An oomycete NLP cytolysin forms transient small pores in lipid membranes

Microbial plant pathogens secrete a range of effector proteins that damage host plants and consequently constrain global food production. Necrosis and ethylene-inducing peptide 1-like proteins (NLPs) are produced by numerous phytopathogenic microbes that cause important crop diseases. Many NLPs are cytolytic, causing cell death and tissue necrosis by disrupting the plant plasma membrane. Here, we reveal the unique molecular mechanism underlying the membrane damage induced by the cytotoxic model NLP. This membrane disruption is a multistep process that includes electrostatic-driven, plant-specific lipid recognition, shallow membrane binding, protein aggregation, and transient pore formation. The NLP-induced damage is not caused by membrane reorganization or large-scale defects but by small membrane ruptures. This distinct mechanism of lipid membrane disruption is highly adapted to effectively damage plant cells.Peer reviewe

Unraveling the Molecular Mechanism of Splicing through Molecular Dynamics Simulation

The genes architecture as made of intron and exons is now a widely accepted fact and a well-established hypothesis. Indeed, the exons regions of a DNA molecule that code for proteins are not a continuous unitary sequence, but silent intervening segments (introns) that must be eliminated in a process known as pre-mRNA splicing. However, the splicing process is far from being fully understood, as the subtle regulatory mechanisms underlying the generation of premature mRNA, hide a large number of unresolved biological questions. The main keeper of this enigma is represented by the spliceosome, a highly dynamic molecular machinery and the main actor of the splicing process. With recent technological advancement in structural biology, crystallographic techniques together with a remarkable and continuous improvement of computational tools, we are witnessing a breakthrough era that allows us to study and to understand at the atomic-scale key functional aspects of the working mechanisms of large biological macromolecules such as the spliceosome. In my Ph.D years, I have tackled mechanistic aspects of splicing process, trying to address to three main questions: (i) discovery of small molecules to target specific splicing factors for treatment of splicing-related diseases; (ii) unraveling the molecular mechanism at the basis of pre-mRNAs recognition and splicing fidelity; (iii) elucidating the structural and dynamical properties of the spliceosome and its allosteric regulatory networks. This have been achieved by the use of classical molecular dynamics simulations (MD), Metadynamics and Virtual screening simulations. In Chapter 2 I introduce you to the biological significance and conservation of the splicing and alternative splicing, how it is used in normal eukaryotic cells, as well as in cancer cells. The impact of deregulated splicing on a plethora of human diseases is also discussed as well. Finally, I will present the structural and molecular biology of the spliceosome machinery, also explaining how it can precisely process different pre-mRNA sequences. Chapter 3 reports a review of all the computational techniques that I have used in this thesis. Namely, a brief introduction to classical molecular dynamics simulations, virtual screening, enhanced sampling methods and network theory analysis is reported. Chapter 4 is entirely dedicated to identifying small molecules inhibitors for a particular kinase that is involved in pre-mRNA splicing and that contributes to the migration propensity of Triple Negative Breast Cancer, one of the most aggressive breast cancer types. Chapter 5 focuses on the early recognition mechanism of specific pre-mRNA sequences by the splicing cofactor U2AF2, that is also involved in the first steps of the spliceosome assembly. The recognition of these sequences represents one of the first pre-mRNA recognition events, and it underlies the alternative splicing of pre-mRNA, directing the spliceosome to generate one specific transcript rather than another, which result into distinct protein isoforms. The effect of cancer-associated mutations on this delicate recognition step is also investigated. Chapter 6 represents the first attempt at understanding the reshaping properties of the spliceosome and the allosteric signaling underlying it. In this chapter I report a MD simulations study based on the cryo-EM structure of a yeast spliceosome solved at near-atomic-level resolution. In particular, I have investigated the structural and dynamical properties of the spliceosome machinery, making use of network theory in order to trace the information exchange pathways at the basis of the characterized functional motions

Is the Rigidity of SARS-CoV-2 Spike Receptor-Binding Motif the Hallmark for Its Enhanced Infectivity? An Answer from All-Atoms Simulations

The latest outbreak of a new pathogenic coronavirus (SARS-CoV-2) is provoking a global health, economic and societal crisis. All-atom simulations enabled us to uncover the key molecular traits underlying the high affinity of SARS-CoV-2 spike glycoprotein towards its human receptor, providing a rationale to its high infectivity. Harnessing this knowledge can boost developing effective medical countermeasures to fight the current global pandemic.</p

