De novo design of allosterically switchable protein assemblies.

Allosteric modulation of protein function, wherein the binding of an effector to a protein triggers conformational changes at distant functional sites, plays a central part in the control of metabolism and cell signalling1-3. There has been considerable interest in designing allosteric systems, both to gain insight into the mechanisms underlying such 'action at a distance' modulation and to create synthetic proteins whose functions can be regulated by effectors4-7. However, emulating the subtle conformational changes distributed across many residues, characteristic of natural allosteric proteins, is a significant challenge8,9. Here, inspired by the classic Monod-Wyman-Changeux model of cooperativity10, we investigate the de novo design of allostery through rigid-body coupling of peptide-switchable hinge modules11 to protein interfaces12 that direct the formation of alternative oligomeric states. We find that this approach can be used to generate a wide variety of allosterically switchable systems, including cyclic rings that incorporate or eject subunits in response to peptide binding and dihedral cages that undergo effector-induced disassembly. Size-exclusion chromatography, mass photometry13 and electron microscopy reveal that these designed allosteric protein assemblies closely resemble the design models in both the presence and absence of peptide effectors and can have ligand-binding cooperativity comparable to classic natural systems such as haemoglobin14. Our results indicate that allostery can arise from global coupling of the energetics of protein substructures without optimized side-chain-side-chain allosteric communication pathways and provide a roadmap for generating allosterically triggerable delivery systems, protein nanomachines and cellular feedback control circuitry.

Oxidation-Induced Protein Cross-Linking in Mammalian Cells.

A proximity-enabled protein cross-linking strategy with additional spatiotemporal control is highly desirable. Here, we report an oxidation-induced protein cross-linking strategy involving the incorporation of a vinyl thioether group into proteins in both Escherichia coli and mammalian cells via genetic code expansion. We demonstrated that vinyl thioether can be selectively induced by exogenously added oxidant or by reactive oxygen species from the cellular environment, as well as by photocatalysts, and converted into a Michael acceptor, enabling fluorescence labeling and protein cross-linking.

Control of protein activity by photoinduced spin polarized charge reorganization.

Considerable electric fields are present within living cells, and the role of bioelectricity has been well established at the organismal level. Yet much remains to be learned about electric-field effects on protein function. Here, we use phototriggered charge injection from a site-specifically attached ruthenium photosensitizer to directly demonstrate the effect of dynamic charge redistribution within a protein. We find that binding of an antibody to phosphoglycerate kinase (PGK) is increased twofold under illumination. Remarkably, illumination is found to suppress the enzymatic activity of PGK by a factor as large as three. These responses are sensitive to the photosensitizer position on the protein. Surprisingly, left (but not right) circularly polarized light elicits these responses, indicating that the electrons involved in the observed dynamics are spin polarized, due to spin filtration by protein chiral structures. Our results directly establish the contribution of electrical polarization as an allosteric signal within proteins. Future experiments with phototriggered charge injection will allow delineation of charge rearrangement pathways within proteins and will further depict their effects on protein function.

Challenges in Discovering Drugs That Target the Protein-Protein Interactions of Disordered Proteins.

Protein-protein interactions (PPIs) outnumber proteins and are crucial to many fundamental processes; in consequence, PPIs are associated with several pathological conditions including neurodegeneration and modulating them by drugs constitutes a potentially major class of therapy. Classically, however, the discovery of small molecules for use as drugs entails targeting individual proteins rather than targeting PPIs. This is largely because discovering small molecules to modulate PPIs has been seen as extremely challenging. Here, we review the difficulties and limitations of strategies to discover drugs that target PPIs directly or indirectly, taking as examples the disordered proteins involved in neurodegenerative diseases.

Precipitate-Supported Thermal Proteome Profiling Coupled with Deep Learning for Comprehensive Screening of Drug Target Proteins.

