Molecular Crowding: The History and Development of a Scientific Paradigm.

It is now generally accepted that macromolecules do not act in isolation but "live" in a crowded environment, that is, an environment populated by numerous different molecules. The field of molecular crowding has its origins in the far 80s but became accepted only by the end of the 90s. In the present issue, we discuss various aspects that are influenced by crowding and need to consider its effects. This Review is meant as an introduction to the theme and an analysis of the evolution of the crowding concept through time from colloidal and polymer physics to a more biological perspective. We introduce themes that will be more thoroughly treated in other Reviews of the present issue. In our intentions, each Review may stand by itself, but the complete collection has the aspiration to provide different but complementary perspectives to propose a more holistic view of molecular crowding.

In-silico guided chemical exploration of KDM4A fragments hits.

BACKGROUND: Lysine demethylase enzymes (KDMs) are an emerging class of therapeutic targets, that catalyse the removal of methyl marks from histone lysine residues regulating chromatin structure and gene expression. KDM4A isoform plays an important role in the epigenetic dysregulation in various cancers and is linked to aggressive disease and poor clinical outcomes. Despite several efforts, the KDM4 family lacks successful specific molecular inhibitors. RESULTS: Herein, starting from a structure-based fragments virtual screening campaign we developed a synergic framework as a guide to rationally design efficient KDM4A inhibitors. Commercial libraries were used to create a fragments collection and perform a virtual screening campaign combining docking and pharmacophore approaches. The most promising compounds were tested in-vitro by a Homogeneous Time-Resolved Fluorescence-based assay developed for identifying selective substrate-competitive inhibitors by means of inhibition of H3K9me3 peptide demethylation. 2-(methylcarbamoyl)isonicotinic acid was identified as a preliminary active fragment, displaying inhibition of KDM4A enzymatic activity. Its chemical exploration was deeply investigated by computational and experimental approaches which allowed a rational fragment growing process. The in-silico studies guided the development of derivatives designed as expansion of the primary fragment hit and provided further knowledge on the structure-activity relationship. CONCLUSIONS: Our study describes useful insights into key ligand-KDM4A protein interaction and provides structural features for the development of successful selective KDM4A inhibitors.

Networks as Biomarkers: Uses and Purposes.

Networks-based approaches are often used to analyze gene expression data or protein-protein interactions but are not usually applied to study the relationships between different biomarkers. Given the clinical need for more comprehensive and integrative biomarkers that can help to identify personalized therapies, the integration of biomarkers of different natures is an emerging trend in the literature. Network analysis can be used to analyze the relationships between different features of a disease; nodes can be disease-related phenotypes, gene expression, mutational events, protein quantification, imaging-derived features and more. Since different biomarkers can exert causal effects between them, describing such interrelationships can be used to better understand the underlying mechanisms of complex diseases. Networks as biomarkers are not yet commonly used, despite being proven to lead to interesting results. Here, we discuss in which ways they have been used to provide novel insights into disease susceptibility, disease development and severity.

Computational design and characterization of a multiepitope vaccine against carbapenemase-producing Klebsiella pneumoniae strains, derived from antigens identified through reverse vaccinology.

Klebsiella pneumoniae is a Gram-negative pathogen of clinical relevance, which can provoke serious urinary and blood infections and pneumonia. This bacterium is a major public health threat due to its resistance to several antibiotic classes. Using a reverse vaccinology approach, 7 potential antigens were identified, of which 4 were present in most of the sequences of Italian carbapenem-resistant K. pneumoniae clinical isolates. Bioinformatics tools demonstrated the antigenic potential of these bacterial proteins and allowed for the identification of T and B cell epitopes. This led to a rational design and in silico characterization of a multiepitope vaccine against carbapenem-resistant K. pneumoniae strains. As adjuvant, the mycobacterial heparin-binding hemagglutinin adhesin (HBHA), which is a Toll-like receptor 4 (TLR-4) agonist, was included, to increase the immunogenicity of the construct. The multiepitope vaccine candidate was analyzed by bioinformatics tools to assess its antigenicity, solubility, allergenicity, toxicity, physical and chemical parameters, and secondary and tertiary structures. Molecular docking binding energies to TLR-2 and TLR-4, two important innate immunity receptors involved in the immune response against K. pneumoniae infections, and molecular dynamics simulations of such complexes supported active interactions. A codon optimized multiepitope sequence cloning strategy is proposed, for production of recombinant vaccine in classical bacterial vectors. Finally, a 3 dose-immunization simulation with the multiepitope construct induced both cellular and humoral immune responses. These results suggest that this multiepitope construct has potential as a vaccination strategy against carbapenem-resistant K. pneumoniae and deserves further validation.

