Acetylation of TIP60 at K104 is essential for metabolic stress-induced apoptosis in cells of hepatocellular cancer.

Tumor cells often encounter hypoglycemic microenvironment due to rapid cell expansion. It remains elusive how tumors reprogram the genome to survive the metabolic stress. The tumor suppressor TIP60 functions as the catalytic subunit of the human NuA4 histone acetyltransferase (HAT) multi-subunit complex and is involved in many different cellular processes including DNA damage response, cell growth and apoptosis. Attenuation of TIP60 expression has been detected in various tumor types. The function of TIP60 in tumor development has not been fully understood. Here we found that suppressing TIP60 inhibited p53 K120 acetylation and thus rescued apoptosis induced by glucose deprivation in hepatocellular cancer cells. Excitingly, Lys-104 (K104), a previously identified lysine acetylation site of TIP60 with unknown function, was observed to be indispensable for inducing p53-mediated apoptosis under low glucose condition. Mutation of Lys-104 to Arg (K104R) impeded the binding of TIP60 to human NuA4 complex, suppressed the acetyltransferase activity of TIP60, and inhibited the expression of pro-apoptotic genes including NOXA and PUMA upon glucose starvation. These findings demonstrate the critical regulation of TIP60/p53 pathway in apoptosis upon metabolic stress and provide a novel insight into the down-regulation of TIP60 in tumor cells.

The NEK1 interactor, C21ORF2, is required for efficient DNA damage repair.

Defective DNA damage response is a threat to genome stability and a proven cause of tumorigenesis. C21ORF2 (chromosome 21 open reading frame 2) is a novel gene on chromosome 21, and the C21ORF2 protein is found to interact with NEK1. Earlier studies showed that C21ORF2 might be associated with some human genetic diseases including Down syndrome. However, the cellular functions of C21ORF2 remain unknown. In the present study, we reported that C21ORF2 affected cell proliferation after DNA damage induced by ionizing radiation, and DNA repair was less efficient in C21ORF2-depleted cells compared with control cells. However, C21ORF2-knockdown cells did not show defects in the activation of the G2-phase DNA damage checkpoint. Furthermore, homologous recombination, but not non-homologous end joining repair, was found to be impaired after C21ORF2 attenuation, which could be rescued by the overexpression of NEK1, indicating that C21ORF2 functions in the same pathway as NEK1 in DNA damage repair.

PKA turnover by the REGγ-proteasome modulates FoxO1 cellular activity and VEGF-induced angiogenesis.

The REGγ-proteasome serves as a short-cut for the destruction of certain intact mammalian proteins in the absence of ubiquitin- and ATP. The biological roles of the proteasome activator REGγ are not completely understood. Here we demonstrate that REGγ controls degradation of protein kinase A catalytic subunit-α (PKAca) both in primary human umbilical vein endothelial cells (HUVECs) and mouse embryonic fibroblast cells (MEFs). Accumulation of PKAca in REGγ-deficient HUVECs or MEFs results in phosphorylation and nuclear exclusion of the transcription factor FoxO1, indicating that REGγ is involved in preserving FoxO1 transcriptional activity. Consequently, VEGF-induced expression of the FoxO1 responsive genes, VCAM-1 and E-Selectin, was tightly controlled by REGγ in a PKA dependent manner. Functionally, REGγ is crucial for the migration of HUVECs. REGγ(-/-) mice display compromised VEGF-instigated neovascularization in cornea and aortic ring models. Implanted matrigel plugs containing VEGF in REGγ(-/-) mice induced fewer capillaries than in REGγ(+/+) littermates. Taken together, our study identifies REGγ as a novel angiogenic factor that plays an important role in VEGF-induced expression of VCAM-1 and E-Selectin by antagonizing PKA signaling. Identification of the REGγ-PKA-FoxO1 pathway in endothelial cells (ECs) provides another potential target for therapeutic intervention in vascular diseases.

