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The COVID-19 pandemic has caused serious concerns for people around the world. The COVID-19 is associated with respiratory failure, generation of reactive oxygen species (ROS), and the lack of antioxidants among patients. Specified ROS levels have an essential role as an adjuster of immunological responses and virus cleaners, but excessive ROS will oxidize membrane lipids and cellular proteins and quickly destroy virus-infected cells. It can also adversely damage normal cells in the lungs and even the heart, resulting in multiple organ failures. Given the above, a highly potent antioxidant therapy can be offered to reduce cardiac loss due to COVID-19. In modern medicine, nanoparticles containing antioxidants can be used as a high-performance therapy in reducing oxidative stress in the prevention and treatment of infectious diseases. This can provide a free and interactive tool to determine whether antioxidants and nanoantioxidants can be administered for COVID-19. More research and studies are needed to investigate and make definitive opinions about their medicinal uses.

The average life cycle of a human RBC is approximately 120 days. Generally, by this point, the cell is worn out and damaged. RBCs pass through both the spleen and liver, where specialised immune cells called macrophages are found. Macrophages recognise when an RBC is spent, and undergo a process called phagocytosis where they digest the cell. In this process, the iron in haemoglobin is recycled for use in new blood cells and the hem molecule is degraded, conjugated to bilirubin, and eliminated from the body. All the other cellular proteins are either recycled or eliminated. Historically, this process was thought to occur exclusively in the spleen, but recent studies have shown that it occurs in the bone marrow. The RBC has been analysed from many perspectives: cytological, haematological, and immunological, as well as from the focus of molecular biology, biophysics, and mathematics. Here we analyse how are red blood cells born and how they live and die in a brief overview of the whole process with special mention of the morphological aspects from bone marrow and spleen provided by transmission and scanning electron microscopy.

New neurons are generated throughout life in distinct regions of the mammalian brain due to the proliferation and differentiation of neural stem cells (NSCs). Ubiquitin, a post-translational modification of cellular proteins, is an important factor in regulating neurogenesis. Deubiquitination is a biochemical process that mediates the removal of ubiquitin moieties from ubiquitin-conjugated substrates. Recent studies have provided growing evidence that deubiquitylases (DUBs) which reverse ubiquitylation process play critical roles in NSCs maintenance, differentiation and maturation. This review mainly focused on the relationship of DUBs and NSCs, and further summarized recent advances in our understanding of DUBs on regulating NSCs biological behaviors.

Deubiquitinases (DUBs) are enzymes that control the stability, interactions or localization of most cellular proteins by removing their ubiquitin modification. In recent years, some DUBs, such as USP7, USP9X and USP10, have been identified as promising therapeutic targets in hematological malignancies. Importantly, some potent inhibitors targeting the oncogenic DUBs have been developed, showing promising inhibitory efficacy in preclinical models, and some have even undergone clinical trials. Different DUBs perform distinct function in diverse hematological malignancies, such as oncogenic, tumor suppressor or context-dependent effects. Therefore, exploring the biological roles of DUBs and their downstream effectors will provide new insights and therapeutic targets for the occurrence and development of hematological malignancies. We summarize the DUBs involved in different categories of hematological malignancies including leukemia, multiple myeloma and lymphoma. We also present the recent development of DUB inhibitors and their applications in hematological malignancies. Together, we demonstrate DUBs as potential therapeutic drug targets in hematological malignancies.

Located at the inner leaflet of the plasma membrane (PM), phosphatidyl-inositol 4,5-bisphosphate [PI(4,5)P2] composes only 1–2 mol% of total PM lipids. With its synthesis and turnover both spatially and temporally regulated, PI(4,5)P2 recruits and interacts with hundreds of cellular proteins to support a broad spectrum of cellular functions. Several factors contribute to the versatile and dynamic distribution of PI(4,5)P2 in membranes. Physiological multivalent cations such as Ca2+ and Mg2+ can bridge between PI(4,5)P2 headgroups, forming nanoscopic PI(4,5)P2–cation clusters. The distinct lipid environment surrounding PI(4,5)P2 affects the degree of PI(4,5)P2 clustering. In addition, diverse cellular proteins interacting with PI(4,5)P2 can further regulate PI(4,5)P2 lateral distribution and accessibility. This review summarizes the current understanding of PI(4,5)P2 behavior in both cells and model membranes, with emphasis on both multivalent cation– and protein-induced PI(4,5)P2 clustering. Understanding the nature of spatially separated pools of PI(4,5)P2 is fundamental to cell biology.

Covalently attaching ubiquitin (Ub) to cellular proteins as a post-translational modification can result in altered function of modified proteins. Enzymes regulating Ub as a post-translational modification, such as ligases and deubiquitinases, are challenging to characterize in part due to the low throughput of in-vitro assays. Single-molecule nanopore based assays have the advantage of detecting proteins with high specificity and resolution, and in a label-free, real-time fashion. Here we demonstrate the use of a MspA nanopore for discriminating and quantifying Ub proteins. We further applied the MspA pore to measure the Ub-chain disassembly activity of UCH37, a proteasome associated deubiquitinase. The implementation of this MspA system into nanopore arrays could enable high throughput characterizations of unknown deubiquitinases as well as drug screening against disease related enzymes.

The most effective way to control newly emerging infectious disease, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, is to strengthen preventative or therapeutic public health strategies before the infection spreads worldwide. However, global health systems remain at the early stages in anticipating effective therapeutics or vaccines to combat the SARS-CoV-2 pandemic. While maintaining social distance is the most crucial metric to avoid spreading the virus, symptomatic therapy given to patients on the clinical manifestations helps save lives. The molecular properties of SARS-CoV-2 infection have been quickly elucidated, paving the way to therapeutics, vaccine development, and other medical interventions. Despite this progress, the detailed biomolecular mechanism of SARS-CoV-2 infection remains elusive. Given virus invasion of cells is a determining factor for virulence, understanding the viral entry process can be a mainstay in controlling newly emerged viruses. Since viral entry is mediated by selective cellular proteases or proteins associated with receptors, identification and functional analysis of these proteins could provide a way to disrupt virus propagation. This review comprehensively discusses cellular machinery necessary for SARS-CoV-2 infection. Understanding multifactorial traits of the virus entry will provide a substantial guide to facilitate antiviral drug development.

