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Mixed ubiquitin chains regulate DNA repair
Rona, Gergely; Pagano, Michele
Diverse linkage in polyubiquitin chain structure gives cells an unparalleled complexity to virtually modulate all aspects of cell biology. Substrates can be covalently modified by ubiquitin chains of different topology. Proper DNA damage response takes advantage of this regulatory system and heavily relies on ubiquitin-based signaling. Moreover, increasing evidence suggests that chain specificity dictates DNA repair outcome. In this issue of Genes & Development, Wu and colleagues (pp. 1702-1717) show that Cezanne and Cezanne2, two paralogous deubiquitinating enzymes that are recruited to sites of DNA damage, ensure proper local polyubiquitin chain composition for downstream DNA repair protein assembly. Their study offers a key insight into the mechanism of crosstalk between linkage-specific ubiquitylation at DNA damage sites, while simultaneously raising important questions for future research.
PMID: 31792015
ISSN: 1549-5477
CID: 4218182
Nrf2 Activation Promotes Lung Cancer Metastasis by Inhibiting the Degradation of Bach1
Lignitto, Luca; LeBoeuf, Sarah E; Homer, Harrison; Jiang, Shaowen; Askenazi, Manor; Karakousi, Triantafyllia R; Pass, Harvey I; Bhutkar, Arjun J; Tsirigos, Aristotelis; Ueberheide, Beatrix; Sayin, Volkan I; Papagiannakopoulos, Thales; Pagano, Michele
Approximately 30% of human lung cancers acquire mutations in either Keap1 or Nfe2l2, resulting in the stabilization of Nrf2, the Nfe2l2 gene product, which controls oxidative homeostasis. Here, we show that heme triggers the degradation of Bach1, a pro-metastatic transcription factor, by promoting its interaction with the ubiquitin ligase Fbxo22. Nrf2 accumulation in lung cancers causes the stabilization of Bach1 by inducing Ho1, the enzyme catabolizing heme. In mouse models of lung cancers, loss of Keap1 or Fbxo22 induces metastasis in a Bach1-dependent manner. Pharmacological inhibition of Ho1 suppresses metastasis in a Fbxo22-dependent manner. Human metastatic lung cancer display high levels of Ho1 and Bach1. Bach1 transcriptional signature is associated with poor survival and metastasis in lung cancer patients. We propose that Nrf2 activates a metastatic program by inhibiting the heme- and Fbxo22-mediated degradation of Bach1, and that Ho1 inhibitors represent an effective therapeutic strategy to prevent lung cancer metastasis.
PMID: 31257023
ISSN: 1097-4172
CID: 3967782
GGTase3 is a newly identified geranylgeranyltransferase targeting a ubiquitin ligase
Kuchay, Shafi; Wang, Hui; Marzio, Antonio; Jain, Kunj; Homer, Harrison; Fehrenbacher, Nicole; Philips, Mark R; Zheng, Ning; Pagano, Michele
Protein prenylation is believed to be catalyzed by three heterodimeric enzymes: FTase, GGTase1 and GGTase2. Here we report the identification of a previously unknown human prenyltransferase complex consisting of an orphan prenyltransferase α-subunit, PTAR1, and the catalytic β-subunit of GGTase2, RabGGTB. This enzyme, which we named GGTase3, geranylgeranylates FBXL2 to allow its localization at cell membranes, where this ubiquitin ligase mediates the polyubiquitylation of membrane-anchored proteins. In cells, FBXL2 is specifically recognized by GGTase3 despite having a typical carboxy-terminal CaaX prenylation motif that is predicted to be recognized by GGTase1. Our crystal structure analysis of the full-length GGTase3-FBXL2-SKP1 complex reveals an extensive multivalent interface specifically formed between the leucine-rich repeat domain of FBXL2 and PTAR1, which unmasks the structural basis of the substrate-enzyme specificity. By uncovering a missing prenyltransferase and its unique mode of substrate recognition, our findings call for a revision of the 'prenylation code'.
PMID: 31209342
ISSN: 1545-9985
CID: 3939022
Cyclin F Controls Cell-Cycle Transcriptional Outputs by Directing the Degradation of the Three Activator E2Fs
Clijsters, Linda; Hoencamp, Claire; Calis, Jorg J A; Marzio, Antonio; Handgraaf, Shanna M; Cuitino, Maria C; Rosenberg, Brad R; Leone, Gustavo; Pagano, Michele
E2F1, E2F2, and E2F3A, the three activators of the E2F family of transcription factors, are key regulators of the G1/S transition, promoting transcription of hundreds of genes critical for cell-cycle progression. We found that during late S and in G2, the degradation of all three activator E2Fs is controlled by cyclin F, the substrate receptor of 1 of 69 human SCF ubiquitin ligase complexes. E2F1, E2F2, and E2F3A interact with the cyclin box of cyclin F via their conserved N-terminal cyclin binding motifs. In the short term, E2F mutants unable to bind cyclin F remain stable throughout the cell cycle, induce unscheduled transcription in G2 and mitosis, and promote faster entry into the next S phase. However, in the long term, they impair cell fitness. We propose that by restricting E2F activity to the S phase, cyclin F controls one of the main and most critical transcriptional engines of the cell cycle.
