Histone Deacetylase 3 Promotes RCAN1 Stability and Nuclear Translocation

Regulator of calcineurin 1 (RCAN1; also referred as DSCR1 or MCIP1) is located in close proximity to a Down syndrome critical region of human chromosome 21. Although RCAN1 is an endogenous inhibitor of calcineurin signaling that controls lymphocyte activation, apoptosis, heart development, skeletal muscle differentiation, and cardiac function, it is not yet clear whether RCAN1 might be involved in other cellular activities. In this study, we explored the extra-functional roles of RCAN1 by searching for novel RCAN1-binding partners. Using a yeast two-hybrid assay, we found that RCAN1 (RCAN1-1S) interacts with histone deacetylase 3 (HDAC3) in mammalian cells. We also demonstrate that HDAC3 deacetylates RCAN1. In addition, HDAC3 increases RCAN1 protein stability by inhibiting its poly-ubiquitination. Furthermore, HDAC3 promotes RCAN1 nuclear translocation. These data suggest that HDAC3, a new binding regulator of RCAN1, affects the protein stability and intracellular localization of RCAN1.     

Nuclear GIT2 Is an ATM Substrate and Promotes DNA Repair

Insults to nuclear DNA induce multiple response pathways to mitigate the deleterious effects of damage and mediate effective DNA repair. G-protein-coupled receptor kinase-interacting protein 2 (GIT2) regulates receptor internalization, focal adhesion dynamics, cell migration, and responses to oxidative stress. Here we demonstrate that GIT2 coordinates the levels of proteins in the DNA damage response (DDR). Cellular sensitivity to irradiation-induced DNA damage was highly associated with GIT2 expression levels. GIT2 is phosphorylated by ATM kinase and forms complexes with multiple DDR-associated factors in response to DNA damage. The targeting of GIT2 to DNA double-strand breaks was rapid and, in part, dependent upon the presence of H2AX, ATM, and MRE11 but was independent of MDC1 and RNF8. GIT2 likely promotes DNA repair through multiple mechanisms, including stabilization of BRCA1 in repair complexes; upregulation of repair proteins, including HMGN1 and RFC1; and regulation of poly(ADP-ribose) polymerase activity. Furthermore, GIT2-knockout mice demonstrated a greater susceptibility to DNA damage than their wild-type littermates. These results suggest that GIT2 plays an important role in MRE11/ATM/H2AX-mediated DNA damage responses.     

Sublethal Caspase Activation Promotes Generation of Cardiomyocytes from Embryonic Stem Cells

Generation of new cardiomyocytes is critical for cardiac repair following myocardial injury, but which kind of stimuli is most important for cardiomyocyte regeneration is still unclear. Here we explore if apoptotic stimuli, manifested through caspase activation, influences cardiac progenitor up-regulation and cardiomyocyte differentiation. Using mouse embryonic stem cells as a cellular model, we show that sublethal activation of caspases increases the yield of cardiomyocytes while concurrently promoting the proliferation and differentiation of c-Kit⁺/α-actinin^low cardiac progenitor cells. A broad-spectrum caspase inhibitor blocked these effects. In addition, the caspase inhibitor reversed the mRNA expression of genes expressed in cardiomyocytes and their precursors. Our study demonstrates that sublethal caspase-activation has an important role in cardiomyocyte differentiation and may have significant implications for promoting cardiac regeneration after myocardial injury involving exogenous or endogenous cell sources.     

