In this issue of Genes & Development, Wei and colleagues (pp. 1204-1216) use elegant genetic approaches to simultaneously delete the essential autophagy gene FIP200 (FAK family-interacting protein of ...200 kDa) and the signaling adaptor p62/SQSTM1 within established murine tumors, which reveals an unexpected synergism between the autophagy pathway and p62 in driving tumor growth. Intriguingly, these observations suggest that the combined targeting of autophagy and p62 may serve as an effective approach to treat specific cancers.
There is great interest in understanding the cellular mechanisms controlling autophagy, a tightly regulated catabolic and stress-response pathway. Prior work has uncovered links between autophagy and ...the Golgi reassembly stacking protein of 55 kDa (GRASP55), but their precise interrelationship remains unclear. Intriguingly, both autophagy and GRASP55 have been functionally and spatially linked to the endoplasmic reticulum (ER)---Golgi interface, broaching this compartment as a site where GRASP55 and autophagy may intersect. Here, we uncover that loss of GRASP55 enhances LC3 puncta formation, indicating that GRASP55 restricts autophagosome formation. Additionally, using proximity-dependent biotinylation, we identify a GRASP55 proximal interactome highly associated with the ER-Golgi interface. Both nutrient starvation and loss of GRASP55 are associated with coalescence of early secretory pathway markers. In light of these findings, we propose that GRASP55 regulates spatial organization of the ER-Golgi interface, which suppresses early autophagosome formation.
Synthetic protein switches controlled with user-defined inputs are powerful tools for studying and controlling dynamic cellular processes. To date, these approaches have relied primarily on ...intermolecular regulation. Here we report a computationally guided framework for engineering intramolecular regulation of protein function. We utilize this framework to develop chemically inducible activator of RAS (CIAR), a single-component RAS rheostat that directly activates endogenous RAS in response to a small molecule. Using CIAR, we show that direct RAS activation elicits markedly different RAS-ERK signaling dynamics from growth factor stimulation, and that these dynamics differ among cell types. We also found that the clinically approved RAF inhibitor vemurafenib potently primes cells to respond to direct wild-type RAS activation. These results demonstrate the utility of CIAR for quantitatively interrogating RAS signaling. Finally, we demonstrate the general utility of our approach in design of intramolecularly regulated protein tools by applying it to the Rho family of guanine nucleotide exchange factors.
Autophagy promotes protein degradation, and therefore has been proposed to maintain amino acid pools to sustain protein synthesis during metabolic stress. To date, how autophagy influences the ...protein synthesis landscape in mammalian cells remains unclear. Here, we utilize ribosome profiling to delineate the effects of genetic ablation of the autophagy regulator, ATG12, on translational control. In mammalian cells, genetic loss of autophagy does not impact global rates of cap dependent translation, even under starvation conditions. Instead, autophagy supports the translation of a subset of mRNAs enriched for cell cycle control and DNA damage repair. In particular, we demonstrate that autophagy enables the translation of the DNA damage repair protein BRCA2, which is functionally required to attenuate DNA damage and promote cell survival in response to PARP inhibition. Overall, our findings illuminate that autophagy impacts protein translation and shapes the protein landscape.
Many viruses have evolved elegant strategies to co-opt cellular autophagic responses to facilitate viral propagation and evasion of immune surveillance. Kaposi's sarcoma-associated herpesvirus (KSHV) ...establishes a life-long persistent infection in its human host, and is etiologically linked to several cancers. KSHV gene products have been shown to modulate autophagy but their contribution to pathogenesis remains unclear. Our recent study demonstrated that KSHV subversion of autophagy promotes bypass of oncogene-induced senescence (OIS), an important host barrier to tumor initiation. These findings suggest that KSHV has evolved to subvert autophagy, at least in part, to establish an optimal niche for infection, concurrently dampening host antiviral defenses and allowing the ongoing proliferation of infected cells.
