Crystal structure and mutagenic analysis of the inhibitor-of-apoptosis protein survivin

The coupling of apoptosis (programmed cell death) to the cell division cycle is essential for homeostasis and genomic integrity. Here, we report the crystal structure of survivin, an inhibitor of apoptosis, which has been implicated in both control of cell death and regulation of cell division. In addition to a conserved N-terminal Zn finger baculovirus IAP repeat, survivin forms a dimer through a symmetric interaction with an intermolecularly bound Zn atom located along the molecular dyad axis. The interaction of the dimer-related C-terminal alpha helices forms an extended surface of approximately 70 A in length. Mutagenesis analysis revealed that survivin dimerization and an extended negatively charged surface surrounding Asp-71 are required to counteract apoptosis and preserve ploidy. These findings may provide a structural basis for a dual role of survivin in inhibition of apoptosis and regulation of cell division.     

Nucleocytoplasmic shuttling and the biological activity of mouse survivin are regulated by an active nuclear export signal

Survivin appears to function as a regulator of cell division and as an apoptosis inhibitor in many species. Here, we characterized the nucleocytoplasmic transport of mouse survivin(140), and its splice variants survivin(121) and survivin(40). We show that the dynamic intracellular localization of survivin(140) is mediated by a Crm1-dependent nuclear export signal (NES) present also in survivin(121), but absent in survivin(40). In contrast, neither survivin nor survivin splice variants contain an active nuclear import signal and seem to enter the nucleus by passive diffusion. The activity of the NES is required for survivin-mediated protection against cell death inducing stimuli and influences protein degradation. During mitosis, NES-deficient survivin variants fail to correctly localize to the mitotic machinery and promote proper cell division. In vivo and in vitro protein interaction assays show that survivin(140) and survivin(121) as well as their export-deficient mutants are able to form homo- as well as heterodimers. The trans-dominant negative phenotype observed upon expression of export-deficient survivin appears, therefore, to be mediated by the formation of inactive survivin heterodimers. The survivin-Crm1 axis is essential for the biological activities of murine survivin, and mouse models will allow investigating its functional implications during development and tumorigenesis.     

Survivin study: What is the next wave?

Survivin, a novel member of inhibitor of apoptosis (IAP) protein family, is aberrantly expressed in cancer but undetectable in normal, differentiated adult tissues. Current studies suggest that survivin is implicated in both control of apoptosis and regulation of cell division. However, due to some inconsistent observations on survivin subcellular localization, there is debate about survivin's function in regulating apoptosis, cell division, or both. This review will discuss concepts, experimental methods, and interesting results that unify the different notions about survivin localization and function or point out gaps of knowledge about controversial issues. The author also intends to review various aspects of survivin studies, which were not emphasized or sufficiently discussed in previous reviews on survivin, and update recent developments that may reveal new applications of disease-oriented therapeutics in the coming years.     

Role of survivin in mitosis

Human survivin is a member of the IAP (Inhibitor of Apoptosis) family. It was reported that survivin expression is associated with drug resistance, cancer progression and low patient survival rate in many cancers. Survivin is implicated in both: inhibition of apoptosis and regulation of cell division. As a member of Chromosomal Passenger Complex (CPC) it is involved in sister chromatids segregation during mitosis. On the other hand, survivin plays an important role in the surveillance mechanism called mitotic spindle assembly checkpoint (MSAC) which regulates metaphase to anaphase transition during mitosis. Additionally, survivin is necessary for cytokinesis progression. The present review is a summary of survivin's functions, focused on its role in cell division in normal and cancer cells, as well as introduction to discussion about anticancer therapies based on survivin depletion.     

Histone deacetylase 6 (HDAC6) deacetylates survivin for its nuclear export in breast cancer

Survivin is an oncogenic protein that is highly expressed in breast cancer and has a dual function that is dependent on its subcellular localization. In the cytosol, survivin blocks programmed cell death by inactivating caspase proteins; however, in the nucleus it facilitates cell division by regulating chromosomal movement and cytokinesis. In prior work, we showed that survivin is acetylated by CREB-binding protein (CBP), which restricts its localization to the nuclear compartment and thereby inhibits its anti-apoptotic function. Here, we identify histone deacetylase 6 (HDAC6) as responsible for abrogating CBP-mediated survivin acetylation in the estrogen receptor (ER)-positive breast cancer cell line, MCF-7. HDAC6 directly binds survivin, an interaction that is enhanced by CBP. In quiescent breast cancer cells in culture and in malignant tissue sections from ER+ breast tumors, HDAC6 localizes to a perinuclear region of the cell, undergoing transport to the nucleus following CBP activation where it then deacetylates survivin. Genetically modified mouse embryonic fibroblasts that lack mhdac6 localize survivin predominantly to the nuclear compartment, whereas wild-type mouse embryonic fibroblasts localize survivin to distinct cytoplasmic structures. Together, these data imply that HDAC6 deacetylates survivin to regulate its nuclear export, a feature that may provide a novel target for patients with ER+ breast cancer.     

