Biochemical and structural studies of ASPP proteins reveal differential binding to p53, p63, and p73

ASPP1 and ASPP2 are activators of p53-dependent apoptosis, whereas iASPP is an inhibitor of p53. Binding assays showed differential binding for C-terminal domains of iASPP and ASPP2 to the core domains of p53 family members p53, p63, and p73. We also determined a high-resolution crystal structure for the C terminus of iASPP, comprised of four ankyrin repeats and an SH3 domain. The crystal lattice revealed an interaction between eight sequential residues in one iASPP molecule and the p53-binding site of a neighboring molecule. ITC confirmed that a peptide corresponding to the crystallographic interaction shows specific binding to iASPP. The contributions of ankyrin repeat residues, in addition to those of the SH3 domain, generate distinctive architecture at the p53-binding site suitable for inhibition by small molecules. These results suggest that the binding properties of iASPP render it a target for antitumor therapeutics and provide a peptide-based template for compound design.     

Regulation of ASPP2 Interaction with p53 Core Domain by an Intramolecular Autoinhibitory Mechanism

ASPP2 is a key protein in regulating apoptosis both in p53-dependent and-independent pathways. The C-terminal part of ASPP2 contains four ankyrin repeats and an SH3 domain (Ank-SH3) that mediate the interactions of ASPP2 with apoptosis related proteins such as p53, Bcl-2 and the p65 subunit of NFκB. p53 core domain (p53CD) binds the n-src loop and the RT loop of ASPP2 SH3. ASPP2 contains a disordered proline rich domain (ASPP2 Pro) that forms an intramolecular autoinhibitory interaction with the Ank-SH3 domains. Here we show how this intramolecular interaction affects the intermolecular interactions of ASPP2 with p53, Bcl-2 and NFkB. We used biophysical methods to obtain better understanding of the relationship between ASPP2 and its partners for getting a comprehensive view on ASPP2 pathways. Fluorescence anisotropy competition experiments revealed that both ASPP2 Pro and p53CD competed for binding the n-src loop of the ASPP2 SH3, indicating regulation of p53CD binding to this loop by ASPP2 Pro. Peptides derived from the ASPP2-binding interface of Bcl-2 did not compete with p53CD or NFkB peptides for binding the ASPP2 n-src loop. However, p53CD displaced the NFκB peptide (residues 303–332) from its complex with ASPP2 Ank-SH3, indicating that NFκB 303–332 and p53CD bind a partly overlapping site in ASPP2 SH3, mostly in the RT loop. These results are in agreement with previous docking studies, which showed that ASPP2 Ank-SH3 binds Bcl-2 and NFκB mostly via distinct sites from p53. However they show some overlap between the binding sites of p53CD and NFkB in ASPP2 Ank-SH3. Our results provide experimental evidence that the intramolecular interaction in ASPP2 regulates its binding to p53CD and that ASPP2 Ank-SH3 binds Bcl-2 and NFκB via distinct sites.     

Solution structure of ASPP2 N-terminal domain (N-ASPP2) reveals a ubiquitin-like fold

Proteins of the ASPP family bind to p53 and regulate p53-mediated apoptosis. Two family members, ASPP1 and ASPP2, have pro-apoptotic functions while iASPP shows anti-apoptotic responses. However, both the mechanism of enhancement/repression of apoptosis and the molecular basis for their different responses remain unknown. To address the role of the N-termini of pro-apoptotic ASPP proteins, we solved the solution structure of N-ASPP2 (1-83) by NMR spectroscopy. The structure of this domain reveals a beta-Grasp ubiquitin-like fold. Our findings suggest a possible role for the N-termini of ASPP proteins in binding to other proteins in the apoptotic response network and thus mediating their selective pro-apoptotic function.     

JNK and NFκB dependence of apoptosis induced by vinblastine in human acute promyelocytic leukaemia cells

The relationship between the mitogen‐activated protein kinase response, nuclear factor‐κB (NFκB) expression and the apoptosis in human acute promyelocytic leukaemia NB4 cells treated with vinblastine was investigated in this work. Cell viability, subdiploid DNA and cell cycle were analysed by propidium iodide permeability and flow cytometry analyses. Apoptosis was determined by annexin V‐Fluorescein isothiocyanate assays. Western‐blot analysis was used for determination of expression levels of apoptotic factors (p53, Bax and Bcl2), intracellular kinases [serine/threonine‐specific protein kinase, extracellular signal‐regulated kinase and c‐Jun N‐terminal kinase (JNK)], NFκB factor and caspases. Electrophoretic mobility shift assay was usefully applied to study DNA‐NFκB interaction. In NB4 cells, vinblastine produces alteration of p53 and DNA fragmentation. Vinblastine treatment had an antiproliferative effect via the induction of apoptosis producing Bax/Bcl‐2 imbalance. Vinblastine treatment suppressed NFκB expression and depressed NFκB‐DNA binding activity while maintaining JNK activation that subsequently resulted in apoptotic response through caspase‐dependent pathway. Our study provides a possible anti‐cancer mechanism of vinblastine action on NB4 cells by deregulation of the intracellular signalling cascade affecting to JNK activation and NFκB expression. Moreover, JNK activation and NFκB depression can be very significant factors in apoptosis induction by vinblastine. Copyright © 2015 John Wiley & Sons, Ltd.     