Atomic-level mechanism of Pre-mRNA splicing in health and disease

Intron removal from premature-mRNA (pre-mRNA splicing) is an essential part of gene expression and regulation that is required for the production of mature, protein-coding mRNA. The spliceosome (SPL), a majestic machine composed of five small nuclear RNAs and hundreds of proteins, behaves as an eminent transcriptome tailor, efficiently performing splicing as a protein-directed metallo-ribozyme. To select and excise long and diverse intronic sequences with single-nucleotide precision, the SPL undergoes a continuous compositional and conformational remodeling, forming eight distinct complexes throughout each splicing cycle. Splicing fidelity is of paramount importance to preserve the integrity of the proteome. Mutations in splicing factors can severely compromise the accuracy of this machinery, leading to aberrant splicing and altered gene expression. Decades of biochemical and genetic studies have provided insights into the SPL's composition and function, but its complexity and plasticity have prevented an in-depth mechanistic understanding. Single-particle cryogenic electron microscopy techniques have ushered in a new era for comprehending eukaryotic gene regulation, providing several near-atomic resolution structures of the SPL from yeast and humans. Nevertheless, these structures represent isolated snapshots of the splicing process and are insufficient to exhaustively assess the function of each SPL component and to unravel particular facets of the splicing mechanism in a dynamic environment.In this Account, building upon our contributions in this field, we discuss the role of biomolecular simulations in uncovering the mechanistic intricacies of eukaryotic splicing in health and disease. Specifically, we showcase previous applications to illustrate the role of atomic-level simulations in elucidating the function of specific proteins involved in the architectural reorganization of the SPL along the splicing cycle. Moreover, molecular dynamics applications have uniquely contributed to decrypting the channels of communication required for critical functional transitions of the SPL assemblies. They have also shed light on the role of carcinogenic mutations in the faithful selection of key intronic regions and the molecular mechanism of splicing modulators. Additionally, we emphasize the role of quantum-classical molecular dynamics in unraveling the chemical details of pre-mRNA cleavage in the SPL and in its evolutionary ancestors, group II intron ribozymes. We discuss methodological pitfalls of multiscale calculations currently used to dissect the splicing mechanism, presenting future challenges in this field. The results highlight how atomic-level simulations can enrich the interpretation of experimental results. We envision that the synergy between computational and experimental approaches will aid in developing innovative therapeutic strategies and revolutionary gene modulation tools to fight the over 200 human diseases associated with splicing misregulation, including cancer and neurodegeneration.J.B. thanks the Slovenian Research Agency (P1-0017 and Z1-1855), and A.M. is thankful for the financial support of the Italian Association for Cancer Research (IG 24514) and of the project “Against bRain cancEr: finding personalized therapies with in Silico and in vitro strategies” (ARES) CUP: D93D19000020007 POR FESR 2014 2020-1.3.b- Friuli Venezia Giulia. L.M. is thankful for the financial support of the Associazione Italiana Ricerca sul Cancro (AIRC) (International Accelerator Program #22796, 5x1000 project #21267, IG 2017 #20125). S.G.M. is supported by the European Union’s H2020 research and innovation programme under Marie Skłodowska-Curie Grant Agreement No. 75442

Disclosing the Impact of Carcinogenic SF3b Mutations on Pre-mRNA Recognition Via All-Atom Simulations.

The spliceosome accurately promotes precursor messenger-RNA splicing by recognizing specific noncoding intronic tracts including the branch point sequence (BPS) and the 3'-splice-site (3'SS). Mutations of Hsh155 (yeast)/SF3B1 (human), which is a protein of the SF3b factor involved in BPS recognition and induces altered BPS binding and 3'SS selection, lead to mis-spliced mRNA transcripts. Although these mutations recur in hematologic malignancies, the mechanism by which they change gene expression remains unclear. In this study, multi-microsecond-long molecular-dynamics simulations of eighth distinct ∼700,000 atom models of the spliceosome Bact complex, and gene sequencing of SF3B1, disclose that these carcinogenic isoforms destabilize intron binding and/or affect the functional dynamics of Hsh155/SF3B1 only when binding non-consensus BPSs, as opposed to the non-pathogenic variants newly annotated here. This pinpoints a cross-talk between the distal Hsh155 mutation and BPS recognition sites. Our outcomes unprecedentedly contribute to elucidating the principles of pre-mRNA recognition, which provides critical insights on the mechanism underlying constitutive/alternative/aberrant splicing

