Molecular basis for DarT ADP-ribosylation of a DNA base

ADP-ribosyltransferases use NAD+ to catalyse substrate ADP-ribosylation1, and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria2–4. Reversible ADP-ribosylation has traditionally been considered a protein-specific modification5, but recent in vitro studies have suggested nucleic acids as targets6–9. Here we present evidence that specific, reversible ADP-ribosylation of DNA on thymidine bases occurs in cellulo through the DarT–DarG toxin–antitoxin system, which is found in a variety of bacteria (including global pathogens such as Mycobacterium tuberculosis, enteropathogenic Escherichia coli and Pseudomonas aeruginosa)10. We report the structure of DarT, which identifies this protein as a diverged member of the PARP family. We provide a set of high-resolution structures of this enzyme in ligand-free and pre- and post-reaction states, which reveals a specialized mechanism of catalysis that includes a key active-site arginine that extends the canonical ADP-ribosyltransferase toolkit. Comparison with PARP–HPF1, a well-established DNA repair protein ADP-ribosylation complex, offers insights into how the DarT class of ADP-ribosyltransferases evolved into specific DNA-modifying enzymes. Together, our structural and mechanistic data provide details of this PARP family member and contribute to a fundamental understanding of the ADP-ribosylation of nucleic acids. We also show that thyminelinked ADP-ribose DNA adducts reversed by DarG antitoxin (functioning as a noncanonical DNA repair factor) are used not only for targeted DNA damage to induce toxicity, but also as a signalling strategy for cellular processes. Using M. tuberculosis as an exemplar, we show that DarT–DarG regulates growth by ADP-ribosylation of DNA at the origin of chromosome replication.Peer reviewedFinal Accepted Versio

Structural insights into the MMACHC-MMADHC protein complex involved in vitamin B12 trafficking

Conversion of vitamin B12 (cobalamin, Cbl) into the cofactor forms methyl-Cbl (MeCbl) and adenosyl-Cbl (AdoCbl) is required for the function of two crucial enzymes, mitochondrial methylmalonyl-CoA mutase and cytosolic methionine synthase, respectively. The intracellular proteins MMACHC and MMADHC play important roles in processing and targeting the Cbl cofactor to its destination enzymes, and recent evidence suggests that they may interact while performing these essential trafficking functions. To better understand the molecular basis of this interaction, we have mapped the crucial protein regions required, indicate that Cbl is likely processed by MMACHC prior to interaction with MMADHC, and identify patient mutations on both proteins that interfere with complex formation, via different mechanisms. We further report the crystal structure of the MMADHC C-terminal region at 2.2 Å resolution, revealing a modified nitroreductase fold with surprising homology to MMACHC despite their poor sequence conservation. Because MMADHC demonstrates no known enzymatic activity, we propose it as the first protein known to repurpose the nitroreductase fold solely for protein-protein interaction. Using small angle x-ray scattering, we reveal the MMACHC-MMADHC complex as a 1:1 heterodimer and provide a structural model of this interaction, where the interaction region overlaps with the MMACHC-Cbl binding site. Together, our findings provide novel structural evidence and mechanistic insight into an essential biological process, whereby an intracellular "trafficking chaperone" highly specific for a trace element cofactor functions via protein-protein interaction, which is disrupted by inherited disease mutations

Identification of a DNA-binding site for the transcription factor Haa1, required for Saccharomyces cerevisiae response to acetic acid stress

