DELTEX E3 ligases ubiquitylate ADP-ribosyl modification on nucleic acids.

Although ubiquitylation had traditionally been considered limited to proteins, the discovery of non-proteinaceous substrates (e.g. lipopolysaccharides and adenosine diphosphate ribose (ADPr)) challenged this perspective. Our recent study showed that DTX2 E3 ligase efficiently ubiquitylates ADPr. Here, we show that the ADPr ubiquitylation activity is also present in another DELTEX family member, DTX3L, analysed both as an isolated catalytic fragment and the full-length PARP9:DTX3L complex, suggesting that it is a general feature of the DELTEX family. Since structural predictions show that DTX3L possesses single-stranded nucleic acids binding ability and given the fact that nucleic acids have recently emerged as substrates for ADP-ribosylation, we asked whether DELTEX E3s might catalyse ubiquitylation of an ADPr moiety linked to nucleic acids. Indeed, we show that DTX3L and DTX2 are capable of ubiquitylating ADP-ribosylated DNA and RNA synthesized by PARPs, including PARP14. Furthermore, we demonstrate that the Ub-ADPr-nucleic acids conjugate can be reversed by two groups of hydrolases, which remove either the whole adduct (e.g. SARS-CoV-2 Mac1 or PARP14 macrodomain 1) or just the Ub (e.g. SARS-CoV-2 PLpro). Overall, this study reveals ADPr ubiquitylation as a general function of the DELTEX family E3s and presents the evidence of reversible ubiquitylation of ADP-ribosylated nucleic acids.

Updated protein domain annotation of the PARP protein family sheds new light on biological function.

AlphaFold2 and related computational tools have greatly aided studies of structural biology through their ability to accurately predict protein structures. In the present work, we explored AF2 structural models of the 17 canonical members of the human PARP protein family and supplemented this analysis with new experiments and an overview of recent published data. PARP proteins are typically involved in the modification of proteins and nucleic acids through mono or poly(ADP-ribosyl)ation, but this function can be modulated by the presence of various auxiliary protein domains. Our analysis provides a comprehensive view of the structured domains and long intrinsically disordered regions within human PARPs, offering a revised basis for understanding the function of these proteins. Among other functional insights, the study provides a model of PARP1 domain dynamics in the DNA-free and DNA-bound states and enhances the connection between ADP-ribosylation and RNA biology and between ADP-ribosylation and ubiquitin-like modifications by predicting putative RNA-binding domains and E2-related RWD domains in certain PARPs. In line with the bioinformatic analysis, we demonstrate for the first time PARP14's RNA-binding capability and RNA ADP-ribosylation activity in vitro. While our insights align with existing experimental data and are probably accurate, they need further validation through experiments.

Novel Calcium-Binding Motif Stabilizes and Increases the Activity of Aspergillus fumigatus Ecto-NADase.

Nicotinamide adenine dinucleotide (NAD) is an essential molecule in all kingdoms of life, mediating energy metabolism and cellular signaling. Recently, a new class of highly active fungal surface NADases was discovered. The enzyme from the opportunistic human pathogen Aspergillus fumigatus was thoroughly characterized. It harbors a catalytic domain that resembles that of the tuberculosis necrotizing toxin from Mycobacterium tuberculosis, which efficiently cleaves NAD+ to nicotinamide and ADP-ribose, thereby depleting the dinucleotide pool. Of note, the A. fumigatus NADase has an additional Ca2+-binding motif at the C-terminus of the protein. Despite the presence of NADases in several fungal divisions, the Ca2+-binding motif is uniquely found in the Eurotiales order, which contains species that have immense health and economic impacts on humans. To identify the potential roles of the metal ion-binding site in catalysis or protein stability, we generated and characterized A. fumigatus NADase variants lacking the ability to bind calcium. X-ray crystallographic analyses revealed that the mutation causes a drastic and dynamic structural rearrangement of the homodimer, resulting in decreased thermal stability. Even though the calcium-binding site is at a long distance from the catalytic center, the structural reorganization upon the loss of calcium binding allosterically alters the active site, thereby negatively affecting NAD-glycohydrolase activity. Together, these findings reveal that this unique calcium-binding site affects the protein fold, stabilizing the dimeric structure, but also mediates long-range effects resulting in an increased catalytic rate.