Induction of Manganese Superoxide Dismutase by Nuclear Translocation and Activation of SIRT1 Promotes Cell Survival in Chronic Heart Failure

Oxidative stress plays a pivotal role in chronic heart failure. SIRT1, an NAD⁺-dependent histone/protein deacetylase, promotes cell survival under oxidative stress when it is expressed in the nucleus. However, adult cardiomyocytes predominantly express SIRT1 in the cytoplasm, and its function has not been elucidated. The purpose of this study was to investigate the functional role of SIRT1 in the heart and the potential use of SIRT1 in therapy for heart failure. We investigated the subcellular localization of SIRT1 in cardiomyocytes and its impact on cell survival. SIRT1 accumulated in the nucleus of cardiomyocytes in the failing hearts of TO-2 hamsters, postmyocardial infarction rats, and a dilated cardiomyopathy patient but not in control healthy hearts. Nuclear but not cytoplasmic SIRT1-induced manganese superoxide dismutase (Mn-SOD), which was further enhanced by resveratrol, and increased the resistance of C2C12 myoblasts to oxidative stress. Resveratrol''s enhancement of Mn-SOD levels depended on the level of nuclear SIRT1, and it suppressed the cell death induced by antimycin A or angiotensin II. The cell-protective effects of nuclear SIRT1 or resveratrol were canceled by the Mn-SOD small interfering RNA or SIRT1 small interfering RNA. The oral administration of resveratrol to TO-2 hamsters increased Mn-SOD levels in cardiomyocytes, suppressed fibrosis, preserved cardiac function, and significantly improved survival. Thus, Mn-SOD induced by resveratrol via nuclear SIRT1 reduced oxidative stress and participated in cardiomyocyte protection. SIRT1 activators such as resveratrol could be novel therapeutic tools for the treatment of chronic heart failure.     

pAkt在衰老细胞中的核转位

磷脂酰肌醇3 激酶（PI3K）/蛋白激酶B（PKB,又称为Akt）/叉头转录因子（FOXO）信号通路在从酵母菌到小鼠的寿命以及衰老的调节上都起着非常重要的作用．有研究发现,血清可以激活年轻细胞、衰老细胞胞浆内的Akt,并且年轻细胞核内磷酸化Akt(pAkt)增多,而衰老细胞核内pAkt没有增多．为了研究衰老细胞膜是否发生转位受损,即pAkt能否通过衰老细胞核膜进入核内,通过激光共聚焦显微镜（CLSM）、Western 印迹等实验方法,发现衰老细胞胞浆中pAkt可以进入核内,进入核内的pAkt很快被去磷酸化灭活．     

Crystal Structure of the Stress-Inducible Human Heat Shock Protein 70 Substrate-Binding Domain in Complex with Peptide Substrate

The HSP70 family of molecular chaperones function to maintain protein quality control and homeostasis. The major stress-induced form, HSP70 (also called HSP72 or HSPA1A) is considered an important anti-cancer drug target because it is constitutively overexpressed in a number of human cancers and promotes cancer cell survival. All HSP70 family members contain two functional domains: an N-terminal nucleotide binding domain (NBD) and a C-terminal protein substrate-binding domain (SBD); the latter is subdivided into SBDα and SBDβ subdomains. The NBD and SBD structures of the bacterial ortholog, DnaK, have been characterized, but only the isolated NBD and SBDα segments of eukaryotic HSP70 proteins have been determined. Here we report the crystal structure of the substrate-bound human HSP70-SBD to 2 angstrom resolution. The overall fold of this SBD is similar to the corresponding domain in the substrate-bound DnaK structures, confirming a similar overall architecture of the orthologous bacterial and human HSP70 proteins. However, conformational differences are observed in the peptide-HSP70-SBD complex, particularly in the loop L_{α, β} that bridges SBDα to SBDβ, and the loop L_L,1 that connects the SBD and NBD. The interaction between the SBDα and SBDβ subdomains and the mode of substrate recognition is also different between DnaK and HSP70. This suggests that differences may exist in how different HSP70 proteins recognize their respective substrates. The high-resolution structure of the substrate-bound-HSP70-SBD complex provides a molecular platform for the rational design of small molecule compounds that preferentially target this C-terminal domain, in order to modulate human HSP70 function.     

