Common Features at the Start of the Neurodegeneration Cascade

A single-molecule study reveals that neurotoxic proteins share common structural features that may trigger neurodegeneration, thus identifying new targets for therapy and diagnosis

The cohesin module is a major determinant of cellulosome mechanical stability

Cellulosomes are bacterial protein complexes that bind and efficiently degrade lignocellulosic substrates. These are formed by multimodular scaffolding proteins known as scaffoldins, which comprise cohesin modules capable of binding dockerin-bearing enzymes and usually a carbohydrate-binding module that anchors the system to a substrate. It has been suggested that cellulosomes bound to the bacterial cell surface might be exposed to significant mechanical forces. Accordingly, the mechanical properties of these anchored cellulosomes may be important to understand and improve cellulosome function. Here we used single-molecule force spectroscopy to study the mechanical properties of selected cohesin modules from scaffoldins of different cellulosomes. We found that cohesins located in the region connecting the cell and the substrate are more robust than those located outside these two anchoring points. This observation applies to cohesins from primary scaffoldins (i.e. those that directly bind dockerin-bearing enzymes) from different cellulosomes despite their sequence differences. Furthermore, we also found that cohesin nanomechanics (specifically, mechanostability and the position of the mechanical clamp of cohesin) are not significantly affected by other cellulosomal components, including linkers between cohesins, multiple cohesin repeats, and dockerin binding. Finally, we also found that cohesins (from both the connecting and external regions) have poor refolding efficiency but similar refolding rates, suggesting that the high mechanostability of connecting cohesins may be an evolutionarily conserved trait selected to minimize the occurrence of cohesin unfolding, which could irreversibly damage the cellulosome. We conclude that cohesin mechanostability is a major determinant of the overall mechanical stability of the cellulosome.This work was supported by a Seventh Framework Programme in Nanosciences, Nanotechnologies, Materials, and New Production Technologies (7PM-NMP 2013–17 and 604530-2) and ERA-IB-ERANET-2013–16 (EIB.12.022) through the Spanish MINECO (PCIN-2013-011-C02-01). The authors declare that they have no conflicts of interest with the contents of this article

Understanding biology by stretching proteins: recent progress

Single molecule manipulation techniques combined with molecular dynamics simulations and protein engineering have enabled, during the last decade, the mechanical properties of proteins to be studied directly, thereby giving birth to the field of protein nanomechanics. Recent data obtained from such techniques have helped gain insight into the structural bases of protein resistance against forced unfolding, as well as revealing structural motifs involved in mechanical stability. Also, important technical developments have provided new perspectives into protein folding. Eventually, new and exciting data have shown that mechanical properties are key factors in cell signaling and pathologies, and have been used to rationally tune these properties in a variety of proteins. Crown Copyright © 2009.Peer Reviewe

Protein engineering approach to enhance activity assays of mono-ADP-ribosyltransferases through proximity

Abstract Human mono-ADP-ribosylating PARP enzymes have been linked to several clinically relevant processes and many of these PARPs have been suggested as potential drug targets. Despite recent advances in the field, efforts to discover inhibitors have been hindered by the lack of tools to rapidly screen for high potency compounds and profile them against the different enzymes. We engineered mono-ART catalytic fragments to be incorporated into a cellulosome-based octavalent scaffold. Compared to the free enzymes, the scaffold-based system results in an improved activity for the tested PARPs due to improved solubility, stability and the proximity of the catalytic domains, altogether boosting their activity beyond 10-fold in the case of PARP12. This allows us to measure their activity using a homogeneous NAD⁺ conversion assay, facilitating its automation to lower the assay volume and costs. The approach will enable the discovery of more potent compounds due to increased assay sensitivity

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

Abstract Human PARP2/ARTD2 is an ADP-ribosyltransferase which, when activated by 5′-phosphorylated DNA ends, catalyses poly-ADP-ribosylation of itself, other proteins and DNA. In this study, a crystal structure of PARP2 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, including reorganization of helical fragments, in the PARP2 regulatory domain. A comparison of PARP1 and PARP2 crystal structures reveals how binding to a DNA damage site leads to formation of a catalytically competent conformation. In this conformation, PARP2 is capable of binding substrate NAD⁺ and histone PARylation factor 1 that changes PARP2 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

Impact of scaffoldin mechanostability on cellulosomal activity

Lignocellulose is the most abundant renewable carbon source in the biosphere. However, the main bottleneck in its conversion to produce second generation biofuels is the saccharification step: the hydrolysis of lignocellulosic material into soluble fermentable sugars. Some anaerobic bacteria have developed an extracellular multi-enzyme complex called the cellulosome that efficiently degrades cellulosic substrates. Cellulosome complexes rely on enzyme-integrating scaffoldins that are large non-catalytic scaffolding proteins comprising several cohesin modules and additional functional modules that mediate the anchoring of the complex to the cell surface and the specific binding to its cellulosic substrate. It was proposed that mechanical forces may affect the cohesins positioned between the cell- and cellulose-anchoring points in the so-called connecting region. Consequently, the mechanical resistance of cohesins within the scaffoldin is of great importance, both to understand cellulosome function and as a parameter of industrial interest, to better mimic natural complexes through the use of the established designer cellulosome technology. Here we study how the mechanical stability of cohesins in a scaffoldin affects the enzymatic activity of a cellulosome. We found that when a cohesin of low mechanical stability is positioned in the connecting region of a scaffoldin, the activity of the resulting cellulosome is reduced as opposed to a cohesin of higher mechanical stability. This observation directly relates mechanical stability of the scaffoldin-borne cohesins to cellulosome activity and provides a rationale for the design of artificial cellulosomes for industrial applications, by incorporating mechanical stability as a new industrial parameter in the biotechnology toolbox.This work was supported by a Seventh Framework Programme in Nanosciences, Nanotechnologies, Materials & New Production Technologies (7PM-NMP 2013–17, 604530-2, CellulosomePlus) and the ERA-IB-ERANET-2013–16 (EIB.12.022, FiberFuel) through the Spanish MINECO (PCIN-2013-011-C02-01). AGP acknowledges financial support from an FPU fellowship of the Spanish MECD.We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

