Large-scale Production And Purification Of Recombinant Protein From An Insect Cell/baculovirus System In Erlenmeyer Flasks: Application To The Chicken Poly(adp-ribose) Polymerase Catalytic Domain

A simple and inexpensive shaker/Erlenmeyer flask system for large-scale cultivation of insect cells is described and compared to a commercial spinner system. On the basis of maximum cell density, average population doubling time and overproduction of recombinant protein, a better result was obtained with a simpler and less expensive bioreactor consisting of Erlenmeyer flasks and an ordinary shaker waterbath. Routinely, about 90 mg of pure poly(ADP-ribose) polymerase catalytic domain was obtained for a total of 3 × 109 infected cells in three liters of culture.308923928Althaus, F.R., Richter, C., ADP-ribosylation of proteins. Enzymology and biological significance (1987) Molecular Biology, Biochemistry and Biophysics, 37, pp. 1-126Lautier, D., Lagueaux, J., Ménard, L., Poirier, G.C., Molecular and biochemical features of poly(ADP-ribose) metabolism (1993) Molecular and Cellular Biochemistry, 122, pp. 171-193Satoh, M.S., Lindahl, T., Role of poly(ADP-ribose) formation in DNA repair (1992) Nature, 356, pp. 356-358Molinete, M., Vermeulen, W., Bürkle, A., Ménissier-de Murcia, J., Küpper, J., Hoeijmakers, J., De Murcia, G., Overproduction of the poly(ADP-ribose) polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells (1993) EMBO Journal, 5, pp. 2109-2117Masson, M., Rolli, V., Dantzer, F., Trucco, C., Schrieber, V., Fribourg, S., Molinete, M., De Murcia, G., Poly(ADP-ribose) polymerase: Structure-function relationship (1995) Biochimie, 77, pp. 456-461Suto, M.L., Suto, M.S., Inhibitors of poly(ADP-ribose) polymerase (ADPRP): Potential chemotherapeutic agents (1991) Drugs of the Future, 16, pp. 723-739Giner, H., Simonin, F., De Murcia, G., Ménissier-de Murcia, J., Overproduction and large-scale purification of the human poly(ADP-ribose) polymerase using a baculovirus expression system (1992) Gene, 114, pp. 279-283Simonin, F., Höfferer, L., Panzeter, P., Muller, S., De Murcia, G., Althaus, F.R., The carboxy-terminal domain of human poly(ADP-ribose) polymerase: Overproduction in Escherichia coli, large scale purification and characterization (1993) Journal of Molecular Biology, 268, pp. 13454-13461Murhammer, D.W., Review and patents literature. The use of insect cell cultures for recombinant protein synthesis: Engineering aspects (1991) Applied Biochemistry and Biotechnology, 31, pp. 283-310Goosen, M.F.A., Large-scale insect cell culture (1992) Current Opinion in Biotechnology, 3, pp. 99-104Kamen, A.A., Tom, R.L., Caron, A.W., Chavarie, C., Massie, B., Archambault, J., Culture of insect cells in a helical ribbon impeller bioreactor (1991) Biotechnology and Bioengineering, 38, pp. 619-628Van Lier, F.L.J., Van Den End, E.J., Gooijer, C.D., Vlak, J.M., Tramper, J., Continuous production of baculovirus in a cascade insect-cell reactor (1990) Applied Microbiology and Biotechnology, 33, pp. 43-47Power, J., Greenfield, P.F., Nielsen, L., Reid, S., Modelling the growth and protein production by insect cells following infection by a recombinant baculovirus in suspension culture (1992) Cytotechnology, 9, pp. 149-155Stavroulakis, D.A., Kalogerakis, N., Behie, L.A., Latrou, K., Kinetic data for the BM-5 insect line in repeated-batch suspension culture (1991) Biotechnology, 38, pp. 116-126Neutra, R., Levi, B.-Z., Shoham, Y., Optimization of protein-production by the baculovirus expression vector system in shake flasks (1992) Applied Microbiology and Biotechnology, 37, pp. 74-78Luckow, V.A., Summers, M.D., Signals important for high level expression of foreign genes in Autographa californica nuclear polyhedrosis virus expression vectors (1989) Virology, 170, pp. 31-39O'Reily, D.R., Miller, L.K., Luckow, V.A., (1992) Baculovirus Expression System: A Laboratory Manual, , Freeman, New YorkScott, R.L., Blanchard, J.H., Fergusson, C.H.R., Effects of oxygen on recombinant protein production by suspension cultures of Spodoptera frugiperda Sf9 insect cell (1992) Enzyme and Microbial Technology, 14, pp. 798-80