Caspase activation of mammalian sterile 20-like kinase 3 (Mst3). Nuclear translocation and induction of apoptosis

Mammalian Sterile 20-like kinase 3 (Mst3), the physiological functions of which are unknown, is a member of the germinal center kinase-III family. It contains a conserved kinase domain at its NH(2) terminus, whereas there is a regulatory domain at its COOH terminus. In this study we demonstrate that endogenous Mst3 is specifically cleaved when Jurkat cells were treated with anti-Fas antibody or staurosporine and that this cleavage is inhibited by the caspase inhibitor, Ac-DEVD-CHO. Using apoptotic Jurkat cell extracts and recombinant caspases, we mapped the caspase cleavage site, AETD(313), which is at the junction of the NH(2)-terminal kinase domain and the COOH-terminal regulatory domain. Caspase-mediated cleavage of Mst3 activates its intrinsic kinase activity, suggesting that the COOH-terminal domain of Mst3 negatively regulates the kinase domain. Furthermore, proteolytic removal of the Mst3 COOH-terminal domain by caspases promotes nuclear translocation. Ectopic expression of either wild-type or COOH-terminal truncated Mst3 in cells results in DNA fragmentation and morphological changes characteristic of apoptosis. By contrast, no such changes were exhibited for catalytically inactive Mst3, implicating the involvement of Mst3 kinase activity for mediation of these effects. Collectively, these results support the notion that caspase-mediated proteolytic activation of Mst3 contributes to apoptosis.     

Nuclear Import and Dimerization of Tomato ASR1, a Water Stress-Inducible Protein Exclusive to Plants

The ASR (for ABA/water stress/ripening) protein family, first described in tomato as nuclear and involved in adaptation to dry climates, is widespread in the plant kingdom, including crops of high agronomic relevance. We show both nuclear and cytosolic localization for ASR1 (the most studied member of the family) in histological plant samples by immunodetection, typically found in small proteins readily diffusing through nuclear pores. Indeed, a nuclear localization was expected based on sorting prediction software, which also highlight a monopartite nuclear localization signal (NLS) in the primary sequence. However, here we prove that such an "NLS" of ASR1 from tomato is dispensable and non-functional, being the transport of the protein to the nucleus due to simple diffusion across nuclear pores. We attribute such a targeting deficiency to the misplacing in that cryptic NLS of two conserved contiguous lysine residues. Based on previous in vitro experiments regarding quaternary structure, we also carried out live cell imaging assays through confocal microscopy to explore dimer formation in planta. We found homodimers in both the cytosol and the nucleus and demonstrated that assembly of both subunits together can occur in the cytosol, giving rise to translocation of preformed dimers. The presence of dimers was further corroborated by means of in vivo crosslinking of nuclei followed by SDS-PAGE.     

跨膜受体核转位通路的研究进展

跨膜受体可从膜表面进入细胞核内直接调控细胞的生命活动,但其核转位的途径至今尚无定论.已有多种模型分析了跨膜受体的核转位过程,它们均强调受体必须从细胞膜或内吞泡"逃脱"到细胞质后,才能进入细胞核内.然而,内吞.分选-浓缩-膜泡融合-释放模型却诠释了一条不同的跨膜受体核转位通路,这将有利于进一步阐明跨膜受体核转位的模式及其分子机制,并为核靶向药物的开发、目的基因的导入、病毒感染的治疗等应用研究提供新的策略.     

