The RNA-Recognition Motifs of TAR DNA-Binding Protein 43 May Play a Role in the Aberrant Self-Assembly of the Protein

The TAR DNA-binding protein 43 (TDP-43) is a nucleic acid-binding protein implicated in gene regulation and RNA processing and shuffling. It is a ribonuclear protein that carries out most of its functions by binding specific nucleic acid sequences with its two RNA-recognition motifs, RRM1 and RRM2. TDP-43 has been identified in toxic cytosolic inclusions in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). The unstructured C-terminus has prion-like behavior and has been considered the driver of the aberrant self-assembly of TDP-43. In this work, we set out to test the hypothesis that the RNA-binding domains could also play a role in protein aggregation. This knowledge could be of important value for understanding TDP-43 aberrant, disease-leading behavior and, in the future, inform the design of small molecules that could prevent or slow down protein aggregation by exploiting the RNA-binding properties of the protein. We investigated the behavior of the two tandem RRM domains separately and linked together and studied their self-assembly properties and RNA-binding ability with a number of biophysical techniques. The picture that emerges from our study suggests that this region of the protein plays an important and so far unexplored role in the aggregation of this protein

A computational approach to investigate TDP-43 C-terminal fragments aggregation in Amyotrophic Lateral Sclerosis

Many of the molecular mechanisms underlying the pathological aggregation of proteins observed in neurodegenerative diseases are still not fully understood. Among the diseases associated with protein aggregates, for example, Amyotrophic Lateral Sclerosis (ALS) is of relevant importance. Although understanding the processes that cause the disease is still an open challenge, its relationship with protein aggregation is widely known. In particular, human TDP-43, an RNA/DNA binding protein, is a major component of pathological cytoplasmic inclusions described in ALS patients. The deposition of the phosphorylated full-length TDP-43 in spinal cord cells has been widely studied, and it has been shown that the brain cortex presents an accumulation of phosphorylated C-terminal fragments (CTFs). Even if it is debated whether CTFs represent a primary cause of ALS, they are a hallmark of TDP-43 related neurodegeneration in the brain. Here, we investigate the CTFs aggregation process, providing a possible computational model of interaction based on the evaluation of shape complementarity at the interfaces. To this end, extensive Molecular Dynamics (MD) simulations were conducted for different types of fragments with the aim of exploring the equilibrium configurations. Adopting a newly developed approach based on Zernike polynomials, for finding complementary regions of the molecular surface, we sampled a large set of exposed portions of the molecular surface of CTFs structures as obtained from MD simulations. The analysis proposes a set of possible associations between the CTFs, which could drive the aggregation process of the CTFs.Comment: 9 pages, 4 figures, 1 tabl

RNA aptamer reveals nuclear TDP-43 pathology is an early aggregation event that coincides with STMN-2 cryptic splicing and precedes clinical manifestation in ALS

Open Access via the Springer Agreement The research leading to this manuscript has been supported by (i) a Target ALS foundation grant to JMG, MHH, GGT, EZ and NS and employing MG and FMW BB-2022-C4-L2; (ii) an NIH grant to JG and MHH, employing HS and FR R01NS127186; (iii) the European Research Council (RIBOMYLOME_309545 and ASTRA_855923) to GGT; and (iv) an MND Association Lady Edith Wolfson Junior Non-Clinical Fellowship to RS Saleeb/Oct22/980-799 (RSS). The authors would also like to thank the University of Aberdeen Microscopy and Histology Core Facility in the Institute of Medical Sciences.Peer reviewe

RRM adjacent TARDBP mutations disrupt RNA binding and enhance TDP-43 proteinopathy

Amyotrophic lateral sclerosis (ALS) presents with focal muscle weakness due to motor neuron degeneration that becomes generalized,leading to death from respiratory failure within 3–5 years from symptom onset. Despite the heterogeneity of aetiology, TDP- 43 proteinopathy is a common pathological feature that is observed in 495% of ALS and tau-negative frontotemporal dementia(FTD) cases. TDP-43 is a DNA/RNA-binding protein that in ALS and FTD translocates from being predominantly nuclear to formdetergent-resistant, hyperphosphorylated aggregates in the cytoplasm of affected neurons and glia. Mutations in TARDBP accountfor 1–4% of all ALS cases and almost all arise in the low complexity C-terminal domain that does not affect RNA binding andprocessing. Here we report an ALS/FTD kindred with a novel K181E TDP-43 mutation that is located in close proximity to the RRM1 domain. To offer predictive gene testing to at-risk family members, we undertook a series of functional studies to characterizethe properties of the mutation. Spectroscopy studies of the K181E protein revealed no evidence of significant misfolding.Although it is unable to bind to or splice RNA, it forms abundant aggregates in transfected cells. We extended our study to includeother ALS-linked mutations adjacent to the RRM domains that also disrupt RNA binding and greatly enhance TDP-43 aggregation,forming detergent-resistant and hyperphosphorylated inclusions. Lastly, we demonstrate that K181E binds to, and sequesters, wild-type TDP-43 within nuclear and cytoplasmic inclusions. Thus, we demonstrate that TDP-43 mutations that disrupt RNAbinding greatly enhance aggregation and are likely to be pathogenic as they promote wild-type TDP-43 to mislocalize andaggregate acting in a dominant-negative manner. This study highlights the importance of RNA binding to maintain TDP-43solubility and the role of TDP-43 aggregation in disease pathogenesis

