多ADP-核糖聚合酶家族

分离鉴定多功能的核基质蛋白及核基质结合蛋白是目前核基质研究的一个重要领域。通过与转录因子、核基质结合元件以及DNA间相互作用,核基质结合蛋白在DNA复制、转录、加工修饰等细胞内事件中起着支持和调节的作用。多ADP-核糖聚合酶[poly(ADP—ribose)polymerase,PARP]是一种高度保守的核基质结合蛋白,在多种活动例如基因组损伤修复、细胞凋亡、信号转导、基因表达调控中都发挥着调节的功能。PARP的潜在生物学功能已越来越引起国内外研究人员的关注。     

Dephosphorylation, Proteolysis, and Reduced Activity of Poly(A) Polymerase Associated with U937 Cell Apoptosis

The apoptotic trend of the widely used cell lines HL-60, U937, HeLa, Molt-3, and K562 has been found to be accompanied and reversibly related with Poly(A) polymerase (PAP; EC 2.7.7.19) activity levels. Moreover, variations in the pattern of multiple enzyme forms are revealed, being most prominent in apoptosis-prone cell lines, HL-60 and U937. Furthermore, in heat-shocked or nutrient-deprived apoptotic U937 Percoll-fractionated subpopulations, PAP lower mobility phosphorylated forms of 106 and 100 kDa as well as enzyme activity were progressively reduced along with the appearance of higher than 80 kDa mobility species. The kinetics of these alterations (dephosphorylation, proteolysis, and activity) coincided with the appearance of DNA fragmentation. In fact, PAP dephosphorylation appears to precede the appearance of DNA fragmentation. In addition, inhibition of PAP dephosphorylation, proteolysis, and decrease in its activity were tightly coupled with the concomitant prevention of apoptosis. This novel finding yields information on a possible involvement of PAP in cell commitment and execution to apoptosis.     

Inhibition of poly(ADP-ribose) Polymerase Interferes with Trypanosoma cruzi Infection and Proliferation of the Parasite

Poly(ADP-ribosylation) is a post-translational covalent modification of proteins catalyzed by a family of enzymes termed poly(ADP-ribose) polymerases (PARPs). In the human genome, 17 different genes have been identified that encode members of the PARP superfamily. Poly (ADP-ribose) metabolism plays a role in a wide range of biological processes. In Trypanosoma cruzi, PARP enzyme appears to play a role in DNA repair mechanisms and may also be involved in controlling the different phases of cell growth. Here we describe the identification of potent inhibitors for T. cruzi PARP with a fluorescence-based activity assay. The inhibitors were also tested on T. cruzi epimastigotes, showing that they reduced ADP-ribose polymer formation in vivo. Notably, the identified inhibitors are able to reduce the growth rate of T. cruzi epimastigotes. The best inhibitor, Olaparib, is effective at nanomolar concentrations, making it an efficient chemical tool for chacterization of ADP-ribose metabolism in T. cruzi. PARP inhibition also decreases drastically the amount of amastigotes but interestingly has no effect on the amount of trypomastigotes in the cell culture. Knocking down human PARP-1 decreases both the amount of amastigotes and trypomastigotes in cell culture, indicating that the effect would be mainly due to inhibition of human PARP-1. The result suggests that the inhibition of PARP could be a potential way to interfere with T. cruzi infection.     

Purification and Properties of Poly(A) Polymerase from Vigna unguiculata

Poly(A) polymerase was purified from germinating Vigna unguiculataseeds by successive column chromatography on phosphocellulose,Toyopearl HW-55S, heparin-Sepharose and TSKgel phenyl-5PW, whichyielded two activity fractions. The first fraction was purifiedas a single polypeptide with a mol wt of 63,000 as estimatedby SDS-PAGE. The enzyme activity was highly specific for ATPand required Mn²⁺ ion; an ATP-Mn complex may be the actual substrate.The polymerization reaction required a primer, with varioustypes of RNAs, poly(A) as well as dinucleoside phosphates having3'—OH, serving as efficient primers. The two forms ofthe enzyme had very similar properties with respect to divalentcation requirement and dependency on ion strength, but theyshowed some difference in primer preference. (Received March 4, 1988; Accepted May 2, 1988)     

