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Lipid transfer proteins (LTPs) are the key contributor of organelle-specific lipid distribution and cellular lipid homeostasis. Here, we report a novel implication of LTPs in phagocytosis, trogocytosis, pinocytosis, biosynthetic secretion, recycling of pinosomes, and motility of the parasitic protist E. histolytica, the etiological agent of human amoebiasis. We show that two StAR-related lipid transfer (START) domain-containing LTPs (named as EhLTP1 and 3) are involved in these biological pathways in an LTP-specific manner. Our findings provide novel implications of LTPs, which are relevant to the elucidation of pathophysiology of the diseases caused by parasitic protists., Competing Interests: The authors have declared that no competing interests exist.

Interest in cryo-Electron Microscopy (EM) imaging has skyrocketed in recent years due to its pristine views of macromolecules and materials. As advances in instrumentation and computing algorithms spurred this progress, there is renewed focus to address specimen-related challenges. Here we contribute a microchip-based toolkit to perform complementary structural and biochemical analysis on low-molecular weight proteins. As a model system, we used the SARS-CoV-2 nucleocapsid (N) protein (48 kDa) due to its stability and important role in therapeutic development. Cryo-EM structures of the N protein monomer revealed a flexible N-terminal "top hat" motif and a helical-rich C-terminal domain. To complement our structural findings, we engineered microchip-based immunoprecipitation assays that led to the discovery of the first antibody binding site on the N protein. The data also facilitated molecular modeling of a variety of pandemic and common cold-related coronavirus proteins. Such insights may guide future pandemic-preparedness protocols through immuno-engineering strategies to mitigate viral outbreaks.

The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Binding of phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported to regulate nucleocytoplasmic N shuttling. All seven isoforms of the human 14-3-3 are abundantly present in tissues vulnerable to SARS-CoV-2, where N can constitute up to ~1% of expressed proteins during infection. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its molecular mechanism and the specific critical phosphosites are unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and deduce the apparent K D to selected isoforms, showing that these are in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, which is conserved among related zoonotic coronaviruses and located within the functionally important, SR-rich region of N. The relatively tight 14-3-3/pN association could regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, and could also hijack cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Lacunary polyoxometalate (POM), [PW 9 O 34 ] 9- , grafts with a boronic acid group attached via an organosilane bridge assemble into microspheres, PW 9 -Si-APBA. The oxygen-rich and hydrophilic surface of POM facilitates the binding of phosphate groups in phosphoproteins and glycans in glycoproteins. While the metal-oxo in POM provides π-π interactions with the phosphate groups of phosphoproteins, the boronic acid group specifically binds to glycoproteins via the cis-diols of glycans. Therefore, these multi-driving forces ensure the selective adsorption of phosphoproteins and glycoproteins by PW 9 -Si-APBA microspheres in biological sample matrixes, even in the presence of very high protein abundance, i.e., BSA, at mass ratio of β-ca/IgG/OVA/BSA = 1 : 1 : 1 : 200.

The current outbreak of the highly infectious COVID-19 respiratory disease is caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). To fight the pandemic, the search for promising viral drug targets has become a cross-border common goal of the international biomedical research community. Within the international Covid19-NMR consortium, scientists support drug development against SARS-CoV-2 by providing publicly available NMR data on viral proteins and RNAs. The coronavirus nucleocapsid protein (N protein) is an RNA-binding protein involved in viral transcription and replication. Its primary function is the packaging of the viral RNA genome. The highly conserved architecture of the coronavirus N protein consists of an N-terminal RNA-binding domain (NTD), followed by an intrinsically disordered Serine/Arginine (SR)-rich linker and a C-terminal dimerization domain (CTD). Besides its involvement in oligomerization, the CTD of the N protein (N-CTD) is also able to bind to nucleic acids by itself, independent of the NTD. Here, we report the near-complete NMR backbone chemical shift assignments of the SARS-CoV-2 N-CTD to provide the basis for downstream applications, in particular site-resolved drug binding studies.

The emerged novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created a global health crisis that warrants an accurate and detailed characterization of the rapidly evolving viral genome for understanding its epidemiology, pathogenesis, and containment. Here, we explored 61,485 sequences of the nucleocapsid (N) protein, a potent diagnostic and prophylactic target, for identifying the mutations to review their roles in real-time polymerase chain reaction based diagnosis and observe consequent impacts. Compared to the Wuhan reference strain, a total of 1034 unique nucleotide mutations were identified in the mutant strains (49.15%, n = 30,221) globally. Of these mutations, 367 occupy primer binding sites including the 3'-end mismatch to the primer-pair of 11 well-characterized primer sets. Noteworthily, CDC (USA) recommended the N2 primer set contained a lower mismatch than the other primer sets. Moreover, 684 amino acid (aa) substitutions were located across 317 (75.66% of total aa) unique positions including 82, 21, and 83 of those in the RNA binding N-terminal domain (NTD), SR-rich region, and C-terminal dimerization domain, respectively. Moreover, 11 in-frame deletions, mostly (n = 10) within the highly flexible linker region, were revealed, and the rest was within the NTD region. Furthermore, we predicted the possible consequence of high-frequency mutations (≥20) and deletions on the tertiary structure of the N protein. Remarkably, we observed that a high frequency (67.94% of mutated sequences) co-occuring mutations (R203K and G204R) destabilized and decreased overall structural flexibility. The N protein of SARS-CoV-2 comprises an average of 1.2 mutations per strain compared to 4.4 and 0.4 in Middle East respiratory syndrome-related coronavirus and SARS-CoV, respectively. Despite being proposed as the alternative target to spike protein for vaccine and therapeutics, the ongoing evolution of the N protein may challenge these endeavors, thus needing further immunoinformatics analyses. Therefore, continuous monitoring is required for tracing the ongoing evolution of the SARS-CoV-2 N protein in prophylactic and diagnostic interventions., (© 2020 Wiley Periodicals LLC.)

Short palate, lung, and nasal epithelium clone 1 (SPLUNC1) is a kind of secretory protein, and gets expressed abundantly in normal respiratory epithelium of humans. As a natural immune molecule, SPLUNC1 is proved to be involved in inflammatory response and airway host defense. This review focuses on summarizing and discussing the role of SPLUNC1 in regulating airway surface liquid (ASL) and participating in airway host defense. PubMed and MEDLINE were used for searching and identifying the data in this review. The domain of bactericidal/permeability-increasing protein in SPLUNC1 and the α-helix, α4, are essential for SPLUNC1 to exert biological activities. As a natural innate immune molecule, SPLUNC1 plays a significant role in inflammatory response and airway host defense. Its special expression patterns are not only observed in physiological conditions, but also in some respiratory diseases. The mechanisms of SPLUNC1 in airway host defense include modulating ASL volume, acting as a surfactant protein, inhibiting biofilm formation, as well as regulating ASL compositions, such as LL-37, mucins, Neutrophil elastase, and inflammatory cytokines. Besides, potential correlations are found among these different mechanisms, especially among different ASL compositions, which should be further explored in more systematical frameworks. In this review, we summarize the structural characteristics and expression patterns of SPLUNC1 briefly, and mainly discuss the mechanisms of SPLUNC1 exerted in host defense, aiming to provide a theoretical basis and a novel target for future studies and clinical treatments.