REGγ Contributes to Regulation of Hemoglobin and Hemoglobin δ Subunit.

Hemoglobin (Hb) is a family of proteins in red blood cells responsible for oxygen transport and vulnerable for oxidative damage. Hemoglobin δ subunit (HBD), a member of Hb family, is normally expressed by cells of erythroid lineage. Expression of Hb genes has been previously reported in nonerythroid and hematopoietic stem cells. Here, we report that Hb and HBD can be degraded via REGγ proteasome in hemopoietic tissues and nonerythroid cells. For this purpose, bone marrow, liver, and spleen hemopoietic tissues from REGγ+/+ and REGγ-/- mice and stable REGγ knockdown cells were evaluated for the degradation of Hb and HBD via REGγ. Western blot and immunohistochemical analyses exhibited downregulation of Hb in REGγ wild-type mouse tissues. This was validated by dynamic analysis following blockade of de novo synthesis of proteins with CHX. Degradation of HBD only occurred in REGγ WT cells but not in REGγN151Y, a dominant-negative REGγ mutant cell. Notably, downregulation of HBD was found in HeLa shN cells with stimulation of phenylhydrazine, an oxidation inducer, suggesting that the REGγ proteasome may target oxidatively damaged Hbs. In conclusion, our findings provide important implications for the degradation of Hb and HBD in hemopoietic tissues and nonerythroid cells via the REGγ proteasome.

Cdc14A and Cdc14B Redundantly Regulate DNA Double-Strand Break Repair.

Cdc14 is a phosphatase that controls mitotic exit and cytokinesis in budding yeast. In mammals, the two Cdc14 homologues, Cdc14A and Cdc14B, have been proposed to regulate DNA damage repair, whereas the mitotic exit and cytokinesis rely on another phosphatase, PP2A-B55α. It is unclear if the two Cdc14s work redundantly in DNA repair and which repair pathways they participate in. More importantly, their target(s) in DNA repair remains elusive. Here we report that Cdc14B knockout (Cdc14B(-/-)) mouse embryonic fibroblasts (MEFs) showed defects in repairing ionizing radiation (IR)-induced DNA double-strand breaks (DSBs), which occurred only at late passages when Cdc14A levels were low. This repair defect could occur at early passages if Cdc14A levels were also compromised. These results indicate redundancy between Cdc14B and Cdc14A in DSB repair. Further, we found that Cdc14B deficiency impaired both homologous recombination (HR) and nonhomologous end joining (NHEJ), the two major DSB repair pathways. We also provide evidence that Cdh1 is a downstream target of Cdc14B in DSB repair.

Oxidative challenge enhances REGγ-proteasome-dependent protein degradation.

Elimination of oxidized proteins is important to cells as accumulation of damaged proteins causes cellular dysfunction, disease, and aging. Abundant evidence shows that the 20S proteasome is largely responsible for degradation of oxidative proteins in both ubiquitin-dependent and ubiquitin-independent pathways. However, the role of the REGγ-proteasome in degrading oxidative proteins remains unclear. Here, we focus on two of the well-known REGγ-proteasome substrates, p21(Waf1/Cip1) and hepatitis C virus (HCV) core protein, to analyze the impact of oxidative stress on REGγ-proteasome functions. We demonstrate that REGγ-proteasome is essential for oxidative stress-induced rapid degradation of p21 and HCV proteins. Silencing REGγ abrogated this response in multiple cell lines. Furthermore, pretreatment with proteasome inhibitor MG132 completely blunted oxidant-induced p21 degradation, indicating a proteasome-dependent action. Cellular oxidation promoted REGγ-proteasome-dependent trypsin-like activity by enhancing the interaction between REGγ and 20S proteasome. Antioxidant could counteract oxidation-induced protein degradation, indicating that REGγ-proteasome activity may be regulated by redox state. This study provides further insights into the actions of a unique proteasome pathway in response to an oxidative stress environment, implying a novel molecular basis for REGγ-proteasome functions in antioxidation.