Investigating the Disordered and Membrane-Active Peptide A-Cage-C Using Conformational Ensembles.

The driving forces and conformational pathways leading to amphitropic protein-membrane binding and in some cases also to protein misfolding and aggregation is the subject of intensive research. In this study, a chimeric polypeptide, A-Cage-C, derived from α-Lactalbumin is investigated with the aim of elucidating conformational changes promoting interaction with bilayers. From previous studies, it is known that A-Cage-C causes membrane leakages associated with the sporadic formation of amorphous aggregates on solid-supported bilayers. Here we express and purify double-labelled A-Cage-C and prepare partially deuterated bicelles as a membrane mimicking system. We investigate A-Cage-C in the presence and absence of these bicelles at non-binding (pH 7.0) and binding (pH 4.5) conditions. Using in silico analyses, NMR, conformational clustering, and Molecular Dynamics, we provide tentative insights into the conformations of bound and unbound A-Cage-C. The conformation of each state is dynamic and samples a large amount of overlapping conformational space. We identify one of the clusters as likely representing the binding conformation and conclude tentatively that the unfolding around the central W23 segment and its reorientation may be necessary for full intercalation at binding conditions (pH 4.5). We also see evidence for an overall elongation of A-Cage-C in the presence of model bilayers.

Keeping the balance in NAD metabolism.

Research over the last few decades has extended our understanding of nicotinamide adenine dinucleotide (NAD) from a vital redox carrier to an important signalling molecule that is involved in the regulation of a multitude of fundamental cellular processes. This includes DNA repair, cell cycle regulation, gene expression and calcium signalling, in which NAD is a substrate for several families of regulatory proteins, such as sirtuins and ADP-ribosyltransferases. At the molecular level, NAD-dependent signalling events differ from hydride transfer by cleavage of the dinucleotide into an ADP-ribosyl moiety and nicotinamide. Therefore, non-redox functions of NAD require continuous biosynthesis of the dinucleotide. Maintenance of cellular NAD levels is mainly achieved by nicotinamide salvage, yet a variety of other precursors can be used to sustain cellular NAD levels via different biosynthetic routes. Biosynthesis and consumption of NAD are compartmentalised at the subcellular level, and currently little is known about the generation and role of some of these subcellular NAD pools. Impaired biosynthesis or increased NAD consumption is deleterious and associated with ageing and several pathologies. Insults to neurons lead to depletion of axonal NAD and rapid degeneration, partial rescue can be achieved pharmacologically by administration of specific NAD precursors. Restoring NAD levels by stimulating biosynthesis or through supplementation with precursors also produces beneficial therapeutic effects in several disease models. In this review, we will briefly discuss the most recent achievements and the challenges ahead in this diverse research field.

Molecular determinants of the N-terminal acetyltransferase Naa60 anchoring to the Golgi membrane.

