that nucleotide-dependent conformational changes in this region are largely responsible for the selective interaction of REP with the GDP-bound form of the Rab substrate.

Lovastatin blocks the biosynthesis of the isoprenoid precursor, mevalonate. When Friend murine erythroleukemia (MEL) cells are cultured in medium containing lovastatin, the precursor of murine leukemia virus envelope glycoprotein (gPr90env) fails to undergo proteolytic processing, which normally occurs in the Golgi complex. Consequently, newly synthesized envelope proteins are not incorporated into viral particles that are shed into the culture medium. gPr90env appears to be localized in a pre-Golgi membrane compartment, based on its enrichment in subcellular fractions containing NADPH-cytochrome c reductase activity and the sensitivity of its carbohydrate chains to digestion with endoglycosidase H. Arrest of gPr90env processing occurs at concentrations of lovastatin that are not cytostatic, and the effect of the inhibitor is prevented by addition of mevalonate to the medium. The low molecular mass GTP-binding proteins, rab1p and rab6p, which are believed to function in early steps of the exocytic pathway, are normally modified posttranslationally by geranylgeranyl isoprenoids. However, in MEL cells treated with 1 microM lovastatin, nonisoprenylated forms of these proteins accumulate in the cytosol prior to arrest of gPr90env processing. These observations suggest that lovastatin may prevent viral envelope precursors from reaching the Golgi compartment by blocking the isoprenylation of rab proteins required for ER to Golgi transport.

The biosynthesis of phosphatidylserine (PS) and its conversion to phosphatidylcholine (PC) are regulated coordinately by inositol and choline in Saccharomyces cerevisiae (G. M. Carman and S. A. Henry, 1989, Annu. Rev. Biochem. 58, 635-669). In this study, PS decarboxylase activity is shown to be partially repressed when inositol is added to the medium of cells in the log phase of growth, and the extent of repression is augmented by the inclusion of choline, but not ethanolamine. The kinetics of repression and derepression of PS decarboxylase, PS synthase, and phospholipid N-methyltransferase (PNMT) activities, as regulatory responses to the availability of exogenous inositol and choline, have been characterized. When inositol was added to the medium of cell cultures growing exponentially, the three biosynthetic enzyme activities reached an intermediate level of repression (50-85% of control) within 60 min. After the addition of the combination of inositol and choline, PS decarboxylase, PS synthase, and PNMT activities decreased to the intermediate levels of repression in 60 min and were subsequently reduced to 15-40% of control values during a later stage of regulation (2-3 h). In a derepression study, the three enzyme activities remained relatively stable for approximately 60 min following the removal of choline and/or inositol from the growth medium, but the specific activities of PS decarboxylase, PS synthase, and PNMT increased to maximally derepressed levels within 2-3 h. The induction of the three biosynthetic activities was blocked by cycloheximide, but not by chloramphenicol. In summary, the level of PS decarboxylase activity in S. cerevisiae is partially and reversibly suppressed by inositol and further diminished by the combination of inositol and choline. The biphasic kinetics of repression by inositol and choline suggest that the effect of choline is dependent on earlier events mediated by inositol and possibly involves a separate regulatory factor(s).

The nucleotide sequence of the RNA of tobacco vein mottling virus, a member of the potyvirus group, was determined. The RNA was found to be 9471 residues in length, excluding a 3'-terminal poly(A) tail. The first three AUG codons from the 5'-terminus were followed by in-frame termination codons. The fourth, at position 206, was the beginning of an open reading frame of 9015 residues which could encode a polyprotein of 340 kDa. No other long open reading frames were present in the sequence or its complement. This AUG was present in the sequence AGGCCAUG, which is similar to the consensus initiation sequence shared by most eukaryotic mRNAs. The chemically-determined amino acid compositions of the helper component and coat proteins were similar to those predicted from the nucleotide sequence. Amino acid sequencing of coat protein from which an amino-terminal peptide had been removed allowed exact location of the coat protein cistron. A consensus sequence of V-(R or K)-F-Q was found on the N-terminal sides of proposed cleavage sites for proteolytic processing of the polyprotein.

A simple assay for phosphatidylserine decarboxylase is described. Following incubation of a mitochondrial fraction from Saccharomyces cerevisiae with purified, exogenous phosphatidyl[3H]serine, the lipid extract is applied to a small DEAE-cellulose column equilibrated in CHCI3-CH3OH (1:1). The unreacted substrate, phosphatidyl[3H]serine, is quantitatively bound by the ion-exchange column while the product, phosphatidyl[3H]ethanolamine, is eluted by sequential washing with CHCI3-CH3OH (1:1) and CH3OH. The organic solvents are evaporated, and the amount of radiolabeled phosphatidyl[3H]ethanolamine formed by enzymatic decarboxylation is determined by liquid scintillation spectrometry. The reliability of this assay was established by showing that several enzymatic properties of the yeast enzyme, defined by the new assay, were essentially identical to the properties characterized by a more tedious paper chromatographic assay described previously. Virtually identical rates of enzymatic decarboxylation of phosphatidyl[3H]serine were also obtained for mitochondrial fractions from pig brain and rat liver when the activities were compared by the column and paper chromatographic methods.

Previous studies have shown that bacteria maintained in a low-nutrient "natural" environment such as swimming pool water are much more resistant to disinfection by various chemical agents than strains maintained on rich media. In the present study a comparison was made of the chlorine (Cl2) susceptibility of hot-water tank isolates of Legionella pneumophila maintained in tap water and strains passaged on either nonselective buffered charcoal-yeast extract or selective differential glycine-vancomycin-polymyxin agar medium. Our earlier work has shown that environmental and clinical isolates of L. pneumophila maintained on agar medium are much more resistant to Cl2 than coliforms are. Under the present experimental conditions (21 degrees C, pH 7.6 to 8.0, and 0.25 mg of free residual Cl2 per liter, we found the tap water-maintained L. pneumophila strains to be even more resistant than the agar-passaged isolates. Under these conditions, 99% kill of tap water-maintained strains of L. pneumophila was usually achieved within 60 to 90 min compared with 10 min for agar-passaged strains. Samples from plumbing fixtures in a hospital yielded legionellae which were "super"-chlorine resistant when assayed under natural conditions. After one agar passage their resistance dropped to levels of comparable strains which had not been previously exposed to additional chlorination. These studies more closely approximate natural conditions than our previous work and show that tap water-maintained L. pneumophila is even more resistant to Cl2 than its already resistant agar medium-passaged counterpart.