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 member

Importin alpha binding and nuclear localization of PARP-2 is dependent on lysine 36, which is located within a predicted classical NLS

BACKGROUND: The enzymes responsible for the synthesis of poly-ADP-ribose are named poly-ADP-ribose polymerases (PARP). PARP-2 is a nuclear protein, which regulates a variety of cellular functions that are mainly controlled by protein-protein interactions. A previously described non-conventional bipartite nuclear localization sequence (NLS) lies in the amino-terminal DNA binding domain of PARP-2 between amino acids 1-69; however, this targeting sequence has not been experimentally examined or validated. RESULTS: Using a site-directed mutagenesis approach, we found that lysines 19 and 20, located within a previously described bipartite NLS, are not required for nuclear localization of PARP-2. In contrast, lysine 36, which is located within a predicted classical monopartite NLS, was required for PARP-2 nuclear localization. While wild type PARP-2 interacted with importin alpha3 and to a very weak extent with importin alpha1 and importin alpha5, the mutant PARP-2 (K36R) did not interact with importin alpha3, providing a molecular explanation why PARP-2 (K36R) is not targeted to the nucleus. CONCLUSION: Our results provide strong evidence that lysine 36 of PARP-2 is a critical residue for proper nuclear targeting of PARP-2 and consequently for the execution of its biological functions

Functional relevance of novel p300-mediated lysine 314 and 315 acetylation of RelA/p65

Nuclear factor kappaB (NF-κB) plays an important role in the transcriptional regulation of genes involved in immunity and cell survival. We show here in vitro and in vivo acetylation of RelA/p65 by p300 on lysine 314 and 315, two novel acetylation sites. Additionally, we confirmed the acetylation on lysine 310 shown previously. Genetic complementation of RelA/p65−/− cells with wild type and non-acetylatable mutants of RelA/p65 (K314R and K315R) revealed that neither shuttling, DNA binding nor the induction of anti-apoptotic genes by tumor necrosis factor α was affected by acetylation on these residues. Microarray analysis of these cells treated with TNFα identified specific sets of genes differently regulated by wild type or acetylation-deficient mutants of RelA/p65. Specific genes were either stimulated or repressed by the acetylation-deficient mutants when compared to RelA/p65 wild type. These results support the hypothesis that site-specific p300-mediated acetylation of RelA/p65 regulates the specificity of NF-κB dependent gene expression

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

A unique transcriptome: 1782 positions of RNA editing alter 1406 codon identities in mitochondrial mRNAs of the lycophyte Isoetes engelmannii

The analysis of the mitochondrial DNA of Isoetes engelmannii as a first representative of the lycophytes recently revealed very small introns and indications for extremely frequent RNA editing. To analyze functionality of intron splicing and the extent of RNA editing in I. engelmannii, we performed a comprehensive analysis of its mitochondrial transcriptome. All 30 groups I and II introns were found to be correctly removed, showing that intron size reduction does not impede splicing. We find that mRNA editing affects 1782 sites, which lead to a total of 1406 changes in codon meanings. This includes the removal of stop codons from 23 of the 25 mitochondrial protein encoding genes. Comprehensive sequence analysis of multiple cDNAs per locus allowed classification of partially edited sites as either inefficiently edited but relevant or as non-specifically edited at mostly low frequencies. Abundant RNA editing was also found to affect tRNAs in hitherto unseen frequency, taking place at 41 positions in tRNA-precursors, including the first identification of U-to-C exchanges in two tRNA species. We finally investigated the four group II introns of the nad7 gene and could identify 27 sites of editing, most of which improve base pairing for proper secondary structure formation

