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Mastering selective molecule trafficking across human cell membranes poses a formidable challenge in healthcare biotechnology while offering the prospect of breakthroughs in drug delivery, gene therapy, and diagnostic imaging. The cholera toxin B-subunit (CTB) has the potential to be a useful cargo transporter for these applications. CTB is a robust protein that is amenable to reengineering for diverse applications; however, protein redesign has mostly focused on modifications of the N- and C-termini of the protein. Exploiting the full power of rational redesign requires a detailed understanding of the contributions of the surface residues to protein stability and binding activity. Here, we employed Rosetta-based computational saturation scans on 58 surface residues of CTB, including the GM1 binding site, to analyze both ligand-bound and ligand-free structures to decipher mutational effects on protein stability and GM1 affinity. Complimentary experimental results from differential scanning fluorimetry and isothermal titration calorimetry provided melting temperatures and GM1 binding affinities for 40 alanine mutants among these positions. The results showed that CTB can accommodate diverse mutations while maintaining its stability and ligand binding affinity. These mutations could potentially allow modification of the oligosaccharide binding specificity to change its cellular targeting, alter the B-subunit intracellular routing, or impact its shelf-life and in vivo half-life through changes to protein stability. We anticipate that the mutational space maps presented here will serve as a cornerstone for future CTB redesigns, paving the way for the development of innovative biotechnological tools. [ABSTRACT FROM AUTHOR]

Influenza virus infections represent an ongoing public health threat as well as an economic burden. Although seasonal influenza vaccines have been available for some decades, efforts are being made to generate new efficient, flexible, and cost-effective technologies to be transferred into production. Our work describes the development of a model influenza hemagglutinin antigen that is capable of inducing protection against viral challenge in mice. High amounts of the H1 hemagglutinin ectodomain, HA18–528, were expressed in a bacterial system as insoluble inclusion bodies. Solubilization was followed by a thorough differential scanning fluorimetry (DSF)-guided optimization of refolding, which allows for fast and reliable screening of several refolding conditions, yielding tens of milligrams/L of folded protein. Structural and functional analysis revealed native-like folding as well as the presence of a mix of monomers and oligomers in solution. Mice immunized with HA18–528 were protected when exposed to influenza A virus as opposed to mice that received full-length denatured protein. Sera of mice immunized with HA18–528 showed both high titers of antigen-specific IgG1 and IgG2a isotypes as well as viral neutralization activity. These results prove the feasibility of the recombinant bacterial expression system coupled with DSF-guided refolding in providing influenza hemagglutinin for vaccine development. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome-causing coronavirus 2 (SARS-CoV-2) papain-like protease (PLpro) and main protease (Mpro) play an important role in viral replication events and are important targets for anti-coronavirus drug discovery. In search of these protease inhibitors, we screened a library of 1300 compounds using a fluorescence thermal shift assay (FTSA) and identified 53 hits that thermally stabilized or destabilized PLpro. The hit compounds structurally belonged to two classes of small molecules: thiazole derivatives and symmetrical disulfide compounds. Compound dissociation constants (Kd) were determined using an enzymatic inhibition method. Seven aromatic disulfide compounds were identified as efficient PLpro inhibitors with Kd values in the micromolar range. Two disulfides displayed six-fold higher potency for PLpro (Kd = 0.5 µM) than for Mpro. The disulfide derivatives bound covalently to both proteases, as confirmed through mass spectrometry. The identified compounds can serve as lead compounds for further chemical optimization toward anti-COVID-19 drugs. [ABSTRACT FROM AUTHOR]

Accurate temperature control within biological and chemical reaction samples and instrument calibration are essential to the diagnostic, pharmaceutical and chemical industries. This is particularly challenging for microlitre-scale reactions typically used in real-time PCR applications and differential scanning fluorometry. Here, we describe the development of a simple, inexpensive ratiometric dual fluorescent protein temperature biosensor (DFPTB). A combination of cycle three green fluorescent protein and a monomeric red fluorescent protein enabled the quantification of relative temperature changes and the identification of temperature discrepancies across a wide temperature range of 4–70 °C. The maximal sensitivity of 6.7% °C−1 and precision of 0.1 °C were achieved in a biologically relevant temperature range of 25–42 °C in standard phosphate-buffered saline conditions at a pH of 7.2. Good temperature sensitivity was achieved in a variety of biological buffers and pH ranging from 4.8 to 9.1. The DFPTB can be used in either purified or mixed bacteria-encapsulated formats, paving the way for in vitro and in vivo applications for topologically precise temperature measurements. [ABSTRACT FROM AUTHOR]

