Cell Cycle Restart Research Articles

A hydrophobic low-complexity region regulates aggregation of the yeast pyruvate kinase Cdc19 into amyloid-like aggregates in vitro

Cells form stress granules (SGs) upon stress stimuli to protect sensitive proteins and RNA from degradation. In the yeast Saccharomyces cerevisiae, specific stresses such as nutrient starvation and heat-shock trigger recruitment of the yeast pyruvate kinase Cdc19 into SGs. This RNA-binding protein was shown to form amyloid-like aggregates that are physiologically reversible and essential for cell cycle restart after stress. Cellular Cdc19 exists in an equilibrium between a homotetramer and monomer state. Here, we show that Cdc19 aggregation in vitro is governed by protein quaternary structure, and we investigate the physical-chemical basis of Cdc19's assembly properties. Equilibrium shift toward the monomer state exposes a hydrophobic low-complexity region (LCR), which is prone to induce intermolecular interactions with surrounding proteins. We further demonstrate that hydrophobic/hydrophilic interfaces can trigger Cdc19 aggregation in vitro Moreover, we performed in vitro biophysical analyses to compare Cdc19 aggregates with fibrils produced by two known dysfunctional amyloidogenic peptides. We show that the Cdc19 aggregates share several structural features with pathological amyloids formed by human insulin and the Alzheimer's disease-associated Aβ42 peptide, particularly secondary β-sheet structure, thermodynamic stability, and staining by the thioflavin T dye. However, Cdc19 aggregates could not seed aggregation. These results indicate that Cdc19 adopts an amyloid-like structure in vitro that is regulated by the exposure of a hydrophobic LCR in its monomeric form. Together, our results highlight striking structural similarities between functional and dysfunctional amyloids and reveal the crucial role of hydrophobic/hydrophilic interfaces in regulating Cdc19 aggregation.

Reversible protein aggregation is a protective mechanism to ensure cell cycle restart after stress.

Protein aggregation is mostly viewed as deleterious and irreversible causing several pathologies. However, reversible protein aggregation has recently emerged as a novel concept for cellular regulation. Here, we characterize stress-induced, reversible aggregation of yeast pyruvate kinase, Cdc19. Aggregation of Cdc19 is regulated by oligomerization and binding to allosteric regulators. We identify a region of low compositional complexity (LCR) within Cdc19 as necessary and sufficient for reversible aggregation. During exponential growth, shielding the LCR within tetrameric Cdc19 or phosphorylation of the LCR prevents unscheduled aggregation, while its dephosphorylation is necessary for reversible aggregation during stress. Cdc19 aggregation triggers its localization to stress granules and modulates their formation and dissolution. Reversible aggregation protects Cdc19 from stress-induced degradation, thereby allowing cell cycle restart after stress. Several other enzymes necessary for G1 progression also contain LCRs and aggregate reversibly during stress, implying that reversible aggregation represents a conserved mechanism regulating cell growth and survival.

ATM/Wip1 activities at chromatin control Plk1 re-activation to determine G2 checkpoint duration.

After DNA damage, the cell cycle is arrested to avoid propagation of mutations. Arrest in G2 phase is initiated by ATM-/ATR-dependent signaling that inhibits mitosis-promoting kinases such as Plk1. At the same time, Plk1 can counteract ATR-dependent signaling and is required for eventual resumption of the cell cycle. However, what determines when Plk1 activity can resume remains unclear. Here, we use FRET-based reporters to show that a global spread of ATM activity on chromatin and phosphorylation of ATM targets including KAP1 control Plk1 re-activation. These phosphorylations are rapidly counteracted by the chromatin-bound phosphatase Wip1, allowing cell cycle restart despite persistent ATM activity present at DNA lesions. Combining experimental data and mathematical modeling, we propose a model for how the minimal duration of cell cycle arrest is controlled. Our model shows how cell cycle restart can occur before completion of DNA repair and suggests a mechanism for checkpoint adaptation in human cells.