The transcription factor Haa1 is the main player in reprogramming yeast genomic expression in response to acetic acid stress. Mapping of the promoter region of one of the Haa1-activated genes, TPO3, allowed the identification of an acetic acid responsive element (ACRE) to which Haa1 binds in vivo. The in silico analysis of the promoter regions of the genes of the Haa1-regulon led to the identification of an Haa1-responsive element (HRE) 5′-GNN(G/C)(A/C)(A/G)G(A/G/C)G-3′. Using surface plasmon resonance experiments and electrophoretic mobility shift assays it is demonstrated that Haa1 interacts with high affinity (KD of 2 nM) with the HRE motif present in the ACRE region of TPO3 promoter. No significant interaction was found between Haa1 and HRE motifs having adenine nucleotides at positions 6 and 8 (KD of 396 and 6780 nM, respectively) suggesting that Haa1p does not recognize these motifs in vivo. A lower affinity of Haa1 toward HRE motifs having mutations in the guanine nucleotides at position 7 and 9 (KD of 21 and 119 nM, respectively) was also observed. Altogether, the results obtained indicate that the minimal functional binding site of Haa1 is 5′-(G/C)(A/C)GG(G/C)G-3′. The Haa1-dependent transcriptional regulatory network active in yeast response to acetic acid stress is proposed

A First Search for coincident Gravitational Waves and High Energy Neutrinos using LIGO, Virgo and ANTARES data from 2007

We present the results of the first search for gravitational wave bursts associated with high energy neutrinos. Together, these messengers could reveal new, hidden sources that are not observed by conventional photon astronomy, particularly at high energy. Our search uses neutrinos detected by the underwater neutrino telescope ANTARES in its 5 line configuration during the period January - September 2007, which coincided with the fifth and first science runs of LIGO and Virgo, respectively. The LIGO-Virgo data were analysed for candidate gravitational-wave signals coincident in time and direction with the neutrino events. No significant coincident events were observed. We place limits on the density of joint high energy neutrino - gravitational wave emission events in the local universe, and compare them with densities of merger and core-collapse events.Comment: 19 pages, 8 figures, science summary page at http://www.ligo.org/science/Publication-S5LV_ANTARES/index.php. Public access area to figures, tables at https://dcc.ligo.org/cgi-bin/DocDB/ShowDocument?docid=p120000

Modular antibodies reveal DNA damage-induced mono-ADP-ribosylation as a second wave of PARP1 signaling

PARP1, an established anti-cancer target that regulates many cellular pathways, including DNA repair signaling, has been intensely studied for decades as a poly(ADP-ribosyl)transferase. Although recent studies have revealed the prevalence of mono-ADP-ribosylation upon DNA damage, it was unknown whether this signal plays an active role in the cell or is just a byproduct of poly-ADP-ribosylation. By engineering SpyTag-based modular antibodies for sensitive and flexible detection of mono-ADP-ribosylation, including fluorescence-based sensors for live-cell imaging, we demonstrate that serine mono-ADP-ribosylation constitutes a second wave of PARP1 signaling shaped by the cellular HPF1/PARP1 ratio. Multilevel chromatin proteomics reveals histone mono-ADP-ribosylation readers, including RNF114, a ubiquitin ligase recruited to DNA lesions through a zinc-finger domain, modulating the DNA damage response and telomere maintenance. Our work provides a technological framework for illuminating ADP-ribosylation in a wide range of applications and biological contexts and establishes mono-ADP-ribosylation by HPF1/PARP1 as an important information carrier for cell signaling. © 2023 The Author(s

Investigating strategies to modify PARP14 function through macrodomain inhibition

