SUMMARY Many cancer cells consume large quantities of glutamine to maintain TCA cycle anaplerosis and support cell survival. It was therefore surprising when RNAi screening revealed that suppression of citrate synthase (CS), the first TCA cycle enzyme, prevented glutamine-withdrawal-induced apoptosis. CS suppression reduced TCA cycle activity and diverted oxaloacetate, the substrate of CS, into production of the nonessential amino acids aspartate and asparagine. We found that asparagine was necessary and sufficient to suppress glutamine-withdrawal-induced apoptosis without restoring the levels of other nonessential amino acids or TCA cycle intermediates. In complete medium, tumor cells exhibiting high rates of glutamine consumption underwent rapid apoptosis when glutamine-dependent asparagine synthesis was suppressed and expression of asparagine synthetase was statistically correlated with poor prognosis in human tumors. Coupled with the success of L-asparaginase as a therapy for childhood leukemia, the data suggest that intracellular asparagine is a critical suppressor of apoptosis in many human tumors.

Multidomain proapoptotic BAX and BAK, once activated, permeabilize mitochondria to trigger apoptosis, whereas antiapoptotic BCL-2 members preserve mitochondrial integrity. The BH3-only molecules (BH3s) promote apoptosis by either activating BAX-BAK or inactivating antiapoptotic members. Here, we present biochemical and genetic evidence that NOXA is a bona fide activator BH3. Using combinatorial gain-of-function and loss-of-function approaches in Bid−/−Bim−/−Puma−/−Noxa−/− and Bax−/−Bak−/− cells, we have constructed an interconnected hierarchical model that accommodates and explains how the intricate interplays between the BCL-2 members dictate cellular survival versus death. BID, BIM, PUMA and NOXA directly induce stepwise, bimodal activation of BAX-BAK. BCL-2, BCL-XL and MCL-1 inhibit both modes of BAX-BAK activation by sequestering activator BH3s and “BH3-exposed” monomers of BAX-BAK, respectively. Furthermore, autoactivation of BAX and BAK can occur independently of activator BH3s through downregulation of BCL-2, BCL-XL and MCL-1. Our studies lay a foundation on targeting the BCL-2 family for treating diseases with dysregulated apoptosis.

Poly(ADP-ribose) polymerase 1 (PARP-1) and p53 are two key proteins in the DNA-damage response. Although PARP-1 is known to poly(ADP-ribosyl)ate p53, the role of this modification remains elusive. Here, we identify the major poly(ADP-ribosyl)ated sites of p53 by PARP-1 and find that PARP-1-mediated poly(ADP-ribosyl)ation blocks the interaction between p53 and the nuclear export receptor Crm1, resulting in nuclear accumulation of p53. These findings molecularly link PARP-1 and p53 in the DNA-damage response, providing the mechanism for how p53 accumulates in the nucleus in response to DNA damage. PARP-1 becomes super-activated by binding to damaged DNA, which in turn poly(ADP-ribosyl)ates p53. The nuclear export machinery is unable to target poly(ADP-ribosyl)ated p53, promoting accumulation of p53 in the nucleus where p53 exerts its transactivational function.

Poly(ADP-ribosyl)ation has been suggested to be involved in regulation of DNA repair, transcription, centrosome duplication, and chromosome stability. However, the regulation of degradation of poly(ADP-ribose) and its significance are not well understood. Here we report a loss-of-function mutant Drosophila with regard to poly(ADP-ribose) glycohydrolase, a major hydrolyzing enzyme of poly(ADP-ribose). The mutant lacks the conserved catalytic domain of poly(ADP-ribose) glycohydrolase, and exhibits lethality in the larval stages at the normal development temperature of 25°C. However, one-fourth of the mutants progress to the adult stage at 29°C but showed progressive neurodegeneration with reduced locomotor activity and a short lifespan. In association with this, extensive accumulation of poly(ADP-ribose) could be detected in the central nervous system. These results suggest that poly(ADPribose) metabolism is required for maintenance of the normal function of neuronal cells. The phenotypes observed in the parg mutant might be useful to understand neurodegenerative conditions such as the Alzheimer's and Parkinson's diseases that are caused by abnormal accumulation of substances in nervous tissue. P oly(ADP-ribosyl)ation process involves a posttranslational modification of target proteins catalyzed by the poly(ADPribose) polymerase (PARP) family of enzymes with NADϩ as the substrate, resulting in formation of long-branched polymers of ADP-ribose (1, 2). The covalently attached and negatively charged poly(ADP-ribose) units significantly affect several important biological functions, including DNA repair (3), transcription (4), regulation of telomere length, cell cycle progression, centrosome duplication (5, 6), and chromosome stability (7). One of the major members of the PARP family is PARP-1, which catalyzes poly(ADP-ribosyl)ation in response to DNA strand breaks. Recently, additional members of the PARP family of enzymes have been characterized; PARP-2, -3, tankyrase-1, -2, VPARP, and Ti-PARP (2). In Drosophila, only two PARP family members, corresponding to PARP-1 and tankyrase, have been reported (8, 9).Poly(ADP-ribose) attached to acceptor proteins is hydrolyzed rapidly to produce free ADP-ribose residues by poly(ADPribose) glycohydrolase (PARG) (10-12). In contrast to PARPs, only one gene for PARG has been detected in mammals and insects (13). Thus, it is likely that the regulation of PARG activity is required to complete protein modification cycles initiated by different PARPs. In fact, PARG has been proposed to shuttle between the nucleus and the cytoplasm and becomes localized to the centrosomes during mitosis (14). There is evidence for an alternative form in the cytoplasm (M. K. Jacobson, personal communication), indicating that regulation of poly(ADPribosyl)ation by PARG is very dynamic.Mutation of the parg gene, tej, in plants alters circadian rhythms (15), and increased sensitivity to DNA damage in parg knockout mouse embryonic stem cells has been reported (3). However, little is known about the effects...