Biology subjects

Maksimainen, M. M.

Publications and source records attributed to Maksimainen, M. M..

8 recordsLinked to original sources

bioRxiv2025

Dimerization of human PARP15 is required for NAD+ binding and automodification

PARP proteins are enzymes catalyzing ADP-ribosylation, a well-conserved post-translational modification. In addition to the catalytic domain present in all PARPs, these proteins have a wide selection of other domains possessing different functions. Additional domains present in human PARP15 are macrodomains, capable of binding ADP-ribose. PARP15 is the least studied of the macrodomain-containing human PARPs, and it is linked to different diseases from infections to cancer. We show that the full-length canonical isoform 1 of PARP15 auto-ADP-ribosylates robustly on glutamate and aspartate residues, which can be hydrolyzed by known ADP-ribosylhydrolases. We have been able to locate several modification sites to the region of tandem macrodomains. In agreement with earlier reports regarding the catalytic domain dimerization being the requirement for enzyme activity, we show this in the full-length context with the recombinant isoform 1 of PARP15. We show that dimerization is required for the efficient substrate NAD+ binding, and we provide structural basis for this requirement with the help of a co-crystal structure of the catalytic domain dimer with an unhydrolyzable substrate analog.

biochemistry↗

bioRxiv2023

Oligomerisation mediated by the D2 domain of DTX3L is critical for DTX3L-PARP9 reading function of mono-ADP-ribosylated androgen receptor

Deltex proteins are a family of E3 ubiquitin ligases that encode C-terminal RING and DTC domains that mediate interactions with E2 ubiquitin-conjugating enzymes and recognise ubiquitination substrates. DTX3L is unique among the Deltex proteins based on its N-terminal domain architecture. The N-terminal D1 and D2 domains of DTX3L mediate homo-oligomerisation, and the D3 domain interacts with PARP9, a protein that contains tandem macrodomains with ADP-ribose reader function. While DTX3L and PARP9 are known to heterodimerize, they assemble into a high molecular weight oligomeric complex, but the nature of the oligomeric structure, including whether this contributes to the ADP-ribose reader function is unknown. Here, we report a crystal structure of the DTX3L N-terminal D2 domain and show that it forms a tetramer with, conveniently, D2 symmetry. We identified two interfaces in the structure: a major, conserved interface with a surface of 973 [A]2 and a smaller one of 415 [A]2. Using native mass spectrometry, we observed molecular species that correspond to monomers, dimers and tetramers of the D2 domain. Reconstitution of DTX3L knockout cells with a D1-D2 deletion mutant showed the domain is dispensable for DTX3L-PARP9 heterodimer formation, but necessary to assemble an oligomeric complex with efficient reader function for ADP-ribosylated androgen receptor. Our results suggest that homo-oligomerisation of DTX3L is important for mono-ADP-ribosylation reading by the DTX3L-PARP9 complex and to a ligand-regulated transcription factor.

biochemistry↗

bioRxiv2023

Discovery of 2-amide-3-methylester thiophenes inhibiting SARS-CoV-2 ADP-ribosyl hydrolysing macrodomain and coronavirus replication

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has made it clear that further development of antiviral therapies will be needed to combat additional SARS-CoV-2 variants or novel CoVs. Here, we describe small molecule inhibitors for SARS-CoV-2 Mac1, which counters ADP-ribosylation mediated innate immune responses. The compounds inhibiting Mac1 were discovered through high-throughput screening (HTS) using a protein FRET-based competition assay and the best hit compound had an IC50 of 14 {micro}M. Three validated HTS hits have the same 2-amide-3-methylester thiophene scaffold and the scaffold was selected for structure-activity relationship (SAR) studies through commercial and synthesized analogs. We studied the compound binding mode in detail using X-ray crystallography and this allowed us to focus on specific features of the compound and design analogs. Compound 27 (MDOLL-0229) had an IC50 of 2.1 {micro}M and was generally selective for CoV Mac1 proteins after profiling for activity against a panel of viral and human ADP-ribose binding proteins. The improved potency allowed testing of its effect on virus replication and indeed, 27 inhibited replication of both MHVa prototype CoV, and SARS-CoV-2. Furthermore, sequencing of a drug-resistant MHV identified mutations in Mac1, further demonstrating the specificity of 27. Compound 27 is the first Mac1 targeted small molecule demonstrated to inhibit coronavirus replication in a cell model. This, together with its well-defined binding mode, makes 27 a good candidate for further hit/lead-optimization efforts.

biochemistry↗

bioRxiv2022

benzothiazole scaffold as versatile nicotinamide mimic allowing nanomolar inhibition of different PARP enzymes

Here we report [1,2,4]triazolo[3,4-b]benzothiazole (TBT) as a new inhibitor scaffold, which competes with nicotinamide in the binding pocket of human poly- and mono-ADP-ribosylating enzymes. The binding mode was studied through analogs and their crystal structures with TNKS2, PARP2, PARP14 and PARP15. Based on the substitution pattern, we were able to identify The 3-amino derivatives 21 (OUL243) and 27 (OUL232), as inhibitors of mono-ARTs PARP7, PARP10, PARP11, PARP12, PARP14 and PARP15 at nM potencies, with compound 27 being the most potent PARP10 inhibitor described to date with an IC50 of 7.8 nM and the first PARP12 inhibitor ever reported. On the contrary, hydroxy derivative 16 (OUL245) inhibits poly-ARTs with a selectivity towards PARP2. The scaffold does not possess inherent cell toxicity and the inhibitors can enter cells and engage with the target protein. This, together with favorable ADME properties, demonstrates the potential of the TBT scaffold for future drug development efforts towards selective inhibitors against specific enzymes.