Macrodomains are conserved protein interaction modules that are present in all domains of life and mediate recognition of sequence motifs harbouring adenosine diphosphate ribose (ADPR) modifications. In addition, some of them are able to control the turnover of ADPR signalling through their catalytic activity removing these modifications. Macrodomains are hence implicated in a variety of cellular processes as well as in diseases including cancer and viral pathogenesis. The polyadenosine-diphosphate-ribose polymerase (PARP) family member PARP14 is one of twelve human macrodomain-containing proteins; it contains three macrodomains in addition to its catalytic PARP domain. PARP14 was shown to be involved in several cellular processes linked to cancer development in for example B-cell lymphoma and hepatocellular carcinoma. Therefore PARP14 including its macrodomains has emerged as a potential therapeutic target. However, the lack of specific small molecule inhibitors has hampered domain-specific target validation studies so far. Current approaches focus on inhibitor development for its PARP domain, yet attaining selectivity of these inhibitors over other PARP enzymes has been challenging. The aim of the work described in this thesis was therefore to evaluate the possibility of targeting PARP14 via inhibition of its macrodomains with small molecule inhibitors as an alternative to PARP domain inhibition. These studies revealed that displacing PARP14 from its target sites requires inhibitor development for both its second and third macrodomain. Druggability of both macrodomains has been demonstrated by the highly selective allosteric macrodomain 2 inhibitor GeA-69 and by several fragment hits targeting the ADPR binding site of macrodomain 3. Finally, PARP14 was confirmed to be implicated in DNA repair mechanisms protecting cells against replication stress, suggesting that a dual PARP14 macrodomain inhibitor may provide the possibility to potentiate genotoxic chemotherapy for cancer treatment.</p

Investigating strategies to modify PARP14 function through macrodomain inhibition

Macrodomains are conserved protein interaction modules that are present in all domains of life and mediate recognition of sequence motifs harbouring adenosine diphosphate ribose (ADPR) modifications. In addition, some of them are able to control the turnover of ADPR signalling through their catalytic activity removing these modifications. Macrodomains are hence implicated in a variety of cellular processes as well as in diseases including cancer and viral pathogenesis. The polyadenosine-diphosphate-ribose polymerase (PARP) family member PARP14 is one of twelve human macrodomain-containing proteins; it contains three macrodomains in addition to its catalytic PARP domain. PARP14 was shown to be involved in several cellular processes linked to cancer development in for example B-cell lymphoma and hepatocellular carcinoma. Therefore PARP14 including its macrodomains has emerged as a potential therapeutic target. However, the lack of specific small molecule inhibitors has hampered domain-specific target validation studies so far. Current approaches focus on inhibitor development for its PARP domain, yet attaining selectivity of these inhibitors over other PARP enzymes has been challenging. The aim of the work described in this thesis was therefore to evaluate the possibility of targeting PARP14 via inhibition of its macrodomains with small molecule inhibitors as an alternative to PARP domain inhibition. These studies revealed that displacing PARP14 from its target sites requires inhibitor development for both its second and third macrodomain. Druggability of both macrodomains has been demonstrated by the highly selective allosteric macrodomain 2 inhibitor GeA-69 and by several fragment hits targeting the ADPR binding site of macrodomain 3. Finally, PARP14 was confirmed to be implicated in DNA repair mechanisms protecting cells against replication stress, suggesting that a dual PARP14 macrodomain inhibitor may provide the possibility to potentiate genotoxic chemotherapy for cancer treatment.</p

Aspartate aminotransferase of Rhizobium leguminosarum has extended substrate specificity and metabolises aspartate to enable N2-fixation in pea nodules

Rhizobium leguminosarum aspartate aminotransferase (AatA) mutants show drastically reduced symbiotic nitrogen fixation in legume nodules. Whilst AatA reversibly transaminates the two major amino-donor compounds aspartate and glutamate the reason for the lack of N2 fixation in the mutant has remained unclear. During our investigations into the role of AatA we found it catalyses an additional transamination reaction between aspartate and pyruvate forming alanine. This secondary reaction runs at around 60% of the canonical aspartate transaminase reaction rate and connects alanine biosynthesis to glutamate via aspartate. This may explain the lack of any glutamate – pyruvate transaminase activity in R. leguminosarum, which is common in eukaryotic and many prokaryotic genomes. However, the aspartate to pyruvate transaminase reaction is not needed for N2 fixation in legume nodules. Consequently, we show that aspartate degradation is required for N2-fixation, rather than biosynthetic transamination to form an amino acid. Consequently, the enzyme aspartase, that catalyses the breakdown of aspartate to fumarate and ammonia, suppressed an AatA mutant and restored N2-fixation in pea nodules