Although thermal proteome profiling (TPP) acts as a popular modification-free approach for drug target deconvolution, some key problems are still limiting screening sensitivity. In the prevailing TPP workflow, only the soluble fractions are analyzed after thermal treatment, while the precipitate fractions that also contain abundant information of drug-induced stability shifts are discarded; the sigmoid melting curve fitting strategy used for data processing suffers from discriminations for a part of human proteome with multiple transitions. In this study, a precipitate-supported TPP (PSTPP) assay was presented for unbiased and comprehensive analysis of protein-drug interactions at the proteome level. In PSTPP, only these temperatures where significant precipitation is observed were applied to induce protein denaturation and the complementary information contained in both supernatant fractions and precipitate fractions was used to improve the screening specificity and sensitivity. In addition, a novel image recognition algorithm based on deep learning was developed to recognize the target proteins, which circumvented the problems that exist in the sigmoid curve fitting strategy. PSTPP assay was validated by identifying the known targets of methotrexate, raltitrexed, and SNS-032 with good performance. Using a promiscuous kinase inhibitor, staurosporine, we delineated 99 kinase targets with a specificity up to 83% in K562 cell lysates, which represented a significant improvement over the existing thermal shift methods. Furthermore, the PSTPP strategy was successfully applied to analyze the binding targets of rapamycin, identifying the well-known targets, FKBP1A, as well as revealing a few other potential targets.

Profiling the regulatory interplay of BET bromodomains and Sirtuins in cancer cell lines.

Alterations in epigenetic marking, due to changes in expression or activity of epigenetic regulators, may affect cancer development and progression and thus, targeting epigenetic regulators provides potential avenues for cancer treatment. Bromodomain and extra terminal domain (BET) proteins, epigenetic readers recognizing histone acetylation, and Sirtuins (SIRT1-7), histone deacetylases or erasers, affect the chromatin acetylation status, and thus have a vital role in transcriptional regulation of a variety of cancer-related genes. Here, the effects of three BET inhibitors on SIRT expression were screened in a broad set of cancer cell lines to study the potential interplay of these distinct epigenetic factors in gene regulation. We show that BET inhibitors have distinct effects on SIRTs and their target gene expression in cancer cell lines derived from several solid tumour cancers. This functional link may open further avenues for epigenetic combination therapies for different cancers.

HIT 2.0: an enhanced platform for Herbal Ingredients' Targets.

Literature-described targets of herbal ingredients have been explored to facilitate the mechanistic study of herbs, as well as the new drug discovery. Though several databases provided similar information, the majority of them are limited to literatures before 2010 and need to be updated urgently. HIT 2.0 was here constructed as the latest curated dataset focusing on Herbal Ingredients' Targets covering PubMed literatures 2000-2020. Currently, HIT 2.0 hosts 10 031 compound-target activity pairs with quality indicators between 2208 targets and 1237 ingredients from more than 1250 reputable herbs. The molecular targets cover those genes/proteins being directly/indirectly activated/inhibited, protein binders, and enzymes substrates or products. Also included are those genes regulated under the treatment of individual ingredient. Crosslinks were made to databases of TTD, DrugBank, KEGG, PDB, UniProt, Pfam, NCBI, TCM-ID and others. More importantly, HIT enables automatic Target-mining and My-target curation from daily released PubMed literatures. Thus, users can retrieve and download the latest abstracts containing potential targets for interested compounds, even for those not yet covered in HIT. Further, users can log into 'My-target' system, to curate personal target-profiling on line based on retrieved abstracts. HIT can be accessible at http://hit2.badd-cao.net.

Systematic analysis of the potential off-target activities of osimertinib by computational target fishing.

Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor used to treat non-small cell lung cancer. However, its off-targets are obscure, and systematic analysis of off-target activities remains to be performed. Here, we identified the off-targets of osimertinib using PharmMapper and DRAR-CPI and analyzed the intersected targets using the GeneMANIA and DAVID servers. A drug-target-pathway network was constructed to visualize the associations. The results showed that osimertinib is associated with 31 off-targets, 40 Kyoto Encyclopedia of Genes and Genomes pathways, and 9 diseases. Network analysis revealed that the targets were involved in cancer and other physiological processes. In addition to EGFR, molecular docking analysis showed that seven proteins, namely Janus kinase 3, peroxisome proliferator-activated receptor alpha, renin, mitogen-activated protein kinases, lymphocyte-specific protein tyrosine kinase, cell division protein kinase 2 and proto-oncogene tyrosine-protein kinase Src, could also be potential targets of osimertinib. In conclusion, osimertinib is predicted to target multiple proteins and pathways, resulting in the formation of an action network via which it exerts systematic pharmacological effects.

Influence of Risk Factors for Male Infertility on Sperm Protein Composition.

Male infertility is a common health problem that can be influenced by a host of lifestyle risk factors such as environment, nutrition, smoking, stress, and endocrine disruptors. These effects have been largely demonstrated on sperm parameters (e.g., motility, numeration, vitality, DNA integrity). In addition, several studies showed the deregulation of sperm proteins in relation to some of these factors. This review inventories the literature related to the identification of sperm proteins showing abundance variations in response to the four risk factors for male infertility that are the most investigated in this context: obesity, diabetes, tobacco smoking, and exposure to bisphenol-A (BPA). First, we provide an overview of the techniques used to identify deregulated proteins. Then, we summarise the main results obtained in the different studies and provide a compiled list of deregulated proteins in relation to each risk factor. Gene ontology analysis of these deregulated proteins shows that oxidative stress and immune and inflammatory responses are common mechanisms involved in sperm alterations encountered in relation to the risk factors.

Proteome-wide mapping of short-lived proteins in human cells.

Rapid protein degradation enables cells to quickly modulate protein abundance. Dysregulation of short-lived proteins plays essential roles in disease pathogenesis. A focused map of short-lived proteins remains understudied. Cycloheximide, a translational inhibitor, is widely used in targeted studies to measure degradation kinetics for short-lived proteins. Here, we combined cycloheximide chase assays with advanced quantitative proteomics to map short-lived proteins under translational inhibition in four human cell lines. Among 11,747 quantified proteins, we identified 1,017 short-lived proteins (half-lives ≤ 8 h). These short-lived proteins are less abundant, evolutionarily younger, and less thermally stable than other proteins. We quantified 103 proteins with different stabilities among cell lines. We showed that U2OS and HCT116 cells express truncated forms of ATRX and GMDS, respectively, which have lower stability than their full-length counterparts. This study provides a large-scale resource of human short-lived proteins under translational arrest, leading to untapped avenues of protein regulation for therapeutic interventions.

Impact of cold plasma on the biomolecules and organoleptic properties of foods: A review.

Cold plasma is formed by the nonthermal ionization of gas into free electrons, ions, reactive atomic and molecular species, and ultraviolet (UV) radiation. This cold plasma can be used to alter the surface of solid and liquid foods, and it offers multiple advantages over traditional thermal treatments, such as no thermal damage and increased output variation (due to the various input parameters gas, power, plasma type, etc.). Cold plasma appears to have limited impact on the sensory and color properties, at lower power and treatment times, but there has been a statistically significant reduction in pH for most of the cold plasma treatments reviewed (p < 0.05). Carbohydrates (cross linking and glycosylation), lipids (oxidation), and proteins (secondary structure) are more significantly impacted due to cold plasma at higher intensities and longer treatment times. Although cold plasma treatments and food matrices can vary considerably, this review has identified the literary evidence of some of the influences and impacts of the vast array of cold plasma treatment parameters on the biomolecular and organoleptic properties of these foods. Due to the rapidly evolving nature of the field, we have also identified that authors prioritize the presentation of different information when publishing from different research areas. Therefore, we have proposed a number of key physical and chemical cold plasma parameters that should be considered for inclusion in all future publications in the field.

Membrane lipid replacement with nano-micelles in human sperm cryopreservation improves post-thaw function and acrosome protein integrity.