Regulation of Stress-Inducible Phosphoprotein 1 Nuclear Retention by Protein Inhibitor of Activated STAT PIAS1

Stress-inducible phosphoprotein 1 (STI1), a cochaperone for Hsp90, has been shown to regulate multiple pathways in astrocytes, but its contributions to cellular stress responses are not fully understood. We show that in response to irradiation-mediated DNA damage stress STI1 accumulates in the nucleus of astrocytes. Also, STI1 haploinsufficiency decreases astrocyte survival after irradiation. Using yeast two-hybrid screenings we identified several nuclear proteins as STI1 interactors. Overexpression of one of these interactors, PIAS1, seems to be specifically involved in STI1 nuclear retention and in directing STI1 and Hsp90 to specific sub-nuclear regions. PIAS1 and STI1 co-immunoprecipitate and PIAS1 can function as an E3 SUMO ligase for STI. Using mass spectrometry we identified five SUMOylation sites in STI1. A STI1 mutant lacking these five sites is not SUMOylated, but still accumulates in the nucleus in response to increased expression of PIAS1, suggesting the possibility that a direct interaction with PIAS1 could be responsible for STI1 nuclear retention. To test this possibility, we mapped the interaction sites between PIAS1 and STI1 using yeast-two hybrid assays and surface plasmon resonance and found that a large domain in the N-terminal region of STI1 interacts with high affinity with amino acids 450–480 of PIAS1. Knockdown of PIAS1 in astrocytes impairs the accumulation of nuclear STI1 in response to irradiation. Moreover, a PIAS1 mutant lacking the STI1 binding site is unable to increase STI1 nuclear retention. Interestingly, in human glioblastoma multiforme PIAS1 expression is increased and we found a significant correlation between increased PIAS1 expression and STI1 nuclear localization. These experiments provide evidence that direct interaction between STI1 and PIAS1 is involved in the accumulation of nuclear STI1. This retention mechanism could facilitate nuclear chaperone activity.Stress-inducible phosphoprotein I (STI1)¹ is a conserved cochaperone protein that assists Hsp90 in managing client proteins, by mediating the transfer of proteins between Hsp70 and Hsp90 (1–3). STI1 contains several tetratricopeptide-repeat domains (TRP) that can serve as interaction modules with Hsp90 and Hsp70 (4). STI1 helps to drive the sequential steps involved in the Hsp90 chaperone machinery (5) and regulates the ATPase activity of Hsp90 (6, 7). STI1 is also secreted by distinct cells (8–12), using a noncanonical mechanism involving extracellular vesicles (11). Secreted STI1 can activate multiple signaling pathways in distinct cell types (8–10, 13–18).Elimination of STI1 in yeast sensitizes cells to Hsp90 inhibitors, but it is not by itself lethal (19). STI1 can also be eliminated in C. elegans, although it results in decreased life span (20). In contrast, STI1 mutant mice do not survive E10.5 and present several morphological defects, owing to decreased levels of several Hsp90-client proteins (21). Mouse embryonic fibroblasts obtained from STI1-deficient embryos also fail to thrive and present increased levels of the DNA damage marker γ-H2AX, suggestive of increased cellular stress (21). Hence, in mammals STI1 seems to play additional roles in cellular survival that are not yet fully understood.STI1 is abundantly expressed in the cytoplasm of cells, but can also be found in the Golgi (22), in vesicles and in multivesicular bodies (11). Moreover, this cochaperone has been shown to shuttle between the cytoplasm and the nucleus in cell lines (23). Cellular stress, arrest in G1/S phase of the cell cycle and phosphorylation are factors that seem to regulate STI1 nuclear localization (23, 24). Presumably nuclear STI1 can regulate chaperone activity, but whether it can interact with nuclear proteins is unknown.Previous experiments using cell lines have shown that knockdown of STI1 increases susceptibility of cells to irradiation (25). Whether changes in STI1 levels in primary differentiated cells, such as astrocytes, may affect their response to irradiation stress is unknown. This is of interest, as astrocytes, which can give rise to distinct tumor cells, are highly radioresistant (26). Indeed, astrocytes have a noncanonical DNA damage response (DDR) to irradiation (26). Here we show that STI1 undergoes nuclear translocation in astrocytes after γ-radiation-induced DNA damage. Moreover, astrocytes haploinsufficient for STI1 are more susceptible to cell death induced by irradiation. To understand potential mechanisms involved with STI1 nuclear retention, we have performed yeast-two hybrid screenings to identify STI1 nuclear partners. We identified protein inhibitor of activated STAT (PIAS1) as a direct interactor of STI1 and provide evidence that it acts as a small ubiquitin-like modifier (SUMO) E3 ligase for STI1. We show this interaction is involved with STI1 nuclear retention after irradiation. Interestingly, tissue microarray analysis demonstrated that higher PIAS1 levels are found in glioblastoma multiforme (GBM) when compared with non-neoplastic tissue. Furthermore, we uncovered a positive relationship between increased PIAS1 expression in GBMs and augmented STI1 nuclear localization. Our results reveal a novel mechanism by which increased expression of PIAS1, as observed in GBM, can increase the retention of nuclear STI1, a critical regulator of the chaperone machinery.     

