Programmed cell death 5 mediates HDAC3 decay to promote genotoxic stress response

The inhibition of p53 activity by histone deacetylase 3 (HDAC3) has been reported, but the precise molecular mechanism is unknown. Here we show that programmed cell death 5 (PDCD5) selectively mediates HDAC3 dissociation from p53, which induces HDAC3 cleavage and ubiquitin-dependent proteasomal degradation. Casein kinase 2 alpha phosphorylates PDCD5 at Ser-119 to enhance its stability and importin 13-mediated nuclear translocation of PDCD5. Genetic deletion of PDCD5 abrogates etoposide (ET)-induced p53 stabilization and HDAC3 cleavage, indicating an essential role of PDCD5 in p53 activation. Restoration of PDCD5(WT) in PDCD5(-/-) MEFs restores ET-induced HDAC3 cleavage. Reduction of both PDCD5 and p53, but not reduction of either protein alone, significantly enhances in vivo tumorigenicity of AGS gastric cancer cells and correlates with poor prognosis in gastric cancer patients. Our results define a mechanism for p53 activation via PDCD5-dependent HDAC3 decay under genotoxic stress conditions.ope

CK2-NCoR signaling cascade promotes prostate tumorigenesis

The aberrant expressions of casein kinase 2 (CK2) was found in prostate cancer patient and cell lines, but little is known of the detailed mechanisms implicated in prostate tumorigenesis. In this study, we report that both CK2 activity and CK2-mediated NCoR phosphorylation are significantly elevated in the androgen-independent prostate cancer cell line DU145 and PC-3 compared with RWPE1 and LNCaP cells. Increased phosphorylation inversely correlates with the mRNA level of the NCoR-regulated gene, interferon-γ-inducible protein 10 (IP-10). CK2 inhibition abrogated NCoR phosphorylation, IP-10 transcriptional repression, and the invasion activity of PC-3 cells. Inhibition of the CK2-NCoR network significantly reduced in vivo PC-3 cell tumorigenicity, likely due to transcriptional derepression of IP-10. Clinicopathological analyses revealed that increased CK2-mediated NCoR phosphorylation significantly correlates with poor survival among prostate cancer patients. These findings elucidate a CK2-modulated oncogenic cascade in prostate tumorigenesis.ope