Nα-Acetyltransferase 60 (Naa60 or NatF) was recently identified as an unconventional N-terminal acetyltransferase (NAT) because it localizes to organelles, in particular the Golgi apparatus, and has a preference for acetylating N termini of the transmembrane proteins. This knowledge challenged the prevailing view of N-terminal acetylation as a co-translational ribosome-associated process and suggested a new mechanistic functioning for the enzymes responsible for this increasingly recognized protein modification. Crystallography studies on Naa60 were unable to resolve the C-terminal tail of Naa60, which is responsible for the organellar localization. Here, we combined modeling, in vitro assays, and cellular localization studies to investigate the secondary structure and membrane interacting capacity of Naa60. The results show that Naa60 is a peripheral membrane protein. Two amphipathic helices within the Naa60 C terminus bind the membrane directly in a parallel position relative to the lipid bilayer via hydrophobic and electrostatic interactions. A peptide corresponding to the C terminus was unstructured in solution and only folded into an α-helical conformation in the presence of liposomes. Computational modeling and cellular mutational analysis revealed the hydrophobic face of two α-helices to be critical for membranous localization. Furthermore, we found a strong and specific binding preference of Naa60 toward membranes containing the phosphatidylinositol PI(4)P, thus possibly explaining the primary residency of Naa60 at the PI(4)P-rich Golgi. In conclusion, we have defined the mode of cytosolic Naa60 anchoring to the Golgi apparatus, most likely occurring post-translationally and specifically facilitating post-translational N-terminal acetylation of many transmembrane proteins.

Peptides derived from α-lactalbumin membrane binding helices oligomerize in presence of lipids and disrupt bilayers.

Helix A and -C of α-lactalbumin, a loosely folded amphitropic protein, perturb lipid monolayers by the formation of amyloid pore-like structures. To investigate whether these helices are able to disrupt fully formed bilayers, we designed peptides comprised of Helix A and -C, and investigated their membrane-perturbing properties. The peptides, designated A-Cage-C and A-Lnk-C, were prepared with tryptophan sites in the helical and the spacer segments in order to monitor which part were involved in membrane association under given conditions. The peptides associate with and disrupt negatively charged bilayers in a pH-dependent manner and α-helical tendencies increased upon membrane association. Both helices and the spacer segment were involved in membrane binding in the case of A-Lnk-C, and there are indications that the two helixes act in synergy to affect the membrane. However, the helices and the spacer segment could not intercalate when present as A-Cage-C at neutral conditions. At acidic pH, both helices could intercalate, but not the central spacer segment. AFM performed on bilayers under aqueous conditions revealed oligomers formed by the peptides. The presence of bilayers and acidic pHs were both drivers for the formation of these, suggestive of models for peptide oligomerization where segments of the peptide are stacked in an electrostatically favorable manner by the surface. Of the two peptides, A-Lnk-C was the more prolific oligomerizer, and also formed amyloid-fibril like structures at acidic pH and elevated concentrations. Our results suggest the peptides perturb membranes not through pore-like structures, but possibly by a thinning mechanism.

Evolution of fungal tuberculosis necrotizing toxin (TNT) domain-containing enzymes reveals divergent adaptations to enhance NAD cleavage.

Tuberculosis necrotizing toxin (TNT) is a protein domain discovered on the outer membrane of Mycobacterium tuberculosis (Mtb), and the fungal pathogen Aspergillus fumigatus. TNT domains have pure NAD(P) hydrolytic activity, setting them apart from other NAD-cleaving domains such as ADP-ribosyl cyclase and Toll/interleukin-1 receptor homology (TIR) domains which form a wider set of products. Importantly, the Mtb TNT domain has been shown to be involved in immune evasion via depletion of the intracellular NAD pool of macrophages. Therefore, an intriguing hypothesis is that TNT domains act as "NAD killers" in host cells facilitating pathogenesis. Here, we explore the phylogenetic distribution of TNT domains and detect their presence solely in bacteria and fungi. Within fungi, we discerned six TNT clades. In addition, X-ray crystallography and AlphaFold2 modeling unveiled clade-specific strategies to promote homodimer stabilization of the fungal enzymes, namely, Ca2+ binding, disulfide bonds, or hydrogen bonds. We show that dimer stabilization is a requirement for NADase activity and that the group-specific strategies affect the active site conformation, thereby modulating enzyme activity. Together, these findings reveal the evolutionary lineage of fungal TNT enzymes, corroborating the hypothesis of them being pure extracellular NAD (eNAD) cleavers, with possible involvement in microbial warfare and host immune evasion.