Introduction : Staphylococcal infections rank high among healthcare­associated infections. Coagulase­negative staphylococci, especially S. epidermidis and S. haemolyticus , often induce inflammatory processes in newborns. In this regard, it is important to study the persistence of these microorganisms in pediatric hospitals using intraspecific differentiation based on the comparison of spectra of their extracellular proteins. Our objective was to study circulation of S. epidermidis and S. haemolyticus strains in a children’s hospital by electrophoretyping of their extracellular proteins. Materials and methods : We studied 277 strains of S. haemolyticus and 267 strains of S. epidermidis isolated from patients and various objects of the hospital environment by obtaining extracellular proteins of the isolates, analyzing them using polyacrylamide gel electrophoresis, and determining their antibiotic resistance and ability to form biofilms. Results : The analysis of electrophoregrams of extra­ cellular proteins of staphylococci revealed groups of identical strains. Isolates from different patients were combined into 21 S. haemolyticus groups comprising of 69 strains and 13 groups of S. epidermidis comprising of 38 strains. All the cultures were methicillin­resistant, with the exception of one group of two S. haemolyticus strains. More than half of the cultures grouped by spectra of extracellular proteins were multidrug resistant. The absolute majority of S. haemolyticus strains (97.2 %) and three quarters of S. epidermidis strains (76.0 %) were able to form biofilms. The average values of the degree of film formation in S. haemolyticus strains were significantly higher than those in S. epidermidis . Conclusions : Of all the studied cultures of staph­ ylococci, 25 % of strains and 14 % of S. epidermidis strains persisted and were endemic in the children’s hospital, including 88.4 % of hemolytic and 42.1 % of epidermal staphylococcus strains for more than a month, and 21.7 % of S. haemolyticu s and 21.1 % of S. epidermidis strains for almost a year. The strains were resistant to methicillin, and the isolates of S. haemolyticus had a high ability to form biofilms.

Self-complementing split fluorescent proteins (split FP1-10/11) have become an important labeling tool in live-cell protein imaging. However, current split FP systems to label multiple proteins in single cells have a fundamental limitation in the number of proteins that can be simultaneously labeled. Here, we describe an approach to expand the number of orthogonal split FP systems with spectrally distinct colors. By combining rational design and cycles of directed evolution, we expand the spectral color palette of FP1-10/11. We also circularly permutate GFP and synthesize the β-strand 7, 8, or 10 system. These split GFP pairs are not only capable of labeling proteins but are also orthogonal to the current FP1-10/11 pairs, offering multiplexed labeling of cellular proteins. Our multiplexing approach, using the new orthogonal split FP systems, demonstrates simultaneous imaging of four distinct proteins in single cells; the resulting images reveal nuclear localization of focal adhesion protein Zyxin., Tamura et al. expand the split fluorescent protein system to multi-colour imaging (six-colours). Their multiplexing approach, using the orthogonal split FP systems, allows simultaneous imaging of four distinct proteins in single cells.

Autonomous parvoviruses encode at least two nonstructural proteins, NS1 and NS2. While NS1 is linked to important nuclear processes required for viral replication, much less is known about the role of NS2. Specifically, the function of canine parvovirus (CPV) NS2 has remained undefined. Here we have used proximity-dependent biotin identification (BioID) to screen for nuclear proteins that associate with CPV NS2. Many of these associations were seen both in noninfected and infected cells, however, the major type of interacting proteins shifted from nuclear envelope proteins to chromatin-associated proteins in infected cells. BioID interactions revealed a potential role for NS2 in DNA remodeling and damage response. Studies of mutant viral genomes with truncated forms of the NS2 protein suggested a change in host chromatin accessibility. Moreover, further studies with NS2 mutants indicated that NS2 performs functions that affect the quantity and distribution of proteins linked to DNA damage response. Notably, mutation in the splice donor site of the NS2 led to a preferred formation of small viral replication center foci instead of the large coalescent centers seen in wild-type infection. Collectively, our results provide insights into potential roles of CPV NS2 in controlling chromatin remodeling and DNA damage response during parvoviral replication. publishedVersion

Advances in proteomic methodologies based on quantitative mass spectrometry are now transforming pharmacology and experimental biology more broadly. The present review will discuss several examples based on work in the author’s laboratory, which focuses on delineating relationships between G protein–coupled receptor signaling and trafficking in the endocytic network. The examples highlighted correspond to those discussed in a talk presented at the 2019 EB/ASPET meeting, which was organized by Professor Joe Beavo to commemorate his receipt of the Julius Axelrod Award. SIGNIFICANCE STATEMENT GPCRs are allosteric machines that signal by interacting with other cellular proteins, and this, in turn, is determined by a complex interplay between the biochemical, subcellular localization, and membrane trafficking properties of receptors relative to transducer and regulatory proteins. The present minireview highlights recent advances and challenges in elucidating this dynamic cell biology and toward delineating the cellular basis of drug action at the level of defined GPCR interaction networks using proteomic approaches enabled by quantitative mass spectrometry.