핵호르몬 수용체 매개 전사억제 조절시 Lis-H/WD-40 domain 함유 단백질들의 기능 규명

의과학과/박사[한글]Nuclear receptor coreprssor (N-CoR)/ silencing mediator for retinoic and thyroid receptors (SMRT)를 주축으로 하는 보조전사억제인자 복합체는 histone deacetylase 3 (HDAC3), transducin beta-like protein 1 (TBL1), transducin beta-likerelated protein 1 (TBLR1), 그리고 G-protein suppressor 2 (GPS2)를 포함한다. 이 중 TBL1과 TBLR1은 N-말단에 Lishomology (Lis-H) domain을, 그리고 C-말단에 WD-40 부위를 가지고 있으며, 이들은 각각 단백질의 이합체 형성과 결합에 중요한 역할을 한다고 보고되어 있다. 현재 N-CoR/SMRT 매개의 표적 유전자 전사억제는 feed-forward working model로 설명되고 있으나 30, 이 model 내에서 TBL1과 TBLR1의 정확한 기능은 아직 밝혀진 바 없다. 본 연구는 TBL1과 TBLR1이 N-말단의 Lis-H domain을 통해 동형 그리고 이형 중합체를 형성함을 밝히고, 이러한 중합체 형성이 N-CoR/SMRT 매개 표적 유전자 전사억제에 어떠한 영향을 미치는가를 관찰함으로써 feed-forward working model에서 TBL1과 TBLR1의 정확한 기능을 규명하는 것을 목적으로 한다. TBL1과 TBLR1은 Lis-H domain의 중합체 형성을 통하여 저아세틸화된 histone H4의 말단에 결합하며, N-CoR/SMRT 복합체 형성을 안정화 시키고, 이들의 크로마틴 표적을 안정화 시킴으로 표적 유전자의 전사억제에 직집적으로 관여한다. 더 나아가 TBL1과 TBLR1의 Lis-H domain내에서 유전적으로 보존된 아미노산의 변형은 TBL1/TBLR1 단백질간의 중합체 형성, N-CoR/SMRT 복합체의 안정화, histone과의 결합, 및 N-CoR/SMRT 복합체의 크리마틴 표적을 불가능하게 한다. 이러한 결과는 N-CoR/SMRT 보조전사억제 복합체 매개의 표적 유전자 전사억제에 있어 TBL1과 TBLR1의 Lis-H domain의 기능적 중요성을 뒷받침해주는 증거가 된다. 또한, C-말단에 WD-40 부위를 가지고 있는 또 다른 단백질인 TBL3가 TBL1과 TBLR1의 N-말단에 결합함을 밝힘으로써, TBL1/TBLR1의 중합체 형성에 WD-40 부위를 지니는 또 다른 단백질이 관여하고 있을 가능성을 제시하고 있다. 이와 반대로 histone deacetylase 1 (HDAC1)이 관여하는 sin3A 보조전사억제인자 복합체의 또 다른 WD-40 부위 함유 단백질인 Rb associated-protein 46 (RbAp46)과 RbAp48은 N-말단을 통한 이형체 및 중합체 형성을 하지 않음을 관찰하였다. 이와 같은 결과는 TBL1/TBLR1 뿐만 아니라, Lis-H domain과 WD-40 부위를 지니는 다양한 단백질간의 중합체 형성이 NCoR/SMRT 보조전사억제인자 복합체에 의한 표적유전자 전사억제에 결정적인 역할을 하며, 이러한 현상은 다른 class 1 histone deacetylase를 함유하는 보조전사억제인자 복합체와는 차별화되는 N-CoR/SMRT 복합체의 특이적인 현상임을 의미한다. [영문]Lis-homology (Lis-H) motifs are involved in protein dimerization, and the discovery of the conserved N-terminal Lis-H domain in transducin beta-like protien 1 and its receptor (TBL1 and TBLR1) led to examine the role of this domain in transcriptional repression. This study shows that multiple beta-transducin (WD-40) repeat containing proteins interact to form a oligomers in solution and that oligomerization depends on the presence of the Lis-H domain in each protein. Gal4-Tk-luciferase reporter gene analysis suggests that oligomerization is a prerequisite for efficient transcriptional repression. Furthermore, this study proves that the Lis-H domain is responsible for the binding to the hypoacetylated histone H4 tail and for stable chromatin targeting by the nuclear receptor corepressor (N-CoR) complex. Mutations of conserved residues in the Lis-H domain of TBL1 and TBLR1 block histone binding, oligomerization, and transcriptional repression, supporting the functional importance of the Lis-H domain in transcriptional repression. These results indicate that another WD-40 protein, TBL3, also preferentially binds to the N-terminal domain of TBL1 and TBLR1, and forms oligomers with other WD-40 proteins. In contrast, the WD-40 proteins RbAp46 and RbAp48 of the sin3A corepressor complex failed to dimerize. Theses data support the hypothesis that the presence of multiple Lis-H/WD-40 repeat containing proteins is exclusive to N-CoR/SMRT complexes compared to other class 1 histone deacetylase-containing corepessor complexes.ope

The chalcone derivative Chana 1 protects against amyloid β peptide-induced oxidative stress and cognitive impairment.