RESEARCH QUESTION: Membrane lipid replacement (MLR) of oxidized membrane lipids can restore sperm cellular membrane functionality and help improve surface protein stability during cryopreservation. What are the effects of MLR with nano-micelles made from a glycerophospholipid (GPL) mixture and cholesterol-loaded cyclodextrin (CLC), on the cryosurvival and expression of acrosome-related proteins in thawed human spermatozoa? DESIGN: Twenty samples were used to determine the optimum level of nano-micelles by incubation of semen with different concentrations of GPL (0.1 and 1%) and CLC (1 and 2 mg/ml) (including GPL-0.1, GPL-1, CLC-1, CLC-2, CLC-1/GPL-0.1, CLC-2/GPL-0.1, CLC-1/GPL-1 and CLC-2/GPL-1) before cryopreservation. Then, 30 semen samples were collected, and each sample was divided into the following three aliquots: fresh, frozen control and frozen incubated with optimum level of nano-micelles (0.1% GPL and 1 mg/ml CLC). RESULTS: CLC-1/GPL-0.1 and GPL-0.1 significantly increased motility parameters. CLC-1, GPL-0.1 and CLC-1/GPL-0.1 significantly improved viability rate compared with frozen control group. Significantly higher mitochondrial activity and acrosome integrity, and a lower rate of apoptosis, were observed in the CLC-1/GPL-0.1 compared with the frozen control group. The expression ratios of arylsulfatase A (ARSA), serine protease 37 (PRSS37), serine protease inhibitor Kazal-type 2 (SPINK2) and equatorin (EQTN) significantly increased compared with the frozen control group. CONCLUSIONS: Modification of membrane cholesterol and GPL mixtures in spermatozoa enhances their acrosome protein integrity by inhibiting early apoptotic changes and spontaneous acrosome reactions.

Use of Mitotic Protein Kinase Inhibitors and Phospho-Specific Antibodies to Monitor Protein Phosphorylation During the Cell Cycle.

Reversible protein phosphorylation regulates the transitions between different phases of the cell cycle ensuring proper segregation of the duplicated genome into two daughter cells. Protein kinases and protein phosphatases establish the appropriate phosphorylation stoichiometries in diverse substrates maintaining genomic stability as a cell undergoes this complex process. Along with regulating common substrates, these opposing enzymes regulate one another by fine-tuning each other's activity both spatially and temporally throughout mitosis. Protein phosphatase catalytic subunits work together with regulatory proteins, which control their localization, activity, and specificity. Protein phosphatase 1 (PP1) recognizes its regulatory proteins via a short linear interaction motif (SLIM) called the "RVxF" motif. A subset of proteins with these "RVxF" motifs contain a phosphorylatable amino acid (S/T) at the 'x' position.Here, we describe methods to generate, affinity purify and utilize phospho-specific antibodies to monitor phosphorylation sites during the cell cycle and the appropriate use of mitotic kinase inhibitors. More specifically, we employ phospho-specific antibodies, which recognize phosphorylated RVp[S/T]F motif-containing proteins, to monitor the phosphorylation status of these motifs throughout the cell cycle. Furthermore, we use mitotic kinase inhibitors to examine the effect of kinase inhibition on the phosphorylation status of multiple RV[S/T]F motifs using these phospho-specific antibodies.

Silver in biology and medicine: opportunities for metallomics researchers.

The antibacterial properties of silver have been known for centuries and the threat of antibiotic-resistant bacteria has led to renewed focus on the noble metal. Silver is now commonly included in a range of household and medical items to imbue them with bactericidal properties. Despite this, the chemical fate of the metal in biological systems is poorly understood. Silver(I) is a soft metal with high affinity for soft donor atoms and displays much similarity to the chemistry of Cu(I). In bacteria, interaction of silver with the cell wall/membrane, DNA, and proteins and enzymes can lead to cell death. Additionally, the intracellular generation of reactive oxygen species by silver is posited to be a significant antimicrobial action. While the antibacterial action of silver is well known, bacteria found in silver mines display resistance against it through use of a protein ensemble thought to have been specifically developed for the metal, highlighting the need for judicious use. In mammals, ∼10-20% of ingested silver is retained by the body and thought to predominantly localize in the liver or kidneys. Chronic exposure can result in argyria, a condition characterized by blue staining of the skin, resulting from subdermal deposition of silver [as Ag(0)/sulfides], but more insidious side effects, such as inclusions in the brain, seizures, liver/kidney damage, and immunosuppression, have also been reported. Here, we hope to highlight the current understanding of the biological chemistry of silver and the necessity for continued study of these systems to fill existing gaps in knowledge.