For a very long time, frequent occurrences of biocrises have wreaked havoc on human beings, animals, and the environment. As a result, it is necessary to develop biocidal agents to destroy or neutralize active agents by releasing large amounts of strong biocides which are obtained upon detonation. Iodine is an efficient biocidal agent for bacteria, fungi, yeasts, viruses, spores, and protozoan parasites, and it is the sole element in the periodic table that can destroy microbes without contaminating the environment. Based on chemical biology, the mechanism of iodine as a bactericide may arise from oxidation and iodination reactions of cellular proteins and nucleic acids. However, because of the high vapor pressure causing elemental iodine to sublime readily at room temperature, it is inconvenient to use this material in its normal solid state directly as a biocidal agent under ambient conditions. Iodine-rich compounds where iodine is firmly bonded in molecules as a C-I or I-O moiety have been observed to be among the most promising energetic biocidal compounds. Gaseous products comprised of large amounts of iodine or iodine-containing components as strong biocides are released in the decomposition or explosion of iodine-rich compounds. Because of the detonation pressure, the iodine species are distributed over a large area greatly improving the efficacy of the system and requiring considerably less effort compared to traditional biocidal methods. The commercially available tetraiodomethane and tetraiodoethene, which possess superb iodine content also have the disadvantages of volatility, light sensitivity, and chemically reactivity, and therefore, are not suitable for use directly as biocidal agents. It is absolutely critical to synthesize new iodine-rich compounds with good thermal and chemical stabilities.In this Account, we describe our strategies for the syntheses of energetic iodine-rich compounds while maintaining the maximum iodine content with concomitant stability and routes for the synthesis of oxygen-containing iodine-rich compounds to improve the oxygen balance and achieve both high-energy and high-iodine content. In the other work, which involves cocrystals, iodine-containing polymers were also summarized. It is hoped that this Account will provide guidelines for the design and syntheses of new iodine-rich compounds and a route for the development of inexpensive, more efficient, and safer iodine-rich antibiological warfare agents of the future.

Human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS). Novel strategies to combat this pandemic include the discovery of cellular proteins targeting distinct steps of the HIV replication cycle. Here, we summarize our current knowledge on antiviral proteins interfering with the infectivity of released HIV particles.

Deubiquitinases are enzymes that remove ubiquitin moieties from the vast majority of cellular proteins, controlling their stability, interactions, and localization. The expression and activity of deubiquitinases are critical for physiology and can go awry in various diseases, including cancer. Based on recent findings in human blood cancers, we discuss the functions of selected deubiquitinases in acute leukemia and efforts to target these enzymes with the aim of blocking leukemia growth and improving disease outcomes. We focus on the emergence of the newest generation of preclinical inhibitors by discussing their modes of inhibition and their effects on leukemia biology.

Following recent hardware and software developments, single particle cryo-electron microscopy (cryo-EM) has become one of the most popular structural biology tools. Many targets, such as viruses, large protein complexes and oligomeric membrane proteins, have been resolved to atomic resolution using single-particle cryo-EM, which relies on the accurate assignment of particle location and orientation from intrinsically noisy projection images. The same image processing procedures are more challenging for smaller proteins due to their lower signal-to-noise ratios. Consequently, though most cellular proteins are less than 50 kDa, so far it has been possible to solve cryo-EM structures near that size range for only a few favorable cases. Here we highlight some of the challenges and recent efforts to break through this lower size limit by engineering large scaffolds to rigidly display multiple small proteins for imaging. Future design efforts are noted.

Significance Pathogenic microbes inject virulence proteins, termed effectors, into the cells of their hosts where they sabotage the cellular machinery and promote the infection process. However, plant and animal cells have evolved the ability to sense the molecular activities of these pathogenic proteins and mount an immune response to protect against infection. Here we have identified a chemical that mimics the immune eliciting activity of a pathogenic effector protein and importantly, it also activates immune responses in plants. We showcase how the intricate molecular dialog of the host–pathogen interface can be used to uncover chemical immunogens that specifically activate the eukaryotic immune system to prevent disease.

Tumors frequently exhibit aberrant glycosylation, which can impact cancer progression and therapeutic responses. The hexosamine biosynthesis pathway (HBP) produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a major substrate for glycosylation in the cell. Prior studies have identified the HBP as a promising therapeutic target in pancreatic ductal adenocarcinoma (PDA). The HBP requires both glucose and glutamine for its initiation. The PDA tumor microenvironment is nutrient poor, however, prompting us to investigate how nutrient limitation impacts hexosamine synthesis. Here, we identify that glutamine limitation in PDA cells suppresses de novo hexosamine synthesis but results in increased free GlcNAc abundance. GlcNAc salvage via N-acetylglucosamine kinase (NAGK) is engaged to feed UDP-GlcNAc pools. NAGK expression is elevated in human PDA, and NAGK deletion from PDA cells impairs tumor growth in mice. Together, these data identify an important role for NAGK-dependent hexosamine salvage in supporting PDA tumor growth., eLife digest Inside tumors, cancer cells often have to compete with each other for food and other resources they need to survive. This is a key factor driving the growth and progression of cancer. One of the resources cells need is a molecule called UDP-GlcNAc, which they use to modify many proteins so they can work properly. Because cancer cells grow quickly, they likely need much more UDP-GlcNAc than healthy cells. Many tumors, including those derived from pancreatic cancers, have very poor blood supplies, so their cells cannot get the nutrients and other resources they need to grow from the bloodstream. This means that tumor cells have to find new ways to use what they already have. One example of this is developing alternative ways to obtain UDP-GlcNAc. Cells require a nutrient called glutamine to produce UDP-GlcNAc. Limiting the supply of glutamine to cells allows researchers to study how cells are producing UDP-GlcNAc in the lab. Campbell et al. used this approach to study how pancreatic cancer cells obtain UDP-GlcNAc when their access to glutamine is limited. They used a technique called isotope tracing, which allows researchers to track how a specific chemical is processed inside the cell, and what it turns into. The results showed that the pancreatic cancer cells do not make new UDP-GlcNAc but use a protein called NAGK to salvage GlcNAc (another precursor of UDP-GlcNAc), which may be obtained from cellular proteins. Cancer cells that lacked NAGK formed smaller tumors, suggesting that the cells grow more slowly because they cannot recycle UDP-GlcNAc fast enough. Pancreatic cancer is one of the most common causes of cancer deaths and is notable for being difficult to detect and treat. Campbell et al. have identified one of the changes that allows pancreatic cancers to survive and grow quickly. Next steps will include examining the role of NAGK in healthy cells and testing whether it could be targeted for cancer treatment.

