Nanosecond pulsed electric fields induce poly(ADP-ribose) formation and non-apoptotic cell death in HeLa S3 cells

Nanosecond pulsed electric fields (nsPEFs) have recently gained attention as effective cancer therapy owing to their potency for cell death induction. Previous studies have shown that apoptosis is a predominant mode of nsPEF-induced cell death in several cell lines, such as Jurkat cells. In this study, we analyzed molecular mechanisms for cell death induced by nsPEFs. When nsPEFs were applied to Jurkat cells, apoptosis was readily induced. Next, we used HeLa S3 cells and analyzed apoptotic events. Contrary to our expectation, nsPEF-exposed HeLa S3 cells exhibited no molecular signs of apoptosis execution. Instead, nsPEFs induced the formation of poly(ADP-ribose) (PAR), a hallmark of necrosis. PAR formation occurred concurrently with a decrease in cell viability, supporting implications of nsPEF-induced PAR formation for cell death. Necrotic PAR formation is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1), and PARP-1 in apoptotic cells is inactivated by caspase-mediated proteolysis. Consistently, we observed intact and cleaved forms of PARP-1 in nsPEF-exposed and UV-irradiated cells, respectively. Taken together, nsPEFs induce two distinct modes of cell death in a cell type-specific manner, and HeLa S3 cells show PAR-associated non-apoptotic cell death in response to nsPEFs.     

Zinc binding catalytic domain of human tankyrase 1

Tankyrases are recently discovered proteins implicated in many important functions in the cell including telomere homeostasis and mitosis. Tankyrase modulates the activity of target proteins through poly(ADP-ribosyl)ation, and here we report the structure of the catalytic poly(ADP-ribose) polymerase (PARP) domain of human tankyrase 1. This is the first structure of a PARP domain from the tankyrase subfamily. The present structure reveals that tankyrases contain a short zinc-binding motif, which has not been predicted. Tankyrase activity contributes to telomere elongation observed in various cancer cells and tankyrase inhibition has been suggested as a potential route for cancer therapy. In comparison with other PARPs, significant structural differences are observed in the regions lining the substrate-binding site of tankyrase 1. These findings will be of great value to facilitate structure-based design of selective PARP inhibitors, in general, and tankyrase inhibitors, in particular.     

Effect of double-strand break DNA sequence on the PARP-1 NHEJ pathway

Efficient repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity. In mammalian cells, DSBs are preferentially repaired by non-homologous end-joining (NHEJ). We have previously described a new DSBs microhomology end-joining pathway depending on PARP-1 and the XRCC1/DNA ligase III complex. In this study we analysed, with recombinant proteins and protein extracts, the effect of DSB end sequences: (i) on the DSB synapsis activity; (ii) on the end-joining activity. We report that PARP-1 DSB synapsis activity is independent of the DSB sequence and could be detected with non-complementary DSBs. We demonstrate also that the efficiency of DSBs repair by PARP-1 NHEJ is strongly dependent on the presence of G:C base pairs at microhomology termini. These results highlight a new role of the PARP-1 protein on the synapsis of DSBs and could explain why the PARP-1 NHEJ pathway is strongly dependent on the DSBs microhomology sequence.     

Discovery of quinazolinone and quinoxaline derivatives as potent and selective poly(ADP-ribose) polymerase-1/2 inhibitors

Two classes of quinazolinone derivatives and quinoxaline derivatives were identified as potent and selective poly(ADP-ribose) polymerase-1 and 2 (PARP-1) and (PARP-2) inhibitors, respectively. In PARP enzyme assays using recombinant PARP-1 and PARP-2, quinazolinone derivatives displayed relatively high selectivity for PARP-1 and quinoxaline derivatives showed superior selectivity for PARP-2. SBDD analysis via a combination of X-ray structural study and homology modeling suggested distinct interactions of inhibitors with PARP-1 and PARP-2. These findings provide a new structural framework for the design of selective inhibitors for PARP-1 and PARP-2.     

