Characterization of Chemical Inhibitors of Brefeldin A-activated Mono-ADP-ribosylation

Abstract

Brefeldin A, a toxin inhibitor of vesicular traffic, induces the selective mono-ADP-ribosylation of two cytosolic proteins, glyceraldehyde-3-phosphate dehydrogenase and the novel GTP-binding protein BARS-50. Here, we have used a new quantitative assay for the characterization of this reaction and the development of specific pharmacological inhibitors. Mono-ADP-ribosylation is activated by brefeldin A with an EC 50 of 17.0 ؎ 3.1 g/ml, but not by biologically inactive analogs including a brefeldin A stereoisomer. Brefeldin A acts by increasing the V max of the reaction, whereas it does not influence the K m of the enzyme for NAD ؉ (154 ؎ 13 M). The enzyme is an integral membrane protein present in most tissues and is modulated by Zn 2؉ , Cu 2؉ , ATP (but not by other nucleotides), pH, temperature, and ionic strength. To identify inhibitors of the reaction, a large number of drugs previously tested as blockers of bacterial ADPribosyltransferases were screened. Two classes of molecules, one belonging to the coumarin group (dicumarol, coumermycin A 1 , and novobiocin) and the other to the quinone group (ilimaquinone, benzoquinone, and naphthoquinone), rather potently and specifically inhibited brefeldin A-dependent mono-ADP-ribosylation. When tested in living cells, these molecules antagonized the tubular reticular redistribution of the Golgi complex caused by brefeldin A at concentrations similar to those active in the mono-ADP-ribosylation assay in vitro, suggesting a role for mono-ADP-ribosylation in the cellular actions of brefeldin A.Mono-ADP-ribosylation is a post-translational modification of proteins whereby the adenosine 5Ј-diphosphoribose moiety of NAD ϩ is transferred to one of a number of amino acid residues (1, 2). Several well characterized mono-ADP-ribosylation reactions are catalyzed by bacterial toxins and result in the permanent modification of GTPases with key regulatory functions in cellular processes (1, 2). Similar reactions are also catalyzed by eukaryotic enzymes, but their cellular significance, despite recent advances (3-5), is less well understood. Recently, we have reported that brefeldin A (BFA), 1 a fungal toxin metabolite of palmitic acid (6) with potent inhibitory effects on intracellular membrane traffic (7), stimulates the selective mono-ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa in mammalian cells (8). The 50-kDa substrate (BARS-50; see Ref. 9) binds GTP and is regulated by ␤␥-subunits of trimeric G proteins; it has therefore been proposed to be a novel G protein involved in membrane transport (9). The p38 substrate is glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a multifunctional protein involved in several cellular processes (10 -16). These observations have raised the question as to whether some of the cellular effects of BFA might be mediated by mono-ADP-ribosylation.The cellular actions of BFA are multiple and complex. BFA selectively blocks constitutive protein secretion and causes a rapid and extensive disruption of the Golgi apparatus consisting of the ...

Architecture of the vimentin cytoskeleton is modified by perturbation of the GTPase ARF1

“…To test this hypothesis, SW13 v+ cells were treated with 10 g/ml BFA or 40 M IQ for either 30 minutes or 1 hour. This dose of IQ was previously shown to perturb Golgi dynamics (Weigert et al, 1997). Treatment with BFA and IQ resulted in fragmentation of the Golgi complex as shown by staining with the Golgi marker GM130 (Fig.…”

“…Therefore, the effects of BFA on vimentin architecture could be due to fragmentation of the Golgi complex. To further explore Golgi fragmentation as a cause of BFA-induced vimentin changes, we used ilimaquinone (IQ), a structurally different drug that exhibits effects similar to BFA in fragmentation of the Golgi complex but differs in the molecular mechanisms underlying this effect (Takizawa et al, 1993;Weigert et al, 1997). In addition to its effects on Golgi fragmentation, IQ has also been shown to result in release of the adaptor COP-I from membranes (Madari et al, 2003;Takizawa et al, 1993).…”

“…To understand whether ADP ribosylation was required for BFA-induced vimentin remodeling, cells were treated with 10 g/ml BFA in the absence or presence of 50 mM nicotinamide. Nicotinamide is a general inhibitor of ADP ribosylation, and this dose is equivalent to 2.5 times the reported IC 50 for inhibition of ADP ribosylation of BARS50 (Weigert et al, 1997). As a control to ensure the efficacy of nicotinamide treatment, SW13 v+ cells were treated with 1 mM hydrogen peroxide to induce nuclear poly-ADP ribosylation (Kupper et al, 1996;Schraufstatter et al, 1986).…”

“…Because vimentin has previously been shown to associate with Golgi fragments and BFA treatment leads to Golgi fragmentation, we treated cells with IQ, a drug that is structurally different from BFA but causes similar effects on fragmentation of the Golgi complex (Takizawa et al, 1993;Weigert et al, 1997). This was particularly important because fragmentation of the Golgi 5).…”

Molecular mechanism and functional role of brefeldin A-mediated ADP-ribosylation of CtBP1/BARS

“…1F). In addition, molecules with a coumarinic moiety, which we previously showed to act as specific inhibitors of ADP-ribosylation (30), and which are known to bind in the Rossmann fold (31), behaved as competitive inhibitors for the binding of BAC to BARS (Fig. S6).…”