Interaction of p53 and ASPPs regulates rhesus monkey embryonic stem cells conversion to neural fate concomitant with apoptosis

The tumor suppressor p53 is a key regulator of cell apoptosis and cell cycle arrest. Recent studies show that the delicate balance of p53 expression is important for neural tube defects, neuronal degeneration, embryonic lethality, as well as differentiation and dedifferentiation. Moreover, p53 showed different regulatory patterns between rodent and primate embryonic stem cells (ESCs). However, the role of p53 and apoptosis stimulating protein of p53 (ASPP) during neural differentiation (ND) from primate ESCs is still unknown. In this study, using an FGF-2 and/or HGF selectively containing ND culture systems for rhesus monkey ESCs (rESCs), the changes of p53 and ASPPs, and p53 targets, i.e. BAX and p21, were analyzed. Our results showed that the expression patterns of ASPP1/ASPP2 and iASPP were opposite in rESCs but similar in differentiated cells, and the expression of p53 was approximately consistent with BAX, but not p21. These findings indicate that the strong expression of iASPP in ESCs and weak expression of ASPP1/ASPP2 maintain the stability of stemness; and in ND niche, unimpaired iASPP may decrease its inhibition of ASPP1/ASPP2 expression, the interaction of p53 and ASPPs causing rESCs to convert towards a neural fate concomitant with apoptosis, but not to cell cycle arrest.     

ASPP2 attenuates triglycerides to protect against hepatocyte injury by reducing autophagy in a cell and mouse model of non‐alcoholic fatty liver disease

ASPP2 is a pro‐apoptotic member of the p53 binding protein family. ASPP2 has been shown to inhibit autophagy, which maintains energy balance in nutritional deprivation. We attempted to identify the role of ASPP2 in the pathogenesis of non‐alcoholic fatty liver disease (NAFLD). In a NAFLD cell model, control treated and untreated HepG2 cells were pre‐incubated with GFP‐adenovirus (GFP‐ad) for 12 hrs and then treated with oleic acid (OA) for 24 hrs. In the experimental groups, the HepG2 cells were pre‐treated with ASPP2‐adenovirus (ASPP2‐ad) or ASPP2‐siRNA for 12 hrs and then treated with OA for 24 hrs. BALB/c mice fed a methionine‐ and choline‐deficient (MCD) diet were used to generate a mouse model of NAFLD. The mice with fatty livers in the control group were pre‐treated with injections of GFP‐ad for 10 days. In the experimental group, the mice that had been pre‐treated with ASPP2‐ad were fed an MCD diet for 10 days. ASPP2‐ad or GFP‐ad was administered once every 5 days. Liver tissue from fatty liver patients and healthy controls were used to analyse the role of ASPP2. Autophagy, apoptosis markers and lipid metabolism mediators, were assessed with confocal fluorescence microscopy, immunohistochemistry, western blot and biochemical assays. ASPP2 overexpression decreased the triglyceride content and inhibited autophagy and apoptosis in the HepG2 cells. ASPP2‐ad administration suppressed the MCD diet‐induced autophagy, steatosis and apoptosis and decreased the previously elevated alanine aminotransferase levels. In conclusion, ASPP2 may participate in the lipid metabolism of non‐alcoholic steatohepatitis and attenuate liver failure.     

