Abstract. The ARF GTP binding proteins are believed to function as regulators of membrane traffic in the secretory pathway. While the ARF1 protein has been shown in vitro to mediate the membrane interaction of the cytosolic coat proteins coatomer (COP1) and 3,-adaptin with the Golgi complex, the functions of the other ARF proteins have not been defined. Here, we show by transient transfection with epitopetagged ARFs, that whereas ARF1 is localized to the Golgi complex and can be shown to affect predictably the assembly of COP1 and 3~-adaptin with Golgi membranes in cells, ARF6 is localized to the endosomal/plasma membrane system and has no effect on these Golgi-associated coat proteins. By immunoelectron microscopy, the wild-type ARF6 protein is observed along the plasma membrane and associated with endosomes, and overexpression of ARF6 does not appear to alter the morphology of the peripheral membrane system. In contrast, overexpression of ARF6 mutants predicted either to hydrolyze or bind GTP poorly shifts the distribution of ARF6 and affects the structure of the endocytic pathway. The GTP hydrolysis-defective mutant is localized to the plasma membrane and its overexpression results in a profound induction of extensive plasma membrane vaginations and a depletion of endosomes. Conversely, the GTP binding-defective ARF6 mutant is present exclusively in endosomal structures, and its overexpression results in a massive accumulation of coated endocytic structures.

Purpose: To determine the safety, dose-limiting toxicity, maximum tolerated dose, and pharmacokinetic and pharmacodynamic profiles of the novel hydroxamate histone deacetylase inhibitor belinostat (previously named PXD101) in patients with advanced refractory solid tumors. Experimental Design: Sequential dose-escalating cohorts of three to six patients received belinostat administered as a 30-min i.v. infusion on days 1to 5 of a 21-day cycle. Pharmacokinetic variables were evaluated at all dose levels. Pharmacodynamic measurements included acetylation of histones extracted from peripheral blood mononuclear cells, caspase-dependent cleavage of cytokeratin-18, and interleukin-6 levels.

Small GTP-binding proteins such as ADP- ribosylation factor 1 (ARF1) and Sar1p regulate the membrane association of coat proteins involved in intracellular membrane trafficking. ARF1 controls the clathrin coat adaptor AP-1 and the nonclathrin coat COPI, whereas Sar1p controls the nonclathrin coat COPII. In this study, we demonstrate that membrane association of the recently described AP-3 adaptor is regulated by ARF1. Association of AP-3 with membranes in vitro was enhanced by GTPγS and inhibited by brefeldin A (BFA), an inhibitor of ARF1 guanine nucleotide exchange. In addition, recombinant myristoylated ARF1 promoted association of AP-3 with membranes. The role of ARF1 in vivo was examined by assessing AP-3 subcellular localization when the intracellular level of ARF1-GTP was altered through overexpression of dominant ARF1 mutants or ARF1- GTPase-activating protein (GAP). Lowering ARF1-GTP levels resulted in redistribution of AP-3 from punctate membrane-bound structures to the cytosol as seen by immunofluorescence microscopy. In contrast, increasing ARF1-GTP levels prevented redistribution of AP-3 to the cytosol induced by BFA or energy depletion. Similar experiments with mutants of ARF5 and ARF6 showed that these other ARF family members had little or no effect on AP-3. Taken together, our results indicate that membrane recruitment of AP-3 is promoted by ARF1-GTP. This finding suggests that ARF1 is not a regulator of specific coat proteins, but rather is a ubiquitous molecular switch that acts as a transducer of diverse signals influencing coat assembly.

The cell surface protein repertoire needs to be regulated in response to changes in the extracellular environment. In this study, we investigate protein turnover of the Saccharomyces cerevisiae plasma membrane copper transporter Ctr1p, in response to a change in extra‐cellular copper levels. As Ctr1p mediates high affinity uptake of copper into the cell, modulation of its expression is expected to be involved in copper homeostasis. We demonstrate that Ctr1p is a stable protein when cells are grown in low concentrations of copper, but that exposure of cells to high concentrations of copper (10 microM) triggers degradation of cell surface Ctr1p. This degradation appears to be specific for Ctr1p and does not occur with another yeast plasma membrane protein tested. Internalization of some Ctr1p can be seen when cells are exposed to copper. However, yeast mutant strains defective in endocytosis (end3, end4 and chc1‐ts) and vacuolar degradation (pep4) exhibit copper‐dependent Ctr1p degradation, indicating that internalization and delivery to the vacuole is not the principal mechanism responsible for degradation. In addition, a variant Ctr1p with a deletion in the cytosolic tail is not internalized upon exposure of cells to copper, but is nevertheless degraded. These observations indicate that proteolysis at the plasma membrane most likely explains copper‐dependent turnover of Ctr1p and point to the existence of a novel pathway in yeast for plasma membrane protein turnover.