PARP14 is a PARP with both ADP-ribosyl transferase and hydrolase activities.

PARP14 is a mono-ADP-ribosyl transferase involved in the control of immunity, transcription, and DNA replication stress management. However, little is known about the ADP-ribosylation activity of PARP14, including its substrate specificity or how PARP14-dependent ADP-ribosylation is reversed. We show that PARP14 is a dual-function enzyme with both ADP-ribosyl transferase and hydrolase activity acting on both protein and nucleic acid substrates. In particular, we show that the PARP14 macrodomain 1 is an active ADP-ribosyl hydrolase. We also demonstrate hydrolytic activity for the first macrodomain of PARP9. We reveal that expression of a PARP14 mutant with the inactivated macrodomain 1 results in a marked increase in mono(ADP-ribosyl)ation of proteins in human cells, including PARP14 itself and antiviral PARP13, and displays specific cellular phenotypes. Moreover, we demonstrate that the closely related hydrolytically active macrodomain of SARS2 Nsp3, Mac1, efficiently reverses PARP14 ADP-ribosylation in vitro and in cells, supporting the evolution of viral macrodomains to counteract PARP14-mediated antiviral response.

Early Evolutionary Selection of NAD Biosynthesis Pathway in Bacteria.

Bacteria use two alternative pathways to synthesize nicotinamide adenine dinucleotide (NAD) from nicotinamide (Nam). A short, two-step route proceeds through nicotinamide mononucleotide (NMN) formation, whereas the other pathway, a four-step route, includes the deamidation of Nam and the reamidation of nicotinic acid adenine dinucleotide (NAAD) to NAD. In addition to having twice as many enzymatic steps, the four-step route appears energetically unfavourable, because the amidation of NAAD includes the cleavage of ATP to AMP. Therefore, it is surprising that this pathway is prevalent not only in bacteria but also in yeast and plants. Here, we demonstrate that the considerably higher chemical stability of the deamidated intermediates, compared with their amidated counterparts, might compensate for the additional energy expenditure, at least at elevated temperatures. Moreover, comprehensive bioinformatics analyses of the available >6000 bacterial genomes indicate that an early selection of one or the other pathway occurred. The mathematical modelling of the NAD pathway dynamics supports this hypothesis, as there appear to be no advantages in having both pathways.

Binding Specificity of ASHH2 CW Domain Toward H3K4me1 Ligand Is Coupled to Its Structural Stability Through Its α1-Helix.

The CW domain binds to histone tail modifications found in different protein families involved in epigenetic regulation and chromatin remodeling. CW domains recognize the methylation state of the fourth lysine on histone 3 and could, therefore, be viewed as a reader of epigenetic information. The specificity toward different methylation states such as me1, me2, or me3 depends on the particular CW subtype. For example, the CW domain of ASHH2 methyltransferase binds preferentially to H3K4me1, and MORC3 binds to both H3K4me2 and me3 modifications, while ZCWPW1 is more specific to H3K4me3. The structural basis for these preferential bindings is not well understood, and recent research suggests that a more complete picture will emerge if dynamical and energetic assessments are included in the analysis of interactions. This study uses fold assessment by NMR in combination with mutagenesis, ITC affinity measurements, and thermal denaturation studies to investigate possible couplings between ASHH2 CW selectivity toward H3K4me1 and the stabilization of the domain and loops implicated in binding. The key elements of the binding site-the two tryptophans and the α1-helix form and maintain the binding pocket- were perturbed by mutagenesis and investigated. Results show that the α1-helix maintains the overall stability of the fold via the I915 and L919 residues and that the correct binding consolidates the loops designated as η1 and η3, as well as the C-terminal. This consolidation is incomplete for H3K4me3 binding to CW, which experiences a decrease in overall thermal stability on binding. Loop mutations not directly involved in the binding site, nonetheless, affect the equilibrium positions of the key residues.

The balance between NAD+ biosynthesis and consumption in ageing.