Identification of small non-coding RNAs from mitochondria and chloroplasts

Small non-protein-coding RNAs (ncRNAs) have been identified in a wide spectrum of organisms ranging from bacteria to humans. In eukarya, systematic searches for ncRNAs have so far been restricted to the nuclear or cytosolic compartments of cells. Whether or not small stable non-coding RNA species also exist in cell organelles, in addition to tRNAs or ribosomal RNAs, is unknown. We have thus generated cDNA libraries from size-selected mammalian mitochondrial RNA and plant chloroplast RNA and searched for small ncRNA species in these two types of DNA-containing cell organelles. In total, we have identified 18 novel candidates for organellar ncRNAs in these two cellular compartments and confirmed expression of six of them by northern blot analysis or RNase A protection assays. Most candidate ncRNA genes map to intergenic regions of the organellar genomes. As found previously in bacteria, the presumptive ancestors of present-day chloroplasts and mitochondria, we also observed examples of antisense ncRNAs that potentially could target organelle-encoded mRNAs. The structural features of the identified ncRNAs as well as their possible cellular functions are discussed. The absence from our libraries of abundant small RNA species that are not encoded by the organellar genomes suggests that the import of RNAs into cell organelles is of very limited significance or does not occur at all

Genomic CDKN2A/2B deletions in adult Ph+ ALL are adverse despite allogeneic stem cell transplantation

We investigated the role of copy number alterations to refine risk stratification in adult Philadelphia chromosome positive (Ph)+ ALL treated with tyrosine kinase inhibitors (TKI) and allogeneic stem cell transplantation (aSCT). 97 Ph+ ALL patients (median age 41 years, range 18-64 years) within the prospective multicenter GMALL studies 06/99 (n=8) and 07/2003 (n=89) were analysed. All patients received TKI and aSCT in first complete remission (CR1). Copy number analysis was performed with SNP arrays and validated by multiplex ligation-dependent probe amplification (MLPA). The frequencies of recurrently deleted genes were: IKZF1, 76%, CDKN2A/2B, 45%, PAX5, 43%, BTG1, 18%, EBF1, 13%, ETV6, 5%, RB, 14%. In univariate analyses, the presence of CDKN2A/2B deletions had a negative impact on all endpoints: overall survival (p=0.023), disease free survival (p=0.012) and remission duration (p=0.036). The negative predictive value of CDKN2A/2B deletions was retained in multivariable analysis along with other factors such as timing of TKI therapy, intensity of conditioning, achieving remission after induction phase I and BTG1 deletions. We therefore conclude that acquired genomic CDKN2A/2B deletions identify a subgroup of Ph+ ALL patients, who have an inferior prognosis despite aSCT in CR1. Their poor outcome was attributable primarily to a high relapse rate after aSCT

Food webs under changing biodiversity - Top-down control

Report on effects of changing predation pressure on benthic and pelagic specie

Crosstalk between SET7/9-dependent methylation and ARTD1-mediated ADP-ribosylation of histone H1.4

BACKGROUND: Different histone post-translational modifications (PTMs) fine-tune and integrate different cellular signaling pathways at the chromatin level. ADP-ribose modification of histones by cellular ADP-ribosyltransferases such as ARTD1 (PARP1) is one of the many elements of the histone code. All 5 histone proteins were described to be ADP-ribosylated in vitro and in vivo. However, the crosstalk between ADP-ribosylation and other modifications is little understood. RESULTS: In experiments with isolated histones, it was found that ADP-ribosylation of H3 by ARTD1 prevents H3 methylation by SET7/9. However, poly(ADP-ribosyl)ation (PARylation) of histone H3 surprisingly allowed subsequent methylation of H1 by SET7/9. Histone H1 was thus identified as a new target for SET7/9. The SET7/9 methylation sites in H1.4 were pinpointed to the last lysine residues of the six KAK motifs in the C-terminal domain (K121, K129, K159, K171, K177 and K192). Interestingly, H1 and the known SET7/9 target protein H3 competed with each other for SET7/9-dependent methylation. CONCLUSIONS: The results presented here identify H1.4 as a novel SET7/9 target protein, and document an intricate crosstalk between H3 and H1 methylation and PARylation, thus implying substrate competition as a regulatory mechanism. Thereby, these results underline the role of ADP-ribosylation as an element of the histone code