Autophagy is an intracellular degradation pathway whose levels are tightly controlled to secure cell homeostasis. Unc-51–like kinase 1 (ULK1) is a conserved serine–threonine kinase that plays a central role in the initiation of autophagy. Here, we report that upon autophagy progression, ULK1 protein levels are specifically down-regulated by the E3 ligase NEDD4L, which ubiquitylates ULK1 for degradation by the proteasome. However, whereas ULK1 protein is degraded, ULK1 mRNA is actively transcribed. Upon reactivation of mTOR-dependent protein synthesis, basal levels of ULK1 are promptly restored, but the activity of newly synthesized ULK1 is inhibited by mTOR. This prepares the cell for a new possible round of autophagy stimulation. Our results thus place NEDD4L and ULK1 in a key position to control oscillatory activation of autophagy during prolonged stress to keep the levels of this process under a safe and physiological threshold.

‘Tissue’ transglutaminase (tTG) selectively accumulates in cells undergoing apoptosis both in vivo and in vitro. Considering the central role played by mitochondria in apoptosis, we investigated the relationships existing amongst tTG expression, apoptosis and mitochondrial function. To this aim we studied the mechanisms of apoptosis in a neuronal cell line (SK‐N‐BE (2)) in which the tTG‐expression was driven by a constitutive promoter. Furthermore, a tet‐off inducible promoter was also used in 3T3 fibroblastic cells used as control. Both cell lines, when expressing tTG, appeared ‘sensitized’ to apoptosis. Strikingly, we found major differences in the morphological features of mitochondria among cell lines in the absence of apoptotic stimuli. In addition, these ultrastructural characteristics were associated with specific functional features: (i) constitutively hyperpolarized mitochondria and (ii) increased reactive oxygen intermediates production. Importantly, after mitochondrial‐mediated apoptosis by stauro‐ sporine, a rapid loss of mitochondrial membrane potential was found in tTG cells only. Taken together, these results seem to suggest that, via hyperpolarization, tTG might act as a ‘sensitizer’ towards apoptotic stimuli specifically targeted to mitochondria. These results could also be of pathogenetic relevance for those diseases that are characterized by increased tTG and apoptotic rate together with impaired mitochondrial function, e.g. in some neurodegenerative disease.

Treatment of the human promonocytic cell line U937 with all-trans-retinoic acid (RA) commits these cells to apoptosis, which can be triggered by simply increasing intracellular calcium levels by the ionophore A23187. RA treatment of U937 cells is characterized by a decrease in Bcl-2 and marked induction of “tissue” transglutaminase (tTG) gene expression. In this study, we show that the inhibition of tTG expression in U937 cells undergoing apoptosis prevents their death. In fact, U937 cell-derived clones transfected with the human tTG gene in the antisense orientation showed a pronounced decrease in apoptosis induced by several stimuli. These findings demonstrate that the Ca²⁺-dependent irreversible cross-linking of intracellular proteins catalyzed by tTG represents an important biochemical event in the gene-regulated cell death in monoblasts. In addition, our data indicate that the apoptotic program in promonocytic cells is strictly regulated by RA and that a key role is played by the free intracellular calcium concentration.

Cisplatin [ cis- diamminedichloroplatinum(II) ( cis -DDP)] is one of the most successful anticancer agents effective against a wide range of solid tumors. However, its use is restricted by side effects and/or by intrinsic or acquired drug resistance. Here, we probed the role of glutathione transferase (GST) P1-1, an antiapoptotic protein often overexpressed in drug-resistant tumors, as a cis -DDP–binding protein. Our results show that cis -DDP is not a substrate for the glutathione (GSH) transferase activity of GST P1-1. Instead, GST P1-1 sequesters and inactivates cisplatin with the aid of 2 solvent-accessible cysteines, resulting in protein subunits cross-linking, while maintaining its GSH-conjugation activity. Furthermore, it is well known that GST P1-1 binding to the c-Jun N-terminal kinase (JNK) inhibits JNK phosphorylation, which is required for downstream apoptosis signaling. Thus, in turn, GST P1-1 overexpression and Pt-induced subunit cross-linking could modulate JNK apoptotic signaling, further confirming the role of GST P1-1 as an antiapoptotic protein.