Nicotinamide adenine dinucleotide (NAD+) is a vital coenzyme in redox reactions. NAD+ is also important in cellular signalling as it is consumed by PARPs, SARM1, sirtuins and CD38. Cellular NAD+ levels regulate several essential processes including DNA repair, immune cell function, senescence, and chromatin remodelling. Maintenance of these cellular processes is important for healthy ageing and lifespan. Interestingly, the levels of NAD+ decline during ageing in several organisms, including humans. Declining NAD+ levels have been linked to several age-related diseases including various metabolic diseases and cognitive decline. Decreasing tissue NAD+ concentrations have been ascribed to an imbalance between biosynthesis and consumption of the dinucleotide, resulting from, for instance, reduced levels of the rate limiting enzyme NAMPT along with an increased activation state of the NAD+-consuming enzymes PARPs and CD38. The progression of some age-related diseases can be halted or reversed by therapeutic augmentation of NAD+ levels. NAD+ metabolism has therefore emerged as a potential target to ameliorate age-related diseases. The present review explores how ageing affects NAD+ metabolism and current approaches to reverse the age-dependent decline of NAD+.

Discovery of fungal surface NADases predominantly present in pathogenic species.

Nicotinamide adenine dinucleotide (NAD) is a key molecule in cellular bioenergetics and signalling. Various bacterial pathogens release NADase enzymes into the host cell that deplete the host's NAD+ pool, thereby causing rapid cell death. Here, we report the identification of NADases on the surface of fungi such as the pathogen Aspergillus fumigatus and the saprophyte Neurospora crassa. The enzymes harbour a tuberculosis necrotizing toxin (TNT) domain and are predominately present in pathogenic species. The 1.6 Å X-ray structure of the homodimeric A. fumigatus protein reveals unique properties including N-linked glycosylation and a Ca2+-binding site whose occupancy regulates activity. The structure in complex with a substrate analogue suggests a catalytic mechanism that is distinct from those of known NADases, ADP-ribosyl cyclases and transferases. We propose that fungal NADases may convey advantages during interaction with the host or competing microorganisms.

The Arabidopsis (ASHH2) CW domain binds monomethylated K4 of the histone H3 tail through conformational selection.

Chromatin post-translational modifications are thought to be important for epigenetic effects on gene expression. Methylation of histone N-terminal tail lysine residues constitutes one of many such modifications, executed by families of histone lysine methyltransferase (HKMTase). One such protein is ASHH2 from the flowering plant Arabidopsis thaliana, equipped with the interaction domain, CW, and the HKMTase domain, SET. The CW domain of ASHH2 is a selective binder of monomethylation at lysine 4 on histone H3 (H3K4me1) and likely helps the enzyme dock correctly onto chromatin sites. The study of CW and related interaction domains has so far been emphasizing lock-key models, missing important aspects of histone-tail CW interactions. We here present an analysis of the ASHH2 CW-H3K4me1 complex using NMR and molecular dynamics, as well as mutation and affinity studies of flexible coils. ß-augmentation and rearrangement of coils coincide with changes in the flexibility of the complex, in particular the η1, η3 and C-terminal coils, but also in the ß1 and ß2 strands and the C-terminal part of the ligand. Furthermore, we show that mutating residues with outlier dynamic behaviour affect the complex binding affinity despite these not being in direct contact with the ligand. Overall, the binding process is consistent with conformational selection. We propose that this binding mechanism presents an advantage when searching for the correct post-translational modification state among the highly modified and flexible histone tails, and also that the binding shifts the catalytic SET domain towards the nucleosome. DATABASES: Structural data are available in the PDB database under the accession code 6QXZ. Resonance assignments for CW42 in its apo- and holo-forms are available in the BMRB database under the accession code 27251.

Spectroscopic and AFM characterization of polypeptide-surface interactions: Controls and lipid quantitative analyses.