Coiled-Coil-Peptide als multivalentes Gerüst für Kohlenhydrate: von Rezeptor- Targeting zum Impfstoff durch Ausnutzung von Zucker-Protein-Wechselwirkungen

Over the past decade, the employment of chemically-synthesized scaffolds for the delivery of biologically-relevant molecules has proven to be a highly advantageous strategy for medicinal purposes, primarily due to stabilization of the cargo and enhancement of site-targeted therapeutic effects. Conjugation to a scaffold has been shown to reduce ligand degradation, impart optimal geometric conformation and increase the likelihood of the active species reaching the target. A wide range of synthetic carriers, including dendrimers, nanoparticles, PNAs, LNAs and cell-penetrating peptides, has been explored; among these, peptides are of particular interest due to their diverse functional groups, folding properties, biocompatibility and low toxicity. In particuar, the structural simplicity and regularity of the α-helical coiledcoil folding motif makes it a suitable scaffold for multivalent ligand display. By changing only a few positions in a coiled-coil sequence, it is possible to influence the behaviour of the resulting helices, obtaining either short dimeric peptides or long, fiber-forming carriers. This thesis explores the coiled-coil motif as a scaffold for the multivalent display of peptide and carbohydrate ligands. We aspired to demonstrate the versatility of the coiled- coil motif in two distinct projects. The first study relied on the dimeric form, providing structural predictability for the rational presentation of carbohydrates for lectin targeting. The second study aimed to increase the efficiency of carbohydrate-antibody recognition by displaying ligands on a self-assembling, fiber-forming peptide. The first project is entitled “Tailored presentation of carbohydrate ligands on a coiled-coil scaffold for asialoglycoprotein receptor targeting”. In this work, we determined the binding of members of a coiled-coil glycopeptide library to hepatocytes and established the optimal distance and orientation of the galactose moieties for interaction with the asialoglycoprotein receptor using flow cytometry. We confirmed that binding occurs through receptor-mediated endocytosis via inhibition studies with cytochalasin D; moreover, fluorescence microscopy studies demonstrated the uptake of the carrier peptides into cells. The second project is entitled “A self-assembling peptide scaffold for the multivalent presentation of antigens”. Here, a coiled coil-based sequence was used to create tunable higher-order structures on the nanometer scale, allowing for the multivalent presentation of a mannose moiety and a peptide epitope, the presence of which did not interfere with selfassembly of the nanostructure. The multivalent display of these ligands led to tighter binding by both mannose-specific lectins and appropriate antibodies. The potential of the novel selfassembling peptide to display antigens in bioanalytical assays that demand high sensitivity was illustrated by decoration with a disaccharide glycotope from the surface of the Leishmania parasite. Anti-Leishmania antibodies present in human and canine sera bind their antigen more effectively in the case of multivalent display on the coiled-coil scaffold. In summary, the α-helical coiled-coil scaffolds investigated here were shown to effectively multivalently present carbohydrate and/or peptide ligands to their specific binding partners, in the context of either a well-defined precision tool (project 1) or self-assembled nanofibers (project 2). Project 1 demonstrated that a coiled-coil carrier decorated with selected ligands may be tailored-made for applications involving other therapeutically-relevant receptors. Project 2 established that a synthetically accessible fiber-forming coiled-coil scaffold can provide the multivalent effect required to enhance binding avidity of specific antibodies and/or receptors.In den letzten Jahren stellt die Anwendung von synthetischen Gerüsten für den Transport biologisch relevanter Moleküle eine vorteilhafte Strategie in der Medizin dar. Dabei wird nicht nur der Wirkstoff stabilisiert sondern auch noch zusätzlich die Wirkungsweise am Zielort moduliert. Es konnte gezeigt werden, dass durch die Konjugation einer biologisch aktiven Spezies