Calmodulin Disrupts the Structure of the HIV-1 MA Protein

The MA protein from HIV-1 is a small, multifunctional protein responsible for regulating various stages of the viral replication cycle. To achieve its diverse tasks, MA interacts with host cell proteins and it has been reported that one of these is the ubiquitous calcium-sensing calmodulin (CaM), which is up-regulated upon HIV-1 infection. The nature of the CaM-MA interaction has been the subject of structural studies, using peptides based on the MA sequence, that have led to conflicting conclusions. The results presented here show that CaM binds intact MA with 1:1 stoichiometry in a Ca²⁺-dependent manner and that the complex adopts a highly extended conformation in solution as revealed by small-angle X-ray scattering. Alterations in tryptophan fluorescence suggest that the two buried tryptophans (W16 and W36) located in the first two alpha-helices of MA mediate the CaM interaction. Major chemical shift changes occur in the NMR spectrum of MA upon complex formation, whereas chemical shift changes in the CaM spectrum are quite modest and are assigned to residues within the normal target protein-binding hydrophobic clefts of CaM. The NMR data indicate that CaM binds MA via its N- and C-terminal lobes and induces a dramatic conformational change involving a significant loss of secondary and tertiary structure within MA. Circular dichroism experiments suggest that MA loses ∼ 20% of its α-helical content upon CaM binding. Thus, CaM binding is expected to impact upon the accessibility of interaction sites within MA that are involved in its various functions.     

The three-dimensional structure of VIM-2, a Zn-beta-lactamase from Pseudomonas aeruginosa in its reduced and oxidised form

The crystal structures of the universally widespread metallo-β-lactamase (MBL) Verona integron-encoded MBL (VIM)-2 from Pseudomonas aeruginosa have been solved in their native form as well as in an unexpected oxidised form. This carbapenem-hydrolysing enzyme belongs to the so-called B1 subfamily of MBLs and shares the folding of αβ/βα sandwich, consisting of a core of β-sheet surrounded by α-helices. Surprisingly, it showed a high tendency to be strongly oxidised at the catalytic cysteine located in the Cys site, Cys221, which, in the oxidised structure, becomes a cysteinesulfonic residue. Its native structure was obtained only in the presence of Tris(2-carboxyethyl)phosphine. This oxidation might be a consequence of a lower affinity for the second Zn located in the Cys site that would also explain the observed susceptibility of VIM-2 to chelating agents. This modification, if present in nature, might play a role in catalytic down-regulation. Comparison between native and oxidised VIM-2 and a predicted model of VIM-1 (which shows one residue different in the Cys site compared with VIM-2) is performed to explain the different activities and antibiotic specificities.     

Control of RecBCD Enzyme Activity by DNA Binding- and Chi Hotspot-Dependent Conformational Changes

Faithful repair of DNA double-strand breaks by homologous recombination is crucial to maintain functional genomes. The major Escherichia coli pathway of DNA break repair requires RecBCD enzyme, a complex protein machine with multiple activities. Upon encountering a Chi recombination hotspot (5′ GCTGGTGG 3′) during DNA unwinding, RecBCD's unwinding, nuclease, and RecA-loading activities change dramatically, but the physical basis for these changes is unknown. Here, we identify, during RecBCD's DNA unwinding, two Chi-stimulated conformational changes involving RecC. One produced a marked, long-lasting, Chi-dependent increase in protease sensitivity of a small patch, near the Chi recognition domain, on the solvent-exposed RecC surface. The other change was identified by crosslinking of an artificial amino acid inserted in this RecC patch to RecB. Small-angle X-ray scattering analysis confirmed a major conformational change upon binding of DNA to the enzyme and is consistent with these two changes. We propose that, upon DNA binding, the RecB nuclease domain swings from one side of RecC to the other; when RecBCD encounters Chi, the nuclease domain returns to its initial position determined by crystallography, where it nicks DNA exiting from RecC and loads RecA onto the newly generated 3′-ended single-stranded DNA during continued unwinding; a crevice between RecB and RecC increasingly narrows during these steps. This model provides a physical basis for the intramolecular “signal transduction” from Chi to RecC to RecD to RecB inferred previously from genetic and enzymatic analyses, and it accounts for the enzymatic changes that accompany Chi's stimulation of recombination.     

