Structural implications of BK polyomavirus sequence variations in the major viral capsid protein Vp1 and large T-antigen: a computational study.

BK polyomavirus (BKPyV) is a double-stranded DNA virus causing nephropathy, hemorrhagic cystitis, and urothelial cancer in transplant patients. The BKPyV-encoded capsid protein Vp1 and large T-antigen (LTag) are key targets of neutralizing antibodies and cytotoxic T-cells, respectively. Our single-center data suggested that variability in Vp1 and LTag may contribute to failing BKPyV-specific immune control and impact vaccine design. We, therefore, analyzed all available entries in GenBank (1516 VP1; 742 LTAG) and explored potential structural effects using computational approaches. BKPyV-genotype (gt)1 was found in 71.18% of entries, followed by BKPyV-gt4 (19.26%), BKPyV-gt2 (8.11%), and BKPyV-gt3 (1.45%), but rates differed according to country and specimen type. Vp1-mutations matched a serotype different than the assigned one or were serotype-independent in 43%, 18% affected more than one amino acid. Notable Vp1-mutations altered antibody-binding domains, interactions with sialic acid receptors, or were predicted to change conformation. LTag-sequences were more conserved, with only 16 mutations detectable in more than one entry and without significant effects on LTag-structure or interaction domains. However, LTag changes were predicted to affect HLA-class I presentation of immunodominant 9mers to cytotoxic T-cells. These global data strengthen single center observations and specifically our earlier findings revealing mutant 9mer epitopes conferring immune escape from HLA-I cytotoxic T cells. We conclude that variability of BKPyV-Vp1 and LTag may have important implications for diagnostic assays assessing BKPyV-specific immune control and for vaccine design. IMPORTANCE: Type and rate of amino acid variations in BKPyV may provide important insights into BKPyV diversity in human populations and an important step toward defining determinants of BKPyV-specific immunity needed to protect vulnerable patients from BKPyV diseases. Our analysis of BKPyV sequences obtained from human specimens reveals an unexpectedly high genetic variability for this double-stranded DNA virus that strongly relies on host cell DNA replication machinery with its proof reading and error correction mechanisms. BKPyV variability and immune escape should be taken into account when designing further approaches to antivirals, monoclonal antibodies, and vaccines for patients at risk of BKPyV diseases.

Protein target highlights in CASP15: Analysis of models by structure providers.

We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.

Target highlights in CASP14: Analysis of models by structure providers.

The biological and functional significance of selected Critical Assessment of Techniques for Protein Structure Prediction 14 (CASP14) targets are described by the authors of the structures. The authors highlight the most relevant features of the target proteins and discuss how well these features were reproduced in the respective submitted predictions. The overall ability to predict three-dimensional structures of proteins has improved remarkably in CASP14, and many difficult targets were modeled with impressive accuracy. For the first time in the history of CASP, the experimentalists not only highlighted that computational models can accurately reproduce the most critical structural features observed in their targets, but also envisaged that models could serve as a guidance for further studies of biologically-relevant properties of proteins.

Global Genomic Analysis of SARS-CoV-2 RNA Dependent RNA Polymerase Evolution and Antiviral Drug Resistance.

A variety of antiviral treatments for COVID-19 have been investigated, involving many repurposed drugs. Currently, the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp, encoded by nsp12-nsp7-nsp8) has been targeted by numerous inhibitors, e.g., remdesivir, the only provisionally approved treatment to-date, although the clinical impact of these interventions remains inconclusive. However, the potential emergence of antiviral resistance poses a threat to the efficacy of any successful therapies on a wide scale. Here, we propose a framework to monitor the emergence of antiviral resistance, and as a proof of concept, we address the interaction between RdRp and remdesivir. We show that SARS-CoV-2 RdRp is under purifying selection, that potential escape mutations are rare in circulating lineages, and that those mutations, where present, do not destabilise RdRp. In more than 56,000 viral genomes from 105 countries from the first pandemic wave, we found negative selective pressure affecting nsp12 (Tajima's D = -2.62), with potential antiviral escape mutations in only 0.3% of sequenced genomes. Potential escape mutations included known key residues, such as Nsp12:Val473 and Nsp12:Arg555. Of the potential escape mutations involved globally, in silico structural models found that they were unlikely to be associated with loss of stability in RdRp. No potential escape mutation was found in a local cohort of remdesivir treated patients. Collectively, these findings indicate that RdRp is a suitable drug target, and that remdesivir does not seem to exert high selective pressure. We anticipate our framework to be the starting point of a larger effort for a global monitoring of drug resistance throughout the COVID-19 pandemic.

