The ASPP interaction network: electrostatic differentiation between pro‐ and anti‐apoptotic proteins

The ASPP proteins are apoptosis regulators: ASPP1 and ASPP2 promote, while iASPP inhibits, apoptosis. The mechanism by which these different outcomes are achieved is still unknown. The C‐terminal ankyrin repeats and SH3 domain (ANK‐SH3) mediate the interactions of the ASPP proteins with major apoptosis regulators such as p53, Bcl‐2, and NFκB. The structure of the complex between ASPP2_ANK‐SH3 and the core domain of p53 (p53CD) was previously determined. We have recently characterized the individual interactions of ASPP2_ANK‐SH3 with Bcl‐2 and NFκB, as well as a regulatory intramolecular interaction with the proline rich domain of ASPP2. Here we compared the ASPP interactions at two levels: ASPP2_ANK‐SH3 with different proteins, and different ASPP family members with each protein partner. We found that the binding sites of ASPP2 to p53CD, Bcl‐2, and NFκB are different, yet lie on the same face of ASPP2_ANK‐SH3. The intramolecular binding site to the proline rich domain overlaps the three intermolecular binding sites. To reveal the basis of functional diversity in the ASPP family, we compared their protein‐binding domains. A subset of surface‐exposed residues differentiates ASPP1 and ASPP2 from iASPP: ASPP1/2 are more negatively charged in specific residues that contact positively charged residues of p53CD, Bcl‐2, and NFκB. We also found a gain of positive charge at the non‐protein binding face of ASPP1/2, suggesting a role in electrostatic direction towards the negatively charged protein binding face. The electrostatic differences in binding interfaces between the ASPP proteins may be one of the causes for their different function. Copyright © 2010 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.     

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.     

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.     

iASPP/p63 autoregulatory feedback loop is required for the homeostasis of stratified epithelia

iASPP, an inhibitory member of the ASPP (apoptosis stimulating protein of p53) family, is an evolutionarily conserved inhibitor of p53 which is frequently upregulated in human cancers. However, little is known about the role of iASPP under physiological conditions. Here, we report that iASPP is a critical regulator of epithelial development. We demonstrate a novel autoregulatory feedback loop which controls crucial physiological activities by linking iASPP to p63, via two previously unreported microRNAs, miR-574-3p and miR-720. By investigating its function in stratified epithelia, we show that iASPP participates in the p63-mediated epithelial integrity program by regulating the expression of genes essential for cell adhesion. Silencing of iASPP in keratinocytes by RNA interference promotes and accelerates a differentiation pathway, which also affects and slowdown cellular proliferation. Taken together, these data reveal iASPP as a key regulator of epithelial homeostasis.     

iASPP contributes to cell cortex rigidity,mitotic cell rounding,and spindle positioning

iASPP is a protein mostly known as an inhibitor of p53 pro-apoptotic activity and a predicted regulatory subunit of the PP1 phosphatase, which is often overexpressed in tumors. We report that iASPP associates with the microtubule plus-end binding protein EB1, a central regulator of microtubule dynamics, via an SxIP motif. iASPP silencing or mutation of the SxIP motif led to defective microtubule capture at the cortex of mitotic cells, leading to abnormal positioning of the mitotic spindle. These effects were recapitulated by the knockdown of the membrane-to-cortex linker Myosin-Ic (Myo1c), which we identified as a novel partner of iASPP. Moreover, iASPP or Myo1c knockdown cells failed to round up upon mitosis because of defective cortical stiffness. We propose that by increasing cortical rigidity, iASPP helps cancer cells maintain a spherical geometry suitable for proper mitotic spindle positioning and chromosome partitioning.     

异构体ΔASPP2可拮抗ASPP2对p53（细胞）凋亡功能的增强作用

p53 凋亡刺激蛋白2(apoptosis stimulating protein 2 of p53, ASPP2)能够与p53 蛋白结合特异性地增强其促细胞凋亡功能,进而发挥肿瘤抑制作用．我们发现的1个比ASPP2少300多个N端氨基酸的异构体ΔASPP2.目前,ΔASPP2对p53起何种作用尚不清楚．在本研究中,我们构建了rAd-ASPP2、rAd-ΔASPP2腺病毒,利用rAd-p53、rAd-ASPP2、rAd-ΔASPP2 感染p53缺失的细胞系H1299,在MMS的作用下研究ASPP2 和 ΔASPP2 对p53介导的细胞凋亡的影响．结果发现,p53自身过表达能明显促进肿瘤细胞的凋亡;ASPP2可显著增强p53介导的MMS引起的H1299细胞凋亡的作用;然而,ΔASPP2对p53介导的细胞凋亡没有明显影响但却显著抑制rAd-ASPP2 增强的rAd-p53的促细胞凋亡作用．p53-ASPP2 复合体可能改变p53 蛋白的构象,促进p53 和增强子Bax的结合活性．p53 转录调控基因的表达研究显示,ΔASPP2的存在可显著抑制ASPP2增强p53 介导的bax基因转录活性, 提示ΔASPP2可能与ASPP2结合后来抑制p53的凋亡基因转录活性．     

Downregulated mRNA expression of ASPP and the hypermethylation of the 5'-untranslated region in cancer cell lines retaining wild-type p53