Potential involvement of environmental triggers in protein aggregation with mercuric chloride as a model.

Heavy metal based toxicity has a direct relation with the perturbation of protein structure. We have investigated the progressive unfolding of ovalbumin, in the presence of increasing concentration mercury (0-6.25 µM) using different spectroscopic techniques. Formation of amorphous aggregate has been observed at the physiological pH. Initial addition of HgCl2 resulted in the association of monomers to oligomers that proceeded to non-fibrillar aggregates on further addition. The sigmoidal curve obtained from the Stern-Volmer plot clearly divided into three stage transition. A strong lag phase is observed indicating the time dependence for the association of competent monomers. The second stage was resolved into non-cooperative binding. These results match very well with the data from atomic force microscopy and the free energy change observed in the regions. Raman spectroscopic studies indicated toxic antiparallel ß-sheets structure. Time dependent atomic force microscopy study revealed the off-pathway nature of amorphous aggregates. At molten globular state, similar quenching behaviour is observed. The atomic force microscopy images clearly indicate at pH 2.2 the initiation of fibril formation occurs at lower concentration of HgCl2 itself. Our results revealed the conformation switch of ovalbumin upon the contact of an environmental toxin and its possible way of toxicity.

Molecular Dynamics Simulation Techniques as Tools in Drug Discovery and Pharmacology: A Focus on Allosteric Drugs.

Allosteric drugs are ligands that when bound to an allosteric site modify the conformational state of the pharmacological target, leading then to a modification of functional response upon binding of the endogenous ligand. Pharmacological targets are defined as biological entities, to which a ligand/drug binds and leads to a functional effect. Pharmacological targets can be proteins or nucleic acids. Computational approaches such as molecular dynamics (MD) sped up discovery and identification of allosteric binding sites and allosteric ligands. Classical all-atom and hybrid classical/quantum MD simulations can be generalized as simulation techniques aimed at analysis of atoms and molecular motion. Main limitations of MD simulations are related to high computational costs, that in turn limit the conformational sampling of biological systems. Indeed, other techniques have been developed to overcome limitations of MD, such as enhanced sampling MD simulations. In this chapter, classical MD and enhanced sampling MD simulations will be described, along with their application to drug discovery, with a focus on allosteric drugs.

Pooled protein tagging, cellular imaging, and in situ sequencing for monitoring drug action in real time.

The levels and subcellular localizations of proteins regulate critical aspects of many cellular processes and can become targets of therapeutic intervention. However, high-throughput methods for the discovery of proteins that change localization either by shuttling between compartments, by binding larger complexes, or by localizing to distinct membraneless organelles are not available. Here we describe a scalable strategy to characterize effects on protein localizations and levels in response to different perturbations. We use CRISPR-Cas9-based intron tagging to generate cell pools expressing hundreds of GFP-fusion proteins from their endogenous promoters and monitor localization changes by time-lapse microscopy followed by clone identification using in situ sequencing. We show that this strategy can characterize cellular responses to drug treatment and thus identify nonclassical effects such as modulation of protein-protein interactions, condensate formation, and chemical degradation.

Context-dependent monoclonal antibodies against protein carbamidomethyl-cysteine.