an ein Gerüst deren Stabilität erhöht wird, die optimale geometrische Konformation vermittelt wird und generell eine Erhöhung der Wahrscheinlichkeit auf ein Erreichen des Wirkstoffes am Wirkungsort stattfindet. Zu diesem Zweck wurden unterschiedlichste synthetische Träger untersucht wie zum Beispiel: Dendrimere, Nanopartikel, PNAs, LNAs und zellpenetrierende Peptide. In diesem Kontext stehen Peptide im besonderen Fokus der Forschung aufgrund ihrer zahlreichen funktionellen Gruppen, ihres vielfältigen Faltungsverhalten und ihrer Biokompatibilität sowie geringen Toxizität. Ein prominenter Vertreter, das α-helikale coiled-coil Faltungsmotif, ermöglicht durch seine strukturelle Einfachheit und Regelmäßigkeit die multivalente Präsentation von Liganden. Durch die Modifikation von wenigen Positionen in einem coiled-coil Motiv ist es möglich, einen Einfluss auf das Oligomerisierungsverhalten der resultierenden Helices auszuüben, wodurch entweder kurze Peptiddimere oder lange fibrillenartige Gerüste erhalten werden. Die vorliegende Arbeit untersucht die Möglichkeit das coiled-coil Motif als ein Gerüst für die multivalente Präsentation von Kohlenhydraten oder Peptiden zu verwenden. Dazu ist es angedacht den Vorteil des coiled-coil Motifs in zwei unterschiedlichen Projekten zu demonstrieren. Im ersten Projekt wird die Dimer-Form genutzt, die eine strukturelle Vorhersagbarkeit besitzt um eine rationale Präsentation von Kohlenhydraten für eine Lektin-Interaktion zu ermöglichen. Die zweite Studie zielt auf eine Erhöhung der Effizienz einer Kohlenhydrat-Antikörper-Erkennung durch die Ligandenpräsentation auf einem selbstorganisierenden Fibrillen bildenden Peptid ab. Das erste Projekt heißt “Tailored presentation of carbohydrate ligands on a coiled-coil scaffold for asialoglycoprotein receptor targeting”. In diesem Teil konnte die Bindung der Mitglieder einer coiled-coil Glykopeptid-Bibliothek zu Heptazyten bestimmt werden und der optimale Abstand sowie die Orientierung der Galactose-Liganden für die Wechselwirkung mit dem Asialoglykoproteinrezeptor (ASGPR) durch Druchflusszytomerie etabliert werden. Anhand von Inhibitionsstudien mit Cytochalasin D wurde bestätigt, dass die Bindung durch die ASGPR vermittelte Endozytose abläuft. Darüber hinaus wurde die Aufnahme der Trägerpeptide in die Zelle mithilfe von fluoreszenz- mikroskopischen Studien nachgewiesen. Im zweiten Teil mit dem Namen “A self- assembling peptide scaffold for the multivalent presentation of antigens” wurde ein coiled-coil-basierende Sequenz genutzt um veränderbare höhergeordnete Strukturen im Nanometerbereich zu generieren. Diese ermöglichten die gleichzeitige Präsentation von Mannose-Liganden und einem Peptid-Epitop, wobei die Selbstorganisation der Helices durch die Modifikationen nicht beeinträchtigt wurde. Die multivalente Präsentation dieser Liganden resultierte in einer stärkeren Bindung durch einerseits Mannose-bindende Lektine und andererseits durch geeignete Antikörper. Weiterhin wurde das Potential dieses neuartigen selbstorganisierenden Peptids untersucht, die eingebauten Antigene in einem bioanalytischen Assay zu präsentieren, welcher einer hohen Empfindlichkeit bedarf. Dazu wurde das Peptid mit einem Disaccharid-Glykotop vom Leishmania Parasit versehen und mit humanen und caninen anti-Leishmania-Antikörpern enthaltenden Seren inkubiert. Die Bindung des Antigens durch die Antikörper erfolgte im Falle der multivalenten Präsentation durch das coiled-coil Gerüst weitaus effektiver. Zusammenfassend wiesen die in der vorliegenden Arbeit untersuchten α-helikalen coiled-coil Gerüste eine effiziente multivalente Präsentation von Kohlenhydraten und/ oder Peptiden für die entsprechenden spezifisch bindenden Partner auf. Dies wurde entweder durch ein gut definiertes Präzisionswerkzeug (Projekt1) oder durch Selbstorganisierenden Nanofasern (Projekt 2) erzielt. In Projekt 1 wurde dargelegt, dass coiled-coil Peptide, welche mit ausgewählten Liganden versehen sind, für die Anwendung zur Untersuchung von therapeutisch relevanten Rezeptoren maßgeschneidert synthetisiert werden können. Im Projekt 2 wurde gezeigt, dass synthetisch zugängliche Fibrillen bildende coiled-coil Gerüste einen multivalenten Effekt aufweisen, welcher für eine Verstärkung der Bindung eines spezifischen Antikörpers und/ oder Rezeptors erforderlich ist