The nucleocapsid protein N from SARS-CoV-2 is one of the most highly expressed proteins by the virus and plays a number of important roles in the transcription and assembly of the virion within the infected host cell. It is expected to be characterized by a highly dynamic and heterogeneous structure as can be inferred by bioinformatics analyses as well as from the data available for the homologous protein from SARS-CoV. The two globular domains of the protein (NTD and CTD) have been investigated while no high-resolution information is available yet for the flexible regions of the protein. We focus here on the 1-248 construct which comprises two disordered fragments (IDR1 and IDR2) in addition to the N-terminal globular domain (NTD) and report the sequence-specific assignment of the two disordered regions, a step forward towards the complete characterization of the whole protein.

Background: The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has generated a pandemic with alarming rates of fatality worldwide. This situation has had a major impact on clinical laboratories that have attempted to answer the urgent need for diagnostic tools, since the identification of coronavirus disease 2019 (COVID-19). Development of a reliable serological diagnostic immunoassay, with high levels of sensitivity and specificity to detect SARS-CoV-2 antibodies with improved differential diagnosis from other circulating viruses, is mandatory., Methods: An enzyme-linked immunosorbent assay (ELISA) using whole inactivated virus cultured in vitro, was developed to detect viral antigens. WB and ELISA investigations were carried out with sera of convalescent patients and negative sera samples. Both analyses were concurrently performed with recombinant MABs to verify the findings., Results: Preliminary data from 10 sera (5 patients with COVID-19, and 5 healthy controls) using this immunoassay are very promising, successfully identifying all of the confirmed SARS-CoV-2-positive individuals., Conclusion: This ELISA appears to be a specific and reliable method for detecting COVID-19 antibodies (IgG, IgM, and IgA), and a useful tool for identifying individuals which have developed immunity to the virus., (© 2021 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Stable isotope labeling by amino acid-based high-resolution phosphoproteomics is a powerful technique that allows for direct comparison of cells stimulated under different experimental conditions. This feature makes it the ideal methodology to identify cytokine signaling networks. Here, we present an optimized protocol for the isolation and identification of phosphopeptides from IL-6-stimulated primary human Th-1 cells. For complete details on the use and execution of this protocol, please refer to Martinez-Fabregas et al. (2020)., Competing Interests: The authors declare no competing interests., (Crown Copyright © 2021.)

The SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA-binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA-binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction.

SF3B1, an essential component of the U2 snRNP, is frequently mutated in cancers. Cancer-associated SF3B1 mutation causes aberrant RNA splicing, mostly at 3' splice sites (3'ss). RNA splicing of DVL2, a regulator of Notch signaling, is affected by SF3B1 mutation. Here, we report that the mutated SF3B1 use an alternative branchpoint sequence (BPS) for the aberrant splicing of DVL2, which has a higher affinity to U2 snRNA than the BPS for the canonical splicing of DVL2. Swapping the position of the alternative BPS with the position of the canonical BPS decreased the aberrant splicing of DVL2, suggesting that the mutated SF3B1 prefers to use BPS with high affinity to U2 snRNA for splicing. Additionally, swapping the positions of two BPSs associated with the canonical splicing of DVL2 demonstrated that both the affinity to the U2 snRNA and the distance to the 3'ss are important to the selection of BPS. Importantly, the aberrant splicing of DVL2 does not require the canonical 3'ss and the canonical polypyrimidine tract, which reveals a novel type of aberrant splicing induced by SF3B1 mutation. These findings provide a more comprehensive understanding of the mechanisms underlying aberrant splicing induced by SF3B1 mutation in cancer., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The huge global expansion of the COVID-19 pandemic caused by the novel SARS-corona virus-2 is an extraordinary public health emergency. The unavailability of specific treatment against SARS-CoV-2 infection necessitates the focus of all scientists in this direction. The reported antiviral activities of guanidine alkaloids encouraged us to run a comprehensive in silico binding affinity of fifteen guanidine alkaloids against five different proteins of SARS-CoV-2, which we investigated. The investigated proteins are COVID-19 main protease (M pro ) (PDB ID: 6lu7), spike glycoprotein (PDB ID: 6VYB), nucleocapsid phosphoprotein (PDB ID: 6VYO), membrane glycoprotein (PDB ID: 6M17), and a non-structural protein (nsp10) (PDB ID: 6W4H). The binding energies for all tested compounds indicated promising binding affinities. A noticeable superiority for the pentacyclic alkaloids particularly, crambescidin 786 ( 5 ) and crambescidin 826 ( 13 ) has been observed. Compound 5 exhibited very good binding affinities against Mpro (ΔG = -8.05 kcal/mol), nucleocapsid phosphoprotein (ΔG = -6.49 kcal/mol), and nsp10 (ΔG = -9.06 kcal/mol). Compound 13 showed promising binding affinities against M pro (ΔG = -7.99 kcal/mol), spike glycoproteins (ΔG = -6.95 kcal/mol), and nucleocapsid phosphoprotein (ΔG = -8.01 kcal/mol). Such promising activities might be attributed to the long ω-fatty acid chain, which may play a vital role in binding within the active sites. The correlation of c Log P with free binding energies has been calculated. Furthermore, the SAR of the active compounds has been clarified. The Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) studies were carried out in silico for the 15 compounds; most examined compounds showed optimal to good range levels of ADMET aqueous solubility, intestinal absorption and being unable to pass blood brain barrier (BBB), non-inhibitors of CYP2D6, non-hepatotoxic, and bind plasma protein with a percentage less than 90%. The toxicity of the tested compounds was screened in silico against five models (FDA rodent carcinogenicity, carcinogenic potency TD 50 , rat maximum tolerated dose, rat oral LD 50 , and rat chronic lowest observed adverse effect level (LOAEL)). All compounds showed expected low toxicity against the tested models. Molecular dynamic (MD) simulations were also carried out to confirm the stable binding interactions of the most promising compounds, 5 and 13, with their targets. In conclusion, the examined 15 alkaloids specially 5 and 13 showed promising docking, ADMET, toxicity and MD results which open the door for further investigations for them against SARS-CoV-2.