Simple Summary: The toolkit for diagnosing the most aggressive primary brain tumor glioblastoma (GBM) is very limited. We recently demonstrated that plasma denaturation profiles (PDPs) of GBM patients and healthy controls obtained with nanoDSF can be automatically classified using artificial intelligence (AI) algorithms. Since PDPs have been shown to be useful for subtype differentiation for lung cancer, we decided to investigate whether nanoDSF-derived PDPs could also be used to discriminate EGFR alterations in GBM, which is important for determining therapy strategies. We found that AI is able to discriminate EGFR alterations in GBM with an 81.5% accuracy. Thus, we have demonstrated that the use of plasma denaturation profiles could answer the unsatisfied neuro-oncology need for a predictive diagnostic biomarker, which could complete MRI and clinical data, allowing for a rapid orientation of patients for a definitive pathological diagnosis and treatment. Glioblastoma (GBM) is the most frequent and aggressive primary brain tumor in adults. Recently, we demonstrated that plasma denaturation profiles of glioblastoma patients obtained using Differential Scanning Fluorimetry can be automatically distinguished from healthy controls with the help of Artificial Intelligence (AI). Here, we used a set of machine-learning algorithms to automatically classify plasma denaturation profiles of glioblastoma patients according to their EGFR status. We found that Adaboost AI is able to discriminate EGFR alterations in GBM with an 81.5% accuracy. Our study shows that the use of these plasma denaturation profiles could answer the unmet neuro-oncology need for diagnostic predictive biomarker in combination with brain MRI and clinical data, in order to allow for a rapid orientation of patients for a definitive pathological diagnosis and then treatment. We complete this study by showing that discriminating another mutation, MGMT, seems harder, and that post-surgery monitoring using our approach is not conclusive in the 48 h that follow the surgery. [ABSTRACT FROM AUTHOR]

The p53 protein is a transcription factor that preserves the integrity of the genome. The TP53 gene has inactivating mutations in about 50% of all human cancers. Some missense mutations lead to decreased thermal stability in the p53 protein, its unfolding and aggregation under physiological conditions. A general understanding of the impact of point mutations on the stability and conformation of mutant p53 is essential for the design and development of small molecules that target specific p53 mutations. In this work, we determined the thermostability properties of some of the most common mutant forms of the p53 protein—p53(R273H), p53(R248Q), p53(R248W) and p53(Y220C)—that are often considered as attractive therapeutic targets. The results showed that these missense mutations lead to destabilization of the p53 protein and a decrease in its melting temperature. [ABSTRACT FROM AUTHOR]

Neuronal calcium sensors (NCSs) are the family of EF-hand proteins mediating Ca2+-dependent signaling pathways in healthy neurons and neurodegenerative diseases. It was hypothesized that the calcium sensor activity of NCSs can be complemented by sensing fluctuation of intracellular zinc, which could further diversify their function. Here, using a set of biophysical techniques, we analyzed the Zn2+-binding properties of five proteins belonging to three different subgroups of the NCS family, namely, VILIP1 and neurocalcin-δ/NCLD (subgroup B), recoverin (subgroup C), as well as GCAP1 and GCAP2 (subgroup D). We demonstrate that each of these proteins is capable of coordinating Zn2+ with a different affinity, stoichiometry, and structural outcome. In the absence of calcium, recoverin and VILIP1 bind two zinc ions with submicromolar affinity, and the binding induces pronounced conformational changes and regulates the dimeric state of these proteins without significant destabilization of their structure. In the presence of calcium, recoverin binds zinc with slightly decreased affinity and moderate conformational outcome, whereas VILIP1 becomes insensitive to Zn2+. NCALD binds Zn2+ with micromolar affinity, but the binding induces dramatic destabilization and aggregation of the protein. In contrast, both GCAPs demonstrate low-affinity binding of zinc independent of calcium, remaining relatively stable even at submillimolar Zn2+ concentrations. Based on these data, and the results of structural bioinformatics analysis, NCSs can be divided into three categories: (1) physiological Ca2+/Zn2+ sensor proteins capable of binding exchangeable (signaling) zinc (recoverin and VILIP1), (2) pathological Ca2+/Zn2+ sensors responding only to aberrantly high free zinc concentrations by denaturation and aggregation (NCALD), and (3) Zn2+-resistant, Ca2+ sensor proteins (GCAP1, GCAP2). We suggest that NCS proteins may therefore govern the interconnection between Ca2+-dependent and Zn2+-dependent signaling pathways in healthy neurons and zinc cytotoxicity-related neurodegenerative diseases, such as Alzheimer's disease and glaucoma. [ABSTRACT FROM AUTHOR]