Solution structure of recombinant Pvfp-5ß reveals insights into mussel adhesion.

Some marine organisms can resist to aqueous tidal environments and adhere tightly on wet surface. This behavior has raised increasing attention for potential applications in medicine, biomaterials, and tissue engineering. In mussels, adhesive forces to the rock are the resultant of proteinic fibrous formations called byssus. We present the solution structure of Pvfp-5ß, one of the three byssal plaque proteins secreted by the Asian green mussel Perna viridis, and the component responsible for initiating interactions with the substrate. We demonstrate that Pvfp-5ß has a stably folded structure in agreement with the presence in the sequence of two EGF motifs. The structure is highly rigid except for a few residues affected by slow local motions in the µs-ms time scale, and differs from the model calculated by artificial intelligence methods for the relative orientation of the EGF modules, which is something where computational methods still underperform. We also show that Pvfp-5ß is able to coacervate even with no DOPA modification, giving thus insights both for understanding the adhesion mechanism of adhesive mussel proteins, and developing of biomaterials.

Investigation on a MMACHC mutant from cblC disease: The c.394C>T variant.

The cblC disease is an inborn disorder of the vitamin B12 (cobalamin, Cbl) metabolism characterized by methylmalonic aciduria and homocystinuria. The clinical consequences of this disease are devastating and, even when early treated with current therapies, the affected children manifest symptoms involving vision, growth, and learning. The illness is caused by mutations in the gene codifying for MMACHC, a 282aa protein that transports and transforms the different Cbl forms. Here we present data on the structural properties of the truncated protein p.R132X resulting from the c.394C > T mutation that, along with c.271dupA and c.331C > T, is among the most common mutations in cblC. Although missing part of the Cbl binding domain, p.R132X is associated to late-onset symptoms and, therefore, it is supposed to retain residual function. However, to our knowledge structural-functional studies on c.394C > T mutant aimed at verifying this hypothesis are still lacking. By using a biophysical approach including Circular Dichroism, fluorescence, Small Angle X-ray Scattering, and Molecular Dynamics, we show that the mutant protein MMACHC-R132X retains secondary structure elements and remains compact in solution, partly preserving its binding affinity for Cbl. Insights on the fragile stability of MMACHC-R132X-Cbl are provided.

"Writing biochips": high-resolution droplet-to-droplet manufacturing of analytical platforms.

The development of high-resolution molecular printing allows the engineering of analytical platforms enabling applications at the interface between chemistry and biology, i.e. in biosensing, electronics, single-cell biology, and point-of-care diagnostics. Their successful implementation stems from the combination of large area printing at resolutions from sub-100 nm up to macroscale, whilst controlling the composition and volume of the ink, and reconfiguring the deposition features in due course. Similar to handwriting pens, the engineering of continuous writing systems tackles the issue of the tedious ink replenishment between different printing steps. To this aim, this review article provides an unprecedented analysis of the latest continuous printing methods for bioanalytical chemistry, focusing on ink deposition systems based on specific sets of technologies that have been developed to this aim, namely nanofountain probes, microcantilever spotting, capillary-based polymer pens and continuous 3D printing. Each approach will be discussed revealing the most important applications in the fields of biosensors, lab-on-chips and diagnostics.

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications.

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium's collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.

Backbone chemical shift spectral assignments of SARS coronavirus-2 non-structural protein nsp9.

As part of an International consortium aiming at the characterization by NMR of the proteins of the SARS-CoV-2 virus, we have obtained the virtually complete assignment of the backbone atoms of the non-structural protein nsp9. This small (12 kDa) protein is encoded by ORF1a, binds to RNA and seems to be essential for viral RNA synthesis. The crystal structures of the SARS-CoV-2 protein and other homologues suggest that the protein is dimeric as also confirmed by analytical ultracentrifugation and dynamic light scattering. Our data constitute the prerequisite for further NMR-based characterization, and provide the starting point for the identification of small molecule lead compounds that could interfere with RNA binding and prevent viral replication.

The Wide World of Coacervates: From the Sea to Neurodegeneration.

The formation of immiscible liquid phases or coacervates is a phenomenon widely observed in biology. Marine organisms, for instance, use liquid-liquid phase separation (LLPS) as the precursor phase to form various fibrillar or crustaceous materials that are essential for surface adhesion. More recently, the importance of LLPS has been realized in the compartmentalization of living cells and in obtaining ordered but dynamic partitions that can be reversed according to necessity. Here, we compare the properties, features, and peculiarities of intracellular and extracellular coacervates, drawing parallels and learning from the differences. A more general view of the phenomenon may in the future inform new studies to allow a better comprehension of its laws.