Feedback-regulated poly(ADP-ribosyl)ation by PARP-1 is required for rapid response to DNA damage in living cells

Genome integrity is constantly threatened by DNA lesions arising from numerous exogenous and endogenous sources. Survival depends on immediate recognition of these lesions and rapid recruitment of repair factors. Using laser microirradiation and live cell microscopy we found that the DNA-damage dependent poly(ADP-ribose) polymerases (PARP) PARP-1 and PARP-2 are recruited to DNA damage sites, however, with different kinetics and roles. With specific PARP inhibitors and mutations, we could show that the initial recruitment of PARP-1 is mediated by the DNA-binding domain. PARP-1 activation and localized poly(ADP-ribose) synthesis then generates binding sites for a second wave of PARP-1 recruitment and for the rapid accumulation of the loading platform XRCC1 at repair sites. Further PARP-1 poly(ADP-ribosyl)ation eventually initiates the release of PARP-1. We conclude that feedback regulated recruitment of PARP-1 and concomitant local poly(ADP-ribosyl)ation at DNA lesions amplifies a signal for rapid recruitment of repair factors enabling efficient restoration of genome integrity

Interaction between ATM and PARP-1 in response to DNA damage and sensitization of ATM deficient cells through PARP inhibition

ATM and PARP-1 are two of the most important players in the cell's response to DNA damage. PARP-1 and ATM recognize and bound to both single and double strand DNA breaks in response to different triggers. Here we report that ATM and PARP-1 form a molecular complex in vivo in undamaged cells and this association increases after γ-irradiation. ATM is also modified by PARP-1 during DNA damage. We have also evaluated the impact of PARP-1 absence or inhibition on ATM-kinase activity and have found that while PARP-1 deficient cells display a defective ATM-kinase activity and reduced γ-H2AX foci formation in response to γ-irradiation, PARP inhibition on itself is able to activate ATM-kinase. PARP inhibition induced γ H2AX foci accumulation, in an ATM-dependent manner. Inhibition of PARP also induces DNA double strand breaks which were dependent on the presence of ATM. As consequence ATM deficient cells display an increased sensitivity to PARP inhibition. In summary our results show that while PARP-1 is needed in the response of ATM to gamma irradiation, the inhibition of PARP induces DNA double strand breaks (which are resolved in and ATM-dependent pathway) and activates ATM kinase

Parp1 facilitates alternative NHEJ, whereas Parp2 suppresses IgH/c-myc translocations during immunoglobulin class switch recombination

Immunoglobulin class switch recombination (CSR) is initiated by DNA breaks triggered by activation-induced cytidine deaminase (AID). These breaks activate DNA damage response proteins to promote appropriate repair and long-range recombination. Aberrant processing of these breaks, however, results in decreased CSR and/or increased frequency of illegitimate recombination between the immunoglobulin heavy chain locus and oncogenes like c-myc. Here, we have examined the contribution of the DNA damage sensors Parp1 and Parp2 in the resolution of AID-induced DNA breaks during CSR. We find that although Parp enzymatic activity is induced in an AID-dependent manner during CSR, neither Parp1 nor Parp2 are required for CSR. We find however, that Parp1 favors repair of switch regions through a microhomology-mediated pathway and that Parp2 actively suppresses IgH/c-myc translocations. Thus, we define Parp1 as facilitating alternative end-joining and Parp2 as a novel translocation suppressor during CSR

Molecular mechanism of poly(ADP-ribosyl)ation by PARP1 and identification of lysine residues as ADP-ribose acceptor sites

Poly(ADP-ribose) polymerase 1 (PARP1) synthesizes poly(ADP-ribose) (PAR) using nicotinamide adenine dinucleotide (NAD) as a substrate. Despite intensive research on the cellular functions of PARP1, the molecular mechanism of PAR formation has not been comprehensively understood. In this study, we elucidate the molecular mechanisms of poly(ADP-ribosyl)ation and identify PAR acceptor sites. Generation of different chimera proteins revealed that the amino-terminal domains of PARP1, PARP2 and PARP3 cooperate tightly with their corresponding catalytic domains. The DNA-dependent interaction between the amino-terminal DNA-binding domain and the catalytic domain of PARP1 increased Vmax and decreased the Km for NAD. Furthermore, we show that glutamic acid residues in the auto-modification domain of PARP1 are not required for PAR formation. Instead, we identify individual lysine residues as acceptor sites for ADP-ribosylation. Together, our findings provide novel mechanistic insights into PAR synthesis with significant relevance for the different biological functions of PARP family members

Ataxia telangiectasia mutated (ATM) signaling network is modulated by a novel poly (ADP-ribose) dependent pathway in the early response to DNA-damaging agents

