INHIBITION OF MACROAUTOPHAGY AND PROTEOLYSIS IN THE ISOLATED RAT HEPATOCYTE BY A NONTRANSPORTABLE DERIVATIVE OF THE MULTIPLE ANTIGEN PEPTIDE LEU8-LYS4 -LYS2-LYS-BETA ALA

The multiple antigen peptide derivative, Leu8-Lys4-Lys2-Lys-beta Ala (Leu8-MAP), was synthesized by attaching the carboxyl of leucine to the NH2 termini of a branched lysine core, termed MAP, creating a molecule of about 1900 Da with 8 leucine residues. On a molar basis (independent of the number of leucine substitutions), Leu8-MAP was as effective as leucine in suppressing macroautophagy and proteolysis; moreover, it exhibited the same apparent Km (about 0.1 mM). The effect was specific for leucine since Ile8-MAP was inactive. It is of interest, though, that Leu8-MAP did not elicit the multiphasic response typical of leucine but instead evoked the single site inhibition normally seen with leucine plus the co-regulator alanine. Some free leucine was produced from Leu8-MAP during hepatocyte incubations, but the amounts were insufficient to account for the inhibition. Although this degradation created species of Leu-MAP that had lost 1-3 residues of leucine, their inhibitory effectiveness was not diminished. Because the extracellular/intracellular distribution ratio of [3H]-Leu8-MAP was 100:1 or greater, the direct transport of Leu8-MAP across the plasma membrane into the cytosolic compartment can be excluded. Hence, cytosolic concentrations of Leu8-MAP will be at least 100-fold smaller than those of leucine under conditions of comparable proteolytic inhibition. For these and related reasons, effects attributable to the recognition of Leu8-MAP cannot be explained by signals generated within the cytosol. They could, however, be mediated from site(s) on the plasma membrane or within associated vesicles

Inactivation of the glutathione peroxidase GPx4 by the ferroptosis-inducing molecule RSL3 requires the adaptor protein 14-3-3 epsilon

RSL3, a drug candidate prototype for cancer chemotherapy, triggers ferroptosis by inactivating GPx4. Here we report the purification of the protein indispensable for GPx4 inactivation by RSL3. MS analysis reveals 14-3-3 isoforms as candidates and recombinant human 14-3-3epsilon confirms the identification. The function of 14-3-3\uf065 is redox-regulated. Moreover, overexpression and silencing of the gene coding for 14-3-3\uf065 consistently control the inactivation of GPx4 by RSL3. The interaction of GPx4 with a redox-regulated adaptor protein, operating in cell signalling, further contributes to frame it within redox-regulated pathways of cell survival and death and opens new therapeutic perspectives

Insight into the mechanism of ferroptosis inhibition by ferrostatin-1

Ferroptosis is a form of cell death primed by iron and lipid hydroperoxides and prevented by GPx4. Ferrostatin-1 (fer-1) inhibits ferroptosis much more efficiently than phenolic antioxidants. Previous studies on the antioxidant efficiency of fer-1 adopted kinetic tests where a diazo compound generates the hydroperoxyl radical scavenged by the antioxidant. However, this reaction, accounting for a chain breaking effect, is only minimally useful for the description of the inhibition of ferrous iron and lipid hydroperoxide dependent peroxidation. Scavenging lipid hydroperoxyl radicals, indeed, generates lipid hydroperoxides from which ferrous iron initiates a new peroxidative chain reaction. We show that when fer-1 inhibits peroxidation, initiated by iron and traces of lipid hydroperoxides in liposomes, the pattern of oxidized species produced from traces of pre-existing hydroperoxides is practically identical to that observed following exhaustive peroxidation in the absence of the antioxidant. This supported the notion that the anti-ferroptotic activity of fer-1 is actually due to the scavenging of initiating alkoxyl radicals produced, together with other rearrangement products, by ferrous iron from lipid hydroperoxides. Notably, fer-1 is not consumed while inhibiting iron dependent lipid peroxidation. The emerging concept is that it is ferrous iron itself that reduces fer-1 radical. This was supported by electroanalytical evidence that fer-1 forms a complex with iron and further confirmed in cells by fluorescence of calcein, indicating a decrease of labile iron in the presence of fer-1. The notion of such as pseudo-catalytic cycle of the ferrostatin-iron complex was also investigated by means of quantum mechanics calculations, which confirmed the reduction of an alkoxyl radical model by fer-1 and the reduction of fer-1 radical by ferrous iron. In summary, GPx4 and fer-1 in the presence of ferrous iron, produces, by distinct mechanism, the most relevant anti-ferroptotic effect, i.e the disappearance of initiating lipid hydroperoxides