Nanopore Enzymology to Study Protein Kinases and Their Inhibition by Small Molecules.

Nanopore enzymology is a powerful single-molecule technique for the label-free study of enzymes using engineered protein nanopore sensors. The technique has been applied to protein kinases, where it has enabled the full repertoire of kinase function to be observed, including: kinetics of substrate binding and dissociation, product binding and dissociation, nucleotide binding, and reversible phosphorylation. Further, minor modifications enable the screening of type I kinase inhibitors and the determination of inhibition constants in a facile and label-free manner. Here, we describe the design and production of suitably engineered protein nanopores and their use for the determination of key mechanistic parameters of kinases. We also provide procedures for the determination of inhibition constants of protein kinase inhibitors.

A Fatty Acid Oxidation-dependent Metabolic Shift Regulates the Adaptation of BRAF-mutated Melanoma to MAPK Inhibitors.

PURPOSE: Treatment of BRAFV600E -mutant melanomas with MAPK inhibitors (MAPKi) results in significant tumor regression, but acquired resistance is pervasive. To understand nonmutational mechanisms underlying the adaptation to MAPKi and to identify novel vulnerabilities of melanomas treated with MAPKi, we focused on the initial response phase during treatment with MAPKi. EXPERIMENTAL DESIGN: By screening proteins expressed on the cell surface of melanoma cells, we identified the fatty acid transporter CD36 as the most consistently upregulated protein upon short-term treatment with MAPKi. We further investigated the effects of MAPKi on fatty acid metabolism using in vitro and in vivo models and analyzing patients' pre- and on-treatment tumor specimens. RESULTS: Melanoma cells treated with MAPKi displayed increased levels of CD36 and of PPARα-mediated and carnitine palmitoyltransferase 1A (CPT1A)-dependent fatty acid oxidation (FAO). While CD36 is a useful marker of melanoma cells during adaptation and drug-tolerant phases, the upregulation of CD36 is not functionally involved in FAO changes that characterize MAPKi-treated cells. Increased FAO is required for BRAFV600E -mutant melanoma cells to survive under the MAPKi-induced metabolic stress prior to acquiring drug resistance. The upfront and concomitant inhibition of FAO, glycolysis, and MAPK synergistically inhibits tumor cell growth in vitro and in vivo. CONCLUSIONS: Thus, we identified a clinically relevant therapeutic approach that has the potential to improve initial responses and to delay acquired drug resistance of BRAFV600E -mutant melanoma.

Single-Molecule Protein Phosphorylation and Dephosphorylation by Nanopore Enzymology.

Reversible protein phosphorylation plays a crucial and ubiquitous role in the control of almost all cellular processes. The interplay of protein kinases and phosphatases acting in opposition ensures tight dynamic control of protein phosphorylation states within the cell. Previously, engineered α-hemolysin pores bearing kinase substrate peptides have been developed as single-molecule stochastic sensors for protein kinases. Here, we have used these pores to observe, label-free, the phosphorylation and dephosphorylation of a single substrate molecule. Further, we investigated the effect of Mg2+ and Mn2+ upon substrate and product binding and found that Mn2+ relaxes active-site specificity toward nucleotides and enhances product binding. In doing so, we demonstrate the power and versatility of nanopore enzymology to scrutinize a critical post-translational modification.

Targeting Pim Kinases and DAPK3 to Control Hypertension.