The proteasome system is a large and complex molecular machinery responsible for the degradation of misfolded, damaged, and redundant cellular proteins. When proteasome function is impaired, unwanted proteins accumulate, which can lead to several diseases including age-related and neurodegenerative diseases. Enhancing proteasome-mediated substrate degradation with small molecules may therefore be a valuable strategy for the treatment of various neurodegenerative diseases such as Parkinson’s, Alzheimer’s, and Huntington’s diseases. In this review, we discuss the structure of proteasome and how proteasome’s proteolytic activity is associated with aging and various neurodegenerative diseases. We also summarize various classes of compounds that are capable of enhancing, directly or indirectly, proteasome-mediated protein degradation.

Assessing the stability and degradation of proteins is central to the study of cellular biological processes. Here, we describe a novel pulse-chase method to determine the half-life of cellular proteins that overcomes the limitations of other commonly used approaches. This method takes advantage of pulse-labeling of nascent proteins in living cells with the bioorthogonal amino acid L-azidohomoalanine (AHA) that is compatible with click chemistry-based modifications. We validate this method in both mammalian and yeast cells by assessing both over-expressed and endogenous proteins using various fluorescent and chemiluminescent click chemistry-compatible probes. Importantly, while cellular stress responses are induced to a limited extent following live-cell AHA pulse-labeling, we also show that this response does not result in changes in cell viability and growth. Moreover, this method is not compromised by the cytotoxicity evident in other commonly used protein half-life measurement methods and it does not require the use of radioactive amino acids. This new method thus presents a versatile, customizable, and valuable addition to the toolbox available to cell biologists to determine the stability of cellular proteins.

Reorganization of cellular proteins into subcellular compartments, such as the rearrangement of RNA-binding proteins into cytoplasmic stress granules and P-bodies, is a well-recognized, widely studied physiological process currently under intense investigation. Using the assembly of a novel, inducible, nuclear granule formed from the yeast RNA-binding transcription termination factors Nab3 and Nrd1, we present a freely-accessible, high-throughput and unbiased algorithm written in MATLAB that detects and measures protein distribution, partitioning, and sequestration into subcellular compartments captured by fluorescence microscopy; an invaluable advancement to current image analysis methods which utilize experiment-specific custom scripts or subjective manual counting. Employing our algorithm, we quantified thousands of cells, ensuring rigorous examination of Nab3 granule formation across strains with reproducible statistical analyses. We document strain differences in Nab3 granule formation and an associated growth defect. Additionally, we applied our algorithm to immunofluorescent images of the inducible polymerization into filaments of an enzyme in human cells, demonstrating the algorithm’s versatility and adaptability.SUMMARY STATEMENTWe describe a computational tool that enables the quantification of protein condensation during assembly of a subnuclear compartment. The algorithm scores assembly of fluorescently tagged proteins in yeast or human cells.

Anthraquinones are privileged chemical scaffolds that have been used for centuries in various therapeutic applications. The anthraquinone moiety forms the core of various anticancer agents. However, the emergence of drug-resistant cancers warrants the development of new anticancer agents. The research endeavours towards new anthraquinone-based compounds are increasing rapidly in recent years. They are used as a core chemical template to achieve structural modifications, resulting in the development of new anthraquinone-based compounds as promising anticancer agents. Mechanistically, most of the anthraquinone-based compounds inhibit cancer progression by targeting essential cellular proteins. Herein, we review new anthraquinone analogues that have been developed in recent years as anticancer agents. This includes a systematic review of the recent literature (2005–2021) on anthraquinone-based compounds in cell-based models and key target proteins such as kinases, topoisomerases, telomerases, matrix metalloproteinases and G-quadruplexes involved in the viability of cancer cells. In addition to this, the developments in PEG-based delivery of anthraquinones and the toxicity aspects of anthraquinone derivatives are also discussed. The review dispenses a compact background knowledge to understanding anthraquinones for future research on the expansion of anticancer therapeutics.

Identification of differentially expressed proteins in antiphospholipid syndrome (APS) is a developing area of research for unique profiles of this pathology. Advances in technologies of mass spectrometry brings improvements in proteomics and results in assessment of soluble or cellular proteins which could be candidates for clinical biomarkers of primary APS. The use of blood as a source of proteins ease the acquisition of samples for proteomics analyses and later for disease diagnosis. We performed a systematic review to explore the proteomics studies carried out in circulating released proteins (serum, plasma) or cellular proteins (monocytes and platelets) of APS patients. The study groups differentiate among clinical APS cases with the aim to translate molecular findings to disease stratification and to improve APS diagnosis and prognosis. These studies also include the unravelling of new autoantibodies in non-criteria APS or how post-translational protein modifications provides clues about the pathological mechanisms of antigen-autoantibody recognition. Herein, we identified 82 proteins that were dysregulated in APS across eleven studies. Enrichment analysis revealed its connection to cellular activation and degranulation that eventually leads to thrombosis as the main biological process highlighted by these studies. Validation of APS-relevant proteins by functional and mechanistic studies will be essential for patient stratification and the development of targeted therapies for every clinical subtype of APS.

Since their discovery, fluorescent probes have found widespread use in biological research. Over time, multiple next-generation probes increased the fluorescence catalog by offering novel capabilities of detection that have been previously difficult or lacking with conventional probes. One of such probes is called a fluorogen-activating protein (FAP). These are bimodular sensors, composed of a single-chain antibody that exhibits high-affinity and selectivity for small-molecule fluorogens. Because fluorogens are inherently nonfluorescent unless sterically restricted, upon the formation of the noncovalent FAP-fluorogen complex the fluorogen module emits fluorescence when excited by light. More interestingly, these bimodular sensors permit improvement of their biophysical properties. For instance, the fluorescence spectra and environmental sensing capabilities of fluorogens may be altered by the method of chemical modification at the fluorogen structural level. Also, optimizations of the single-chain antibody scaffold, via amino acid substitutions at the selectivity regions, may improve the detection brightness and affinities of fluorogens; this may also improve the biophysical stability of FAPs in different cellular environments. Additionally, when utilized as biological discovery probes, FAP biosensors exhibit functional activity as genetic fusion tags with cellular proteins; this results in high fluorescent sensitivities of cell surface and intracellular targets. Also, FAPs allow the monitoring of cellular traffic of surface receptors by fluorescence methods of real-time color switching, or signal onset and offset. They find application as biological probes integrated into biomaterials, or as soluble affinity reagents for whole live animal studies. Overall, this noncovalent activation of fluorogen particles results in advanced strategies of fluorescence detection.