The interferon gamma-inducible protein 16 (IFI16) and its murine homologous protein p204 function in non-sequence specific dsDNA sensing; however, the exact dsDNA recognition mechanisms of IFI16/p204, which harbour two HIN domains, remain unclear. In the present study, we determined crystal structures of p204 HINa and HINb domains, which are highly similar to those of other PYHIN family proteins. Moreover, we obtained the crystal structure of p204 HINab domain in complex with dsDNA and provided insights into the dsDNA binding mode. p204 HINab binds dsDNA mainly through α2 helix of HINa and HINb, and the linker between them, revealing a similar HIN:DNA binding mode. Both HINa and HINb are vital for HINab recognition of dsDNA, as confirmed by fluorescence polarization assays. Furthermore, a HINa dimerization interface was observed in structures of p204 HINa and HINab:dsDNA complex, which is involved in binding dsDNA. The linker between HINa and HINb reveals dynamic flexibility in solution and changes its direction at ∼90° angle in comparison with crystal structure of HINab:dsDNA complex. These structural information provide insights into the mechanism of DNA recognition by different HIN domains, and shed light on the unique roles of two HIN domains in activating the IFI16/p204 signaling pathway., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

This work builds upon the record-breaking speed and generous immediate release of new experimental three-dimensional structures of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins and complexes, which are crucial to downstream vaccine and drug development. We have surveyed those structures to catch the occasional errors that could be significant for those important uses and for which we were able to provide demonstrably higher-accuracy corrections. This process relied on new validation and correction methods such as CaBLAM and ISOLDE, which are not yet in routine use. We found such important and correctable problems in seven early SARS-CoV-2 structures. Two of the structures were soon superseded by new higher-resolution data, confirming our proposed changes. For the other five, we emailed the depositors a documented and illustrated report and encouraged them to make the model corrections themselves and use the new option at the worldwide Protein Data Bank for depositors to re-version their coordinates without changing the Protein Data Bank code. This quickly and easily makes the better-accuracy coordinates available to anyone who examines or downloads their structure, even before formal publication. The changes have involved sequence misalignments, incorrect RNA conformations near a bound inhibitor, incorrect metal ligands, and cis-trans or peptide flips that prevent good contact at interaction sites. These improvements have propagated into nearly all related structures done afterward. This process constitutes a new form of highly rigorous peer review, which is actually faster and more strict than standard publication review because it has access to coordinates and maps; journal peer review would also be strengthened by such access., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The spindle pole body (SPB) provides microtubule-organizing functions in yeast and duplicates exactly once per cell cycle. The first step in SPB duplication is the half-bridge to bridge conversion via the antiparallel dimerization of the centrin (Cdc31)-binding protein Sfi1 in anaphase. The bridge, which is anchored to the old SPB on the proximal end, exposes free Sfi1 N-termini (N-Sfi1) at its distal end. These free N-Sfi1 promote in G1 the assembly of the daughter SPB (dSPB) in a yet unclear manner. This study shows that N-Sfi1 including the first three Cdc31 binding sites interacts with the SPB components Spc29 and Spc42, triggering the assembly of the dSPB. Cdc31 binding to N-Sfi1 promotes Spc29 recruitment and is essential for satellite formation. Furthermore, phosphorylation of N-Sfi1 has an inhibitory effect and delays dSPB biogenesis until G1. Taking these data together, we provide an understanding of the initial steps in SPB assembly and describe a new function of Cdc31 in the recruitment of dSPB components., (© 2021 Rüthnick et al.)

The recent outbreak of coronavirus disease (COVID-19) in China caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to worldwide human infections and deaths. The nucleocapsid (N) protein of coronaviruses (CoVs) is a multifunctional RNA binding protein necessary for viral RNA replication and transcription. Therefore, it is a potential antiviral drug target, serving multiple critical functions during the viral life cycle. This study addresses the potential to repurpose antiviral compounds approved or in development for treating human CoV induced infections against SARS-CoV-2 N. For this purpose, we used the docking methodology to better understand the inhibitory mechanism of this protein with the existing 34 antiviral compounds. The results of this analysis indicate that rapamycin, saracatinib, camostat, trametinib, and nafamostat were the top hit compounds with binding energy (-11.87, -10.40, -9.85, -9.45, -9.35 kcal/mol, respectively). This analysis also showed that the most common residues that interact with the compounds are Phe66, Arg68, Gly69, Tyr123, Ile131, Trp132, Val133, and Ala134. Subsequently, protein-ligand complex stability was examined with molecular dynamics simulations for these five compounds, which showed the best binding affinity. According to the results of this study, the interaction between these compounds and crucial residues of the target protein were maintained. These results suggest that these residues are potential drug targeting sites for the SARS-CoV-2 N protein. This study information will contribute to the development of novel compounds for further in vitro and in vivo studies of SARS-CoV-2, as well as possible new drug repurposing strategies to treat COVID-19 disease., (© 2020 American Institute of Chemical Engineers.)

The pandemic of COVID-19 is still ongoing, and many studies on serum antibodies have been reported, however, there are few studies about asymptomatic and mild patients. In this study, we enrolled 44 COVID-19 patients with relatively mild disease and 48 pre-pandemic controls. We measured serum antibodies against extracellular domain, S1 domain, and receptor-binding domain of Spike and N protein, examined neutralization titers by authentic virus neutralization assay and newly-developed bead/cell-based Spike-ACE2 inhibition assay, and compared them with clinical features. Most of these antibodies, including neutralizing titers, were mutually correlated, and the production of antibodies were associated with low Ct values of PCR test, disease severity, symptoms especially pneumonia, lymphopenia, and serological test including CRP, LD, D-dimer, and procalcitonin. Notably, 87.5% of asymptomatic and 23.5% of mild patients did not have antibody against SARS-CoV-2. Our results revealed the inadequate acquisition of humoral immunity in patients with asymptomatic and mild COVID-19 patients., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Recently, SARS-CoV-2 has been identified as the causative factor of viral infection called COVID-19 that belongs to the zoonotic beta coronavirus family known to cause respiratory disorders or viral pneumonia, followed by an extensive attack on organs that express angiotensin-converting enzyme II (ACE2). Human transmission of this virus occurs via respiratory droplets from symptomatic and asymptomatic patients, which are released into the environment after sneezing or coughing. These droplets are capable of staying in the air as aerosols or surfaces and can be transmitted to persons through inhalation or contact with contaminated surfaces. Thus, there is an urgent need for advanced theranostic solutions to control the spread of COVID-19 infection. The development of such fit-for-purpose technologies hinges on a proper understanding of the transmission, incubation, and structural characteristics of the virus in the external environment and within the host. Hence, this article describes the development of an intrinsic model to describe the incubation characteristics of the virus under varying environmental factors. It also discusses on the evaluation of SARS-CoV-2 structural nucleocapsid protein properties via computational approaches to generate high-affinity binding probes for effective diagnosis and targeted treatment applications by specific targeting of viruses. In addition, this article provides useful insights on the transmission behavior of the virus and creates new opportunities for theranostics development., (© 2020 American Institute of Chemical Engineers.)

The T cell receptor (TCR) signaling pathway is an ensemble of numerous proteins that are crucial for an adequate immune response. Disruption of any protein involved in this pathway leads to severe immunodeficiency and unfavorable clinical outcomes. Here, we describe an infant with severe immunodeficiency who was found to have novel biallelic mutations in SLP76. SLP76 is a key protein involved in TCR signaling and in other hematopoietic pathways. Previous studies of this protein were performed using Jurkat-derived human leukemic T cell lines and SLP76-deficient mice. Our current study links this gene, for the first time, to a human immunodeficiency characterized by early-onset life-threatening infections, combined T and B cell immunodeficiency, severe neutrophil defects, and impaired platelet aggregation. Hereby, we characterized aspects of the patient's immune phenotype, modeled them with an SLP76-deficient Jurkat-derived T cell line, and rescued some consequences using ectopic expression of wild-type SLP76. Understanding human diseases due to SLP76 deficiency is helpful in explaining the mixed T cell and neutrophil defects, providing a guide for exploring human SLP76 biology., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Lev et al.)