PMCID:6588466
PMID: 31130363
ISSN: 1097-4164
CID: 3941422
Two Distinct E2F Transcriptional Modules Drive Cell Cycles and Differentiation
Cuitiño, Maria C; Pécot, Thierry; Sun, Daokun; Kladney, Raleigh; Okano-Uchida, Takayuki; Shinde, Neelam; Saeed, Resham; Perez-Castro, Antonio J; Webb, Amy; Liu, Tom; Bae, Soo In; Clijsters, Linda; Selner, Nicholas; Coppola, Vincenzo; Timmers, Cynthia; Ostrowski, Michael C; Pagano, Michele; Leone, Gustavo
Orchestrating cell-cycle-dependent mRNA oscillations is critical to cell proliferation in multicellular organisms. Even though our understanding of cell-cycle-regulated transcription has improved significantly over the last three decades, the mechanisms remain untested in vivo. Unbiased transcriptomic profiling of G0, G1-S, and S-G2-M sorted cells from FUCCI mouse embryos suggested a central role for E2Fs in the control of cell-cycle-dependent gene expression. The analysis of gene expression and E2F-tagged knockin mice with tissue imaging and deep-learning tools suggested that post-transcriptional mechanisms universally coordinate the nuclear accumulation of E2F activators (E2F3A) and canonical (E2F4) and atypical (E2F8) repressors during the cell cycle in vivo. In summary, we mapped the spatiotemporal expression of sentinel E2F activators and canonical and atypical repressors at the single-cell level in vivo and propose that two distinct E2F modules relay the control of gene expression in cells actively cycling (E2F3A-8-4) and exiting the cycle (E2F3A-4) during mammalian development.
PMID: 31130414
ISSN: 2211-1247
CID: 4000142
Cryptochromes-Mediated Inhibition of the CRL4Cop1-Complex Assembly Defines an Evolutionary Conserved Signaling Mechanism
Rizzini, Luca; Levine, Daniel C; Perelis, Mark; Bass, Joseph; Peek, Clara B; Pagano, Michele
In plants, cryptochromes are photoreceptors that negatively regulate the ubiquitin ligase CRL4Cop1. In mammals, cryptochromes are core components of the circadian clock and repressors of the glucocorticoid receptor (GR). Moreover, mammalian cryptochromes lost their ability to interact with Cop1, suggesting that they are unable to inhibit CRL4Cop1. Contrary to this assumption, we found that mammalian cryptochromes are also negative regulators of CRL4Cop1, and through this mechanism, they repress the GR transcriptional network both in cultured cells and in the mouse liver. Mechanistically, cryptochromes inactivate Cop1 by interacting with Det1, a subunit of the mammalian CRL4Cop1 complex that is not present in other CRL4s. Through this interaction, the ability of Cop1 to join the CRL4 complex is inhibited; therefore, its substrates accumulate. Thus, the interaction between cryptochromes and Det1 in mammals mirrors the interaction between cryptochromes and Cop1 in planta, pointing to a common ancestor in which the cryptochromes-Cop1 axis originated.
PMID: 31155351
ISSN: 1879-0445
CID: 3923312
The F-Box Domain-Dependent Activity of EMI1 Regulates PARPi Sensitivity in Triple-Negative Breast Cancers
Marzio, Antonio; Puccini, Joseph; Kwon, Youngho; Maverakis, Natalia K; Arbini, Arnaldo; Sung, Patrick; Bar-Sagi, Dafna; Pagano, Michele
The BRCA1-BRCA2-RAD51 axis is essential for homologous recombination repair (HRR) and is frequently disrupted in breast cancers. PARP inhibitors (PARPis) are used clinically to treat BRCA-mutated breast tumors. Using a genetic screen, we identified EMI1 as a modulator of PARPi sensitivity in triple-negative breast cancer (TNBC) cells. This function requires the F-box domain of EMI1, through which EMI1 assembles a canonical SCF ubiquitin ligase complex that constitutively targets RAD51 for degradation. In response to genotoxic stress, CHK1-mediated phosphorylation of RAD51 counteracts EMI1-dependent degradation by enhancing RAD51's affinity for BRCA2, leading to RAD51 accumulation. Inhibition of RAD51 degradation restores HRR in BRCA1-depleted cells. Human breast cancer samples display an inverse correlation between EMI1 and RAD51 protein levels. A subset of BRCA1-deficient TNBC cells develop resistance to PARPi by downregulating EMI1 and restoring RAD51-dependent HRR. Notably, reconstitution of EMI1 expression reestablishes PARPi sensitivity both in cellular systems and in an orthotopic mouse model.