SummaryThe bactericidal/permeability-increasing protein (BPI) of polymorphonuclear leukocytes (PMN) is a potent cytotoxin, specific for Gram-negative bacteria, that also inhibits endotoxin activity by neutralizing isolated bacterial lipopolysaccharides (LPS) . We have previously shown that an isolated 25 kD N-terminal fragment of human BPI carries all the antibacterial activities of the parent 55-60 kD molecule . In this study we have compared the LPS-neutralizing activities of human holo-BPI, the N-terminal fragment and a 30 kD C-terminal fragment that we have now isolated. We show that the N-terminal fragment also has LPS-neutralizing activity as detected by inhibition (up to 95%) of (a) activation by LPS of procoagulant proteases in Limulus amebocyte lysates, (b) LPS "priming" of PMN, and (c) LPS-mediated production of tumor necrosis factor in whole human blood. Holo-BPI and the 25 kD fragment have similar neutralizing potency (in nanomolar range) in all assays toward "smooth" LPS from Escherichia coli 0111:B4 and 055 :B5 (possessing long chain polysaccharide or 0-antigen), and "deep rough" LPS from Salmonella minnesota 8,595 mutant (possessing no 0-antigen) . The C-terminal fragment of BPI is devoid of antibacterial activity when tested against BPI-sensitive E. coli J5, but does have endotoxinneutralizing activity. This activity is weak relative to holo-BPI and the 25 kD N-terminal fragment in the Limulus and PMN-priming assay, but is comparable for inhibition of TNF production in whole blood. We conclude that the principal determinants for LPS recognition and neutralization, like those for antibacterial action, reside in the N-terminal half of the BPI molecule, but that sites within the C-terminal half can also contribute to BPI-LPS interaction once LPS is detached from the bacterial envelope .

Recent studies have described a widely expressed adaptor-like complex, named AP-3, which is likely involved in protein sorting in exocytic/endocytic pathways. The AP-3 complex is composed of four distinct subunits. Here, we report the identification of one of the subunits of this complex, which we call ␤3A-adaptin. The predicted amino acid sequence of ␤3A-adaptin reveals that the protein is closely related to the neuronspecific protein ␤-NAP (61% overall identity) and more distantly related to the ␤1-and ␤2-adaptin subunits of the clathrin-associated adaptor complexes AP-1 and AP-2, respectively. Sequence comparisons also suggest that ␤3A-adaptin has a domain organization similar to ␤-NAP and to ␤1-and ␤2-adaptins. ␤3A-adaptin is expressed in all tissues and cells examined. Co-purification and co-precipitation analyses demonstrate that ␤3A-adaptin corresponds to the ϳ140-kDa subunit of the ubiquitous AP-3 complex, the other subunits being ␦-adaptin, p47A (now called 3A) and 3 (A or B). ␤3A-adaptin is phosphorylated on serine residues in vivo while the other subunits of the complex are not detectably phosphorylated. ␤3A-adaptin is not present in significant amounts in clathrin-coated vesicles. The characteristics of ␤3A-adaptin reported here lend support to the idea that AP-3 is a structural and functional homolog of the clathrin-associated adaptors AP-1 and AP-2.Cytosolic protein coats function as mediators of vesicle budding and selection of cargo molecules at different stages of the secretory and endocytic pathways (reviewed in Refs. 1 and 2). Among the various protein coats that have been described to date, those containing the protein clathrin are the most extensively characterized (reviewed in Refs. 3-5). Clathrin coats are found in association with the cytosolic aspect of the trans-Golgi network and the plasma membrane, and with coated vesicles that originate from these organelles. In addition to clathrin, these coats contain specific protein complexes known as adaptors or APs. One of these adaptors, AP-1, is a component of trans-Golgi network clathrin coats and consists of four subunits: ␥-and ␤1-adaptins (ϳ100 kDa), 1 (ϳ47 kDa), and 1 (ϳ19 kDa). Another adaptor, AP-2, is associated with plasma membrane clathrin coats and is also composed of four subunits: ␣-and ␤2-adaptins (ϳ100 kDa), 2 (ϳ50 kDa), and 2 (ϳ17 kDa). The analogous subunits of AP-1 and AP-2 display significant homology to each other; in addition, the adaptor complexes themselves exhibit a similar overall structure of a "head" with two protruding "ears," each separated from the head by a flexible "hinge" (3-5, 46).A major function of AP-1 and AP-2 is to link clathrin lattices to the corresponding membranes. This role is fulfilled by the ␥-and ␤1-adaptin subunits of AP-1 and the ␣-and ␤2-adaptin subunits of AP-2 (6 -9). The adaptors are also responsible for the recognition of sorting signals present in the cytosolic domains of integral membrane proteins (10 -21), an event that leads to the concentration of these proteins within clathrincoated ...

Intravenous belinostat at 600, 900 and 1000 mg/m(2)/d is well tolerated by patients with hematological malignancies. The study was carried out in parallel to a similar dose-finding study in patients with solid tumors, in which the MTD was determined to be 1000 mg/m(2)/d days 1-5 in a 21-d cycle. This dose can also be recommended for phase II studies in patients with hematological neoplasms.

Affinity purification of crude acid extracts of rabbit polymorphonuclear leukocytes using Escherichia coli (J5) as adsorbent yields the bactericidal/permeability-increasing protein (BPI), two 15-kD species (pl5s), and the two most potent (cationic) defensin species (neutrophil peptides [NP] -1 and -2). Tested in buffered isotonic medium, the relative antibacterial potency of these proteins against E. coli J5 is BPI (IC50 0.2 nM) > pl5A (10 nM) > NP -1 (400 nM). Sublethal doses of pl5A or NP-1 can synergize with BPI to decrease the dose required to inhibit the growth of E. coli by up to 50-fold. BPI and pl5A display similar features of antibacterial action distinct from defensin NP-1, but NP-1 acts synergistically only with BPI and not with pl5A. All aspects of the combined action of BPI and NP-1 resemble those observed with higher concentrations of BPI alone, implying that NP-1 enhances BPI potency. Neither NP-1 nor pi5A alter the amount of BPI binding to E. coli but BPI enhances binding of pi5A to E. col, raising the possibility that synergy between these two proteins may occur at least partially at the level of binding. The potent synergistic actions of these proteins can also be demonstrated against serum-resistant clinical isolates of encapsulated E. coli tested in whole blood and plasma ex vivo, suggesting that such combined action may contribute to host defense in vivo. (J. Clin. Invest. 1994. 94:672-682.)