The outbreak of a novel coronavirus associated with acute respiratory disease, called COVID-19, marked the introduction of the third spillover of an animal coronavirus (CoV) to humans in the last two decades. The genome analysis with various bioinformatics tools revealed that the causative pathogen (SARS-CoV-2) belongs to the subgenus Sarbecovirus of the genus Betacoronavirus, with highly similar genome as bat coronavirus and receptor-binding domain (RBD) of spike glycoprotein as Malayan pangolin coronavirus. Based on its genetic proximity, SARS-CoV-2 is likely to have originated from bat-derived CoV and transmitted to humans via an unknown intermediate mammalian host, probably Malayan pangolin. Further, spike protein S1/S2 cleavage site of SARS-CoV-2 has acquired polybasic furin cleavage site which is absent in bat and pangolin suggesting natural selection either in an animal host before zoonotic transfer or in humans following zoonotic transfer. In the current review, we recapitulate a preliminary opinion about the disease, origin and life cycle of SARS-CoV-2, roles of virus proteins in pathogenesis, commonalities, and differences between different corona viruses. Moreover, the crystal structures of SARS-CoV-2 proteins with unique characteristics differentiating it from other CoVs are discussed. Our review also provides comprehensive information on the molecular aspects of SARS-CoV-2 including secondary structures in the genome and protein-protein interactions which can be useful to understand the aggressive spread of the SARS-CoV-2. The mutations and the haplotypes reported in the SARS-CoV-2 genome are summarized to understand the virus evolution., (© 2021 Wiley-VCH GmbH.)

Developing optimal T-cell response assays to severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is critical for measuring the duration of immunity to this disease and assessing the efficacy of vaccine candidates. These assays need to target conserved regions of SARS-CoV-2 global variants and avoid cross-reactivity to seasonal human coronaviruses. To contribute to this effort, we employed an in silico immunoinformatics analysis pipeline to identify immunogenic peptides resulting from conserved and highly networked regions with topological importance from the SARS-CoV-2 nucleocapsid and spike proteins. A total of 57 highly networked T-cell epitopes that are conserved across geographic viral variants were identified from these viral proteins, with a binding potential to diverse HLA alleles and 80 to 100% global population coverage. Importantly, 18 of these T-cell epitope derived peptides had limited homology to seasonal human coronaviruses making them promising candidates for SARS-CoV-2-specific T-cell immunity assays. Moreover, two of the NC-derived peptides elicited effector/polyfunctional responses of CD8 + T cells derived from SARS-CoV-2 convalescent patients. IMPORTANCE The development of specific and validated immunologic tools is critical for understanding the level and duration of the cellular response induced by SARS-CoV-2 infection and/or vaccines against this novel coronavirus disease. To contribute to this effort, we employed an immunoinformatics analysis pipeline to define 57 SARS-CoV-2 immunogenic peptides within topologically important regions of the nucleocapsid (NC) and spike (S) proteins that will be effective for detecting cellular immune responses in 80 to 100% of the global population. Our immunoinformatics analysis revealed that 18 of these peptides had limited homology to circulating seasonal human coronaviruses and therefore are promising candidates for distinguishing SARS-CoV-2-specific immune responses from pre-existing coronavirus immunity. Importantly, CD8 + T cells derived from SARS-CoV-2 survivors exhibited polyfunctional effector responses to two novel NC-derived peptides identified as HLA-binders. These studies provide a proof of concept that our immunoinformatics analysis pipeline identifies novel immunogens which can elicit polyfunctional SARS-CoV-2-specific T-cell responses., (Copyright © 2021 Lee et al.)

SARS-CoV-2 is a highly contagious coronavirus causing the ongoing pandemic. Very recently its genomic RNA of ∼30 kb was decoded to be packaged with nucleocapsid (N) protein into phase separated condensates. Interestingly, viruses have no ability to generate ATP but host cells have very high ATP concentrations of 2-12 mM. A key question thus arises whether ATP modulates liquid-liquid phase separation (LLPS) of the N protein. Here we discovered that ATP not only biphasically modulates LLPS of the viral N protein as we previously found on human FUS and TDP-43, but also dissolves the droplets induced by oligonucleic acid. Residue-specific NMR characterization showed ATP specifically binds the RNA-binding domain (RBD) of the N protein with the average Kd of 3.3 ± 0.4 mM. The ATP-RBD complex structure was constructed by NMR-derived constraints, in which ATP occupies a pocket within the positive-charged surface utilized for binding nucleic acids. Our study suggests that ATP appears to be exploited by SARS-CoV-2 to promote its life cycle by facilitating the uncoating, localizing and packing of its genomic RNA. Therefore the interactions of ATP with the viral RNA and N protein might represent promising targets for design of drugs and vaccines to terminate the pandemic., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The phosphorylation of proteins modulates various functions of proteins and plays an important role in the regulation of cell signaling. In recent years, label-free quantitative (LFQ) phosphoproteomics has become a powerful tool to analyze the phosphorylation of proteins within complex samples. Despite the great progress, the studies of protein phosphorylation are still limited in throughput, robustness, and reproducibility, hampering analyses that involve multiple perturbations, such as those needed to follow the dynamics of phosphoproteomes. To address these challenges, we introduce here the LFQ phosphoproteomics workflow that is based on Fe-IMAC phosphopeptide enrichment followed by strong anion exchange (SAX) and porous graphitic carbon (PGC) fractionation strategies. We applied this workflow to analyze the whole-cell phosphoproteome of the fission yeast Schizosaccharomyces pombe . Using this strategy, we identified 8353 phosphosites from which 1274 were newly identified. This provides a significant addition to the S. pombe phosphoproteome. The results of our study highlight that combining of PGC and SAX fractionation strategies substantially increases the robustness and specificity of LFQ phosphoproteomics. Overall, the presented LFQ phosphoproteomics workflow opens the door for studies that would get better insight into the complexity of the protein kinase functions of the fission yeast S. pombe .