Subtle Interplay between Synaptotagmin and Complexin Binding to the SNARE Complex

Ca^2 +-triggered neurotransmitter release depends on the formation of SNARE complexes that bring the synaptic vesicle and plasma membranes together, on the Ca^2 + sensor synaptotagmin-1 and on complexins, which play active and inhibitory roles. Release of the complexin inhibitory activity by binding of synaptotagmin-1 to the SNARE complex, causing complexin displacement, was proposed to trigger exocytosis. However, the validity of this model was questioned based on the observation of simultaneous binding of complexin-I and a fragment containing the synaptotagmin-1 C₂ domains (C₂AB) to membrane-anchored SNARE complex. Using diverse biophysical techniques, here we show that C₂AB and complexin-I do not bind to each other but can indeed bind simultaneously to the SNARE complex in solution. Hence, the SNARE complex contains separate binding sites for both proteins. However, total internal reflection fluorescence microscopy experiments show that C₂AB can displace a complexin-I fragment containing its central SNARE-binding helix and an inhibitory helix (Cpx26-83) from membrane-anchored SNARE complex under equilibrium conditions. Interestingly, full-length complexin-I binds more tightly to membrane-anchored SNARE complex than Cpx26-83, and it is not displaced by C₂AB. These results show that interactions of N- and/or C-terminal sequences of complexin-I with the SNARE complex and/or phospholipids increase the affinity of complexin-I for the SNARE complex, hindering dissociation induced by C₂AB. We propose a model whereby binding of synaptotagmin-1 to the SNARE complex directly or indirectly causes a rearrangement of the complexin-I inhibitory helix without inducing complexin-I dissociation, thus relieving the inhibitory activity and enabling cooperation between synaptotagmin-1 and complexin-I in triggering release.     

DNA packaging motor assembly intermediate of bacteriophage phi29

Unraveling the structure and assembly of the DNA packaging ATPases of the tailed double-stranded DNA bacteriophages is integral to understanding the mechanism of DNA translocation. Here, the bacteriophage phi29 packaging ATPase gene product 16 (gp16) was overexpressed in soluble form in Bacillus subtilis (pSAC), purified to near homogeneity, and assembled to the phi29 precursor capsid (prohead) to produce a packaging motor intermediate that was fully active in in vitro DNA packaging. The formation of higher oligomers of the gp16 from monomers was concentration dependent and was characterized by analytical ultracentrifugation, gel filtration, and electron microscopy. The binding of multiple copies of gp16 to the prohead was dependent on the presence of an oligomer of 174- or 120-base prohead RNA (pRNA) fixed to the head-tail connector at the unique portal vertex of the prohead. The use of mutant pRNAs demonstrated that gp16 bound specifically to the A-helix of pRNA, and ribonuclease footprinting of gp16 on pRNA showed that gp16 protected the CC residues of the CCA bulge (residues 18-20) of the A-helix. The binding of gp16 to the prohead/pRNA to constitute the complete and active packaging motor was confirmed by cryo-electron microscopy three-dimensional reconstruction of the prohead/pRNA/gp16 complex. The complex was capable of supercoiling DNA-gp3 as observed previously for gp16 alone; therefore, the binding of gp16 to the prohead, rather than first to DNA-gp3, represents an alternative packaging motor assembly pathway.     

Influence of poly(ADP-ribose) polymerase-1 and its apoptotic 24-kD fragment on repair of DNA duplexes in bovine testis nuclear extract