Inhibitory action of isovaleryl-L-carnitine on proteolysis in perfused rat liver.

Isovaleryl-l-carnitine inhibits the proteolysis induced by amino acid deprivation in the perfused rat liver to an extent equivalent, or, below 0.4 mM, even greater than that previously found for 1-leucine (Ref. 1). Also the typical concentration-response curve previously found for leucine (Ref. 1) is mimicked by isovaleryl-l-carnitine. The maximum inhibition (approximately 50% of the control) occurred for both l-leucine and isovaleryl-l-carnitine above 0.8 mM. Only at these high concentrations also 1-carnitine and isobutyryl-l-carnitine exhibit a significant, albeit lower, degree of inhibition. The possible mechanism of this proteolysis inhibition is discussed

Ganglioside GM1 protection from apoptosis of rat heart fibroblasts

Ceramide is involved as a mediator of apoptosis induced by a variety of signaling molecules or stressful events, Ceramide-derived sphingosine 1-phosphate behaves as an antiapoptotic agent. The ganglioside GM1 is known to protect neuronal cell lines from apoptosis induced by serum/growth factor withdrawal and its effect is mediated in part by the direct activation of the trkA NGF receptor [G. Ferrari et al. (1995) J. Biol; Chem, 270, 3074-3080]. We show that GM1, similarly to sphingosine 1-phosphate, protects rat heart fibroblasts from apoptosis induced by the protein kinase C inhibitor staurosporine and by C2-ceramide. Furthermore, we show that GM1 induces the synthesis of sphingosine 1-phosphate and that this effect is partially prevented by the sphingosine kinase inhibitor N,N-dimethylsphingosine. We conclude that the antiapoptotic action of GM1 is largely to be ascribed to an increased sphingosine kinase activity

De novo autophagic vacuole formation in hepatocytes permeabilized by Staphylococcus aureus alpha-toxin. Inhibition by nonhydrolyzable GTP analogs.

The role of GTP-binding proteins in autophagic vacuole formation was investigated in isolated rat hepatocytes permeabilized by alpha-toxin from Staphylococcus aureus, an agent which creates stable plasma membrane channels allowing exchange of small (less-than-or-equal-to 1000 Da) molecules. Vacuole formation was monitored from the uptake of I-125-tyramine-cellobiitol ((ITC)-I-125) into osmotically sensitive vacuoles isolated on colloidal silica density gradients. Separation was based on an established observation that autophagic vacuoles are retained in a heavy midgradient band when samples are layered, but are selectively shifted to dense fractions when they are previously dispersed in the gradient material. The vacuolar uptake of (ITC)-I-125 was concentration-dependent and required exogenous ATP: 94% was directly mediated by sequestration; 6% was acquired by fluid-phase endocytosis as monitored by [carboxyl-C-14]dextran-carboxyl. Although the amino acid control of proteolysis was lost, addition of the nonhydrolyzable GTP analog GTPgammaS (as well as GMP-PNP) decreased fractional rates of direct vacuolar (ITC)-I-125 uptake and long-lived proteolysis by similar amounts (1.02-1.03% h-1), substantiating the notion that the effects were the direct result of autophagic inhibition. These and associated findings, supported by quantitative electron microscopy, indicate the presence of ongoing macro- and microautophagy in alpha-toxin-permeabilized cells and suggest that one or more GTP-binding proteins is required in macroautophagic vacuole formation

Autophagy.