Acute ablation of survivin uncovers p53-dependent mitotic checkpoint functions and control of mitochondrial apoptosis

Survivin is a member of the Inhibitor of Apoptosis gene family that has been implicated in cell division and suppression of apoptosis. Here, we show that preferential ablation of the nuclear pool of survivin by RNA interference produces a mitotic arrest followed by re-entry into the cell cycle and polyploidy. Survivin ablation causes multiple centrosomal defects, aberrant multipolar spindle formation, and chromatin missegregation, and these phenotypes are exacerbated by loss of the cell cycle regulator, p21(Waf1/Cip1) in p21(-/-) cells. The mitotic checkpoint activated by loss of survivin is mediated by induction of p53 and associated with increased expression of its downstream target, p21(Waf1/Cip1). Accordingly, p53(-/-) cells exhibit reduced mitotic arrest and enhanced polyploidy upon survivin ablation as compared with their p53(+/+) counterparts. Partial reduction of the cytosolic pool of survivin by RNA interference sensitizes cells to ultraviolet B-mediated apoptosis and results in enhanced caspase-9 proteolytic cleavage, whereas complete ablation of cytosolic survivin causes loss of mitochondrial membrane potential and spontaneous apoptosis. These data demonstrate that survivin has separable checkpoint functions at multiple phases of mitosis and in the control of mitochondrial-dependent apoptosis.     

Developmental control of apoptosis by the immunophilin aryl hydrocarbon receptor-interacting protein (AIP) involves mitochondrial import of the survivin protein

Survivin is a multifunctional protein with essential roles in cell division and inhibition of apoptosis, but the molecular underpinnings of its cytoprotective properties are poorly understood. Here we show that homozygous deletion of the aryl hydrocarbon receptor-interacting protein (AIP), a survivin-associated immunophilin, causes embryonic lethality in mice by embryonic day 13.5-14, increased apoptosis of Ter119(-)/CD71(-) early erythropoietic progenitors, and loss of survivin expression in its cytosolic and mitochondrial compartments in vivo. In import assays using recombinant proteins, AIP directly mediated the import of survivin to mitochondria, thus enabling its anti-apoptotic function, whereas a survivin 1-141 mutant that does not bind AIP was not imported to mitochondria and failed to inhibit apoptosis. AIP-directed mitochondrial import of survivin did not affect cell division, was independent of the organelle transmembrane potential, did not require the chaperone Heat Shock Protein 90 (Hsp90), and was inhibited by cytosolic factor(s) present in normal cells. shRNA knockdown of the mitochondrial import receptor Tom20 abolished mitochondrial import of survivin and sensitized tumor cells to apoptosis, whereas silencing of Tom70 had no effect. Therefore, an AIP-Tom20 recognition contributes to cell survival in development and cancer by mediating the mitochondrial import of survivin.     

Inhibition of Survivin Influences the Biological Activities of Canine Histiocytic Sarcoma Cell Lines

Canine histiocytic sarcoma (CHS) is an aggressive malignant neoplasm that originates from histiocytic lineage cells, including dendritic cells and macrophages, and is characterized by progressive local infiltration and a very high metastatic potential. Survivin is as an apoptotic inhibitory factor that has major functions in cell proliferation, including inhibition of apoptosis and regulation of cell division, and is expressed in most types of human and canine malignant neoplasms, including melanoma and osteosarcoma. To investigate whether survivin was expressed at high levels in CHS and whether its expression was correlated with the aggressive biological behavior of CHS, we assessed relation between survivin expression and CHS progression, as well as the effects of survivin inhibition on the biological activities of CHS cells. We comparatively analyzed the expression of 6 selected anti-apoptotic genes, including survivin, in specimens from 30 dogs with histiocytic sarcoma and performed annexin V staining to evaluate apoptosis, methylthiazole tetrazolium assays to assess cell viability and chemosensitivity, and latex bead assays to measure changes in phagocytic activities in 4 CHS cell lines and normal canine fibroblasts transfected with survivin siRNA. Survivin gene expression levels in 30 specimens were significantly higher than those of the other 6 genes. After transfection with survivin siRNA, apoptosis, cell growth inhibition, enhanced chemosensitivity, and weakened phagocytic activities were observed in all CHS cell lines. In contrast, normal canine fibroblasts were not significantly affected by survivin knockdown. These results suggested that survivin expression may mediate the aggressive biological activities of CHS and that survivin may be an effective therapeutic target for the treatment of CHS.     