Effects of exogenous proteins poly(ADP-ribose) polymerase-1 (PARP1) and its 24-kD proteolytic fragment (p24) on the repair of DNA duplexes containing a one nucleotide gap with furan phosphate or phosphate group at the 5'-end of the downstream primer were studied in bovine testis nuclear extract. These damaged DNAs are repaired by the long-patch or short-patch subpathways of base excision repair (BER), respectively. Exogenous PARP1 and p24 decreased the efficiency of gap filling DNA synthesis for both duplexes, but did not influence the ligation stage in the repair of DNA duplex by the short-patch subpathway. Under the same conditions, these proteins inhibited strand-displacement DNA synthesis and decreased the efficiency of the flap endonuclease 1 (FEN1)-catalyzed endonuclease reaction in the nuclear extract, blocking repair of DNA duplex by the long-patch subpathway. Addition of exogenous PARP1 and p24 also reduced the efficiency of UV light crosslinking of extract BER proteins to the photoreactive BER intermediates carrying a nick. Thus, PARP1 and p24 interact with DNA intermediates of BER and compete with nuclear extract proteins for binding to DNA. The interaction of PARP1 and p24 with DNA intermediates of the long-patch subpathway of BER resulted in inhibition of subsequent stages of the repair mediated by this mechanism. However, on recovery of the intact structure of DNA duplex by the short-patch subpathway, PARP1 and p24 suppressed the repair of the one nucleotide gap less efficiently and failed to influence the final stage of the repair, ligation.     

Involvement of polynucleotide kinase in a poly(ADP-ribose) polymerase-1-dependent DNA double-strand breaks rejoining pathway

Efficient DNA double-strand break (DSB) repair is critical for the maintenance of genomic integrity. In mammalian cells, DSBs are preferentially repaired by the non-homologous end-joining pathway relying on DNA-PK activity, but other mechanisms may promote end-joining. We previously described a DSB repair pathway that requires synapsis of DNA ends by poly(ADP-ribose) polymerase-1 (PARP-1) and ligation by the XRCC1/DNA ligase III complex (XL). Here, the repair of non-ligatable DNA ends by this pathway was examined in human cell extracts. The phosphorylation of the 5'-terminal end was shown to represent a limiting step for the repair process. Polynucleotide kinase (hPNK) was identified as the 5'-DNA kinase associated with the PARP-1-dependent end-joining pathway because (i) hPNK was co-recruited to DNA ends together with PARP-1 and XL, (ii) ligation of 5'-OH terminal breaks was compromised in hPNK-depleted extracts and restored upon addition of recombinant hPNK, and (iii) recombinant hPNK was necessary for end-joining of 5'-OH terminal breaks reconstituted with the PARP-1/XL complex. Also, using an assay enabling us to follow the ligation kinetics of each strand of a DSB, we established that the two strands at the junction can be processed and joined independently, so that one strand can be ligated without a ligatable nick on the other strand at the DSB site. Taken together these results reveal functional parallels between the PARP-1 and DNA-PK-dependent end-joining processes.     

The 40 kDa carboxy-terminal domain of poly(ADP-ribose) polymerase-1 forms catalytically competent homo- and heterodimers in the absence of DNA

The 40 kDa carboxy-terminal catalytic domain (CD) of avian poly(ADP-ribose) polymerase (PARP-1) was cloned, expressed in a baculovirus expression system, and purified to homogeneity by affinity chromatography. The purified polypeptide synthesized covalent CD-poly(ADP-ribose) conjugates in the absence of DNA. Electrophoretic analysis of the ADP-ribose chain length distribution generated indicated that recombinant CD was able to catalyze the initiation, elongation, and branching reactions of poly(ADP-ribose) synthesis, although at a 500-fold lower efficiency than wild-type PARP-1. Kinetic evaluation of poly(ADP-ribose) synthesis showed that the enzymatic activities of CD increased for up to 60 minutes in a time-dependent manner. Moreover, the rates of CD auto-poly(ADP-ribosyl)ation increased with second-order kinetics as a function of the protein concentration with either betaNAD(+) or 3'-deoxyNAD(+) as a substrate. Furthermore, the formation of catalytically competent CD-[PARP-1] heterodimers was also observed in specific ultrafiltration experiments. Thus, we conclude that the 40 kDa carboxy terminus of PARP-1 forms a competent catalytic dimer in the absence of DNA, and that its automodification reaction is intermolecular.     