Monitoring acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic diversity and emerging mutations in this ongoing pandemic is crucial for understanding its evolution and assuring the performance of diagnostic tests, vaccines, and therapies against coronavirus disease (COVID-19). This study reports on the amino acid (aa) conservation degree and the global and regional temporal evolution by epidemiological week for each residue of the following four structural SARS-CoV-2 proteins: spike, envelope, membrane, and nucleocapsid. All, 105,276 worldwide SARS-CoV-2 complete and partial sequences from 117 countries available in the Global Initiative on Sharing All Influenza Data (GISAID) from 29 December 2019 to 12 September 2020 were downloaded and processed using an in-house bioinformatics tool. Despite the extremely high conservation of SARS-CoV-2 structural proteins (>99%), all presented aa changes, i.e., 142 aa changes in 65 of the 75 envelope aa, 291 aa changes in 165 of the 222 membrane aa, 890 aa changes in 359 of the 419 nucleocapsid aa, and 2671 changes in 1132 of the 1273 spike aa. Mutations evolution differed across geographic regions and epidemiological weeks (epiweeks). The most prevalent aa changes were D614G (81.5%) in the spike protein, followed by the R203K and G204R combination (37%) in the nucleocapsid protein. The presented data provide insight into the genetic variability of SARS-CoV-2 structural proteins during the pandemic and highlights local and worldwide emerging aa changes of interest for further SARS-CoV-2 structural and functional analysis.

Mononegavirales phosphoproteins (P) are essential co-factors of the viral polymerase by serving as a linchpin between the catalytic subunit and the ribonucleoprotein template. They have highly diverged, but their overall architecture is conserved. They are multidomain proteins, which all possess an oligomerization domain that separates N - and C -terminal domains. Large intrinsically disordered regions constitute their hallmark. Here, we exemplify their structural features and interaction potential, based on the Pneumoviridae P proteins. These P proteins are rather small, and their oligomerization domain is the only part with a defined 3D structure, owing to a quaternary arrangement. All other parts are either flexible or form short-lived secondary structure elements that transiently associate with the rest of the protein. Pneumoviridae P proteins interact with several viral and cellular proteins that are essential for viral transcription and replication. The combination of intrinsic disorder and tetrameric organization enables them to structurally adapt to different partners and to act as adaptor-like platforms to bring the latter close in space. Transient structures are stabilized in complex with protein partners. This class of proteins gives an insight into the structural versatility of non-globular intrinsically disordered protein domains.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally. Although measures to control SARS-CoV-2, namely, vaccination, medication, and chemical disinfectants are being investigated, there is an increase in the demand for auxiliary antiviral approaches using natural compounds. Here we have focused on hydroxytyrosol (HT)-rich aqueous olive pulp extract (HIDROX ® ) and evaluated its SARS-CoV-2-inactivating activity in vitro. We showed that the HIDROX solution exhibits time- and concentration-dependent SARS-CoV-2-inactivating activities, and that HIDROX has more potent virucidal activity than pure HT. The evaluation of the mechanism of action suggested that both HIDROX and HT induced structural changes in SARS-CoV-2, which changed the molecular weight of the spike proteins. Even though the spike protein is highly glycosylated, this change was induced regardless of the glycosylation status. In addition, HIDROX or HT treatment disrupted the viral genome. Moreover, the HIDROX-containing cream applied on film showed time- and concentration-dependent SARS-CoV-2-inactivating activities. Thus, the HIDROX-containing cream can be applied topically as an antiviral hand cream. Our findings suggest that HIDROX contributes to improving SARS-CoV-2 control measures.

Although severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) infection have emerged globally, findings related to ocular involvement and reported cases are quite limited. Immune reactions against viral infections are closely related to viral and host proteins sequence similarity. Molecular Mimicry has been described for many different viruses; sequence similarities of viral and human tissue proteins may trigger autoimmune reactions after viral infections due to similarities between viral and human structures. With this study, we aimed to investigate the protein sequence similarity of SARS CoV-2 with retinal proteins and retinal pigment epithelium (RPE) surface proteins. Retinal proteins involved in autoimmune retinopathy and retinal pigment epithelium surface transport proteins were analyzed in order to infer their structural similarity to surface glycoprotein (S), nucleocapsid phosphoprotein (N), membrane glycoprotein (M), envelope protein (E), ORF1ab polyprotein (orf1ab) proteins of SARS CoV-2. Protein similarity comparisons, 3D protein structure prediction, T cell epitopes-MHC binding prediction, B cell epitopes-MHC binding prediction and the evaluation of the antigenicity of peptides assessments were performed. The protein sequence analysis was made using the Pairwise Sequence Alignment and the LALIGN program. 3D protein structure estimates were made using Swiss Model with default settings and analyzed with TM-align web server. T-cell epitope identification was performed using the Immune Epitope Database and Analysis (IEDB) resource Tepitool. B cell epitopes based on sequence characteristics of the antigen was performed using amino acid scales and HMMs with the BepiPred 2.0 web server. The predicted peptides/epitopes in terms of antigenicity were examined using the default settings with the VaxiJen v2.0 server. Analyses showed that, there is a meaningful similarities between 6 retinal pigment epithelium surface transport proteins (MRP-4, MRP-5, RFC1, SNAT7, TAUT and MATE) and the SARS CoV-2 E protein. Immunoreactive epitopic sites of these proteins which are similar to protein E epitope can create an immune stimulation on T cytotoxic and T helper cells and 6 of these 9 epitopic sites are also vaxiJen. These result imply that autoimmune cross-reaction is likely between the studied RPE proteins and SARS CoV-2 E protein. The structure of SARS CoV-2, its proteins and immunologic reactions against these proteins remain largely unknown. Understanding the structure of SARS CoV-2 proteins and demonstration of similarity with human proteins are crucial to predict an autoimmune response associated with immunity against host proteins and its clinical manifestations as well as possible adverse effects of vaccination., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The emergence of the COVID-19 has caused public health problems worldwide and there is no effective pharmacological treatment for this disease. Research on 3D models of proteins and the search for active molecular sites are important tools to assist in the discovery of effective antiviral drugs to combat COVID-19. To address this problem, the 3D protein structures of SARS-CoV 2 were analyzed and submitted to cavities research, evaluation of their druggabillity and liganbility, and applied to molecular docking studies with potential ligand candidates actually assayed against COVID-19. Eight druggable potential cavity sites were determined in model structures' PDB code, 6W4B, 6VWW, 6W01, 6M3M, and 6VYO, and these are the good alternatives to be characterized as targets for antiviral compounds. The good cavity model of the protease 3D structure was used in molecular docking, and this allowed verifying the theoric interactions of this protein and lopinavir and ritonavir antiviral drugs. These results may assist in the use of 3D protein models in drug design studies aiming to develop drugs against the COVID-19 pandemic., (© 2020 Wiley-VCH GmbH.)

