Beclin 1 was originally identified as a novel Bcl-2-interacting protein, but co-immunoprecipitation studies suggest that the major physiological partner for is mammalian class III phosphatidylinositol 3-kinase (PI 3-kinase) Vps34. has been proposed to function tumor suppressor by promoting cellular macroautophagy, process known depend on However, an alternative role in modulating normal Vps34-dependent protein trafficking pathways not ruled out. This possibility examined U-251 glioblastoma...

Abstract Expression of activated Ras in glioblastoma cells induces accumulation large phase-lucent cytoplasmic vacuoles, followed by cell death. This was previously described as autophagic However, unlike autophagosomes, the Ras-induced vacuoles are not bounded a double membrane and do sequester organelles or cytoplasm. Moreover, they acidic contain autophagosomal protein LC3-II. Here we show that enlarged macropinosomes. They rapidly incorporate extracellular fluid-phase tracers but...

Methuosis is a novel caspase-independent form of cell death in which massive accumulation vacuoles derived from macropinosomes ultimately causes cells to detach the substratum and rupture. We recently described chalcone-like compound, 3-(2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (i.e., MIPP), can induce methuosis glioblastoma other types cancer cells. Herein, we describe synthesis structure–activity relationships directed library related compounds, providing insights into...

Abstract Background Methuosis is a unique form of non-apoptotic cell death triggered by alterations in the trafficking clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture cell. Results Here we describe novel chalcone-like molecule, 3-(2- m ethyl-1H- i ndol-3-yl)-1-(4- p yridinyl)-2- ropen-1-one (MIPP) that induces with hallmarks methuosis. MIPP causes rapid accumulation vacuoles derived from macropinosomes, based on time-lapse microscopy labeling...

Abstract Membrane localization of p21ras is dependent upon its posttranslational modification by a 15-carbon farnesyl group. The isoprenoid linked to cysteine located within conserved carboxyl-terminal sequence termed the CAAX box (where C cysteine, A an aliphatic amino acid, and X any acid). We now show that three GTP-binding proteins encoded recently identified rac1, rac2, ralA genes also undergo modification. cDNAs coding for each protein were transcribed in vitro, RNAs translated...

p21ras and several other ras-related GTP-binding proteins are modified post-translationally by addition of 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoids to cysteines within a conserved carboxyl-terminal sequence motif, Caa(M/S/L), where is an aliphatic amino acid. Proteins ending with M S substrates for farnesyltransferase, whereas those L preferentially geranylgeranyltransferase. We recently reported that encoded rab1B (GGCC), rab2 rab5 (CCSN) isoprenyl derivatives...

Abstract In the presence of lovastatin (mevinolin), an inhibitor endogenous mevalonate synthesis, C1300 murine neuroblastoma cells incorporated (2‐ 14 C)mevalonate into several discrete polypeptides that were separable by SDS‐PAGE. The electrophoretic pattern labeled proteins did not vary substantially when homogenized with Ca ++ , Mg high concentrations NaCl or phosphatase inhibitor, lysed immediately in trichloroacetic acid. When had been prelabeled ( incubated and simultaneously deprived...

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase catalyzes the formation of mevalonate, an essential precursor for isoprenoid compounds in mammalian cells. Recent studies have shown that mevinolin, a competitive inhibitor reductase, inhibits cell proliferation and induces differentiation cultured C1300 (Neuro-2A) murine neuroblastoma We now report mevinolin can inhibit growth vivo. The specific activity HMG-CoA subcutaneous neuroblastomas increased more than 20-fold between fifth...

The human type III phosphatidylinositol 3-kinase, hVps34, converts (PtdIns) to 3-phosphate [PtdIns(3)P]. Studies using inhibitors of phosphatidylinositide 3-kinases have indicated that production PtdIns(3)P is important for a variety vesicle-mediated trafficking events, including endocytosis, sorting receptors in multivesicular endosomes, and transport lysosomal enzymes from the trans-Golgi network (TGN) endosomes lysosomes. This study utilizes small interfering (si)RNA-mediated gene...

Several proteins in mammalian cells are modified post-translationally by the isoprenoid, farnesol. Incubation of cultured with [3H]mevalonate, an isoprenoid precursor, results labeling multiple polypeptides, most prominent which migrate range 21-26 kDa on sodium dodecyl sulfate-polyacrylamide gels. In Rat-6 fibroblasts transformed H-ras, one farnesylated was identified as p21ras two-dimensional immunoblotting. However, this protein accounted for only a small proportion [3H]mevalonate-derived...