Poly(Adenylate) Polymerase Activity of Rat Brain

Abstract: The poly(adenylate)[poly(A)] polymerase of rat brain, as in rat liver, is located primarily in the nuclear sap when nuclei are prepared under hypertonic conditions. The enzyme can be released from nuclei in two forms. Form I is prepared by gentle incubation of nuclei at 0°C in hypotonic buffer. It has a Mn optimum of 0.6 mM and a pH optimum between 8 and 9. The ATP concentration curve plateaus at 0.2 mM. The optimal poly(A) primer concentration is 600 μg/ml, which is three times higher than that for the enzyme similarly prepared from liver. The time course of the reaction for the form I enzyme is increasing over the first 40 min and becomes nearly linear thereafter. Form I is not stimulated by either calcium or cyclic nucleotides, but is inhibited by polyamines, pyrophosphate, and high concentrations of GTP. Form II enzyme is prepared by homogenization of nuclei in hypotonic buffer. It has the same ATP and poly(A) optima as the form I enzyme but displays linear kinetics over a 60-min time course. It is slightly stimulated by cGMP and cAMP and strongly inhibited by spermine, sodium pyrophosphate, and high concentrations of GTP.     

Crystal Structure of Human Poly(A) Polymerase Gamma Reveals a Conserved Catalytic Core for Canonical Poly(A) Polymerases

In eukaryotes, the poly(A) tail added at the 3′ end of an mRNA precursor is essential for the regulation of mRNA stability and the initiation of translation. Poly(A) polymerase (PAP) is the enzyme that catalyzes the poly(A) addition reaction. Multiple isoforms of PAP have been identified in vertebrates, which originate from gene duplication, alternative splicing or post-translational modifications. The complexity of PAP isoforms suggests that they might play different roles in the cell. Phylogenetic studies indicate that vertebrate PAPs are grouped into three clades termed α, β and γ, which originated from two gene duplication events. To date, all the available PAP structures are from the PAPα clade. Here, we present the crystal structure of the first representative of the PAPγ clade, human PAPγ bound to cordycepin triphosphate (3′dATP) and Ca^2 +. The structure revealed that PAPγ closely resembles its PAPα ortholog. An analysis of residue conservation reveals a conserved catalytic binding pocket, whereas residues at the surface of the polymerase are more divergent.     

Targeting Poly (ADP-Ribose) Polymerase Partially Contributes to Bufalin-Induced Cell Death in Multiple Myeloma Cells

Despite recent pharmaceutical advancements in therapeutic drugs, multiple myeloma (MM) remains an incurable disease. Recently, ploy(ADP-ribose) polymerase 1 (PARP1) has been shown as a potentially promising target for MM therapy. A previous report suggested bufalin, a component of traditional Chinese medicine (“Chan Su”), might target PARP1. However, this hypothesis has not been verified. We here showed that bufalin could inhibit PARP1 activity in vitro and reduce DNA–damage-induced poly(ADP-ribosyl)ation in MM cells. Molecular docking analysis revealed that the active site of bufalin interaction is within the catalytic domain of PAPR1. Thus, PARP1 is a putative target of bufalin. Furthermore, we showed, for the first time that the proliferation of MM cell lines (NCI-H929, U266, RPMI8226 and MM.1S) and primary CD138⁺ MM cells could be inhibited by bufalin, mainly via apoptosis and G₂-M phase cell cycle arrest. MM cell apoptosis was confirmed by apoptotic cell morphology, Annexin-V positive cells, and the caspase3 activation. We further evaluated the role of PARP1 in bufalin-induced apoptosis, discovering that PARP1 overexpression partially suppressed bufalin-induced cell death. Moreover, bufalin can act as chemosensitizer to enhance the cell growth-inhibitory effects of topotecan, camptothecin, etoposide and vorinostat in MM cells. Collectively, our data suggest that bufalin is a novel PARP1 inhibitor and a potentially promising therapeutic agent against MM alone or in combination with other drugs.     

Poly(ADP-ribose) Polymerase Activation and Cell Injury in the Course of Rat Heart Heterotopic Transplantation

Free radicals and other reactive species generated during reperfusion of ischemic tissues may cause DNA damage and, consequently, the activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP). An excessive PARP activation may result in a depletion of intracellular NAD + and ATP, hence cell suffering and, ultimately, cell death. The present study is aimed at clarifying the role of PARP in a heart transplantation procedure and the contribution of myocyte necrosis and/or apoptosis to this process. In our experimental model, rat heart subjected to heterotopic transplantation, low temperature global ischemia (2 h) was followed by an in vivo reperfusion (30 or 60 &#117 min). Under these conditions clear signs of oxidative stress, such as lipoperoxidation and DNA strand breaks, were evident. In addition to a marked activation, accompanied by a significant NAD + and ATP depletion, PARP protein levels significantly increased after 60 &#117 min of reperfusion. Ultrastructural analysis showed nuclear clearings, intracellular oedema and plasma membrane discontinuity. Other relevant observations were the absence of typical signs of apoptosis like caspase-3 activation and PARP cleavage, random DNA fragmentation, rise in serum levels of heart damage markers. Our results suggest that during heart transplantation, the activation of PARP, causing energy depletion, results in myocardial cell injury whose dominant feature, at least in our experimental model, is necrosis rather than apoptosis.     