The SH2 domain-containing protein of 76 kDa, SLP76, is an important adaptor protein that coordinates a complex protein network downstream of T-cell receptors (TCR), ultimately regulating the immune response. Upon phosphorylation on Ser376, SLP76 interacts with 14-3-3 adaptor proteins, which leads to its proteolytic degradation. This provides a negative feedback mechanism by which TCR signalling can be controlled. To gain insight into the 14-3-3/SLP76 protein-protein interaction (PPI), we have determined a high-resolution crystal structure of a SLP76 synthetic peptide containing Ser376 with 14-3-3σ. We then characterized its binding to 14-3-3 proteins biophysically by means of fluorescence polarization and isothermal titration calorimetry. Furthermore, we generated two recombinant SLP76 protein constructs and characterized their binding to 14-3-3. Our work lays the foundation for drug design efforts aimed at targeting the 14-3-3/SLP76 interaction and, thereby, TCR signalling., (© 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Unprecedented by number of casualties and socio-economic burden occurring worldwide, the coronavirus disease 2019 (Covid-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the worst health crisis of this century. In order to develop adequate countermeasures against Covid-19, identification and structural characterization of suitable antiviral targets within the SARS-CoV-2 protein repertoire is urgently needed. The nucleocapsid phosphoprotein (N) is a multifunctional and highly immunogenic determinant of virulence and pathogenicity, whose main functions consist in oligomerizing and packaging the single-stranded RNA (ssRNA) viral genome. Here we report the structural and biophysical characterization of the SARS-CoV-2 N C-terminal domain (CTD), on which both N homo-oligomerization and ssRNA binding depend. Crystal structures solved at 1.44 Å and 1.36 Å resolution describe a rhombus-shape N CTD dimer, which stably exists in solution as validated by size-exclusion chromatography coupled to multi-angle light scattering and analytical ultracentrifugation. Differential scanning fluorimetry revealed moderate thermal stability and a tendency towards conformational change. Microscale thermophoresis demonstrated binding to a 7-bp SARS-CoV-2 genomic ssRNA fragment at micromolar affinity. Furthermore, a low-resolution preliminary model of the full-length SARS-CoV N in complex with ssRNA, obtained by cryo-electron microscopy, provides an initial understanding of self-associating and RNA binding functions exerted by the SARS-CoV-2 N., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Development of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n = 115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.

Antibody responses are important in the control of viral respiratory infection in the human host. What is not clear for SARS-CoV-2 is how rapidly this response occurs, or when antibodies with protective capability evolve. Hence, defining the events of SARS-CoV-2 seroconversion and the time frame for the development of antibodies with protective potential may help to explain the different clinical presentations of COVID-19. Furthermore, accurate descriptions of seroconversion are needed to inform the best use of serological assays for diagnostic testing and serosurveillance studies. Here, we describe the humoral responses in a cohort of hospitalised COVID-19 patients (n = 19) shortly following the onset of symptoms. Commercial and 'in-house' serological assays were used to measure IgG antibodies against different SARS-CoV-2 structural antigens-Spike (S) S1 sub-unit and Nucleocapsid protein (NP)-and to assess the potential for virus neutralisation mediated specifically by inhibition of binding between the viral attachment protein (S protein) and cognate receptor (ACE-2). Antibody response kinetics varied amongst the cohort, with patients seroconverting within 1 week, between 1-2 weeks, or after 2 weeks, following symptom onset. Anti-NP IgG responses were generally detected earlier, but reached maximum levels slower, than anti-S1 IgG responses. The earliest IgG antibodies produced by all patients included those that recognised the S protein receptor-binding domain (RBD) and were capable of inhibiting binding to ACE-2. These data revealed events and patterns of SARS-CoV-2 seroconversion that may be important predictors of the outcome of infection and guide the delivery of clinical services in the COVID-19 response., Competing Interests: The authors have declared that no competing interests exist.

This study aims to highlight SARS-COV-2 mutations which are associated with increased or decreased viral virulence. We utilize genetic data from all strains available from GISAID and countries' regional information, such as deaths and cases per million, as well as COVID-19-related public health austerity measure response times. Initial indications of selective advantage of specific mutations can be obtained from calculating their frequencies across viral strains. By applying modelling approaches, we provide additional information that is not evident from standard statistics or mutation frequencies alone. We therefore, propose a more precise way of selecting informative mutations. We highlight two interesting mutations found in genes N (P13L) and ORF3a (Q57H). The former appears to be significantly associated with decreased deaths and cases per million according to our models, while the latter shows an opposing association with decreased deaths and increased cases per million. Moreover, protein structure prediction tools show that the mutations infer conformational changes to the protein that significantly alter its structure when compared to the reference protein., Competing Interests: The authors have declared that no competing interests exist.

Collection of nasopharyngeal samples using swabs followed by the transfer of the virus into a solution and an RNA extraction step to perform reverse transcription polymerase chain reaction (PCR) is the primary method currently used for the diagnosis of COVID-19. However, the need for several reagents and steps and the high cost of PCR hinder its worldwide implementation to contain the outbreak. Here, we report a cotton-tipped electrochemical immunosensor for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus antigen. Unlike the reported approaches, we integrated the sample collection and detection tools into a single platform by coating screen-printed electrodes with absorbing cotton padding. The immunosensor was fabricated by immobilizing the virus nucleocapsid (N) protein on carbon nanofiber-modified screen-printed electrodes which were functionalized by diazonium electrografting. The detection of the virus antigen was achieved via swabbing followed by competitive assay using a fixed amount of N protein antibody in the solution. A square wave voltammetric technique was used for the detection. The limit of detection for our electrochemical biosensor was 0.8 pg/mL for SARS-CoV-2, indicating very good sensitivity for the sensor. The biosensor did not show significant cross-reactivity with other virus antigens such as influenza A and HCoV, indicating high selectivity of the method. Moreover, the biosensor was successfully applied for the detection of the virus antigen in spiked nasal samples showing excellent recovery percentages. Thus, our electrochemical immunosensor is a promising diagnostic tool for the direct rapid detection of the COVID-19 virus that requires no sample transfer or pretreatment.

Na + /H + exchange factor-1 (NHERF1), a multidomain PDZ scaffolding phosphoprotein, is required for the type II sodium-dependent phosphate cotransporter (NPT2A)-mediated renal phosphate absorption. Both PDZ1 and PDZ2 domains are involved in NPT2A-dependent phosphate uptake. Though harboring identical core-binding motifs, PDZ1 and PDZ2 play entirely different roles in hormone-regulated phosphate transport. PDZ1 is required for the interaction with the C-terminal PDZ-binding sequence of NPT2A (-TRL). Remarkably, phosphocycling at Ser 290 distant from PDZ1, the penultimate step for both parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) regulation, controls the association between NHERF1 and NPT2A. PDZ2 interacts with the C-terminal PDZ-recognition motif (-TRL) of G Protein-coupled Receptor Kinase 6A (GRK6A), and that promotes phosphorylation of Ser 290 . The compelling biological puzzle is how PDZ1 and PDZ2 with identical GYGF core-binding motifs specifically recognize distinct binding partners. Binding determinants distinct from the canonical PDZ-ligand interactions and located "outside the box" explain PDZ domain specificity. Phosphorylation of NHERF1 by diverse kinases and associated conformational changes in NHERF1 add more complexity to PDZ-binding diversity.

The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80-90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that the N protein's central disordered domain drives phase separation with RNA, and that phosphorylation of an adjacent serine/arginine rich region modulates the physical properties of the resulting condensates. In cells, N forms condensates that recruit the stress granule protein G3BP1, highlighting a potential role for N in G3BP1 sequestration and stress granule inhibition. The SARS-CoV-2 membrane (M) protein independently induces N protein phase separation, and three-component mixtures of N + M + RNA form condensates with mutually exclusive compartments containing N + M or N + RNA, including annular structures in which the M protein coats the outside of an N + RNA condensate. These findings support a model in which phase separation of the SARS-CoV-2 N protein contributes both to suppression of the G3BP1-dependent host immune response and to packaging genomic RNA during virion assembly.