Triolein from Coix Lacryma-Jobi Induces Cell Cycle Arrest through P53/P21Signaling Pathway

p53, a tumor suppressor protein, has important roles in DNA repair, cell cycle and apoptosis, is a one of the key events in cancer development. Coix lacryma-jobi seed has been used as a food and traditional medicine plant with anti-oxidant, anti-cancer and anti-diabetic effects. In currently research, we identified the most potent p53-increasing compound among 4 compounds (1-4) found in Coix lacryma-jobi and demonstrated its molecular mechanism in MCF-7 cells. Among the four isolated compounds (1-4), triolein most increased p53. Triolein treatment induced p53, p21, p27 and Bax in MCF-7 cells. Moreover, triolein caused S phase arrest through suppression of CDK1, phopho-Rb and E2F1 in dose-dependent manner. We also observed the decreasing of DNA synthesis by triolein. These data suggest that triolein may induced cell cycle restart involve DNA synthesis and apoptosis pathway in MCF-7 cells. (Less)

Abstract 946: SGN-CD70A, a pyrrolobenzodiazepine (PBD) dimer linked ADC, mediates DNA damage pathway activation and G2 cell cycle arrest leading to cell death

Abstract Seattle Genetics is utilizing the highly potent DNA cross-linking molecule, pyrrolobenzodiazepine (PBD) dimer, as the payload across multiple antibody drug conjugate (ADC) platforms. Previous studies have demonstrated that PBD dimers bind to the minor groove of DNA causing an interstrand crosslink; however, it is poorly understood how the cells recognize and respond to this insult. The DNA damage pathway exists to ensure genomic fidelity by either repairing damaged DNA or forcing programmed cell death if the damage is too extensive. Cells do this in an orchestrated manner that involves sensing the damage and eliciting a signaling cascade to induce repair and cell cycle arrest and ultimately either cell cycle re-start, death, or senescence. Using SGN-CD70A, an ADC consisting of a CD70-directed engineered cysteine antibody linked to two PBD dimers, we have examined the DNA damage response and cell death kinetics in a panel of renal cell carcinoma (RCC) and non-Hodgkin lymphoma (NHL) cell lines after treatment with free PBD and SGN-CD70A. We examined the central DNA damage sensing protein kinases, ATM, ATR, and DNA-PK and find that all three are phosphorylated on activation residues within 24 hours at EC50 doses in both RCC and NHL cell lines. Similarly, the downstream checkpoint kinases, Chk1 and Chk2 are also activated, within 24 hours after treatment with SGN-CD70A. The kinetics of pChk2 activation mirror Caspase 3/7 activation, with both peaking 48-72 hours after treatment. To confirm the involvement of the DNA damage pathway in the PBD mechanism, we used small molecules to inhibit ATM (KU-60019), ATR (VE-821), DNA-PK (NU-7441), and Chk1/2 (AZD7762, LY2603618) and found that all were synergistic with SGN-CD70A and accelerated cell death kinetics. We also conducted a small molecule screen to identify other processes involved in SGN-CD70A's activity. The Mps1 inhibitor (AZ 3146), which shortens mitosis and abrogates the mitotic checkpoint, was strongly synergistic. Conversely, molecules that cause mitotic arrest, such as microtubule inhibitors and Plk1 inhibitors did not show the same degree of synergy. To follow up on this finding we looked at cell cycle kinetics. We found that SGN-CD70A treatment alone causes a G2 arrest and a modest increase in polyploid cells whereas synergistic combinations with SGN-CD70A and DNA damage kinase and Mps1 inhibitors decrease the number of cells in G2 while markedly increasing polyploid cells. These data suggest induction of mitotic catastrophe may be part of the cellular response to SGN-CD70A treatment. Based on these results, we are currently testing whether sequencing SGN-CD70A and various mitotic inhibitors can increase synergy. Citation Format: Sharsti L. Sandall, Renee McCormick, Jamie Miyamoto, Travis Biechele, Che-Leung Law, Timothy S. Lewis. SGN-CD70A, a pyrrolobenzodiazepine (PBD) dimer linked ADC, mediates DNA damage pathway activation and G2 cell cycle arrest leading to cell death. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 946. doi:10.1158/1538-7445.AM2015-946

Effect of insulin on the cell cycle of germinating maize seeds (Zea maysL.)