Experimental methods to study protein–nucleic acid interactions

The interactions between proteins and nucleic acids regulate gene expression at transcriptional, post-transcriptional, and translational levels and often dictate the fate of cellular metabolism in physiological conditions and disease. To appreciate more in-depth the regulatory implications of these interactions and the pathological consequences of their disruption, it is necessary to identify the highest possible number of protein domains and DNA/RNA sequences and structures involved and to reveal the signaling cascades they activate. The tools in our hands are becoming increasingly sophisticated and precise in helping determine the main actors involved and how they modulate each other. Starting from in vitro single-molecule interaction techniques to their validation and implementations in cells, this chapter wants to offer an overview of the state-of-the-art experimental methods available to the scientific community to identify and investigate proteins and nucleic-acid-binding partners

RNA sequestration driven by amyloid formation: the alpha synuclein case

Nucleic acids can act as potent modulators of protein aggregation, and RNA has the ability to either hinder or facilitate protein assembly, depending on the molecular context. In this study, we utilized a computational approach to characterize the physico-chemical properties of regions involved in amyloid aggregation. In various experimental datasets, we observed that while the core is hydrophobic and highly ordered, external regions, which are more disordered, display a distinct tendency to interact with nucleic acids. To validate our predictions, we performed aggregation assays with alpha-synuclein (aS140), a non-nucleic acid-binding amyloidogenic protein, and a mutant truncated at the acidic C-terminus (aS103), which is predicted to have a higher tendency to interact with RNA. For both aS140 and aS103, we observed an acceleration of aggregation upon RNA addition, with a significantly stronger effect for aS103. Due to favorable electrostatics, we noted an enhanced nucleic acid sequestration ability for the aggregated aS103, allowing it to entrap a larger amount of RNA compared to the aggregated wild-type counterpart. Overall, our research suggests that RNA sequestration might be a common phenomenon linked to protein aggregation, constituting a gain-of-function mechanism that warrants further investigation

The RNA-recognition motifs of TAR DNA-binding protein 43 may play a role in the aberrant self-assembly of the protein

The TAR DNA-binding protein 43 (TDP-43) is a nucleic acid-binding protein implicated in gene regulation and RNA processing and shuffling. It is a ribonuclear protein that carries out most of its functions by binding specific nucleic acid sequences with its two RNA-recognition motifs, RRM1 and RRM2. TDP-43 has been identified in toxic cytosolic inclusions in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). The unstructured C-terminus has prion-like behavior and has been considered the driver of the aberrant self-assembly of TDP-43. In this work, we set out to test the hypothesis that the RNA-binding domains could also play a role in protein aggregation. This knowledge could be of important value for understanding TDP-43 aberrant, disease-leading behavior and, in the future, inform the design of small molecules that could prevent or slow down protein aggregation by exploiting the RNA-binding properties of the protein. We investigated the behavior of the two tandem RRM domains separately and linked together and studied their self-assembly properties and RNA-binding ability with a number of biophysical techniques. The picture that emerges from our study suggests that this region of the protein plays an important and so far unexplored role in the aggregation of this protein

Zooming in on protein-RNA interactions: a multi-level workflow to identify interaction partners

Interactions between proteins and RNA are at the base of numerous cellular regulatory and functional phenomena. The investigation of the biological relevance of non-coding RNAs has led to the identification of numerous novel RNA-binding proteins (RBPs). However, defining the RNA sequences and structures that are selectively recognised by an RBP remains challenging, since these interactions can be transient and highly dynamic, and may be mediated by unstructured regions in the protein, as in the case of many non-canonical RBPs. Numerous experimental and computational methodologies have been developed to predict, identify and verify the binding between a given RBP and potential RNA partners, but navigating across the vast ocean of data can be frustrating and misleading. In this mini-review, we propose a workflow for the identification of the RNA binding partners of putative, newly identified RBPs. The large pool of potential binders selected by in-cell experiments can be enriched by in silico tools such as catRAPID, which is able to predict the RNA sequences more likely to interact with specific RBP regions with high accuracy. The RNA candidates with the highest potential can then be analysed in vitro to determine the binding strength and to precisely identify the binding sites. The results thus obtained can furthermore validate the computational predictions, offering an all-round solution to the issue of finding the most likely RNA binding partners for a newly identified potential RBP.The research leading to this work has been supported by European Research Council (RIBOMYLOME_309545 and ASTRA_855923), by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 754490 and under projects IASIS_727658 and INFORE_825080, by the Spanish Ministry of Economy and Competitiveness BFU2017-86970-P as well as the collaboration with Peter St. George-Hyslop financed by the Wellcome Trust