Background: The latest coronavirus SARS-CoV-2, discovered in China and rapidly spread Worldwide. COVID-19 affected millions of people and killed hundreds of thousands worldwide. There are many ongoing studies investigating drug(s) suitable for preventing and/or treating this pandemic; however, there are no specific drugs or vaccines available to treat or prevent SARS-CoV-2 as of today., Methods: Fifty-eight fragrance materials, which are classified as allergen fragrance molecules, were selected and used in this study. Docking simulations were carried out using four functional proteins; the Covid19 Main Protase (MPro), Receptor binding domain (RBD) of spike protein, Nucleocapsid, and host Bromodomain protein (BRD2), as target macromolecules. Three different software, AutoDock, AutoDock Vina (Vina), and Molegro Virtual Docker (MVD), running a total of four different docking protocol with optimized energy functions were used. Results were compared with the five molecules reported in the literature as potential drugs against COVID-19. Virtual screening was carried out using Vina, molecules satisfying our cut-off (- 6.5 kcal/mol) binding affinity was confirmed by MVD. Selected molecules were analyzed using the flexible docking protocol of Vina and AutoDock default settings., Results: Ten out of 58 allergen fragrance molecules were selected for further docking studies. MPro and BRD2 are potential targets for the tested allergen fragrance molecules, while RBD and Nucleocapsid showed weak binding energies. According to AutoDock results, three molecules, Benzyl Cinnamate, Dihydroambrettolide, and Galaxolide, had good binding affinities to BRD2. While Dihydroambrettolide and Galaxolide showed the potential to bind to MPro, Sclareol and Vertofix had the best calculated binding affinities to this target. When the flexible docking results analyzed, all the molecules tested had better calculated binding affinities as expected. Benzyl Benzoate and Benzyl Salicylate showed good binding affinities to BRD2. In the case of MPro, Sclareol had the lowest binding affinity among all the tested allergen fragrance molecules., Conclusion: Allergen fragrance molecules are readily available, cost-efficient, and shown to be safe for human use. Results showed that several of these molecules had comparable binding affinities as the potential drug molecules reported in the literature to target proteins. Thus, these allergen molecules at correct doses could have significant health benefits.

As an important post-translational modification (PTM), protein phosphorylation is involved in the regulation of almost all of biological processes in eukaryotes. Due to the rapid progress in mass spectrometry-based phosphoproteomics, a large number of phosphorylation sites (p-sites) have been characterized but remain to be curated. Here, we briefly summarized the current progresses in the development of data resources for the collection, curation, integration and annotation of p-sites in eukaryotic proteins. Also, we designed the eukaryotic phosphorylation site database (EPSD), which contained 1 616 804 experimentally identified p-sites in 209 326 phosphoproteins from 68 eukaryotic species. In EPSD, we not only collected 1 451 629 newly identified p-sites from high-throughput (HTP) phosphoproteomic studies, but also integrated known p-sites from 13 additional databases. Moreover, we carefully annotated the phosphoproteins and p-sites of eight model organisms by integrating the knowledge from 100 additional resources that covered 15 aspects, including phosphorylation regulator, genetic variation and mutation, functional annotation, structural annotation, physicochemical property, functional domain, disease-associated information, protein-protein interaction, drug-target relation, orthologous information, biological pathway, transcriptional regulator, mRNA expression, protein expression/proteomics and subcellular localization. We anticipate that the EPSD can serve as a useful resource for further analysis of eukaryotic phosphorylation. With a data volume of 14.1 GB, EPSD is free for all users at http://epsd.biocuckoo.cn/., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Protein phosphorylation is one of the most studied post-translational modifications (PTMs). Despite the remarkable advances in phosphoproteomics, a chemically less-stable subset of the phosphosites, which we call the crypto-phosphoproteome, has remained underexplored due to technological challenges. In this Viewpoint, we briefly summarize the current understanding of these elusive protein phosphorylations and identify the missing pieces for future studies., (© 2020 Wiley-VCH GmbH.)

Mutations of Odontogenesis-Associated Phosphoprotein (ODAPH, OMIM *614829) cause autosomal recessive amelogenesis imperfecta, however, the function of ODAPH during amelogenesis is unknown. Here we characterized normal Odaph expression by in situ hybridization, generated Odaph truncation mice using CRISPR/Cas9 to replace the TGC codon encoding Cys41 into a TGA translation termination codon, and characterized and compared molar and incisor tooth formation in Odaph +/+ , Odaph +/C41* , and Odaph C41*/C41* mice. We also searched genomes to determine when Odaph first appeared phylogenetically. We determined that tooth development in Odaph +/+ and Odaph +/C41* mice was indistinguishable in all respects, so the condition in mice is inherited in a recessive pattern, as it is in humans. Odaph is specifically expressed by ameloblasts starting with the onset of post-secretory transition and continues until mid-maturation. Based upon histological and ultrastructural analyses, we determined that the secretory stage of amelogenesis is not affected in Odaph C41*/C41* mice. The enamel layer achieves a normal shape and contour, normal thickness, and normal rod decussation. The fundamental problem in Odaph C41*/C41* mice starts during post-secretory transition, which fails to generate maturation stage ameloblasts. At the onset of what should be enamel maturation, a cyst forms that separates flattened ameloblasts from the enamel surface. The maturation stage fails completely.

To date, the effects of specific modification types and sites on protein lifetime have not been systematically illustrated. Here, we describe a proteomic method, DeltaSILAC, to quantitatively assess the impact of site-specific phosphorylation on the turnover of thousands of proteins in live cells. Based on the accurate and reproducible mass spectrometry-based method, a pulse labeling approach using stable isotope-labeled amino acids in cells (pSILAC), phosphoproteomics, and a unique peptide-level matching strategy, our DeltaSILAC profiling revealed a global, unexpected delaying effect of many phosphosites on protein turnover. We further found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness, enriched in Cyclin-dependent kinase substrates, and evolutionarily conserved, whereas the glutamic acids surrounding phosphosites significantly delay protein turnover. Our method represents a generalizable approach and provides a rich resource for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Recent advances in mass spectrometry (MS)-based quantitative proteomics now allow the identification and quantification of deep proteomes and post-translational modifications (PTMs) in relatively short times. Therefore, in the last few years, this technology has proven successful in the circadian field to characterize temporal oscillations of the proteome and more recently PTMs in cellular systems and in tissues. In this chapter, we describe a robust and simple protocol, based on the EasyPhos workflow, to enable preparation of large number of proteomes and phosphoproteomes from mouse tissues for MS-based quantitative analysis. We additionally discuss computational methods to analyze proteome and phosphoproteome time series to determine circadian oscillations.