AbstractDuring seed germination, metabolism is reactivated, DNA is repaired and cell division is restarted in the meristems. The mechanisms that co-ordinate cell growth and division in maize embryonic axes during germination are not well understood. However, the presence of a factor similar to IGF (insulin-like growth factor) that accelerates germination has been reported. In the present work, the regulation of the cell-cycle restart by bovine insulin [which has been demonstrated to produce similar effects as insulin-like growth factor of maize (ZmIGF) in maize seeds] was studied in germinating embryonic axes. Our results showed that bovine insulin differentially stimulates growth, S6K phosphorylation,S6rptranscript accumulation on the polysomal fraction, as well asde novoDNA synthesis in the radicles and the coleoptiles of the embryonic axis. A stronger and earlier effect was observed in radicles compared to coleoptiles; therefore, the effect of insulin on the cell cycle of the root meristem was studied by flow cytometry. The G1–S transition was stimulated and cell proliferation was induced. Furthermore, it was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) that bovine insulin increased E2F and PCNA (proliferating cell nuclear antigen) transcription after 15 h of germination and PCNAde novosynthesis at 15 h of germination. These results show that bovine insulin preferentially stimulates growth in the radicles of germinating embryonic axes and suggest that its effect on the G1–S transition and the activation of cell proliferation is mediated by the induction of E2F and PCNA transcription.

Loss of H3 K79 Trimethylation Leads to Suppression of Rtt107-dependent DNA Damage Sensitivity through the Translesion Synthesis Pathway

Genomic integrity is maintained by the coordinated interaction of many DNA damage response pathways, including checkpoints, DNA repair processes, and cell cycle restart. In Saccharomyces cerevisiae, the BRCA1 C-terminal domain-containing protein Rtt107/Esc4 is required for restart of DNA replication after successful repair of DNA damage and for cellular resistance to DNA-damaging agents. Rtt107 and its interaction partner Slx4 are phosphorylated during the initial phase of DNA damage response by the checkpoint kinases Mec1 and Tel1. Because the natural chromatin template plays an important role during the DNA damage response, we tested whether chromatin modifications affected the requirement for Rtt107 and Slx4 during DNA damage repair. Here, we report that the sensitivity to DNA-damaging agents of rtt107Δ and slx4Δ mutants was rescued by inactivation of the chromatin regulatory pathway leading to H3 K79 trimethylation. Further analysis revealed that lack of Dot1, the H3 K79 methyltransferase, led to activation of the translesion synthesis pathway, thereby allowing the survival in the presence of DNA damage. The DNA damage-induced phosphorylation of Rtt107 and Slx4, which was mutually dependent, was not restored in the absence of Dot1. The antagonistic relationship between Rtt107 and Dot1 was specific for DNA damage-induced phenotypes, whereas the genomic instability caused by loss of Rtt107 was not rescued. These data revealed a multifaceted functional relationship between Rtt107 and Dot1 in the DNA damage response and maintenance of genome integrity.

Essential Role of Nuclear Localization for Yeast Ulp2 SUMO Protease Function

The SUMO protein is covalently attached to many different substrates throughout the cell. This modification is rapidly reversed by SUMO proteases. The Saccharomyces cerevisiae SUMO protease Ulp2 is a nuclear protein required for chromosome stability and cell cycle restart after checkpoint arrest. Ulp2 is related to the human SENP6 protease, also a nuclear protein. All members of the Ulp2/SENP6 family of SUMO proteases have large but poorly conserved N-terminal domains (NTDs) adjacent to the catalytic domain. Ulp2 also has a long C-terminal domain (CTD). We show that CTD deletion has modest effects on yeast growth, but poly-SUMO conjugates accumulate. In contrast, the NTD is essential for Ulp2 function and is required for nuclear targeting. Two short, widely separated sequences within the NTD confer nuclear localization. Efficient Ulp2 import into the nucleus requires the beta-importin Kap95, which functions on classical nuclear-localization signal (NLS)-bearing substrates. Remarkably, replacement of the entire >400-residue NTD by a heterologous NLS results in near-normal Ulp2 function. These data demonstrate that nuclear localization of Ulp2 is crucial in vivo, yet only small segments of the NTD provide the key functional elements, explaining the minimal sequence conservation of the NTDs in the Ulp2/SENP6 family of enzymes.