biochemistry↗

bioRxiv2021

Analogs of TIQ-A as inhibitors of human mono-ADP-ribosylating PARPs

The scaffold of TIQ-A, a previously known inhibitor of human poly-ADP-ribosyltransferase PARP1, was utilized to develop inhibitors against human mono-ADP-ribosyltransferases through structure-guided design and activity profiling. By supplementing the TIQ-A scaffold with small structural changes, based on a PARP10 inhibitor OUL35, selectivity changed from poly-ADP-ribosyltransferases towards mono-ADP-ribosyltransferases. Binding modes of analogs were experimentally verified by determining complex crystal structures with mono-ADP-ribosyltransferase PARP15 and with poly-ADP-ribosyltransferase TNKS2. The best analogs of the study achieved 10 - 20-fold selectivity towards mono-ADP-ribosyltransferases PARP10 and PARP15 while maintaining micromolar potencies. The work demonstrates a route to differentiate compound selectivity between mono- and poly-ribosyltransferases of the human ARTD family.

biochemistry↗

bioRxiv2021

A molecular toolbox for ADP-ribosyl binding proteins

Proteins interacting with ADP-ribosyl groups are often involved in disease-related pathways or in viral infections, which makes them attractive targets for the development of inhibitors. Our goal was to develop a robust and accessible assay technology that is suitable for high-throughput screening and applicable to a wide range of proteins acting as either hydrolysing or non-hydrolysing binders of mono- and poly-ADP-ribosyl groups. As a foundation of our work, we developed a C-terminal protein fusion tag based on a Gi protein alpha subunit peptide (GAP), which allows for site-specific introduction of cysteine-linked mono- and poly-ADP-ribosyl groups as well as chemical ADP-ribosyl analogs. By fusion of the GAP-tag and ADP-ribosyl binders to fluorescent proteins, we were able to generate robust FRET signals and the interaction with 22 previously described ADP-ribosyl-binders was confirmed. To demonstrate the applicability of this binding assay for high-throughput screening, we utilized it to screen for inhibitors of the SARS-CoV-2 nsp3 macrodomain and identified the drug suramin as a moderate yet unspecific inhibitor of this protein. To complement the binding technology, we prepared high-affinity ADP-ribosyl binders fused to a nanoluciferase, which enabled simple blot-based detection of mono- and poly-ADP-ribosylated proteins. These tools can be expressed recombinantly in E. coli using commonly available agents and will help to investigate ADP-ribosylation systems and aid in drug discovery.

biochemistry↗

bioRxiv2020

Activation of ARTD2/PARP2 by DNA damage induces conformational changes relieving enzyme autoinhibition

Human ARTD2/PARP2 is an ADP-ribosyltransferase which, when activated by 5- phosphorylated DNA ends, catalyzes poly-ADP-ribosylation of itself, other proteins and DNA. A crystal structure of ARTD2 in complex with an activating 5-phosphorylated DNA shows that the WGR domain bridges the dsDNA gap and joins the DNA ends. This DNA binding results in major conformational changes, reorganization of helical fragments, in the ARTD2 regulatory domain. Comparison of ARTD1-3 crystal structures reveal how binding to a DNA damage site leads to formation of a catalytically competent conformation capable of binding substrate NAD+ and histone PARylation factor 1 changing the ARTD2 residue specificity from glutamate to serine when initiating DNA repair processes. The structure also reveals how the conformational changes in the autoinhibitory regulatory domain would promote the flexibility needed by the enzyme to reach the target macromolecule for ADP-ribosylation.

biochemistry↗

bioRxiv2019

FMN-dependent oligomerization of putative lactate oxidase from Pediococcus acidilactici

Lactate oxidases belong to a group of FMN-dependent enzymes and they catalyze a conversion of lactate to pyruvate with a release of hydrogen peroxide. Hydrogen peroxide is also utilized as a read out in biosensors to quantitate lactate levels in biological samples. Aerococcus viridans lactate oxidase is the best characterized lactate oxidase and our knowledge of lactate oxidases relies largely to studies conducted with that particular enzyme. Pediococcus acidilactici lactate oxidase is also commercially available for e.g. lactate measurements, but this enzyme has not been characterized before in detail. Here we report structural characterization of the recombinant enzyme and its co-factor dependent oligomerization. The crystal structures revealed two distinct conformations in the loop closing the active site, consistent with previous biochemical studies implicating the role of loop in catalysis. Despite the structural conservation of active site residues when compared to Aerococcus viridans lactate oxidase we were not able to detect either oxidase or monooxygenase activity when L-lactate or other potential alpha hydroxyl acids were used as a substrate. Pediococcus acidilactici lactate oxidase is therefore an example of a misannotation of an FMN-dependent enzyme, which catalyzes likely a so far unknown oxidation reaction.

biochemistry↗