Sustained vascular smooth muscle hypercontractility promotes hypertension and cardiovascular disease. The etiology of hypercontractility is not completely understood. New therapeutic targets remain vitally important for drug discovery. Here we report that Pim kinases, in combination with DAPK3, regulate contractility and control hypertension. Using a co-crystal structure of lead molecule (HS38) in complex with DAPK3, a dual Pim/DAPK3 inhibitor (HS56) and selective DAPK3 inhibitors (HS94 and HS148) were developed to provide mechanistic insight into the polypharmacology of hypertension. In vitro and ex vivo studies indicated that Pim kinases directly phosphorylate smooth muscle targets and that Pim/DAPK3 inhibition, unlike selective DAPK3 inhibition, significantly reduces contractility. In vivo, HS56 decreased blood pressure in spontaneously hypertensive mice in a dose-dependent manner without affecting heart rate. These findings suggest including Pim kinase inhibition within a multi-target engagement strategy for hypertension management. HS56 represents a significant step in the development of molecularly targeted antihypertensive medications.

Metabotypes of breast cancer cell lines revealed by non-targeted metabolomics.

We present an analysis of intracellular metabolism by non-targeted, high-throughput metabolomics profiling of 18 breast cell lines. We profiled >900 putatively annotated metabolite ions for >100 samples collected under both normoxic and hypoxic conditions and revealed extensive heterogeneity across all metabolic pathways and cell lines. Cell line-specific metabolome profiles dominated over patterns associated with malignancy or with the clinical nomenclature of breast cancer cells. Such characteristic metabolome profiles were reproducible across different laboratories and experiments and exhibited mild to robust changes with change in experimental conditions. To extract a functional overview of cell line heterogeneity, we devised an unsupervised metabotyping procedure that for each pathway automatically recognized metabolic types from metabolome data and assigned cell lines. Our procedure provided a condensed yet global representation of cell line metabolism, revealing the fine structure of metabolic heterogeneity across all tested pathways and cell lines. In follow-up experiments on selected pathways, we confirmed that different metabolic types correlated to differences in the underlying fluxes and difference sensitivity to gene knockdown or pharmacological inhibition. Thus, the identified metabotypes recapitulated functional differences at the pathway level. Metabotyping provides a powerful compression of multi-dimensional data that preserves functional information and serves as a resource for reconciling or understanding heterogeneous metabolic phenotypes or response to inhibition of metabolic pathways.

Genome-wide RNAi Screening Identifies Protein Modules Required for 40S Subunit Synthesis in Human Cells.

Ribosome biogenesis is a highly complex process requiring many assisting factors. Studies in yeast have yielded comprehensive knowledge of the cellular machinery involved in this process. However, many aspects of ribosome synthesis are different in higher eukaryotes, and the global set of mammalian ribosome biogenesis factors remains unexplored. We used an imaging-based, genome-wide RNAi screen to find human proteins involved in 40S ribosomal subunit biogenesis. Our analysis identified ∼ 300 factors, many part of essential protein modules such as the small subunit (SSU) processome, the eIF3 and chaperonin complexes, and the ubiquitin-proteasome system. We demonstrate a role for the vertebrate-specific factor RBIS in ribosome synthesis, uncover a requirement for the CRL4 E3 ubiquitin ligase in nucleolar ribosome biogenesis, and reveal that intracellular glutamine synthesis supports 40S subunit production.

Type II Inhibitors Targeting CDK2.

Kinases can switch between active and inactive conformations of the ATP/Mg(2+) binding motif DFG, which has been explored for the development of type I or type II inhibitors. However, factors modulating DFG conformations remain poorly understood. We chose CDK2 as a model system to study the DFG in-out transition on a target that was thought to have an inaccessible DFG-out conformation. We used site-directed mutagenesis of key residues identified in structural comparisons in conjunction with biochemical and biophysical characterization of the generated mutants. As a result, we identified key residues that facilitate the DFG-out movement, facilitating binding of type II inhibitors. However, surprisingly, we also found that wild type CDK2 is able to bind type II inhibitors. Using protein crystallography structural analysis of the CDK2 complex with an aminopyrimidine-phenyl urea inhibitor (K03861) revealed a canonical type II binding mode and the first available type II inhibitor CDK2 cocrystal structure. We found that the identified type II inhibitors compete with binding of activating cyclins. In addition, analysis of the binding kinetics of the identified inhibitors revealed slow off-rates. The study highlights the importance of residues that may be distant to the ATP binding pocket in modulating the energetics of the DFG-out transition and hence inhibitor binding. The presented data also provide the foundation for a new class of slow off-rate cyclin-competitive CDK2 inhibitors targeting the inactive DFG-out state of this important kinase target.