Substrate Binding Promotes Formation of the Skp1-Cul1-Fbxl3 (SCFFbxl3) Protein Complex

The Skp1–Cul1–F-box protein (SCF) complex is one of the most well characterized types of ubiquitin ligase (E3), with the E3 activity of the complex being regulated in part at the level of complex formation. Fbxl3 is an F-box protein that is responsible for the ubiquitylation and consequent degradation of cryptochromes (Crys) and thus regulates oscillation of the circadian clock. Here we show that formation of the SCF^Fbxl3 complex is regulated by substrate binding in vivo. Fbxl3 did not associate with Skp1 and Cul1 to a substantial extent in transfected mammalian cells. Unexpectedly, however, formation of the SCF^Fbxl3 complex was markedly promoted by forced expression of its substrate Cry1 in these cells. A mutant form of Fbxl3 that does not bind to Cry1 was unable to form an SCF complex, suggesting that interaction of Cry1 with Fbxl3 is essential for formation of SCF^Fbxl3. In contrast, recombinant Fbxl3 associated with recombinant Skp1 and Cul1 in vitro even in the absence of recombinant Cry1. Domain-swap analysis revealed that the COOH-terminal leucine-rich repeat domain of Fbxl3 attenuates the interaction of Skp1, suggesting that a yet unknown protein associated with the COOH-terminal domain of Fbxl3 and inhibited SCF complex formation. Our results thus provide important insight into the regulation of both SCF ubiquitin ligase activity and circadian rhythmicity.     

Stress-Induced Nuclear-to-Cytoplasmic Translocation of Cyclin C Promotes Mitochondrial Fission in Yeast

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PAC-1 Activates Procaspase-3 in Vitro through Relief of Zinc-Mediated Inhibition

The direct induction of apoptosis has emerged as a powerful anticancer strategy, and small molecules that either inhibit or activate certain proteins in the apoptotic pathway have great potential as novel chemotherapeutic agents. Central to apoptosis is the activation of the zymogen procaspase-3 to caspase-3. Caspase-3 is the key “executioner” caspase, catalyzing the hydrolysis of a multitude of protein substrates within the cell. Interestingly, procaspase-3 levels are often elevated in cancer cells, suggesting a compound that directly stimulates the activation of procaspase-3 to caspase-3 could selectively induce apoptosis in cancer cells. We recently reported the discovery of a compound, PAC-1, which enhances procaspase-3 activity in vitro and induces apoptotic death in cancer cells in culture and in mouse xenograft models. Described herein is the mechanism by which PAC-1 activates procaspase-3 in vitro. We show that zinc inhibits the enzymatic activity of procaspase-3 and that PAC-1 strongly activates procaspase-3 in buffers that contain zinc. PAC-1 and zinc form a tight complex with one another, with a dissociation constant of approximately 42 nM. The combined data indicate that PAC-1 activates procaspase-3 in vitro by sequestering inhibitory zinc ions, thus allowing procaspase-3 to autoactivate itself to caspase-3. The small-molecule-mediated activation of procaspases has great therapeutic potential and thus this discovery of the in vitro mechanism of action of PAC-1 is critical to the development and optimization of other procaspase-activating compounds.     