Quantifying the thermodynamics of protein unfolding using 2D NMR spectroscopy.

A topic that has attracted considerable interest in recent years is the possibility to perform thermodynamic studies of proteins directly in-cell or in complex environments which mimic the cellular interior. Nuclear magnetic resonance (NMR) could be an attractive technique for these studies but its applicability has so far been limited by technical issues. Here, we demonstrate that 2D NMR methods can be successfully applied to measure thermodynamic parameters provided that a suitable choice of the residues used for the calculation is made. We propose a new parameter, named RAD, which reflects the level of protection of a specific amide proton in the protein core and can guide through the selection of the resonances. We also suggest a way to calibrate the volumes to become independent of technical limitations. The methodology we propose leads to stability curves comparable to that calculated from CD data and provides a new tool for thermodynamic measurements in complex environments.

Recombinant mussel protein Pvfp-5ß: A potential tissue bioadhesive.

During their lifecycle, many marine organisms rely on natural adhesives to attach to wet surfaces for movement and self-defense in aqueous tidal environments. Adhesive proteins from mussels are biocompatible and elicit only minimal immune responses in humans. Therefore these proteins have received increased attention for their potential applications in medicine, biomaterials, and biotechnology. The Asian green mussel Perna viridis secretes several byssal plaque proteins, molecules that help anchoring the mussel to surfaces. Among these proteins, protein-5ß (Pvfp-5ß) initiates interactions with the substrate, displacing interfacial water molecules before binding to the surface. Here, we established the first recombinant expression in Escherichia coli of Pvfp-5ß. We characterized recombinant Pvfp-5ß, finding that despite displaying a CD spectrum consistent with features of a random coil, the protein is correctly folded as indicated by MS and NMR analyses. Pvfp-5ß folds as a ß-sheet-rich protein as expected for an epidermal growth factor-like module. We examined the effects of Pvfp-5ß on cell viability and adhesion capacity in NIH-3T3 and HeLa cell lines, revealing that Pvfp-5ß has no cytotoxic effects at the protein concentrations used and provides good cell-adhesion strength on both glass and plastic plates. Our findings suggest that the adhesive properties of recombinant Pvfp-5ß make it an efficient surface-coating material, potentially suitable for biomedical applications including regeneration of damaged tissues.

RNA as a key factor in driving or preventing self-assembly of the TAR DNA-binding protein 43.

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration are incurable motor neuron diseases associated with muscle weakness, paralysis and respiratory failure. Accumulation of TAR DNA-binding protein 43 (TDP-43) as toxic cytoplasmic inclusions is one of the hallmarks of these pathologies. TDP-43 is an RNA-binding protein responsible for regulating RNA transcription, splicing, transport and translation. Aggregated TDP-43 does not retain its physiological function. Here, we exploit the ability of TDP-43 to bind specific RNA sequences to validate our hypothesis that the native partners of a protein can be used to interfere with its ability to self-assemble into aggregates. We propose that binding of TDP-43 to specific RNA can compete with protein aggregation. This study provides a solid proof of concept to the hypothesis that natural interactions can be exploited to increase protein solubility and could be adopted as a more general rational therapeutic strategy.

Structural and Functional Insights into Bacillus subtilis Sigma Factor Inhibitor, CsfB.

Global changes in bacterial gene expression can be orchestrated by the coordinated activation/deactivation of alternative sigma (σ) factor subunits of RNA polymerase. Sigma factors themselves are regulated in myriad ways, including via anti-sigma factors. Here, we have determined the solution structure of anti-sigma factor CsfB, responsible for inhibition of two alternative sigma factors, σG and σE, during spore formation by Bacillus subtilis. CsfB assembles into a symmetrical homodimer, with each monomer bound to a single Zn2+ ion via a treble-clef zinc finger fold. Directed mutagenesis indicates that dimer formation is critical for CsfB-mediated inhibition of both σG and σE, and we have characterized these interactions in vitro. This work represents an advance in our understanding of how CsfB mediates inhibition of two alternative sigma factors to drive developmental gene expression in a bacterium.

Thrombopoietin mutation in congenital amegakaryocytic thrombocytopenia treatable with romiplostim.