肿瘤中与survivin有关的信号通路及分子以及靶向survivin的治疗前景

Survivin在细胞内环境稳定和肿瘤的形成中起重要的作用,在肿瘤的治疗中,survivivin的靶向治疗调节与一些典型的信号通路和一系列生长因子有关。众所周知,survivin是一个小的凋亡蛋白抑制因子,也是一个主要的抗癌靶标,与细胞分裂和凋亡抑制有关,它在大部分正常组织中缺失但在大部分癌组织中过表达。Survivin是一个与众多细胞信号通路有关的节点蛋白,这些通路协调各种细胞因子、转录网络和修饰基因,通过调节癌细胞内环境稳定直接或间接促进细胞增殖。临床前研究数据表明,survivin的抑制可以降低细胞增殖促进凋亡,增加细胞对细胞毒药物和放疗的敏感性,其过表达与不良预后和治疗耐受有关。因此对于癌症治疗,survivin是一个潜在的靶标。     

抗凋亡蛋白survivin研究与癌治疗新策略的进展

survivin是凋亡抑制蛋白家族中的一个新成员,在近乎所有的人类肿瘤中都显示高表达,而在终末分化的正常组织中未能检测出表达。survivin表达的另一个独特性质是,其表达受细胞周期调控,在G2／M期有异常高的特异性表达。survivin的功能意义在于参与细胞凋亡调控和细胞分裂调控。引人兴趣的是,新近癌生物学基础与应用研究则更关注它是一个枢纽癌基因。本文将重点讨论与survivin在细胞死亡和细胞分裂功能相联系的、当前一些新的癌治疗策略的进展。     

Production of affinity purified antipeptide antibodies to survivin for structural and functional studies of the protein

Tumor-associated protein survivin is a bifunctional protein that can participate either in the regulation of cell division or in the inhibition of apoptosis depending on its location in the cell and its structural state. The aim of the present work was to obtain monospecific antibodies to survivin targeting various sites in the protein and suitable for the study of its structural and functional features. The ability of the antibodies to detect survivin in tumor cells and mammary tumor tissues has been assessed. Antibodies targeting the (1–22) and (95–105) fragments of the protein have been shown to have the highest specific activity. Antibodies targeting the (1–22) site preferentially bound to survivin-containing complex (presumably a dimer of survivin) in immunoblotting, whereas antibodies targeting the (95–105) site detected only the monomeric form of the protein. Analysis of malignant mammary tumor samples showed that monomeric survivin was present only in tumor tissue samples, whereas survivin-containing complex was expressed both in tumor tissues and in the tissues adjacent to the tumor. Antibodies targeting the (1–22) site were shown to preferentially detect survivin localized in the cell nuclei (and involved in the regulation of mitosis) in immunocytochemistry, while those targeting the (95–105) site stained the nucleoplasm and cytoplasm at all stages of the cell cycle. Therefore, the antibodies obtained can serve as a useful tool for structural and functional research on survivin.     

An anti-apoptotic protein human survivin is a direct inhibitor of caspase-3 and -7

Survivin, an apoptosis inhibitor/cell-cycle regulator, is critically required for suppression of apoptosis and ensuring normal cell division in the G2/M phase of the cell cycle. It is highly expressed in a cell cycle-regulated manner and localizes together with caspase-3 on microtubules within centrosomes. Whether survivin is a physiologically relevant caspase inhibitor has been unclear due to the difficulties with obtaining correctly folded survivin and finding the right conditions for inhibition assay. In this study, recombinant, active human survivin was expressed in Escherichia coli and purified to homogeneity. The protein, existing as a homodimer in solution, binds caspase-3 and -7 tightly with dissociation constants of 20.9 and 11.5 nM, respectively, when evaluated by surface plasmon resonance spectroscopy. Consistently, survivin potently inhibits the cleavage of a physiological substrate poly(ADP-ribose) polymerase and an artificial tetrapeptide by caspase-3 and -7 in vitro with apparent inhibition constants of 36.0 and 16.5 nM, respectively. The data suggest that sequestering caspase-3 and -7 in inhibited states on microtubules is at least one mechanism of survivin in the suppression of default apoptosis in the G2/M phase. The localization of survivin on microtubules, which is essential for its function, should increase the protective activity at the action site.     