Brain‐derived neurotrophic factor protects cementoblasts from serum starvation‐induced cell death

Our previous studies have shown that brain‐derived neurotrophic factor (BDNF) enhances bone/cementum‐related protein gene expression through the TrkB‐c‐Raf‐ERK1/2‐Elk‐1 signaling pathway in cementoblasts, which play a critical role in the establishment of a functional periodontal ligament. To clarify how BDNF regulates survival in cementoblasts, we examined its effects on cell death induced by serum starvation in immortalized human cementoblast‐like (HCEM) cells. BDNF inhibited the death of HCEM cells. Small‐interfering RNA (siRNA) for TRKB, a high affinity receptor for BDNF, and for Bcl‐2, countered the BDNF‐induced decrease in dead cell number. In addition, LY294002, a PI3‐kinase inhibitor; SH‐6, an Akt inhibitor; and PDTC, a nuclear factor kappa B (NF‐κB) inhibitor, but not PD98059, an ERK1/2 inhibitor, abolished the protective effect of BDNF against cell death. BDNF enhanced phosphorylated Akt levels, NF‐κB activity in the nucleus, Bcl‐2 mRNA levels, and mitochondrial membrane potential. The blocking of BDNF's actions by treatment with siRNA in all cases for TRKB and Bcl‐2, LY294002, SH‐6, and PDTC suppressed the enhancement. These findings provide the first evidence that a TrkB‐PI3‐kinase‐Akt‐NF‐κB‐Bcl‐2 signaling pathway triggered by BDNF and the subsequent protective effect of BDNF on mitochondrial membrane potential are required to rescue HCEM cells from serum starvation‐induced cell death. Furthermore, the survival and increased expression of bone/cementum‐related proteins induced by BDNF in HCEM cells occur through different signaling pathways. J. Cell. Physiol. 221: 696–706, 2009. © 2009 Wiley‐Liss, Inc.     

Antiapoptotic Bcl‐2 homolog CED‐9 in Caenorhabditis elegans: Dynamics of BH3 and CED‐4 binding regions and comparison with mammalian antiapoptotic Bcl‐2 proteins

Proteins belonging to Bcl‐2 family regulate intrinsic cell death pathway. Although mammalian antiapoptotic Bcl‐2 members interact with multiple proapoptotic proteins, the Caenorhabditis elegans Bcl‐2 homolog CED‐9 is known to have only two proapoptotic partners. The BH3‐motif of proapoptotic proteins bind to the hydrophobic groove of prosurvival proteins formed by the Bcl‐2 helical fold. CED‐9 is also known to interact with CED‐4, a homolog of the human cell death activator Apaf1. We have performed molecular dynamics simulations of CED‐9 in two forms and compared the results with those of mammalian counterparts Bcl‐X_L, Bcl‐w, and Bcl‐2. Our studies demonstrate that the region forming the hydrophobic cleft is more flexible compared with the CED‐4‐binding region, and this is generally true for all antiapoptotic Bcl‐2 proteins studied. CED‐9 is the most stable protein during simulations and its hydrophobic pocket is relatively rigid explaining the absence of functional redundancy in CED‐9. The BH3‐binding region of Bcl‐2 is less flexible among the mammalian proteins and this lends support to the studies that Bcl‐2 binds to less number of BH3 peptides with high affinity. The C‐terminal helix of CED‐9 lost its helical character because of a large number of charged residues. We speculate that this region probably plays a role in intracellular localization of CED‐9. The BH4‐motif accessibility in CED‐9 and Bcl‐w is controlled by the loop connecting the first two helices. Although CED‐9 adopts the same Bcl‐2 fold, our studies highlight important differences in the dynamic behavior of CED‐9 and mammalian antiapoptotic homologs. Proteins 2014; 82:1035–1047. © 2013 Wiley Periodicals, Inc.     

The structure and interactions of the proline-rich domain of ASPP2

ASPP2 is a pro-apoptotic protein that stimulates the p53-mediated apoptotic response. The C terminus of ASPP2 contains ankyrin (Ank) repeats and a SH3 domain, which mediate its interactions with numerous partner proteins such as p53, NFkappaB, and Bcl-2. It also contains a proline-rich domain (ASPP2 Pro), whose structure and function are unclear. Here we used biophysical and biochemical methods to study the structure and the interactions of ASPP2 Pro, to gain insight into its biological role. We show, using biophysical and computational methods, that the ASPP2 Pro domain is natively unfolded. We found that the ASPP2 Pro domain interacts with the ASPP2 Ank-SH3 domains, and mapped the interaction sites in both domains. Using a combination of peptide array screening, biophysical and biochemical techniques, we found that ASPP2 Ank-SH3, but not ASPP2 Pro, mediates interactions of ASPP2 with peptides derived from its partner proteins. ASPP2 Pro-Ank-SH3 bound a peptide derived from its partner protein NFkappaB weaker than ASPP2 Ank-SH3 bound this peptide. This suggested that the presence of the proline-rich domain inhibited the interactions mediated by the Ank-SH3 domains. Furthermore, a peptide from ASPP2 Pro competed with a peptide derived from NFkappaB on binding to ASPP2 Ank-SH3. Based on our results, we propose a model in which the interaction between the ASPP2 domains regulates the intermolecular interactions of ASPP2 with its partner proteins.