Activity-based screening assay for mono-ADP-Ribosylhydrolases

Abstract ADP-ribosylation is a post-translational modification involved in the regulation of many vital cellular processes. This posttranslational modification is carried out by ADP-ribosyltransferases converting β-NAD+ into nicotinamide and a protein-linked ADP-ribosyl group or a chain of PAR. The reverse reaction, release of ADP-ribose from the acceptor molecule, is catalyzed by ADP-ribosylhydrolases. Several hydrolases contain a macrodomain fold, and activities of human macrodomain protein modules vary from reading or erasing mono- and poly-ADP-ribosylation. Macrodomains have been linked to diseases such as cancer, making them potential drug targets. Discovery of inhibitors requires robust biochemical tools mostly lacking for hydrolases, and here we describe an inhibitor screening assay against mono-ADP-ribosylhydrolyzing enzymes. The activity-based assay uses an α-NAD⁺, anomer of β-NAD⁺, which is accepted as a substrate by MacroD1, MacroD2, and ARH3 due to its resemblance to the protein-linked ADP-ribose. The amount of α-NAD⁺ present after hydrolysis is measured by chemically converting it on a microtiter plate to a fluorescent compound. We optimized the assay for MacroD2 and performed a proof-of-concept compound screening. Three compounds were identified as screening hits with micromolar potency. However, further characterization of the compounds identified them as protein destabilizers, excluding further follow-up studies. Validation and screening demonstrated the usability of the in vitro assay for MacroD2, and we also demonstrate the applicability of the assay as a tool for other human ADP-ribosylhydrolase

An evolutionary perspective on the origin, conservation and binding partner acquisition of tankyrases

Abstract Tankyrases are poly-ADP-ribosyltransferases that regulate many crucial and diverse cellular processes in humans such as Wnt signaling, telomere homeostasis, mitotic spindle formation and glucose metabolism. While tankyrases are present in most animals, functional differences across species may exist. In this work, we confirm the widespread distribution of tankyrases throughout the branches of multicellular animal life and identify the single-celled choanoflagellates as earliest origin of tankyrases. We further show that the sequences and structural aspects of TNKSs are well-conserved even between distantly related species. We also experimentally characterized an anciently diverged tankyrase homolog from the sponge Amphimedon queenslandica and show that the basic functional aspects, such as poly-ADP-ribosylation activity and interaction with the canonical tankyrase binding peptide motif, are conserved. Conversely, the presence of tankyrase binding motifs in orthologs of confirmed interaction partners varies greatly between species, indicating that tankyrases may have different sets of interaction partners depending on the animal lineage. Overall, our analysis suggests a remarkable degree of conservation for tankyrases, and that their regulatory functions in cells have likely changed considerably throughout evolution

Molecular basis of Orb2 amyloidogenesis and blockade of memory consolidation

Amyloids are ordered protein aggregates that are typically associated with neurodegenerative diseases and cognitive impairment. By contrast, the amyloid-like state of the neuronal RNA binding protein Orb2 in Drosophila was recently implicated in memory consolidation, but it remains unclear what features of this functional amyloid-like protein give rise to such diametrically opposed behaviour. Here, using an array of biophysical, cell biological and behavioural assays we have characterized the structural features of Orb2 from the monomer to the amyloid state. Surprisingly, we find that Orb2 shares many structural traits with pathological amyloids, including the intermediate toxic oligomeric species, which can be sequestered in vivo in hetero-oligomers by pathological amyloids. However, unlike pathological amyloids, Orb2 rapidly forms amyloids and its toxic intermediates are extremely transient, indicating that kinetic parameters differentiate this functional amyloid from pathological amyloids. We also observed that a well-known anti-amyloidogenic peptide interferes with long-term memory in Drosophila. These results provide structural insights into how the amyloid-like state of the Orb2 protein can stabilize memory and be nontoxic. They also provide insight into how amyloid-based diseases may affect memory processes.This research was supported by funds from SAF2013-49179-C2-1-R JPND_CD_FP-688- 059 (AC14/00037 ISCIII) to MCV, SIMR to KS, SAF2013-49179-C2-2-R JPND_CD_FP-688-059 (AC14/00037 ISCIII) to DVL, BFU2012-36825, S2011/BMD-2457 (Comunidad de Madrid)Peer Reviewe

A molecular toolbox for ADP-ribosyl binding proteins

Summary Proteins interacting with ADP-ribosyl groups are often involved in disease-related pathways or viral infections, making them attractive drug targets. We present a robust and accessible assay applicable to both hydrolyzing or non-hydrolyzing binders of mono- and poly-ADP-ribosyl groups. This technology relies on a C-terminal 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 or analogs. By fusing the GAP-tag and ADP-ribosyl binders to fluorescent proteins, we generate robust FRET partners and confirm the interaction with 22 known ADP-ribosyl binders. The applicability for high-throughput screening of inhibitors is demonstrated with the SARS-CoV-2 nsp3 macrodomain, for which we identify suramin as a moderate-affinity yet non-specific inhibitor. High-affinity ADP-ribosyl binders fused to nanoluciferase complement this technology, enabling simple blot-based detection of ADP-ribosylated proteins. All these tools can be produced in Escherichia coli and will help in ADP-ribosylation research and drug discovery