The p53 protein is one of the best-known tumour suppressors. Recently discovered ASPP1 and ASPP2 are specific activators of p53. To understand, if apoptosis-stimulating protein of p53 (ASPP) inactivation offers a selective advantage to tumors that have wild-type p53, we measured the mRNA expression of ASPP1 and ASPP2 in tumor cell lines retaining wide-type p53. In addition, the CpG island methylation status of ASPP1 gene and ASPP2 gene in the 5'-untranslated region was also investigated in order to understand the possible cause of abnormal expression of ASPP1 and ASPP2 in the tumor cell lines retaining wide-type p53. The data showed that mRNA expression of ASPP1 and ASPP2 is downregulated and CpG island tested is hypermethylated. These results indicated that ASPP CpG island aberrant methylation could be one molecular and genetic alteration in wild-type p53 tumours.     

Disruption of serine/threonine protein phosphatase 5 (PP5:PPP5c) in mice reveals a novel role for PP5 in the regulation of ultraviolet light-induced phosphorylation of serine/threonine protein kinase Chk1 (CHEK1)

PP5 is a ubiquitously expressed Ser/Thr protein phosphatase. High levels of PP5 have been observed in human cancers, and constitutive PP5 overexpression aids tumor progression in mouse models of tumor development. However, PP5 is highly conserved among species, and the roles of PP5 in normal tissues are not clear. Here, to help evaluate the biological actions of PP5, a Cre/loxP-conditional mouse line was generated. In marked contrast to the early embryonic lethality associated with the genetic disruption of other PPP family phosphatases (e.g. PP2A and PP4), intercrosses with mouse lines that ubiquitously express Cre recombinase starting early in development (e.g. MeuCre40 and ACTB-Cre) produced viable and fertile PP5-deficient mice. Phenotypic differences caused by the total disruption of PP5 were minor, suggesting that small molecule inhibitors of PP5 will not have widespread systemic toxicity. Examination of roles for PP5 in fibroblasts generated from PP5-deficient embryos (PP5(-/-) mouse embryonic fibroblasts) confirmed some known roles and identified new actions for PP5. PP5(-/-) mouse embryonic fibroblasts demonstrated increased sensitivity to UV light, hydroxyurea, and camptothecin, which are known activators of ATR (ataxia-telangiectasia and Rad3-related) kinase. Further study revealed a previously unrecognized role for PP5 downstream of ATR activation in a UV light-induced response. The genetic disruption of PP5 is associated with enhanced and prolonged phosphorylation of a single serine (Ser-345) on Chk1, increased phosphorylation of the p53 tumor suppressor protein (p53) at serine 18, and increased p53 protein levels. A comparable role for PP5 in the regulation of Chk1 phosphorylation was also observed in human cells.     

RNA interference-mediated silencing of iASPP induces cell proliferation inhibition and G0/G1 cell cycle arrest in U251 human glioblastoma cells

iASPP is an evolutionally conserved inhibitory member of the ASPP (apoptosis-stimulating protein of p53) protein family. Overexpression of iASPP was observed in several types of human tumors, however, its role in tumorigenesis has not been fully clarified. To investigate the role of iASPP in human glioblastoma multiforme (GMB) progression, the authors employed lentivirus-mediated shRNA to silence endogenous iASPP expression and elucidated iASPP function by analysis of viability, colony formation, DNA synthesis, and cell cycle in p53-mutant glioblastoma cell line U251. iASPP was significantly and sustainably knocked down by iASPP-specific shRNA in U251 cells. Stable down-regulation of iASPP expression-induced cell proliferation inhibition and G0/G1 cell cycle arrest by down-regulation of cyclin D1 and up-regulation of p21(Waf1/Cip1). Thus, the findings not only provide a molecular basis for the role of iASPP in cell cycle progression of glioblastoma cells but also suggest a novel therapeutic target for the treatment of GBM.     

Molecular interactions of ASPP1 and ASPP2 with the p53 protein family and the apoptotic promoters PUMA and Bax

The apoptosis stimulating p53 proteins, ASPP1 and ASPP2, are the first two common activators of the p53 protein family that selectively enable the latter to regulate specific apoptotic target genes, which facilitates yes yet unknown mechanisms for discrimination between cell cycle arrest and apoptosis. To better understand the interplay between ASPP- and p53-family of proteins we investigated the molecular interactions between them using biochemical methods and structure-based homology modelling. The data demonstrate that: (i) the binding of ASPP1 and ASPP2 to p53, p63 and p73 is direct; (ii) the C-termini of ASPP1 and ASPP2 interact with the DNA-binding domains of p53 protein family with dissociation constants, K_d, in the lower micro-molar range; (iii) the stoichiometry of binding is 1:1; (iv) the DNA-binding domains of p53 family members are sufficient for these protein–protein interactions; (v) EMSA titrations revealed that while tri-complex formation between ASPPs, p53 family of proteins and PUMA/Bax is mutually exclusive, ASPP2 (but not ASPP1) formed a complex with PUMA (but not Bax) and displaced p53 and p73. The structure-based homology modelling revealed subtle differences between ASPP2 and ASPP1 and together with the experimental data provide novel mechanistic insights.