Pleiotropic cell-division defects and apoptosis induced by interference with survivin function

Here we investigate the role of the control of apoptosis in normal cell division. We show that interference with the expression or function of the apoptosis inhibitor survivin causes caspase-dependent cell death in the G2/M phase of the cell cycle, and a cell-division defect characterized by centrosome dysregulation, multipolar mitotic spindles and multinucleated, polyploid cells. Use of a dominant-negative survivin mutant or antisense survivin complementary DNA disrupts a supramolecular assembly of survivin, caspase-3 and the cyclin-dependent-kinase inhibitor p21Waf1/Cip1 within centrosomes, and results in caspase-dependent cleavage of p21. Polyploidy induced by survivin antagonists is accentuated in p21-deficient cells, and corrected by exogenous expression of p21. These findings show that control of apoptosis and preservation of p21 integrity within centrosomes by survivin are required for normal mitotic progression.     

Preparation of antibodies capable of recognizing the Glu/Lys amino acid substitution at position 129 of the survivin sequence

Survivin is an oncofetal protein involved in the inhibition of apoptosis and the regulation of cell division. The functions of survivin are determined by its structural state (monomer or dimer). Owing to the natural polymorphism, either the Glu or the Lys residue can be at position 129 of the amino acid sequence of survivin. Lys has the capability for acetylation, and only the protein containing the acetylated residue Lys¹²⁹ tends to form a dimer. Thus, antibodies recognizing the amino acid substitution Glu129Lys can serve as a tool in the structural and functional investigations of survivin. For preparing the target antibodies, survivin fragments containing residue 129 were synthesized, rabbits were immunized with synthetic peptides, and the antibodies were purified by affinity chromatography on Sepharose conjugated with the corresponding peptides. It was shown by ELISA and immunoblotting that the affinity-purified antibodies are capable of recognizing the amino acid substitution Glu129Lys in the sequence of recombinant and endogenous survivin.     

抗凋亡因子Survivin研究进展

Survivin是在肿瘤组织及胚胎中发现的一类细胞因子,它是IAPs(inhibitorsofapoptosisprotein)家族的成员之一,具有其独特的分子结构和组织表达特异性,在细胞中参与细胞周期的调控,主要在细胞周期的G2/M期通过抑制caspase-3及caspase-7的活性发挥作用.Survivin在细胞中的活性可能受p53的调节.Survivin也是胚胎发育早期过程中调节细胞分裂分化的一类重要的因子.对Survivin的研究对于肿瘤治疗的研究及揭示胚胎早期的发育机制有重要的意义.     

Molecular circuits of apoptosis regulation and cell division control: the survivin paradigm

The coupling of cell proliferation to cell death is thought to function as a pivotal crossroad, essential to preserve normal homeostasis and to eliminate dangerous cells before they divide. Survivin is a prototype molecule at this crossroad, intercalated in protection against mitochondrial cell death and orchestrating various aspects of cell division. Dramatically exploited in cancer and an unfavorable gene signature for disease outcome, the survivin pathway has now provided tangible opportunities for targeted, rational cancer therapy.     

Survivin acts as anti-apoptotic factor during the development of bovine pre-implantation embryos

Survivin, an inhibitor of apoptotic protein containing a single baculoviral inhibit apoptotic protein repeat domain, is a bifunctional protein that suppresses apoptosis and regulates cell division. Thus, we used double stranded RNA (dsRNA) interference to manipulate survivin expression in bovine embryos and analyze its role in blocking apoptosis and facilitating development of pre-implantation embryos. In vitro fertilized embryos (1-cell) were injected with survivin dsRNA, and expression of survivin mRNA was evaluated by real-time quantitative RT-PCR. To analyze survivin protein expression, we performed immunocytochemistry using a rabbit anti-bovine suvivin antibody. Expression levels of survivin mRNA and protein were decreased in the dsRNA group compared to the sham group. Rates of in vitro blastocyst development were lower in the survivin dsRNA-injected group than in the sham-injected group. Also, the total cell number seen in blastocysts was decreased in the dsRNA group. TUNEL assays of DNA fragmentation indicated an increased apoptotic index in the dsRNA group compared to the sham group. These results indicate that survivin is important for optimal development of bovine blastocysts and confirm that survivin expression suppresses apoptosis of pre-implantation embryos.