Human butyrylcholinesterase is a performant stoichiometric bioscavenger of organophosphorous nerve agents. It is either isolated from outdated plasma or functionally expressed in eukaryotic systems. Here, we report the production of active human butyrylcholinesterase in a prokaryotic system after optimization of the primary sequence through the Protein Repair One Stop Shop process, a structure- and sequence-based algorithm for soluble bacterial expression of difficult eukaryotic proteins. The mutant enzyme was purified to homogeneity. Its kinetic parameters with substrate are similar to the endogenous human butyrylcholinesterase or recombinants produced in eukaryotic systems. The isolated protein was prone to crystallize and its 2.5-Å X-ray structure revealed an active site gorge region identical to that of previously solved structures. The advantages of this alternate expression system, particularly for the generation of butyrylcholinesterase variants with nerve agent hydrolysis activity, are discussed. [ABSTRACT FROM AUTHOR]

Ricin is a potent cytotoxin easily purified in large quantities. It presents a significant public health concern due to its potential use as a bioterrorism agent. For this reason, extensive efforts have been underway to develop antidotes against this deadly poison. The catalytic A subunit of the heterodimeric toxin has been biochemically and structurally well characterized, and is an attractive target for structure-based drug design. Aided by computer docking simulations, several ricin toxin A chain (RTA) inhibitors have been identified; the most promising leads belonging to the pterin family. Development of these lead compounds into potent drug candidates is a challenging prospect for numerous reasons, including poor solubility of pterins, the large and highly polar secondary binding pocket of RTA, as well as the enzyme's near perfect catalytic efficiency and tight binding affinity for its natural substrate, the eukaryotic ribosome. To date, the most potent RTA inhibitors developed using this approach are only modest inhibitors with apparent IC50 values in the 10-4 M range, leaving significant room for improvement. This review highlights the variety of techniques routinely employed in structure-based drug design projects, as well as the challenges faced in the design of RTA inhibitors. [ABSTRACT FROM AUTHOR]

Protein kinase CK2 is a highly pleiotropic protein kinase capable of phosphorylating hundreds of protein substrates. It is involved in numerous cellular functions, including cell viability, apoptosis, cell proliferation and survival, angiogenesis, or ER-stress response. As CK2 activity is found perturbed in many pathological states, including cancers, it becomes an attractive target for the pharma. A large number of low-mass ATP-competitive inhibitors have already been developed, the majority of them halogenated. We tested the binding of six series of halogenated heterocyclic ligands derived from the commercially available 4,5-dihalo-benzene-1,2-diamines. These ligand series were selected to enable the separation of the scaffold effect from the hydrophobic interactions attributed directly to the presence of halogen atoms. In silico molecular docking was initially applied to test the capability of each ligand for binding at the ATP-binding site of CK2. HPLC-derived ligand hydrophobicity data are compared with the binding affinity assessed by low-volume differential scanning fluorimetry (nanoDSF). We identified three promising ligand scaffolds, two of which have not yet been described as CK2 inhibitors but may lead to potent CK2 kinase inhibitors. The inhibitory activity against CK2α and toxicity against four reference cell lines have been determined for eight compounds identified as the most promising in nanoDSF assay. [ABSTRACT FROM AUTHOR]

U2AF65 (U2AF2) and PUF60 (PUF60) are splicing factors important for recruitment of the U2 small nuclear ribonucleoprotein to lariat branch points and selection of 3′ splice sites (3′ss). Both proteins preferentially bind uridine-rich sequences upstream of 3′ss via their RNA recognition motifs (RRMs). Here, we examined 36 RRM substitutions reported in cancer patients to identify variants that alter 3′ss selection, RNA binding and protein properties. Employing PUF60- and U2AF65-dependent 3′ss previously identified by RNA-seq of depleted cells, we found that 43% (10/23) and 15% (2/13) of independent RRM mutations in U2AF65 and PUF60, respectively, conferred splicing defects. At least three RRM mutations increased skipping of internal U2AF2 (~9%, 2/23) or PUF60 (~8%, 1/13) exons, indicating that cancer-associated RRM mutations can have both cis- and trans-acting effects on splicing. We also report residues required for correct folding/stability of each protein and map functional RRM substitutions on to existing high-resolution structures of U2AF65 and PUF60. These results identify new RRM residues critical for 3′ss selection and provide relatively simple tools to detect clonal RRM mutations that enhance the mRNA isoform diversity. [ABSTRACT FROM AUTHOR]