The high-risk Human Papillomavirus (HPV) E6 oncoprotein is known to contribute to human malignancy by targeting several of its cellular substrates through the ubiquitin-mediated degradation pathway. Previous studies have revealed that E6 interacts with the E6AP ubiquitin-protein ligase and directs its ubiquitylation activity toward several specific cellular proteins, one of the most important of which is p53. However, the role of E6AP in the degradation of many other E6 substrates is still ambiguous because loss of E6AP also induces a loss of E6 expression. To examine this further, we used CRISPR-edited E6AP knockout cells to perform E6 degradation assays in the presence of a catalytically inactive mutant form of E6AP, thus ensuring the stabilization of E6 but with the ligase itself being functionally inactive. Using this system, we found that E6 can mediate the degradation of several PDZ domain-containing proteins independently of E6AP ubiquitin ligase activity. This study thus opens up ways to investigate other possible components of the cellular ubiquitin proteasome pathway that E6 might utilize to target these substrates.

A large section of cellular proteins in both prokaryotic and eukaryotic systems have oligomeric property. Intently, oligomerization of protein is an invaluable phenomenon from the point of view of protein evolution. This review comprises an overview on modulation of protein oligomerization. The comprehensive modulation of protein oligomerization can be supportive for effective drug designing in the future. The common mechanisms of protein oligomerization are domain swapping and ligand induced dimerization. Infrequent mechanism of protein oligomerization involves point mutations at the dimer interface, post-translational modification and insertion/deletion at the interface. Predominantly, ligand induced oligomerization is the most useful method to regulate the protein oligomerization that can act as a modulator. Thus, functional modulation of oligomeric proteins can be done, both in-vitro and in-vivo, using various artificial and natural modulators, respectively. Though, the biophysical methods, like microscopy and spectroscopy, have strong potential to characterize the oligomeric proteins. Oligomeric proteins can be characterized biochemically too. Hence, this review illustrates the regulation of protein oligomerization using several modulators, in the future, these can be used for effective drug designing to cure several diseases associated with oligomeric proteins.

As the largest family of E3 ligases, the Skp1-cullin 1-F-box (SCF) E3 ligase complex is comprised of Cullins, Skp1 and F-box proteins. And the SCF E3 ubiquitin ligases play an important role in regulating critical cellular processes, which promote degradation of many cellular proteins, including signal transducers, cell cycle regulators, and transcription factors. We review the biological roles of the SCF ubiquitin-ligase complex in gametogenesis, oocyte-to-embryo transition, embryo development and the regulation for estrogen and progestin. We find that researches about the SCF ubiquitin-ligase complex at the beginning of life are not comprehensive, thus more in-depth researches will promote its eventual clinical application.

Human immunodeficiency virus-1 capsid (HIV-1 CA) is involved in different stages of the viral replication cycle. During virion assembly, CA drives the formation of the hexameric lattice in immature viral particles, while in mature virions CA monomers assemble in cone-shaped cores surrounding the viral RNA genome and associated proteins. In addition to its functions in late stages of the viral replication cycle, CA plays key roles in a number of processes during early phases of HIV-1 infection including trafficking, uncoating, recognition by host cellular proteins and nuclear import of the viral pre-integration complex. As a result of efficient cooperation of CA with other viral and cellular proteins, integration of the viral genetic material into the host genome, which is an essential step for productive viral infection, successfully occurs. In this review, we will summarize available data on CA functions in HIV-1 replication, describing in detail its roles in late and early phases of the viral replication cycle.

Isolevuglandins (IsoLGs) are a family of highly reactive 4-ketoaldehydes formed by lipid peroxidation that modify the lysyl residues of cellular proteins. Modification of proteins by IsoLGs have been shown to contribute to disease processes such as the development of hypertension. Accurate quantitation of the extent of protein modification by IsoLGs is essential for understanding the mechanisms whereby these modifications contribute to disease and the efficacy of interventions designed to prevent this modification. The previously described LC/MS assay to quantitate IsoLG protein adducts was extremely labor-intensive and time consuming, and while it offered reasonably low intra-day variation for replicate samples, variation when replicate samples were processed on separate days was significant. These limitations significantly restricted utilization of this approach. We therefore performed a series of studies to optimize the assay. We now report a significantly simplified LC/MS assay for measurement of IsoLG protein adducts with increased sensitivity and lower intra-day and inter-day variability.

Assessment of the fidelity of gene expression is crucial to understand cell homeostasis. Here we present a highly sensitive method for the systematic Quantification of Rare Amino acid Substitutions (QRAS) using absolute quantification by targeted mass spectrometry after chromatographic enrichment of peptides with missense amino acid substitutions. By analyzing incorporation of near- and non-cognate amino acids in a model protein EF-Tu, we show that most of missense errors are too rare to detect by conventional methods, such as DDA, and are estimated to be between

Within the last decade, active research on circular RNAs (circRNAs) has dramatically improved our understanding of the expression and function of these non-coding RNAs. While several mechanisms for circRNA function have been proposed, including sequestration of microRNAs and regulation of cellular proteins, studies provide evidence that circRNAs can regulate transcription and may also serve as biomarkers. Due to the heterogeneous nature of the brain, and the dynamic transcriptional mechanisms that support neurobiological pathways, the influence of circRNAs is potentially extensive. Understanding how circRNAs contribute to key neurological pathways will fill gaps in our understanding of brain function and provide valuable insight into novel therapeutic approaches to treat neurological diseases. Here, we review recent research on circRNA expression in the brain, describe the proposed functions of circRNAs, and evaluate the role of circRNAs in neurological diseases. Keywords: circRNA, Non-coding RNA, Brain, Transcriptional regulation, Neurological disease, microRNA