Decrypting the information exchange pathways across the spliceosome machinery

Intron splicing of a nascent mRNA transcript by spliceosome (SPL) is a hallmark of gene regulation in eukaryotes. SPL is a majestic molecular machine composed of an entangled network of proteins and RNAs that meticulously promotes intron splicing through the formation of eight intermediate complexes. Cross-communication among the critical distal proteins of the SPL assembly is pivotal for fast and accurate directing of the compositional and conformational readjustments necessary to achieve high splicing fidelity. Here, molecular dynamics (MD) simulations of an 800 000 atom model of SPL C complex from yeast Saccharomyces cerevisiae and community network analysis enabled us to decrypt the complexity of this huge molecular machine, by identifying the key channels of information transfer across long distances separating key protein components. The reported study represents an unprecedented attempt in dissecting cross-communication pathways within one of the most complex machines of eukaryotic cells, supporting the critical role of Clf1 and Cwc2 splicing cofactors and specific domains of the PrpS protein as signal conveyors for pre-mRNA maturation. Our findings provide fundamental advances into mechanistic aspects of SPL, providing a conceptual basis for controlling the SPL via small-molecule modulators able to tackle splicing-associated diseases by altering/obstructing information-exchange paths

All-Atom Simulations Elucidate the Impact of U2AF2 Cancer-Associated Mutations on Pre-mRNA Recognition

The U2AF2 splicing factor, made of two tandem RNA recognition motifs (RRMs) joined by a flexible linker, selects the intronic polypyrimidine sequence of premature mRNA, thus ensuring splicing fidelity. Increasing evidence links mutations of key splicing factors, including U2AF2, to a variety of cancers. Nevertheless, the impact of U2AF2 cancer-associated mutations on polypyrimidine recognition remains unclear. Here, we combined extensive (18 μs-long) all-atom molecular dynamics simulations and dynamical network theory analysis (NWA) of U2AF2, in its wild-type form and in the presence of the six most frequent cancer-associated mutations, bound to a poly-U strand. Our results reveal that the selected mutations affect the pre-mRNA binding at two hot spot regions, irrespectively of where these mutants are placed on the distinct U2AF2 domains. Complementarily, NWA traced the existence of cross-communication pathways, connecting each mutation site to these recognition hot spots, whose strength is altered by the mutations. Our outcomes suggest the existence of a structural/dynamical interplay of the two U2AF2’s RRMs underlying the recognition of the polypyrimidine tract and reveal that the cancer-associated mutations affect the polypyrimidine selection by altering the RRMs’ cooperativity. This mechanism may be shared by other RNA binding proteins hallmarked, like U2AF2, by multidomain architecture and high plasticity

The human−animal relationship in dairy animals

AbstractThe present study aims to identify margins for the improvement of dairy animal welfare and production based on the quality of the human−animal relationship (HAR). The main tool proposed to improve the quality of HAR in dairy animals is training of stock-people by targeting their attitude and behaviour. Given that a good quality HAR may benefit the welfare of dairy animals and productivity, new technologies, by monitoring the handling routine on farm, may be more effective in promoting good practices. In particular, the implementation of new technologies may allow identification of specific inappropriate behaviours to be targeted at stockperson level, thus increasing the efficacy of training. However, an issue related to the introduction of new technologies in the farms, particularly in those that follow traditional farming practices, is the resistance to innovation which may be encountered

An oomycete NLP cytolysin forms transient small pores in lipid membranes

Microbial plant pathogens secrete a range of effector proteins that damage host plants and consequently constrain global food production. Necrosis and ethylene-inducing peptide 1-like proteins (NLPs) are produced by numerous phytopathogenic microbes that cause important crop diseases. Many NLPs are cytolytic, causing cell death and tissue necrosis by disrupting the plant plasma membrane. Here, we reveal the unique molecular mechanism underlying the membrane damage induced by the cytotoxic model NLP. This membrane disruption is a multistep process that includes electrostatic-driven, plant-specific lipid recognition, shallow membrane binding, protein aggregation, and transient pore formation. The NLP-induced damage is not caused by membrane reorganization or large-scale defects but by small membrane ruptures. This distinct mechanism of lipid membrane disruption is highly adapted to effectively damage plant cells.Peer reviewe