Congenital amegakaryocytic thrombocytopenia (CAMT) is an inherited disorder characterized at birth by thrombocytopenia with reduced megakaryocytes, which evolves into generalized bone marrow aplasia during childhood. Although CAMT is genetically heterogeneous, mutations of MPL, the gene encoding for the receptor of thrombopoietin (THPO), are the only known disease-causing alterations. We identified a family with three children affected with CAMT caused by a homozygous mutation (p.R119C) of the THPO gene. Functional studies showed that p.R119C affects not only ability of the cytokine to stimulate MPL but also its release, which is consistent with the relatively low serum THPO levels measured in patients. In all the three affected children, treatment with the THPO-mimetic romiplostim induced trilineage hematological responses, remission of bleeding and infections, and transfusion independence, which were maintained after up to 6.5 years of observation. Recognizing patients with THPO mutations among those with juvenile bone marrow failure is essential to provide them with appropriate substitutive therapy and prevent the use of invasive and unnecessary treatments, such as hematopoietic stem cell transplantation or immunosuppression.

A novel RNA polymerase-binding protein that interacts with a sigma-factor docking site.

Sporulation in Bacillus subtilis is governed by a cascade of alternative RNA polymerase sigma factors. We previously identified a small protein Fin that is produced under the control of the sporulation sigma factor σF to create a negative feedback loop that inhibits σF -directed gene transcription. Cells deleted for fin are defective for spore formation and exhibit increased levels of σF -directed gene transcription. Based on pull-down experiments, chemical crosslinking, bacterial two-hybrid experiments and nuclear magnetic resonance chemical shift analysis, we now report that Fin binds to RNA polymerase and specifically to the coiled-coil region of the ß' subunit. The coiled-coil is a docking site for sigma factors on RNA polymerase, and evidence is presented that the binding of Fin and σF to RNA polymerase is mutually exclusive. We propose that Fin functions by a mechanism distinct from that of classic sigma factor antagonists (anti-σ factors), which bind directly to a target sigma factor to prevent its association with RNA polymerase, and instead functions to inhibit σF by competing for binding to the ß' coiled-coil.

An optimized strategy to measure protein stability highlights differences between cold and hot unfolded states.

Macromolecular crowding ought to stabilize folded forms of proteins, through an excluded volume effect. This explanation has been questioned and observed effects attributed to weak interactions with other cell components. Here we show conclusively that protein stability is affected by volume exclusion and that the effect is more pronounced when the crowder's size is closer to that of the protein under study. Accurate evaluation of the volume exclusion effect is made possible by the choice of yeast frataxin, a protein that undergoes cold denaturation above zero degrees, because the unfolded form at low temperature is more expanded than the corresponding one at high temperature. To achieve optimum sensitivity to changes in stability we introduce an empirical parameter derived from the stability curve. The large effect of PEG 20 on cold denaturation can be explained by a change in water activity, according to Privalov's interpretation of cold denaturation.

Expression and Purification of ZASP Subdomains and Clinically Important Isoforms: High-Affinity Binding to G-Actin.

Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP) is a principal component of the sarcomere. The three prevalent isoforms of ZASP in skeletal muscle are generated by alternative splicing of exons 9 and 10. The long isoforms, either having (ZASP-L) or lacking exon 10 (ZASP-LΔex10), include an N-terminal PDZ domain, an actin-binding region (ABR) with a conserved motif (ZM), and three C-terminal LIM domains. The short isoform (ZASP-S) lacks the LIM domains. Mutations, A147T and A165V, within the ZM of ZASP-LΔex10 cause myofibrillar myopathy, but the mechanism is unknown. We have prepared these proteins, their ABR, and the respective mutant variants in recombinant form, characterized them biophysically, and analyzed their actin-binding properties by surface plasmon resonance and electron microscopy. All the proteins were physically homogeneous and monomeric and had circular dichroic spectra consistent with partially folded conformations. Comparison of the NMR HSQC spectra of ZASP-S and the PDZ domain showed that the ABR is unstructured. ZASP-S and its mutant variants and ZASP-LΔex10 all bound to immobilized G-actin with high affinity (Kd ≈ 10-8 to 10-9 M). Constructs of the isolated actin-binding region missing exon 10 (ABRΔ10) bound with lower affinity (Kd ≈ 10-7 M), but those retaining exon 10 (ABR+10) did so only weakly (Kd ≈ 10-5 M). ZASP-S, and the ABRΔ10, also induced F-actin and array formation, even in conditions of low ionic strength and in the absence of KCl and Mg2+ ions. Interestingly, the ZM mutations A147T and A165V did not affect any of the results described above.