Autophagy or autophagocytosis are terms given to a membrane-mediated process in eukaryotic cells in which portions of cytoplasm are sequestered within vacuoles and degraded by acid hydrolases that are acquired by fusion with lysosomes. Although vacuoles of this type may be formed under pathologic conditions, autophagy is fundamentally a physiologic process which plays indispensible roles in cell restructuring and in the ongoing turnover of cytoplasmic macromolecules. Sequestration is the one step in this pathway that separates autophagy from other degradative processes in the cell. As a volume uptake mechanism it is relatively nonselective (one exception is discussed insection 3.1.2.) and, accordingly, will sequester most organelles and macromolecules in proportion to their cytoplasmic abundance. Moreover, it permits the simultaneous handling of more than one class of macromolecule. This is illustrated in the perfused rat liver by the striking similarity in the accelerated responses of protein and RNA degradation to amino acid deprivation (see Table I). Although the appearance of the vacuoles varies widely among cells, it is, nevertheless, highly conserved and found in nearly all lower plants and animals as well as in higher species. In yeast, for example, a vacuole that expresses autophagic function (Takeshige et al., 1992; reviewed by Jones and Murdock, 1994) plays a major role in the supply of endogenous amino acids. A similar role for the vacuole is found in germinating seeds (Nishimura and Beevers, 1979) and in the turnover of intracellular proteins in protoplasts of cultured plant cells (Canut et al., 1985). An interesting variant of autophagy is utilized in cell remodeling where irreversible alterations are involved (Marty, 1978; Paavola, 1978 a,b), and an enzymatically unique type degrades intracellular membranes in the amoeba Tetrahymena pyriformis to provide lipid substrate for gluconeogenesis by the glyoxalate pathway (May et al., 1982). Finally, in the mammalian heptocyte, where both protein turnover and the need for endogenous amino acids are large, autophagy is highly expressed and closely regulated by complex amino acid feedback and hormonal mechanisms (reviewed by Mortimore and P\uf6s\uf6, 1987). Taken together, these findings attest to a fundamental role of autophagy in cellular homeostasis. In this chapter the authors will discuss the main features of general intracellular protein and RNA degradation and the major classes of autophagy and present a current overview of autophagic regulation and its mechanism, focusing primarily on the mammalian hepatocyte which has been extensively studied as a model for the pathway

Calpain activation and death of isolated cardiomyocytes exposed to intracellular calcium overload

The causal relationships linking cell death with calpain activation are still elusive. To this aim calpain activation was investigated in Hl-5 cardiomyocyte added with 1 \u3bcM A23187, a calcium ionophore. To obtain a persistent intracellular Ca2+ overload, as detected by fluo-3 fluorescence, NaCl of the incubation buffer was replaced with KCl and 1 mM vanadate was added to inhibit Ca2+ ATPases. Fluo3 fluorescence dropped immediately to background levels when EGTA was added. In this way it was possible to expose HL-5 cells to various durations of calcium overload. Calpain activation was investigated by means of immunoblot analyses of desmin degradation and fluorescence increases reflecting the hydrolysis of the synthetic peptide Suc-LLVY-AMC, a calpain substrate. Cell death was assessed as lactic dehydrogenase (LDH) release. Calpain activation became detectable after 20 min of calcium overload and was followed by the increase in LDH release, which approached to plateau after 40 min. The addition of EGTA after 30 min was no longer able to block the progression in cell death and calpain activation. More importantly, calpain inhibition by 10 \u3bcM PD150606 or 100 \u3bcM calpeptin reduced significantly LDH release although at a lesser extent than calpain mediated proteolysis. In conclusion, the present findings suggest that (i) calpain activation precedes the onset of cell death; (ii) intracellular calcium overload hampers cell viability in a process that eventually becomes independent of Ca2+; (iii) calpain activation is causally related to cell death, although the severity of the present protocol limits the protective efficacy of calpain inhibition