SIAH1介导TRB3的泛素化和降解

目的利用酵母回转实验和免疫共沉淀实验验证SIAHI和TRB3之间的相互作用并探讨其功能相关性。方法将全长形式的TRB3基因和SIAH1基因分别克隆入酵母表达载体pDBLeu和pPC86中,共转化至MaV203酵母感受态细胞,验证其相互作用,然后分别构建至真核表达载体pCMV—Myc和pFLAG—CMV-2中,采用免疫共沉淀实验进行进一步验证。通过体内泛素化实验检测SIAH1对TRB3蛋白稳定性及泛素化修饰的影响。结果通过在酵母细胞中的回转实验和HEK293rr细胞中的免疫共沉淀实验证实了TRB3与SIAH1之间的相互作用。通过体内泛素化实验证实了S1AH1介导了TRB3的泛素化修饰和降解。结论证实了TRB3与SIAH1之间的相互作用并发现SIAH1介导了TRB3的泛素化修饰和降解,为TRB3蛋白的功能研究提供了新的线索。     

TRB3 mediates homocysteine-induced inhibition of endothelial cell proliferation

Hyperhomocysteinemia (HHcy) has been shown to induce endothelial dysfunction, an early event in the progression of atherosclerosis. However, the underlying mechanism of endothelial cell injury in HHcy has not been clearly elucidated. In this study, we examined the effect of homocysteine on tribbles‐related protein 3 (TRB3)‐mediated cell‐cycle arrest in human umbilical vein endothelial cells (HUVECs). Treatment of HUVECs with homocysteine (0–250 µmol/L) resulted in inhibition of cell proliferation assessed by [³H]‐thymidine incorporation into DNA. Homocysteine induced cell‐cycle arrest in the G1 phase by up‐regulating the protein levels of p27^kip1. Under these conditions, homocysteine did not induce endoplasmic reticulum stress. However, homocysteine up‐regulated the expression of TRB3, thus leading to the dephosphorylation of Akt (Thr308). Knock‐down of endogenous TRB3 using siRNA significantly suppressed the inhibitory effect of homocysteine on the proliferation of HUVECs. Homocysteine‐induced TRB3 expression was mediated by the cAMP/cAMP response element‐binding protein (CREB) pathway. These results demonstrate that TRB3 is a critical molecule in the homocysteine‐mediated cell‐cycle arrest in endothelial cells. J. Cell. Physiol. 226: 2782–2789, 2011. © 2011 Wiley‐Liss, Inc.     

小鼠短暂前脑缺血海马中半胱天冬酶-３酶原表达的变化

通过测定脑缺血再灌注时海马中半胱天冬酶-3酶原(procaspase-3)的表达变化, 从细胞凋亡的角度探讨脑缺血再灌注损伤的分子生物学机制及procaspase-3的活化机制.将C57BL/6N小鼠随机分为假手术组(正常对照组)、缺血再灌注组(I/R组), 后者夹闭双侧颈总动脉20 min后再通血流, 建立前脑缺血再灌注模型, 分别于再灌注6 h、12 h、24 h和48 h取海马.采用蛋白免疫印迹(Western blotting)方法检测海马中procaspase-3的表达变化.结果显示, 12 h I/R及24hI/R组海马中总procaspase-3水平与假手术组相比有明显升高, 且差异有统计学意义(P<0.05),24 h I/R组海马中去磷酸化水平与假手术组相比有明显升高, 且差异有统计学意义(P<0.05),而各组procaspase-3磷酸化水平与假手术组相比差异无统计学意义.结果提示, 脑缺血再灌注损伤诱发procaspase-3表达增加,其中procaspase-3去磷酸化水平高明显, 提示脑缺血再灌注损伤可能诱发procaspase-3去磷酸化, 继而促进procaspase-3转化为活性形式.