The attachment of a single O-linked b-N-acetylglucosamine (O-GlcNAc) to serine and threonine residues of numerous proteins in the nucleus, cytoplasm, and mitochondria is a reversible post-translational modification (PTM) and plays an important role as a regulator of various cellular processes in both healthy and disease states. Advances in strategies and tools that allow for the detection of dynamic O-GlcNAcylation on cellular proteins have helped to enhance our initial and ongoing understanding of its dynamic effects on cellular stimuli and given insights into its link to the pathogenesis of several chronic diseases. Furthermore, chemical genetic strategies and related tools have been successfully applied to a myriad of biological systems with a new level of spatiotemporal and molecular precision. These strategies have started to be used in studying and controlling O-GlcNAcylation both in vivo and in vitro. In this mini-review, overviews of recent advances in molecular tools being applied to the detection and identification of O-GlcNAcylation on cellular proteins as well as on individual proteins are provided. In addition, chemical genetic strategies that have already been applied or are potentially usable in O-GlcNAc functional are also discussed.

73 p.-12 fig.-1 tab.-1 graph. abst., Viral infections are a major health problem; therefore, there is an urgent need for novel therapeutic strategies.Antivirals used to target proteins encoded by the viral genome usually enhance drug resistance generated by the virus. A potentialsolution may be drugs acting at host-based targets since viruses are dependent on numerous cellular proteins and phosphorylationevents that are crucial during their life cycle. Repurposing existing kinase inhibitors as antiviral agents would help in the cost andeffectiveness of the process, but this strategy usually does not provide much improvement, and specific medicinal chemistryprograms are needed in thefield. Anyway, extensive use of FDA-approved kinase inhibitors has been quite useful in deciphering therole of host kinases in viral infection. The present perspective aims to review the state of the art of kinase inhibitors that target viralinfections in different development stages., Funding from“la Caixa”Banking Foundation (LCF/PR/HR19/52160012), CSIC (202020E103 and 202080E293),and AEI (Grant PID2019-105600RB-I00) is acknowledged.E.C. holds a JAE Intro fellowship (JAEINT_20_01339) from CSIC., postprint

Author(s): Lam, Yi-Li | Advisor(s): Lew, John | Abstract: One of the hallmarks of Alzheimer’s disease is pathological tau aggregation. We previously reported that cinnamaldehyde, found in cinnamon, could inhibit tau aggregation in vitro, by reacting with one or both of the two cysteines in tau. In my thesis, I use spectrophotometric methods to show that both cysteines display hyperreactivity over model sulfhydryl compounds by as much as four fold. The hyperreactivity is specific to cysteines in tau as opposed to those in other sulfhydryl-containing cellular proteins. Cinnamaldehyde (CA) displays several characteristics that make it favorable as a possible inhibitor of aggregation in vivo: it is a water-soluble, brain-permeable molecule that is non-toxic and inexpensive. I now show in my thesis that the hyperreactivity of CA toward tau specifically, as described in this thesis, is an additional favorable property towards the goal of being a potential therapeutic.

Tailoring stress responses When faced with environmental stress, cells respond by shutting down cellular processes such as translation and nucleocytoplasmic transport. At the same time, cells preserve cytoplasmic messenger RNAs in structures known as stress granules, and many cellular proteins are modified by the covalent addition of ubiquitin, which has long been presumed to reflect degradation of stress-damaged proteins (see the Perspective by Dormann). Maxwell et al. show that cells generate distinct patterns of ubiquitination in response to different stressors. Rather than reflecting the degradation of stress-damaged proteins, this ubiquitination primes cells to dismantle stress granules and reinitiate normal cellular activities once the stress is removed. Gwon et al. show that persistent stress granules are degraded by autophagy, whereas short-lived granules undergo a process of disassembly that is autophagy independent. The mechanism of this disassembly depends on the initiating stress. Science , abc3593 and abf6548, this issue p. eabc3593 and p. eabf6548 ; see also abj2400, p. 1393

The coronavirus disease 2019 (COVID-19) pandemic has caused more than 2.3 million casualties worldwide and the lack of effective treatments is a major health concern. The development of targeted drugs is held back due to a limited understanding of the molecular mechanisms underlying the perturbation of cell physiology observed after viral infection. Recently, several approaches, aimed at identifying cellular proteins that may contribute to COVID-19 pathology, have been reported. Albeit valuable, this information offers limited mechanistic insight as these efforts have produced long lists of cellular proteins, the majority of which are not annotated to any cellular pathway. We have embarked in a project aimed at bridging this mechanistic gap by developing a new bioinformatic approach to estimate the functional distance between a subset of proteins and a list of pathways. A comprehensive literature search allowed us to annotate, in the SIGNOR 2.0 resource, causal information underlying the main molecular mechanisms through which severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and related coronaviruses affect the host–cell physiology. Next, we developed a new strategy that enabled us to link SARS-CoV-2 interacting proteins to cellular phenotypes via paths of causal relationships. Remarkably, the extensive information about inhibitors of signaling proteins annotated in SIGNOR 2.0 makes it possible to formulate new potential therapeutic strategies. The proposed approach, which is generally applicable, generated a literature-based causal network that can be used as a framework to formulate informed mechanistic hypotheses on COVID-19 etiology and pathology.

Mammalian cells process information through coordinated spatiotemporal regulation of proteins. Engineering cellular networks thus relies on efficient tools for regulating protein levels in specific subcellular compartments. To address the need to manipulate the extent and dynamics of protein localization, we developed a platform technology for the target-specific control of protein destination. This platform is based on bifunctional molecules comprising a target-specific nanobody and universal sequences determining target subcellular localization or degradation rate. We demonstrate that nanobody-mediated localization depends on the expression level of the target and the nanobody, and the extent of target subcellular localization can be regulated by combining multiple target-specific nanobodies with distinct localization or degradation sequences. We also show that this platform for nanobody-mediated target localization and degradation can be regulated transcriptionally and integrated within orthogonal genetic circuits to achieve the desired temporal control over spatial regulation of target proteins. The platform reported in this study provides an innovative tool to control protein subcellular localization, which will be useful to investigate protein function and regulate large synthetic gene circuits.