Poly(A) Tail Length Is Controlled by the Nuclear Poly(A)-binding Protein Regulating the Interaction between Poly(A) Polymerase and the Cleavage and Polyadenylation Specificity Factor

Poly(A) tails of mRNAs are synthesized in the cell nucleus with a defined length, ∼250 nucleotides in mammalian cells. The same type of length control is seen in an in vitro polyadenylation system reconstituted from three proteins: poly(A) polymerase, cleavage and polyadenylation specificity factor (CPSF), and the nuclear poly(A)-binding protein (PABPN1). CPSF, binding the polyadenylation signal AAUAAA, and PABPN1, binding the growing poly(A) tail, cooperatively stimulate poly(A) polymerase such that a complete poly(A) tail is synthesized in one processive event, which terminates at a length of ∼250 nucleotides. We report that PABPN1 is required to restrict CPSF binding to the AAUAAA sequence and to permit the stimulation of poly(A) polymerase by AAUAAA-bound CPSF to be maintained throughout the elongation reaction. The stimulation by CPSF is disrupted when the poly(A) tail has reached a length of ∼250 nucleotides, and this terminates processive elongation. PABPN1 measures the length of the tail and is responsible for disrupting the CPSF-poly(A) polymerase interaction.The poly(A) tails present at the 3′ end of almost all eukaryotic mRNAs have two major functions. The first function is in the control of mRNA decay; degradation of the poly(A) tail by a 3′ exonuclease (deadenylation) is the first step in both of the two main pathways of mRNA decay, and the completion of deadenylation triggers the second step, either cap hydrolysis or further 3′–5′ degradation. Because the rate of deadenylation is governed by sequence elements in the mRNA, it is specific for each mRNA species and serves as a major determinant of mRNA half-life (1–3). Obviously, a control of mRNA stability by the rate of deadenylation requires a defined poly(A) length as a starting point. The second function of the poly(A) tail is in the initiation of translation; the cytoplasmic poly(A)-binding protein associated with the poly(A) tail promotes the initiation of translation by an interaction with the initiation factor eIF4G and probably through additional mechanisms (4–7). In this process, poly(A) tail length can also be important. For example, gene regulation during oocyte maturation and early embryonic development of animals depends on translational regulation of maternal mRNAs, and changes in poly(A) tail lengths of specific mRNAs, determined both by deadenylation and by regulated cytoplasmic poly(A) extension, play a major role in this translational regulation. Long poly(A) tails favor translation, whereas a shortening of the tail promotes translational inactivation of the message (8, 9). Similar mechanisms seem to operate in neurons (10, 11) and possibly in other somatic cells (12).Because the length of the poly(A) tail is important for its function, it is not surprising that poly(A) tails are generally synthesized with a defined length, which is species-specific, ∼70–90 nucleotides in Saccharomyces cerevisiae (13, 14) and ∼250 nucleotides in mammalian cells (15). Subtle differences between newly made poly(A) tails of different mRNAs have been described (13), and there is even a class of mRNAs that never receives more than an oligo(A) tail (16, 17). However, the heterogeneous length distribution seen in the steady-state mRNA population is the result of cytoplasmic shortening starting from a relatively well defined initial tail length; heterogeneity of tail length reflects age differences of the mRNA molecules. The oligo(A) tails present on inactive mRNAs in oocytes or embryos are also generated by shortening of full-length tails made in the cell nucleus (18).The poly(A) tail is added during 3′ end processing of mRNA precursors in the cell nucleus (19–21). This reaction consists of two steps: an endonucleolytic cleavage followed by the addition of the poly(A) tail to the upstream cleavage product. Whereas a large protein machinery of some 20 or more polypeptides (22) is required for the cleavage reaction, subsequent polyadenylation has much simpler protein requirements. In the mammalian system, it can be reconstituted from three proteins: poly(A) polymerase, the enzyme catalyzing primer-dependent polymerization of AMP using ATP as a precursor (23–25); the cleavage and polyadenylation specificity factor (CPSF),⁶ which binds the cleavage and polyadenylation