Pim Kinase Inhibitors Evaluated with a Single-Molecule Engineered Nanopore Sensor.

Protein kinases are critical therapeutic targets. Pim kinases are implicated in several leukaemias and cancers. Here, we exploit a protein nanopore sensor for Pim kinases that bears a pseudosubstrate peptide attached by an enhanced engineering approach. Analyte binding to the sensor peptide is measured through observation of the modulation of ionic current through a single nanopore. We observed synergistic binding of MgATP and kinase to the sensor, which was used to develop a superior method to evaluate Pim kinase inhibitors featuring label-free determination of inhibition constants. The procedure circumvents many sources of bias or false-positives inherent in current assays. For example, we identified a potent inhibitor missed by differential scanning fluorimetry. The approach is also amenable to implementation on high throughput chips.

Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrate.

In stochastic sensing, the association and dissociation of analyte molecules is observed as the modulation of an ionic current flowing through a single engineered protein pore, enabling the label-free determination of rate and equilibrium constants with respect to a specific binding site. We engineered sensors based on the staphylococcal α-hemolysin pore to allow the single-molecule detection and characterization of protein kinase-peptide interactions. We enhanced this approach by using site-specific proteolysis to generate pores bearing a single peptide sensor element attached by an N-terminal peptide bond to the trans mouth of the pore. Kinetics and affinities for the Pim protein kinases (Pim-1, Pim-2, and Pim-3) and cAMP-dependent protein kinase were measured and found to be independent of membrane potential and in good agreement with previously reported data. Kinase binding exhibited a distinct current noise behavior that forms a basis for analyte discrimination. Finally, we observed unusually high association rate constants for the interaction of Pim kinases with their consensus substrate Pimtide (~10(7) to 10(8) M(-1) · s(-1)), the result of electrostatic enhancement, and propose a cellular role for this phenomenon.

Arginine methylation-dependent reader-writer interplay governs growth control by E2F-1.

The mechanisms that underlie and dictate the different biological outcomes of E2F-1 activity have yet to be elucidated. We describe the residue-specific methylation of E2F-1 by the asymmetric dimethylating protein arginine methyltransferase 1 (PRMT1) and symmetric dimethylating PRMT5 and relate the marks to different functional consequences of E2F-1 activity. Methylation by PRMT1 hinders methylation by PRMT5, which augments E2F-1-dependent apoptosis, whereas PRMT5-dependent methylation favors proliferation by antagonizing methylation by PRMT1. The ability of E2F-1 to prompt apoptosis in DNA damaged cells coincides with enhanced PRMT1 methylation. In contrast, cyclin A binding to E2F-1 impedes PRMT1 methylation and augments PRMT5 methylation, thus ensuring that E2F-1 is locked into its cell-cycle progression mode. The Tudor domain protein p100-TSN reads the symmetric methylation mark, and binding of p100-TSN downregulates E2F-1 apoptotic activity. Our results define an exquisite level of precision in the reader-writer interplay that governs the biological outcome of E2F-1 activity.

Effect of single amino acid substitution observed in cancer on Pim-1 kinase thermodynamic stability and structure.

Pim-1 kinase, a serine/threonine protein kinase encoded by the pim proto-oncogene, is involved in several signalling pathways such as the regulation of cell cycle progression and apoptosis. Many cancer types show high expression levels of Pim kinases and particularly Pim-1 has been linked to the initiation and progression of the malignant phenotype. In several cancer tissues somatic Pim-1 mutants have been identified. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of Pim-1 kinase. We expressed and purified some of the mutants of Pim-1 kinase that are expressed in cancer tissues and reported in the single nucleotide polymorphisms database. The point mutations in the variants significantly affect the conformation of the native state of Pim-1. All the mutants, expressed as soluble recombinant proteins, show a decreased thermal and thermodynamic stability and a lower activation energy values for kinase activity. The decreased stability accompanied by an increased flexibility suggests that Pim-1 variants may be involved in a wider network of protein interactions. All mutants bound ATP and ATP mimetic inhibitors with comparable IC50 values suggesting that the studied Pim-1 kinase mutants can be efficiently targeted with inhibitors developed for the wild type protein.