Role of bundle helices in a regulatory crosstalk in the trimeric betaine transporter BetP

The Na⁺-coupled betaine symporter BetP regulates transport activity in response to hyperosmotic stress only in its trimeric state, suggesting a regulatory crosstalk between individual protomers. BetP shares the overall fold of two inverted structurally related five-transmembrane (TM) helix repeats with the sequence-unrelated Na⁺-coupled symporters LeuT, vSGLT, and Mhp1, which are neither trimeric nor regulated in transport activity. Conformational changes characteristic for this transporter fold involve the two first helices of each repeat, which form a four-TM-helix bundle. Here, we identify two ionic networks in BetP located on both sides of the membrane that might be responsible for BetP's unique regulatory behavior by restricting the conformational flexibility of the four-TM-helix bundle. The cytoplasmic ionic interaction network links both first helices of each repeat in one protomer to the osmosensing C-terminal domain of the adjacent protomer. Moreover, the periplasmic ionic interaction network conformationally locks the four-TM-helix bundle between the same neighbor protomers. By a combination of site-directed mutagenesis, cross-linking, and betaine uptake measurements, we demonstrate how conformational changes in individual bundle helices are transduced to the entire bundle by specific inter-helical interactions. We suggest that one purpose of bundle networking is to assist crosstalk between protomers during transport regulation by specifically modulating the transition from outward-facing to inward-facing state.     

Salvianolic acid B protects cardiomyocytes from angiotensin II-induced hypertrophy via inhibition of PARP-1

Salvianolic acid B (SalB), one of the major bioactive components in Salviamiltiorrhiza, has plenty of cardioprotective effects. The present study was designed to investigate the effect of SalB on angiotensin II (AngII)-induced hypertrophy in neonatal rat cardiomyocytes, and to find out whether or not this effect is attributed to inhibition of poly (ADP-ribose) polymerase-1 (PARP-1), which plays a key role in cardiac hypertrophy. Our results showed that SalB prevented the cardiomyocytes from AngII-induced hypertrophy, associated with attenuation of the mRNA expressions of atrial natriuretic factor and brain natriuretic peptide, and reduction in the cell surface area. SalB inhibited the activity of PARP-1. The inhibitory effect was comparable to that of the PARP-1 inhibitor 3-Aminobenzamide (3-AB). In addition, SalB reversed the depletion of cellular NAD⁺ induced by AngII. Moreover, overexpression of PARP-1 attenuated the anti-hypertrophic effect of SalB. These observations suggested that SalB prevented the cardiomyocytes from AngII-induced hypertrophy, at least partially through inhibition of PARP-1. Moreover, SalB attenuated the generation of oxidative stress via suppression of NADPH oxidase 2 and 4, which might probably contribute to the inhibition of PARP-1. These present findings may shed new light on the understanding of the cardioprotective effect of SalB.     

Effect of Metal Loading and Subcellular pH on Net Charge of Superoxide Dismutase-1

The net charge of a folded protein is hypothesized to influence myriad biochemical processes (e.g., protein misfolding, electron transfer, molecular recognition); however, few tools exist for measuring net charge and this elusive property remains undetermined—at any pH—for nearly all proteins. This study used lysine-acetyl “protein charge ladders” and capillary electrophoresis to measure the net charge of superoxide dismutase-1 (SOD1)—whose aggregation causes amyotrophic lateral sclerosis (ALS)—as a function of coordinated metal ions and pH. The net negative charge of apo-SOD1 was similar to predicted values; however, the binding of a single Zn^2 + or Cu^2 + ion reduced the net negative charge by a greater magnitude than predicted (i.e., ~ 4 units, instead of 2), whereas the SOD1 protein underwent charge regulation upon binding 2–4 metal ions. From pH5 to pH8 (i.e., a range consistent with the multiple subcellular loci of SOD1), the holo-SOD1 protein underwent smaller fluctuations in net negative charge than predicted (i.e., ~ 3 units, instead of ~ 14) and did not undergo charge inversion at its isoelectric point (pI = 5.3) but remained anionic. The regulation of SOD1 net charge along its pathways of metal binding, and across solvent pH, provides insight into its metal-induced maturation and enzymatic activity (which remains diffusion-limited across pH5–8). The anionic nature of holo-SOD1 across subcellular pH suggests that ~ 45 different ALS-linked mutations to SOD1 will reduce its net negative charge regardless of subcellular localization.