Historically, covalent drugs were avoided in drug development process due to the possible toxicity linked to the covalent binding of such drugs and cellular proteins. However, recent years have witnessed the fast resurgence of the discovery of covalent drugs because of the realization of the advantages of covalent drugs in efficacy, duration of action, therapy-induced resistance, and targeting hard targets. Since December 2019, SARS-CoV-2 has caused nearly 40 million COVID-19 patients and over one million deaths in the whole world as of October 19, 2020. In contrast, effective drugs have not been found at the same time. Therefore, it is of great value to discover and design drugs for the prevention and cure of SARS-CoV-2 infection. Recently, we developed a simple but efficient method named as SCARdock for the discovery of covalent drugs. In this work, we present a detailed protocol of this method in discovering potential anti-SARS-CoV-2 drugs.

The ATG8 family of proteins regulates autophagy in a variety of ways. Recently, ATG8s were demonstrated to conjugate directly to cellular proteins in a process termed “ATG8ylation,” which is amplified by mitochondrial damage and antagonized by ATG4 proteases. ATG8s may have an emerging role as small protein modifiers. Refereed/Peer-reviewed

Extracellular vesicles (EVs) are secreted nano‐sized vesicles that contain cellular proteins, lipids, and nucleic acids. Although EVs are expected to be biologically diverse, current analyses cannot adequately characterize this diversity because most are ensemble methods that inevitably average out information from diverse EVs. Here we describe a single vesicle analysis, which directly visualizes marker expressions of individual EVs using a total internal‐reflection microscopy and analyzes their co‐localization to investigate EV subpopulations. The single‐vesicle imaging and co‐localization analysis successfully illustrated the diversity of EVs and revealed distinct patterns of tetraspanin expressions. Application of the analysis demonstrated similarities and dissimilarities between the EV fractions that had been acquired from different conventional EV isolation methods. The analysis method developed in this study will provide a new and reliable tool for investigating characteristics of single EVs, and the findings of the analysis might increase understanding of the characteristics of EVs.

PROteolysis Targeting Chimeras (PROTACs) are a family of heterobifunctional small molecules that specifically target cellular proteins for degradation. Given that their mode of action is distinct from that of small-molecule inhibitors widely used in clinical practice, PROTACs have the potential to improve current cancer therapies. Multiple studies have suggested that PROTACs exhibit enhanced pharmacodynamics and reduced toxicity both in vitro and in vivo compared to clinically relevant small-molecule kinase inhibitors. In addition, PROTACs have been reported to be less prone to mutation-mediated drug resistance in specific disease settings. Since its development in 2001, the field of targeted protein degradation, in which PROTACs are used, has expanded rapidly. However, earlier studies focused on the advancement of the technology itself, while preclinical and clinical data on the disease-modifying effect of PROTACs have only recently been reported. As preclinical and clinical evidence accumulates, the efficacy of PROTACs as targeted therapeutics-distinct from that of small-molecule kinase inhibitors-suggests potential translational benefit in the clinical setting. In this short review, we aim to describe translational potentials of PROTACs. We offer our perspectives as practicing oncologists on the preclinical and clinical data on PROTACs as novel therapeutics for both solid and hematological malignancies.

Membrane (M) proteins of coronaviruses are the most abundant component of the virus envelope and play crucial roles in virus assembly, virus budding and the regulation of host immunity. To understand more about these functions in the context of PEDV M protein, forty host cell proteins interacting with the M protein were identified in the present study by exploiting the proximity-labeling enzyme APEX2 (a mutant soybean ascorbate peroxidase). Bioinformatic analysis showed that the identified host cell proteins were related to fifty-four signal pathways and a wide diversity of biological processes. Interaction between M and five of the identified proteins (RIG-I, PPID, NHE-RF1, S100A11, CLDN4) was confirmed by co-immunoprecipitation (Co-IP). In addition, knockdown of PPID and S100A11 genes by siRNA significantly improved virus production, indicating that the proteins encoded by the two genes were interfering with or down-regulating virus replication in infected cells. Identification of the host cell proteins accomplished in this study provides new information about the mechanisms underlying PEDV replication and immune evasion. Significance PEDV M protein is an essential structural protein implicated in viral infection, replication and assembly although the precise mechanisms underlying these functions remain enigmatic. In this study, we have identified 40 host cell proteins that interact with PEDV M protein using the proximity-labeling enzyme APEX2. Co-immunoprecipitation subsequently confirmed interactions between PEDV M protein and five host cell proteins, two of which (S100A11 and PPID) were involved in down-regulating virus replication in infected cells. This study is significant in that it formulates a strategy to provide new information about the mechanisms relating to the novel functions of PEDV M protein., Graphical abstract Unlabelled Image

SummarySince the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, investigation of the SARS-CoV-2 infection in the native cellular context is scarce, and there is a lack of comprehensive knowledge on SARS-CoV-2 replicative cycle. Understanding the genome replication, assembly and egress of SARS-CoV-2, a multistage process that involves different cellular compartments and the activity of many viral and cellular proteins, is critically important as it bears the means of medical intervention to stop infection. Here, we investigated SARS-CoV-2 replication in Vero cells under the near-native frozen-hydrated condition using a unique correlative multi-modal, multi-scale cryo-imaging approach combining soft X-ray cryo-tomography and serial cryoFIB/SEM volume imaging of the entire SARS-CoV-2 infected cell with cryo-electron tomography (cryoET) of cellular lamellae and cell periphery, as well as structure determination of viral components by subtomogram averaging. Our results reveal at the whole cell level profound cytopathic effects of SARS-CoV-2 infection, exemplified by a large amount of heterogeneous vesicles in the cytoplasm for RNA synthesis and virus assembly, formation of membrane tunnels through which viruses exit, and drastic cytoplasm invasion into nucleus. Furthermore, cryoET of cell lamellae reveals how viral RNAs are transported from double-membrane vesicles where they are synthesized to viral assembly sites; how viral spikes and RNPs assist in virus assembly and budding; and how fully assembled virus particles exit the cell, thus stablishing a model of SARS-CoV-2 genome replication, virus assembly and egress pathways.