PMID: 30554948
ISSN: 1097-4164
CID: 3556882
The ULK1-FBXW5-SEC23B nexus controls autophagy
Jeong, Yeon-Tae; Simoneschi, Daniele; Keegan, Sarah; Melville, David; Adler, Natalia S; Saraf, Anita; Florens, Laurence; Washburn, Michael P; Cavasotto, Claudio N; Fenyö, David; Cuervo, Ana Maria; Rossi, Mario; Pagano, Michele
In response to nutrient deprivation, the cell mobilizes an extensive amount of membrane to form and grow the autophagosome, allowing the progression of autophagy. By providing membranes and stimulating LC3 lipidation, COPII (Coat Protein Complex II) promotes autophagosome biogenesis. Here, we show that the F-box protein FBXW5 targets SEC23B, a component of COPII, for proteasomal degradation and that this event limits the autophagic flux in the presence of nutrients. In response to starvation, ULK1 phosphorylates SEC23B on Serine 186, preventing the interaction of SEC23B with FBXW5 and, therefore, inhibiting SEC23B degradation. Phosphorylated and stabilized SEC23B associates with SEC24A and SEC24B, but not SEC24C and SEC24D, and they re-localize to the ER-Golgi intermediate compartment, promoting autophagic flux. We propose that, in the presence of nutrients, FBXW5 limits COPII-mediated autophagosome biogenesis. Inhibition of this event by ULK1 ensures efficient execution of the autophagic cascade in response to nutrient starvation.
PMID: 30596474
ISSN: 2050-084x
CID: 3563252
NS5A Promotes Constitutive Degradation of IP3R3 to Counteract Apoptosis Induced by Hepatitis C Virus
Kuchay, Shafi; Saeed, Mohsan; Giorgi, Carlotta; Li, Jie; Hoffmann, Hans-Heinrich; Pinton, Paolo; Rice, Charles M; Pagano, Michele
FBXL2 targets IP3R3 for ubiquitin-mediated degradation to limit Ca2+ flux to mitochondria and, consequently, apoptosis. Efficient replication of hepatitis C virus (HCV) requires geranylgeranylation of FBXL2. Here, we show that the viral protein NS5A forms a trimeric complex with IP3R3 and FBXL2, unmasking IP3R3's degron in the absence of inositol 1,4,5-trisphosphate (IP3) stimulation. FBXL2 knockdown or expression of a stable IP3R3 mutant causes persistent Ca2+ flux and sensitizes cells to apoptosis, resulting in the inhibition of viral replication. Importantly, the effect of FBXL2 silencing is rescued by depleting IP3R3, but not p85β, another established FBXL2 substrate, indicating that the anti-HCV effect of FBXL2 knockdown is largely due to IP3R3 stabilization. Finally, disruption of the FBXL2-NS5A-IP3R3 complex using somatic cell genetics or pharmacologic inhibition results in IP3R3 stabilization and suppression of HCV replication. This study reveals an IP3-independent molecular mechanism through which HCV promotes IP3R3 degradation, thereby inhibiting virus-induced apoptosis and establishing chronic infection.
PMID: 30355490
ISSN: 2211-1247
CID: 3385972
β-TrCP- and Casein Kinase II-Mediated Degradation of Cyclin F Controls Timely Mitotic Progression
Mavrommati, Ioanna; Faedda, Roberta; Galasso, Giovanni; Li, Jie; Burdova, Kamila; Fischer, Roman; Kessler, Benedikt M; Carrero, Zunamys I; Guardavaccaro, Daniele; Pagano, Michele; D'Angiolella, Vincenzo
Orderly progressions of events in the cell division cycle are necessary to ensure the replication of DNA and cell division. Checkpoint systems allow the accurate execution of each cell-cycle phase. The precise regulation of the levels of cyclin proteins is fundamental to coordinate cell division with checkpoints, avoiding genome instability. Cyclin F has important functions in regulating the cell cycle during the G2 checkpoint; however, the mechanisms underlying the regulation of cyclin F are poorly understood. Here, we observe that cyclin F is regulated by proteolysis through β-TrCP. β-TrCP recognizes cyclin F through a non-canonical degron site (TSGXXS) after its phosphorylation by casein kinase II. The degradation of cyclin F mediated by β-TrCP occurs at the G2/M transition. This event is required to promote mitotic progression and favors the activation of a transcriptional program required for mitosis.
PMID: 30257202
ISSN: 2211-1247
CID: 3314352