Emerging data indicate that SARS-CoV-2-specific CD8 + T cells targeting different viral proteins are detectable in up to 70% of convalescent individuals 1-5 . However, very little information is currently available about the abundance, phenotype, functional capacity and fate of pre-existing and induced SARS-CoV-2-specific CD8 + T cell responses during the natural course of SARS-CoV-2 infection. Here, we define a set of optimal and dominant SARS-CoV-2-specific CD8 + T cell epitopes. We also perform a high-resolution ex vivo analysis of pre-existing and induced SARS-CoV-2-specific CD8 + T cells, applying peptide-loaded major histocompatibility complex class I (pMHCI) tetramer technology. We observe rapid induction, prolonged contraction and emergence of heterogeneous and functionally competent cross-reactive and induced memory CD8 + T cell responses in cross-sectionally analyzed individuals with mild disease following SARS-CoV-2 infection and three individuals longitudinally assessed for their T cells pre- and post-SARS-CoV-2 infection. SARS-CoV-2-specific memory CD8 + T cells exhibited functional characteristics comparable to influenza-specific CD8 + T cells and were detectable in SARS-CoV-2 convalescent individuals who were seronegative for anti-SARS-CoV-2 antibodies targeting spike (S) and nucleoprotein (N). These results define cross-reactive and induced SARS-CoV-2-specific CD8 + T cell responses as potentially important determinants of immune protection in mild SARS-CoV-2 infection.

In pancreatic β-cells, the expression of the splicing factor SRSF6 is regulated by GLIS3, a transcription factor encoded by a diabetes susceptibility gene. SRSF6 down-regulation promotes β-cell demise through splicing dysregulation of central genes for β-cells function and survival, but how RNAs are targeted by SRSF6 remains poorly understood. Here, we define the SRSF6 binding landscape in the human pancreatic β-cell line EndoC-βH1 by integrating individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) under basal conditions with RNA sequencing after SRSF6 knockdown. We detect thousands of SRSF6 bindings sites in coding sequences. Motif analyses suggest that SRSF6 specifically recognizes a purine-rich consensus motif consisting of GAA triplets and that the number of contiguous GAA triplets correlates with increasing binding site strength. The SRSF6 positioning determines the splicing fate. In line with its role in β-cell function, we identify SRSF6 binding sites on regulated exons in several diabetes susceptibility genes. In a proof-of-principle, the splicing of the susceptibility gene LMO7 is modulated by antisense oligonucleotides. Our present study unveils the splicing regulatory landscape of SRSF6 in immortalized human pancreatic β-cells., (© 2020 Alvelos et al.)

Tremendous effort has been given to the development of diagnostic tests, preventive vaccines, and therapeutic medicines for coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much of this development has been based on the reference genome collected on January 5, 2020. Based on the genotyping of 15 140 genome samples collected up to June 1, 2020, we report that SARS-CoV-2 has undergone 8309 single mutations which can be clustered into six subtypes. We introduce mutation ratio and mutation h -index to characterize the protein conservativeness and unveil that SARS-CoV-2 envelope protein, main protease, and endoribonuclease protein are relatively conservative, while SARS-CoV-2 nucleocapsid protein, spike protein, and papain-like protease are relatively nonconservative. In particular, we have identified mutations on 40% of nucleotides in the nucleocapsid gene in the population level, signaling potential impacts on the ongoing development of COVID-19 diagnosis, vaccines, and antibody and small-molecular drugs.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene, encoding for a chloride ion channel. Membrane expression of CFTR is negatively regulated by CFTR-associated ligand (CAL). We previously showed that inhibition of the CFTR/CAL interaction with a cell-permeable peptide improves the function of rescued F508del-CFTR. In this study, optimization of the peptidyl inhibitor yielded PGD97, which exhibits a K D value of 6 nM for the CAL PDZ domain, ≥ 130-fold selectivity over closely related PDZ domains, and a serum t 1/2 of >24 h. In patient-derived F508del homozygous cells, PGD97 (100 nM) increased short-circuit currents by ∼3-fold and further potentiated the therapeutic effects of small-molecule correctors (e.g., VX-661) by ∼2-fold (with an EC 50 of ∼10 nM). Our results suggest that PGD97 may be used as a novel treatment for CF, either as a single agent or in combination with small-molecule correctors/potentiators.

YtvA from Bacillus subtilis is a sensor protein that responds to blue light stress and regulates the activity of transcription factor σ B . It is composed of the N-terminal LOV (light-oxygen-voltage) domain, the C-terminal STAS (sulfate transporter and anti-sigma factor antagonist) domain, and a linker region connecting them. In this study, the photoreaction and kinetics of full-length YtvA and the intermolecular interaction with a downstream protein, RsbRA, were revealed by the transient grating method. Although N-YLOV-linker, which is composed of the LOV domain of YtvA with helices A'α and Jα, exhibits a diffusion change due to the rotational motion of the helices, the YtvA dimer does not show the diffusion change. This result suggests that the STAS domain inhibits the rotational movement of helices A'α and Jα. We found that the YtvA dimer formed a heterotetramer with the RsbRA dimer probably via the interaction between the STAS domains, and we showed the diffusion change upon blue light illumination with a time constant faster than 70 μs. This result suggests a conformational change of the STAS domains; i.e., the interface between the STAS domains of the proteins changes to enhance the friction with water by the rotation structural change of helices A'α and Jα of YtvA.

The nucleocapsid (N) protein of coronaviruses serves two major functions: compaction of the RNA genome in the virion and regulation of viral gene transcription. It is not clear how the N protein mediates such distinct functions. The N protein contains two RNA-binding domains surrounded by regions of intrinsic disorder. Phosphorylation of the central disordered region promotes the protein's transcriptional function, but the underlying mechanism is not known. Here, we show that the N protein of SARS-CoV-2, together with viral RNA, forms biomolecular condensates. Unmodified N protein forms partially ordered gel-like condensates and discrete 15-nm particles based on multivalent RNA-protein and protein-protein interactions. Phosphorylation reduces these interactions, generating a more liquid-like droplet. We propose that distinct oligomeric states support the two functions of the N protein: unmodified protein forms a structured oligomer that is suited for nucleocapsid assembly, and phosphorylated protein forms a liquid-like compartment for viral genome processing., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Tightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and assemble within viral factories, dynamic compartments formed within the host cells associated with human stress granules. Here, we test the possibility that the multivalent RNA-binding nucleocapsid protein (N) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) condenses with RNA via liquid-liquid phase separation (LLPS) and that N protein can be recruited in phase-separated forms of human RNA-binding proteins associated with SG formation. Robust LLPS with RNA requires two intrinsically disordered regions (IDRs), the N-terminal IDR and central-linker IDR, as well as the folded C-terminal oligomerization domain, while the folded N-terminal domain and the C-terminal IDR are not required. N protein phase separation is induced by addition of non-specific RNA. In addition, N partitions in vitro into phase-separated forms of full-length human hnRNPs (TDP-43, FUS, hnRNPA2) and their low-complexity domains (LCs). These results provide a potential mechanism for the role of N in SARS-CoV-2 viral genome packing and in host-protein co-opting necessary for viral replication and infectivity., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)