This article is related to http://dx.doi.org/10.1016/j.bbamem.2017.01.005 (Ø. Strømland, Ø.S. Handegård, M.L. Govasli, H. Wen, Ø. Halskau, 2017) [1]. In protein and polypeptide-membrane interaction studies, negatively charged lipids are often used as they are a known driver for membrane interaction. When using fluorescence spectroscopy and CD as indicators of polypeptide binding and conformational change, respectively, the effect of zwitterionic lipids only should be documented. The present data documents several aspects of how two engineered polypeptides (A-Cage-C and A-Lnk-C) derived from the membrane associating protein alpha-Lactalbumin affects and are affected by the presence of zwitterionic bilayers in the form of vesicles. We here document the behavior or the Cage and Lnk segments with respect to membrane interaction and their residual fold, using intrinsic tryptophan fluorescence assays. This data description also documents the coverage of solid-supported bilayers prepared by spin-coating mica using binary lipid mixes, a necessary step to ensure that AFM is performed on areas that are covered by lipid bilayers when performing experiments. Uncovered patches are detectable by both force curve measurements and height measurements. We tested naked mica׳s ability to cause aggregation as seen by AFM, and found this to be low compared to preparations containing negatively charged lipids. Work with lipids also carries the risk of chemical degradation taking place during vesicles preparation or other handling of the lipids. We therefor use 31P NMR to quantify the head-group content of commonly used commercial extracts before and after a standard protocol for vesicle production is applied.

Detection of misfolded protein aggregates from a clinical perspective.

Neurodegenerative Protein Misfolding Diseases (PMDs), such as Alzheimer's (AD), Parkinson's (PD) and prion diseases, are generally difficult to diagnose before irreversible damage to the central nervous system damage has occurred. Detection of the misfolded proteins that ultimately lead to these conditions offers a means for providing early detection and diagnosis of this class of disease. In this review, we discuss recent developments surrounding protein misfolding diseases with emphasis on the cytotoxic oligomers implicated in their aetiology. We also discuss the relationship of misfolded proteins with biological membranes. Finally, we discuss how far techniques for providing early diagnoses for PMDs have advanced and describe promising clinical approaches. We conclude that antibodies with specificity towards oligomeric species of AD and PD and lectins with specificity for particular glycosylation, show promise. However, it is not clear which approach may yield a reliable clinical test first. Relevance for patients: Individuals suffering from protein misfolding diseases will likely benefit form earlier, less- or even non-invasive diagnosis techniques. The current state and possible future directions for these are subject of this review.

α-Lactalbumin:Oleic Acid Complex Spontaneously Delivers Oleic Acid to Artificial and Erythrocyte Membranes.

Human α-lactalbumin made lethal to tumor cells (HAMLET) is a tumoricidal complex consisting of human α-lactalbumin and multiple oleic acids (OAs). OA has been shown to play a key role in the activity of HAMLET and its related complexes, generally known as protein-fatty acid (PFA) complexes. In contrast to what is known about the fate of the protein component of such complexes, information about what happens to OA during their action is still lacking. We monitored the membrane, OA and protein components of bovine α-lactalbumin complexed with OA (BLAOA; a HAMLET-like substance) and how they associate with each other. Using ultracentrifugation, we found that the OA and lipid components follow each other closely. We then firmly identify a transfer of OA from BLAOA to both artificial and erythrocyte membranes, indicating that natural cells respond similarly to BLAOA treatment as artificial membranes. Uncomplexed OA is unable to similarly affect membranes at the conditions tested, even at elevated concentrations. Thus, BLAOA can spontaneously transfer OA to a lipid membrane. After the interaction with the membrane, the protein is likely to have lost most or all of its OA. We suggest a mechanism for passive import of mainly uncomplexed protein into cells, using existing models for OA's effect on membranes. Our results are consistent with a membrane destabilization mediated predominantly by OA insertion being a significant contribution to PFA cytotoxicity.