Direct interaction between intrinsically disordered proteins (IDPs) is often difficult to characterize hampering the elucidation of their binding mechanism. Particularly challenging is the study of fuzzy complexes, in which the intrinsically disordered proteins or regions retain conformational freedom within the assembly. To date, nuclear magnetic resonance spectroscopy has proven to be one of the most powerful techniques to characterize at the atomic level intrinsically disordered proteins and their interactions, including those cases where the formed complexes are highly dynamic. Here, we present the characterization of the interaction between a viral protein, the Early region 1A protein from Adenovirus (E1A), and a disordered region of the human CREB-binding protein, namely the fourth intrinsically disordered linker CBP-ID4. E1A was widely studied as a prototypical viral oncogene. Its interaction with two folded domains of CBP was mapped, providing hints for understanding some functional aspects of the interaction with this transcriptional coactivator. However, the role of the flexible linker connecting these two globular domains of CBP in this interaction was never explored before.

Deubiquitinating enzymes (DUBs) control the removal of ubiquitin and ubiquitin-like proteins from cellular proteins. There are approximately 100 DUBs in the human genome, and they regulate diverse biochemical, cellular and physiological processes. Notably, they are known to control many pathways which are misregulated and affected in human diseases, such as cancer, immunology and neurodegeneration. Due to the broad scope of DUB biology, they are emerging as a target class for inhibitor development. In this chapter, we will describe the promise of targeting DUB in different disease contexts, describe practices for identifying and validating small-molecule inhibitors and physiologically relevant substrates of DUBs and review recent examples of well-characterized DUB inhibitors. These advances underscore the excitement and potential in targeting DUBs for both therapeutic and research purposes.

Covalent attachment of ubiquitin, a small globular polypeptide, to protein substrates is a key post-translational modification that determines the fate, function, and turnover of most cellular proteins. Ubiquitin modification exists as mono- or polyubiquitin chains involving multiple ways how ubiquitin C-termini are connected to lysine, perhaps other amino acid side chains, and N-termini of proteins, often including branching of the ubiquitin chains. Understanding this enormous complexity in protein ubiquitination, the so-called ‘ubiquitin code’, in combination with the ∼1000 enzymes involved in controlling ubiquitin recognition, conjugation, and deconjugation, calls for novel developments in analytical techniques. Here, we review different headways in the field mainly driven by mass spectrometry and chemical biology, referred to as “ubiquitomics”, aiming to understand this system’s biological diversity.

Cavezzi A, Troiani E, Corrao S (2020) COVID-19: hemoglobin, iron, and hypoxia beyond inflammation. A narrative review. Clin Pract 10(2). https://doi.org/10.4081/cp.2020.1271 Garrick M, Ghio A (2020) Iron chelation may harm patients with COVID-19. Eur J Clin Pharmacol Herbert V, Jayatilleke E, Shaw S, Rosman A, Giardina P, Grady R et al (1997) Serum ferritin iron, a new test, measures human body iron stores unconfounded by inflammation. Stem Cells 15(4):291–296 Article Google Scholar Perricone C, Bartoloni E, Bursi R, Cafaro G, Guidelli G, Shoenfeld Y et al (2020) COVID-19 as part of the hyperferritinemic syndromes: the role of iron depletion therapy. Immunol Res 68(4):213–224 Article Google Scholar Edeas M, Saleh J, Peyssonnaux C (2020) Iron: Innocent bystander or vicious culprit in COVID-19 pathogenesis? Int J Infect Dis 97:303–305 Article Google Scholar Liu W, Zhang S, Nekhai S, Liu S (2020) Depriving iron supply to the virus represents a promising adjuvant therapeutic against viral survival. Curr Clin Microbiol Rep 7(2):13–19 Article Google Scholar Shi S, Lai M (2005) Viral and cellular proteins involved in coronavirus replication. Curr Top Microbiol Immunol 287:95–131 PubMed PubMed Central Google Scholar Juang H (2004) Modulation of iron on mitochondrial aconitase expression in human prostatic carcinoma cells. Mol Cell Biochem 265(1/2):185–194 Article Google Scholar Download references Spire Fylde Coast Hospital, St Walburgas Road, Blackpool, FY3 8BP, UK Anis Abobaker You can also search for this author in PubMed Google Scholar Correspondence to Anis Abobaker. The author declares that he has no conflict of interest. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Reprints and Permissions Abobaker, A. Reply: Iron chelation may harm patients with COVID-19. Eur J Clin Pharmacol (2020). https://doi.org/10.1007/s00228-020-02988-9 Download citation Received: 12 August 2020 Accepted: 27 August 2020 Published: 01 September 2020 DOI: https://doi.org/10.1007/s00228-020-02988-9

Targeted Protein Degradation (TPD) has emerged as an exciting new era in chemical biology and drug discovery. PROteolysis TArgeting Chimera (PROTAC) technology targets cellular proteins for degradation by co-opting the Ubiquitin Proteasome System. Over the last five years, numerous studies have expanded our understanding of the unique mode of action and advantages of PROTACs, which has in turn spurred interest in both academia and industry to explore PROTACs as a novel therapeutic strategy. In this review, we first highlight the key advantages of PROTACs and then discuss the spatiotemporal regulation of protein degradation. Next, we explore current chemically tractable E3 ligases focusing on expanding the existing repertoire with novel E3 ligases to uncover the full potential of TPD. Collectively, these studies are guiding the development of the PROTAC technology as it emerges as a new modality in precision medicine.