Alzheimer's disease (AD) is the most common neurodegenerative disease to cause dementia in the elderly. Amyloid β (Aβ)-peptide induced oxidative stress causes the initiation and progression of AD. Recently, new chalcone derivatives termed the Chana series were synthesized. Among them, Chana 1 showed high free radical scavenging activity (72.5%), as measured by a DPPH (1,1-diphenyl-2-picrylhydrazyl) assay. In this study, we investigated the effect of Chana 1 against Aβ-induced cytotoxicity and cognitive deficits. Additionally, we sought to estimate the lethal dose, 50% (LD50) of Chana 1 in mice using an acute oral toxicity test. We found that Chana 1 significantly protected against Aβ-induced neuronal cell death in PC12 cells. Oral administration of Chana 1 at a dose of 50 mg/kg body weight/day significantly improved Aβ-induced learning and memory impairment in mice, as measured in Y-maze and passive avoidance tests. In acute toxicity tests, the LD50 in mice was determined to be 520.44 mg/kg body weight. The data are valuable for future studies and suggest that Chana 1 has therapeutic potential for the management of neurodegenerative disease.ope

EGCG suppresses prostate cancer cell growth modulating acetylation of androgen receptor by anti-histone acetyltransferase activity.

Manipulating acetylation status of key gene targets is likely to be crucial for effective cancer therapy. In this study, we utilized green tea catechins, epicatechin (EC), epigallocatechin (EGC) and epigallocatechin-3-gallate (EGCG) to examine the regulation of androgen receptor acetylation in androgen-dependent prostate cancer cells by histone acetyl-transferase (HAT) activity. EC, EGC and EGCG induced prostate cancer cell death, suppressed agonist-dependent androgen receptor (AR) activation and AR-regulated gene transcription. These results demonstrated a similar tendency to HAT inhibitory activities; EGCG>EGC>EC. The strongest HAT inhibitor among them, EGCG (50 µM), downregulated AR acetylation and finally, AR protein translocation to nucleus from the cytoplasmic compartment was effectively inhibited in the presence of the agonist. These results suggest another mechanism to develop effective therapeutics based on green tea catechins.ope

PKA negatively regulates PP2Cβ to activate NF-κB-mediated inflammatory signaling

Protein phosphatase 2Cβ (PP2Cβ) was found to act as a negative regulator of NF-κB-mediated inflammatory signaling; however, its regulatory mechanism has not been examined. Here, we show that protein kinase A (PKA) phosphorylates the PP2Cβ, which was inhibited by PKA-specific inhibitor, H89. Mutation analysis of serine residues in PP2Cβ revealed that Ser-195 in PP2Cβ is phosphorylated by PKA. Importantly, PKA inhibition by H89 abrogated the Forskolin-induced destabilization of PP2Cβ against ubiquitin-dependent proteosomal degradation pathway. Furthermore, H89 treatment efficiently reversed the negative effect of Forskolin on the anti-inflammatory function of PP2Cβ. Collectively, these data suggest that PKA destabilizes PP2Cβ upon inflammatory stimuli via phosphorylation of Ser-195 in PP2Cβ.ope

Deubiquitinase OTUD5 mediates the sequential activation of PDCD5 and p53 in response to genotoxic stress

Programmed cell death 5 (PDCD5) positively regulates p53-mediated apoptosis and rapidly accumulates upon DNA damage. However, the underlying mechanism of PDCD5 upregulation during the DNA damage response remains unknown. Here, we found that OTU deubiquitinase 5 (OTUD5) was bound to PDCD5 in response to etoposide treatment and increased the stability of PDCD5 by mediating deubiquitination of PDCD5 at Lys-97/98. Overexpression of OTUD5 efficiently enhanced the activation of both PDCD5 and p53. Conversely, PDCD5 knockdown greatly attenuated the effect of OTUD5 on p53 activation. In addition, when OTUD5 was depleted, PDCD5 failed to facilitate p53 activation, demonstrating an essential role for the PDCD5-OTUD5 network in p53 activation. Importantly, we found that OTUD5-dependent PDCD5 stabilization was required for sequential activation of p53 in response to genotoxic stress. The sequential activation of PDCD5 and p53 was abrogated by knockdown of OTUD5. Finally, impairment of the genotoxic stress response upon PDCD5 ablation was substantially rescued by reintroducing PDCD5(WT) but not PDCD5(E94D) (defective for OTUD5 interaction) or PDCD5(E16D) (defective for p53 interaction). Together, our findings have uncovered an apoptotic signaling cascade linking PDCD5, OTUD5, and p53 during genotoxic stress responses.ope