signal AAUAAA (26, 27); and the nuclear poly(A)-binding protein (PABPN1), which binds the growing poly(A) tail (28, 29). Note that PABPN1 is distinct from the family of cytoplasmic poly(A)-binding proteins (30). Roles of poly(A) polymerase and CPSF in polyadenylation in vivo have been most clearly demonstrated by genetic analysis of the orthologues in S. cerevisiae (21, 31). PABPN1 has no functional orthologue in budding yeast (32); its function in polyadenylation has been confirmed in mammalian cells (33) and in Drosophila (34).Whereas PABPN1 and poly(A) polymerase are monomeric proteins, CPSF is a hetero-oligomer, which has not yet been reconstituted from recombinant proteins (22, 26, 35–40). Poly(A) polymerase on its own is barely active because of a low affinity for its RNA substrate and thus acts distributively, i.e. it dissociates from the RNA after each polymerization step, and presumably often before it has incorporated any nucleotide; the enzyme also has no significant sequence specificity and will elongate any RNA with a free 3′ OH (24). Both CPSF and PABPN1 enhance the activity of the polymerase by recruiting the enzyme to its substrate through direct interactions (38, 41). Sequence specificity of poly(A) addition reflects the RNA binding specificities of the two stimulatory factors: CPSF recruits the polymerase to RNAs containing the AAUAAA sequence in the vicinity of their 3′ ends (24, 42, 43), and PABPN1 recruits the enzyme to substrate RNAs carrying a terminal oligo(A) tract (29). Each factor alone endows the polymerase with modest processivity, such that it can incorporate maybe two to five nucleotides before dissociating (44). RNAs containing both the AAUAAA sequence and an oligo(A) tail and thus resembling intermediates of the polyadenylation reaction support a cooperative or synergistic stimulation of poly(A) polymerase by both CPSF and PABPN1. Under these conditions, addition of the poly(A) tail occurs in a processive manner, i.e. without intermittent dissociation of the protein complex from its substrate RNA (29, 44).Interestingly, the reconstituted polyadenylation reaction also shows proper length control, generating poly(A) tails of the same length as seen in vivo; tails grow to a relatively well defined length of 250–300 nucleotides in a rapid, processive reaction (29, 44). Length control is due to termination of this processive elongation; extension beyond 250 A residues is largely distributive and therefore slow (45). These kinetics of in vitro poly(A) tail synthesis are fully consistent with the in vivo kinetics derived from pulse-labeling studies (46). In vitro, poly(A) tail elongation rates beyond 250 A residues are similar when either CPSF or PABPN1 or both are present. In other words, substrates with long poly(A) tails no longer support the cooperative stimulation of poly(A) polymerase by both CPSF and PABPN1 that is the basis of processive elongation (45). The termination of processive elongation must be mediated by a change in the RNA-protein complex that remains to be defined. When RNAs carrying poly(A) tails of different lengths are used as substrates for polyadenylation, the tails are always elongated processively to 250 nucleotides, independently of the initial length, whereas extension of a tail of 250 or more nucleotides in length is slow and distributive from the start of the reaction. Thus, poly(A) tail length control is based on some kind of AMP residue counting or length measurement, not on a kinetic mechanism (45).In this paper, we address the two problems outlined above: first, how does the polyadenylation complex change to terminate processive poly(A) tail elongation, and second, how is the length of the tail measured? We provide evidence that PABPN1 is the active component in the mechanism of length control. The protein promotes the interaction between CPSF and poly(A) polymerase when bound to a short poly(A) tail. PABPN1 no longer promotes or even actively disrupts this interaction when bound to a poly(A) tail of 250 nucleotides or longer and thereby terminates the cooperative, processive elongation reaction in a poly(A) tail length-dependent manner. Only poly(A) sequences are counted as part of the tail. Because this reflects the binding specificity of PABPN1 and because disruption of the CPSF-poly(A) polymerase interaction requires complete coverage of the poly(A) tail by this protein, PABPN1 is also the protein that measures the length of the tail.     