Low molecular mass GTP-binding proteins encoded by the mammalian rab genes are found in membranes of Golgi complex and endosomes, suggesting that they play a role movement exocytic endocytic vesicles. The basis for membrane association these has not been defined. Herein, we demonstrate terminal cysteine residues rab1B, rab2, rab5 undergo thioether modification isoprenyl groups when translated vitro presence radiolabeled isoprenoid precursor, [3H]mevalonate. Results gel permeation...

The predicted amino acid sequences for the Gi alpha 1 and G gamma 6 subunits of brain heterotrimeric G-proteins both contain C-terminal Cys-A-A-X elements (A is an aliphatic residue X any acid). This domain represents site Cys thioether modification by isoprenoids in p21ras, nuclear lamins, fungal mating factors. We now show that 6, translated reticulocyte lysate, efficiently labeled with isoprenoid precursor, [3H]mevalonate. Alteration sequence so a Gly was substituted element rendered...

Cells incorporate isoprenoid products derived from mevalonate (MVA) into several unique proteins. The aim of this study was to delineate the effects blocking MVA synthesis on covalent isoprenylation these proteins in murine erythroleukemia cells. Inhibition protein with cycloheximide prevented incorporation [3H]MVA proteins, suggesting that normally occurs immediately after polypeptides. However, incubation cells lovastatin, a competitive inhibitor 3-hydroxy-3-methylglutaryl coenzyme A...

Abstract Mevinolin, a competitive inhibitor of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase, stimulates neurite outgrowth and acetylcholinesterase (ACE) activity in C1300 (Neuro‐2A) murine neuroblastoma cells. Sprouting neurites began within 4–8 h, before changes cell proliferation could be detected by [ 3 H]thymidine incorporation or flow cytometry. In contrast, the increase ACE was temporally correlated with suppression DNA synthesis, which occurred after 8 h. The membrane...

Methuosis is a unique form of nonapoptotic cell death triggered by alterations in the trafficking clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture cell. can be induced glioblastoma cells expression constitutively active Ras. This study identifies small GTPases, Rac1 Arf6, Arf6 GTPase-activating protein, GIT1, as key downstream components signaling pathway underlying Ras-induced methuosis. The extent which graded H-Ras(G12V) triggers cytoplasmic...

Synthetic indolyl- pyridinyl- propenones (IPPs) induce methuosis, a form of non-apoptotic cell death, in glioblastoma and other cancer lines. Methuosis is characterized by accumulation cytoplasmic vacuoles derived from macropinosomes late endosomes, followed metabolic failure rupture the plasma membrane. However, not all IPPs that cause vacuolization are cytotoxic. The main goals present study were to identify key signaling pathways contribute methuosis induced cytotoxic evaluate anti-tumor...

ras proteins undergo posttranslational modification by a 15-carbon farnesyl isoprenoid at cysteine within defined COOH-terminal amino acid motif; i.e., Cys-Ali-Ali-Ser/Met (where Ali represents an aliphatic residue). In other low molecular mass GTP-binding proteins, cysteines are modified 20-carbon geranylgeranyl groups Cys-Ali-Ali-Leu motif. We changed the terminal Ser-189 of Ha-ras p21 to Leu-189 site-directed mutagenesis and found that protein was [3H]geranylgeranyl instead [3H]farnesyl...

Because many cancers harbor mutations that confer resistance to apoptosis, there is a need for therapeutic agents can trigger alternative forms of cell death. Methuosis novel form non-apoptotic death characterized by accumulation 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), induces methuosis in human cancer cells. Herein, we describe the synthesis related...

Methuosis is a form of nonapoptotic cell death characterized by an accumulation macropinosome-derived vacuoles with eventual loss 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 chemical substitutions at 2- and 5-indolyl positions our lead compound 3-(5-methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one (MOMIPP)....

We have identified a Rab1B effector-domain mutant (D44N) that, when geranylgeranylated by Rab:geranylgeranyltransferase (GGTase II) in cell-free systems or intact cells, fails to form detectable complexes with GDP-dissociation inhibitors (GDIs). GDI-Rab were collected on anti-FLAG affinity beads after incubating recombinant FLAG epitope-tagged GDI vitro, transiently coexpressing Myc-tagged FLAG-GDI-α FLAG-GDI-2 human embryonal kidney 293 cells. [3H]Mevalonate labeling and immunoprecipitation...