Poly(ADP-ribosyl)ation is a post-translational modification that is instantly stimulated by DNA strand breaks creating a unique signal for the modulation of protein functions in DNA repair and cell cycle checkpoint pathways. Here we report that lack of poly(ADP-ribose) synthesis leads to a compromised response to DNA damage. Deficiency in poly(ADP-ribosyl)ation metabolism induces profound cellular sensitivity to DNA-damaging agents, particularly in cells deficient for the protein kinase ataxia telangiectasia mutated (ATM). At the biochemical level, we examined the significance of poly(ADP-ribose) synthesis on the regulation of early DNA damage-induced signaling cascade initiated by ATM. Using potent PARP inhibitors and PARP-1 knock-out cells, we demonstrate a functional interplay between ATM and poly(ADP-ribose) that is important for the phosphorylation of p53, SMC1, and H2AX. For the first time, we demonstrate a functional and physical interaction between the major DSB signaling kinase, ATM and poly(ADP-ribosyl)ation by PARP-1, a key enzyme of chromatin remodeling. This study suggests that poly(ADP-ribose) might serve as a DNA damage sensory molecule that is critical for early DNA damage signaling

Poly(ADP-ribose) polymerase 1 at the crossroad of metabolic stress and inflammation in aging

Poly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated nuclear protein, which functions as molecular stress sensor. Reactive oxygen species, responsible for the most plausible and currently acceptable global mechanism to explain the aging process, strongly activate the enzymatic activity of PARP1 and the formation of poly(ADP-ribose) (PAR) from NAD+. Consumption of NAD+ links PARP1 to energy metabolism and to a large number of NAD+-dependent enzymes, such as the sirtuins. As transcriptional cofactor for NF-κB-dependent gene expression, PARP1 is also connected to the immune response, which is implicated in almost all age-related or associated diseases. Accordingly, numerous experimental studies have demonstrated the beneficial effects of PARP inhibition for several age-related diseases. This review summarizes recent findings on PARP1 and puts them in the context of metabolic stress and inflammation in aging

The BRCT Domain of PARP-1 Is Required for Immunoglobulin Gene Conversion

During affinity maturation, genomic integrity is maintained through specific targeting of DNA mutations. The DNA damage sensor PARP-1 helps determine whether a DNA lesion results in faithful or mutagenic repair

PolyADP-Ribosylation Is Required for Pronuclear Fusion during Postfertilization in Mice

BACKGROUND: During fertilization, pronuclear envelope breakdown (PNEB) is followed by the mingling of male and female genomes. Dynamic chromatin and protein rearrangements require posttranslational modification (PTM) for the postfertilization development. METHODOLOGY/PRINCIPAL FINDINGS: Inhibition of poly(ADP-ribose) polymerase activity (PARylation) by either PJ-34 or 5-AIQ resulted in developmental arrest of fertilized embryos at the PNEB. PARylation inhibition affects spindle bundle formation and phosphorylation of Erk molecules of metaphase II (MII) unfertilized oocytes. We found a frequent appearance of multiple pronuclei (PN) in the PARylation-inhibited embryos, suggesting defective polymerization of tubulins. Attenuated phosphorylation of lamin A/C by PARylation was detected in the PARylation-inhibited embryos at PNEB. This was associated with sustained localization of heterodomain protein 1 (HP1) at the PN of the one-cell embryos arrested by PARylation inhibition. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that PARylation is required for pronuclear fusion during postfertilization processes. These data further suggest that PARylation regulates protein dynamics essential for the beginning of mouse zygotic development. PARylation and its involving signal-pathways may represent potential targets as contraceptives

Targeting poly(ADP-ribose) polymerase activity for cancer therapy

Poly(ADP-ribosyl)ation is a ubiquitous protein modification found in mammalian cells that modulates many cellular responses, including DNA repair. The poly(ADP-ribose) polymerase (PARP) family catalyze the formation and addition onto proteins of negatively charged ADP-ribose polymers synthesized from NAD+. The absence of PARP-1 and PARP-2, both of which are activated by DNA damage, results in hypersensitivity to ionizing radiation and alkylating agents. PARP inhibitors that compete with NAD+ at the enzyme’s activity site are effective chemo- and radiopotentiation agents and, in BRCA-deficient tumors, can be used as single-agent therapies acting through the principle of synthetic lethality. Through extensive drug-development programs, third-generation inhibitors have now entered clinical trials and are showing great promise. However, both PARP-1 and PARP-2 are not only involved in DNA repair but also in transcription regulation, chromatin modification, and cellular homeostasis. The impact on these processes of PARP inhibition on long-term therapeutic responses needs to be investigated