Protein Kinase A phosphorylates NCoR to enhance its nuclear translocation and repressive function in human prostate cancer cells

Protein kinase A (PKA) phosphorylates diverse protein substrates to modulate their function. In this study, we found that PKA specifically phosphorylates the RD1 (repression domain 1) domain of nuclear receptor corepressor (NCoR). We demonstrated that the Serine-70 of NCoR is identified the critical amino acid for PKA-dependent NCoR phosphorylation. Importantly, we found that PKA-dependent phosphorylation enhances the nuclear translocation of NCoR. More importantly, the activation of PKA enhanced the repressive activity of NCoR in a reporter assay and potentiated the antagonist activity in the androgen receptor (AR)-mediated transcription. Taken together, these results uncover a regulatory mechanism by which PKA positively modulates NCoR function in transcriptional regulation in prostate cancer.restrictio

PINK1 positively regulates HDAC3 to suppress dopaminergic neuronal cell death

Deciphering the molecular basis of neuronal cell death is a central issue in the etiology of neurodegenerative diseases, such as Parkinson's and Alzheimer's. Dysregulation of p53 levels has been implicated in neuronal apoptosis. The role of histone deacetylase 3 (HDAC3) in suppressing p53-dependent apoptosis has been recently emphasized; however, the molecular basis of modulation of p53 function by HDAC3 remains unclear. Here we show that PTEN-induced putative kinase 1 (PINK1), which is linked to autosomal recessive early-onset familial Parkinson's disease, phosphorylates HDAC3 at Ser-424 to enhance its HDAC activity in a neural cell-specific manner. PINK1 prevents H2O2-induced C-terminal cleavage of HDAC3 via phosphorylation of HDAC3 at Ser-424, which is reversed by protein phosphatase 4c. PINK1-mediated phosphorylation of HDAC3 enhances its direct association with p53 and causes subsequent hypoacetylation of p53. Genetic deletion of PINK1 partly impaired the suppressive role of HDAC3 in regulating p53 acetylation and transcriptional activity. However, depletion of HDAC3 fully abolished the PINK1-mediated p53 inhibitory loop. Finally, ectopic expression of phosphomometic-HDAC3(S424E) substantially overcomes the defective action of PINK1 against oxidative stress in dopaminergic neuronal cells. Together, our results uncovered a mechanism by which PINK1-HDAC3 network mediates p53 inhibitory loop in response to oxidative stress-induced damage.ope

YAF2 promotes TP53-mediated genotoxic stress response via stabilization of PDCD5

Programmed cell death 5 (PDCD5) plays a crucial role in TP53-mediated apoptosis, but the regulatory mechanism of PDCD5 itself during apoptosis remains obscure. We identified YY1-associated factor 2 (YAF2) as a novel PDCD5-interacting protein in a yeast two-hybrid screen for PDCD5-interacting proteins. We found that YY1-associated factor 2 (YAF2) binds to and increases PDCD5 stability by inhibiting the ubiquitin-dependent proteosomal degradation pathway. However, knocking-down of YAF2 diminishes the levels of PDCD5 protein but not the levels of PDCD5 mRNA. Upon genotoxic stress response, YAF2 promotes TP53 activation via association with PDCD5. Strikingly, YAF2 failed to promote TP53 activation in the deletion of PDCD5, whereas restoration of wild-type PDCD5WT efficiently reversed the ineffectiveness of YAF2 on TP53 activation. Conversely, PDCD5 efficiently overcame the knockdown effect of YAF2 on ET-induced TP53 activation. Finally, impaired apoptosis upon PDCD5 ablation was substantially rescued by restoration of PDCD5WT but not YAF2-interacting defective PDCD5E4D nor TP53-interacting defective PDCD5E16D mutant. Our findings uncovered an apoptotic signaling cascade linking YAF2, PDCD5, and TP53 during genotoxic stress responses.ope