Protein sulfhydryl residues participate in key structural and biochemical functions. Alterations in sulfhydryl status, regulated by either reversible redox reactions or by permanent covalent capping, may be challenging to identify. To advance the detection of protein sulfhydryl groups, we describe the production of new Rabbit monoclonal antibodies that react with carbamidomethyl-cysteine (CAM-cys), a product of iodoacetamide (IAM) labeling of protein sulfhydryl residues. These antibodies bind to proteins labeled with IAM (but not N-ethylmaleimide (NEM) or acrylamide) and identify multiple protein bands when applied to Western blots of cell lysates treated with IAM. The monoclonal antibodies label a subset of CAM-cys modified peptide sequences and purified proteins (human von Willebrand Factor (gene:vWF), Jagged 1 (gene:JAG1), Laminin subunit alpha 2 (gene:LAMA2), Thrombospondin-2 (gene:TSP2), and Collagen IV (gene:COL4)) but do not recognize specific proteins such as Bovine serum albumin (gene:BSA) and human Thrombospondin-1 (gene:TSP1), Biglycan (gene:BGN) and Decorin (gene:DCN). Scanning mutants of the peptide sequence used to generate the CAM-cys antibodies elucidated residues required for context dependent reactivity. In addition to recognition of in vitro labeled proteins, the antibodies were used to identify selected sulfhydryl-containing proteins from living cells that were pulse labeled with IAM. Further development of novel CAM-cys monoclonal antibodies in conjunction with other biochemical tools may complement current methods for sulfhydryl detection within specific proteins. Moreover, CAM-cys reactive reagents may be useful when there is a need to label subpopulations of proteins.

Polyol and sugar osmolytes can shorten protein hydrogen bonds to modulate function.

Polyol and sugar osmolytes are commonly used in therapeutic protein formulations. How they may affect protein structure and function is an important question. In this work, through NMR measurements, we show that glycerol and sorbitol (polyols), as well as glucose (sugar), can shorten protein backbone hydrogen bonds. The hydrogen bond shortening is also captured by molecular dynamics simulations, which suggest a hydrogen bond competition mechanism. Specifically, osmolytes weaken hydrogen bonds between the protein and solvent to strengthen those within the protein. Although the hydrogen bond change is small, with the average experimental cross hydrogen bond 3hJNC' coupling of two proteins GB3 and TTHA increased by ~ 0.01 Hz by the three osmolytes (160 g/L), its effect on protein function should not be overlooked. This is exemplified by the PDZ3-peptide binding where several intermolecular hydrogen bonds are formed and osmolytes shift the equilibrium towards the bound state.

Effects of low and high doses of fenofibrate on protein, amino acid, and energy metabolism in rat.

A feared adverse effect of dyslipidaemia therapy by fibrates is myopathy. We examined the effect of fenofibrate (FF) on protein and amino acid metabolism. Rats received a low (50 mg/kg, LFFD) or high (300 mg/kg, HFFD) dose of FF or vehicle daily by oral gavage. Blood plasma, liver, and soleus and extensor digitorum longus muscles were analysed after 10 days. The FF-treated rats developed hepatomegaly associated with increased hepatic carnitine and ATP and AMP concentrations, decreased protein breakdown, and decreased concentrations of DNA and triglycerides. HFFD increased plasma ALT and AST activities. The weight and protein content of muscles in the HFFD group were lower compared with controls. In muscles of the LFFD group there were increased ATP and decreased AMP concentrations; in the HFFD group AMP was increased. In both FF-treated groups there were increased glycine, phenylalanine, and citrulline and decreased arginine and branched-chain keto acids (BCKA) in blood plasma. After HFFD there were decreased levels of branched-chain amino acids (BCAA; valine, leucine and isoleucine), methionine, and lysine and increased homocysteine. Decreased arginine and increased glycine concentrations were found in both muscles in FF-treated animals; in HFFD-treated animals lysine, methionine, and BCAA were decreased. We conclude that FF exerts protein-anabolic effects on the liver and catabolic effects on muscles. HFFD causes signs of hepatotoxicity, impairs energy and protein balance in muscles, and decreases BCAA, methionine, and lysine. It is suggested that increased glycine and decreased lysine and methionine levels are due to activated carnitine synthesis; decreased BCAA and BCKA levels are due to increased BCAA oxidation.