► Herpesviruses induce DNA damage responses (DDRs) that facilitate lytic replication. ► Oncogenic stress caused by latent infection with EBV or KSHV also activates DDRs. ► DDR signaling can trigger ...cellular senescence, permanently arresting the cell cycle. ► Auxiliary viral gene products allow ongoing proliferation of latently infected cells. ► Viral usurpation of DDR signaling and autophagy remodels secretory pathways.
A common feature of herpesvirus infection is activation of DNA damage responses (DDRs) that are essential for efficient lytic replication. Latent infection with Epstein–Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) also elicit DDRs via the action of latent viral oncoproteins that deregulate cell proliferation and initiate a host anti-proliferative defense known as oncogene-induced senescence (OIS). These viruses encode auxiliary latent proteins that undermine OIS to allow the ongoing proliferation of infected cells despite robust DDR signaling. Persistent DDRs have also been linked to the aberrant secretion of pathogenetically important inflammatory mediators from infected cells. The accumulating evidence indicates that herpesviruses have evolved ways to co-opt DDR signaling to manage both latent and lytic phases of infection, and that DDR subversion may contribute to herpesvirus-associated disease states.
Cellular stress and DNA damage up-regulate and activate p53, fundamental for cell cycle control, senescence, DNA repair and apoptosis. The specific mechanism(s) that determine whether p53-dependent ...cell cycle arrest or p53-dependent apoptosis prevails in response to specific DNA damage are poorly understood. In this study, we investigated two types of DNA damage, chromium treatment and gamma irradiation (IR) that induced similar levels of p53, but that mediated two distinct p53-dependent cell fates. Chromium exposure induced a robust DNA-dependent protein kinase (DNA-PK)-mediated apoptotic response that was accompanied by the rapid loss of the cyclin-dependent kinase inhibitor 1A (p21) protein, whereas IR treatment-induced cell cycle arrests that was supported by the rapid induction of p21. Inhibition of DNA-PK effectively blocked chromium-, but not IR-induced p53 stabilization and activation. In contrast, inhibition of ATM and ATR by caffeine had the inverse effect of blocking IR-, but not chromium-induced p53 stabilization and activation. Chromium exposure ablated
p21 transcription but
PUMA and
Bax transcription was significantly enhanced compared to non-damaged cells. In contrast, IR treatment triggered significant p21 mRNA synthesis in addition to PUMA and Bax mRNA production. While chromium treatment enhanced the binding of p53 and RNA polymerase II (RNA Pol II) to both the
p21 and
PUMA promoters, RNA Pol II elongation was only observed along the
PUMA gene and not the
p21 gene. In contrast, following IR treatment, RNA Pol II elongation was observed on both
p21 and
PUMA. Chromium-induced apoptosis therefore involves DNA-PK-mediated p53 activation followed by preferential transcription of pro-apoptotic
PUMA over anti-apoptotic
p21 genes.
The cyclin-dependent kinase inhibitor p21
CIP1/WAF1 is a key component in cell cycle control and apoptosis, directing an anti-apoptotic response following DNA damage. Chromium exposure resulted in a ...500–1000 fold increase in apoptosis-induced cell death in p21−/− HCT116 cells compared to wild-type or p53−/− cells. p53 shRNA (or transient p53 siRNA) into p21−/− HCT116 cells reduced Cr(VI) sensitivity, suggesting the enhanced apoptosis in p21−/− cells is p53-dependent. Under non-DNA damage conditions, the p53 level in p21−/− cells was significantly higher than in wild-type cells, due to enhanced p53 phosphorylation and stabilization rather than elevated p53 transcription. Wild-type cells showed significant p53 protein induction upon DNA damage whereas p21−/− cells showed no p53 increase. p21−/− cells display the constitutive activation of upstream p53 kinases (ATM, DNA-PK, ATR, AKT and p38). 2D gel analysis revealed p53 patterns in p21−/− cells were distinct from those in wild-type cells
before and after chromium exposure. Our results suggest that p21 has an important role in the cellular response to normal replicative stress and its absence leads to a “chronic DNA damage” state that primes the cell for p53-dependent apoptosis.
PDF