A Novel and Selective Poly (ADP-Ribose) Polymerase Inhibitor Ameliorates Chemotherapy-Induced Painful Neuropathy

Background

Chemotherapy-induced neuropathy is the principle dose limiting factor requiring discontinuation of many chemotherapeutic agents, including cisplatin and oxaliplatin. About 30 to 40% of patients receiving chemotherapy develop pain and sensory changes. Given that poly (ADP-ribose) polymerase (PARP) inhibition has been shown to provide neuroprotection, the current study was developed to test whether the novel PARP inhibitor compound 4a (analog of ABT-888) would attenuate pain in cisplatin and oxaliplatin-induced neuropathy in mice.

Results

An established chemotherapy-induced painful neuropathy model of two weekly cycles of 10 intraperitoneal (i.p.) injections separated by 5 days rest was used to examine the therapeutic potential of the PARP inhibitor compound 4a. Behavioral testing using von Frey, paw radiant heat, cold plate, and exploratory behaviors were taken at baseline, and followed by testing at 3, 6, and 8 weeks from the beginning of drug treatment.

Conclusion

Cisplatin-treated mice developed heat hyperalgesia and mechanical allodynia while oxaliplatin-treated mice exhibited cold hyperalgesia and mechanical allodynia. Co-administration of 50 mg/kg or 25 mg/kg compound 4a with platinum regimen, attenuated cisplatin-induced heat hyperalgesia and mechanical allodynia in a dose dependent manner. Similarly, co-administration of 50 mg/kg compound 4a attenuated oxaliplatin-induced cold hyperalgesia and mechanical allodynia. These data indicate that administration of a novel PARP inhibitor may have important applications as a therapeutic agent for human chemotherapy-induced painful neuropathy.     

Nitric Oxide Interferes with Islet Cell Zinc Homeostasis

Zinc is crucial for the biosynthesis, storage, and secretion of insulin in pancreatic islet cells. We have previously presented evidence that NO interferes with cellular Zn(2+) homeostasis and we therefore investigated the influence of chronic NO exposure on the labile islet cell Zn(2+) content. A strong fluorescence activity in a large islet cell subpopulation was found after staining with the Zn(2+)-specific fluorophore Zinquin. Culture for 24 h in the presence of nontoxic concentrations of the slow-releasing NO donor DETA/NO resulted in a significantly reduced Zn(2+)-dependent fluorescence. This appears to be islet specific as in endothelial cells DETA/NO exposure enhanced the Zn(2+)-dependent fluorescence activity in a concentration-dependent manner. These results suggest that NO interferes with cellular Zn(2+) homeostasis, which in islet cells is crucial for proper hormone delivery and thus special cell function.     

Natural Ageing: Poly(A) Polymerase in Germinating Embryos of Triticum durum Wheat

The viability of seeds is associated with ageing and storageconditions. A loss of viability is accompanied by slow germination,reduced growth, and a decline in protein and poly(A)⁺RNA synthesis.This paper reports on the activity of poly(A) polymerase indry and germinating embryos of Triticum durum Desf. cv. Cappellicaryopses of different ages and viability. The enzyme was presentas a single form during ageing and germination. The poly(A)polymerase was active at decreasing levels in all aged dry embryos,in parallel with loss of viability. Its activity strongly increasedduring the germination only in viable embryos. The observedincrease was due to de novo synthesis of the enzyme. Poly(A)polymerase synthesis was low during germination of less viableembryos and absent in older ones. Reduced poly(A) polymeraseactivity in dry or germinated wheat embryos may cause a shorteningof poly(A) chains in vitro and a decline in poly(A)⁺RNA synthesis.Copyright1995, 1999 Academic Press Triticum durum Desf. cv. Cappelli, wheat, embryo, natural ageing, poly(A) polymerase     

Possible Involvement of Poly(ADP-Ribosyl) Polymerase in Triggering Stress-Induced Apoptosis

U937 human myeloid leukemia cells respond to mild treatment with hydrogen peroxide and hyperthermia by undergoing apoptosis, an active mode of cell suicide. Higher concentrations of hydrogen peroxide, or longer incubation at the hyperthermic temperature, change the mode of cell death from apoptosis to the passive necrosis. Stress treatments cause a severe drop in the intracellular NAD concentration. 3-Aminobenzamide (3-ABA), a specific inhibitor of poly(ADP-ribosyl) polymerase (PARP), a nuclear enzyme which is activated by breaks in DNA to catabolize intracellular NAD, is capable of relieving such a drop. This suggests that breaks in DNA have been induced by both oxidative stress and heat shock, thereby activating PARP. Upon stress, NAD concentration has a first initial sharp drop; then, for mild stress treatments, it recovers, just when apoptosis begins to be detectable (8 h of recovery). At 20 h, when the apoptotic ladder-like pattern of DNA is visible, NAD concentration has dropped again, probably because of a second PARP activation due to the extensive DNA degradation that accompanies apoptosis. The presence of 3-ABA, concomitantly with the preservation of the intracellular NAD content, reduces the extent of apoptosis upon oxidative stress and strongly enhances cell survival, thus suggesting a role for PARP in triggering stress-induced apoptosis. All apoptotic U937 cells have a reduced NAD content, independently of the inducing agent; however, upon treatments which do not cause immediate DNA breaks, the drop in NAD concentration occurs only after the apoptotic ladder is detectable and can be ascribed to the activation of PARP by the free ends of DNA formed during the endonucleolitic degradation. Moreover, in these instances the inhibition of PARP, although effective in blocking the drop in NAD concentration, has no effect on apoptosis, thus being only circumstantial.