Virus-like Particles Identify an HIV V1V2 Apex-Binding Neutralizing Antibody that Lacks a Protruding Loop.

Most HIV-1-specific neutralizing antibodies isolated to date exhibit unusual characteristics that complicate their elicitation. Neutralizing antibodies that target the V1V2 apex of the HIV-1 envelope (Env) trimer feature unusually long protruding loops, which enable them to penetrate the HIV-1 glycan shield. As antibodies with loops of requisite length are created through uncommon recombination events, an alternative mode of apex binding has been sought. Here, we isolated a lineage of Env apex-directed neutralizing antibodies, N90-VRC38.01-11, by using virus-like particles and conformationally stabilized Env trimers as B cell probes. A crystal structure of N90-VRC38.01 with a scaffolded V1V2 revealed a binding mode involving side-chain-to-side-chain interactions that reduced the distance the antibody loop must traverse the glycan shield, thereby facilitating V1V2 binding via a non-protruding loop. The N90-VRC38 lineage thus identifies a solution for V1V2-apex binding that provides a more conventional B cell pathway for vaccine design.

Antibacterial photosensitization through activation of coproporphyrinogen oxidase.

Gram-positive bacteria cause the majority of skin and soft tissue infections (SSTIs), resulting in the most common reason for clinic visits in the United States. Recently, it was discovered that Gram-positive pathogens use a unique heme biosynthesis pathway, which implicates this pathway as a target for development of antibacterial therapies. We report here the identification of a small-molecule activator of coproporphyrinogen oxidase (CgoX) from Gram-positive bacteria, an enzyme essential for heme biosynthesis. Activation of CgoX induces accumulation of coproporphyrin III and leads to photosensitization of Gram-positive pathogens. In combination with light, CgoX activation reduces bacterial burden in murine models of SSTI. Thus, small-molecule activation of CgoX represents an effective strategy for the development of light-based antimicrobial therapies.

Genome-wide identification and analysis of Nrf2 binding sites - Antioxidant response elements in zebrafish.

In the post-genomic era, deciphering the Nrf2 binding sites - antioxidant response elements (AREs) is an essential task that underlies and governs the Keap1-Nrf2-ARE pathway - a cell survival response pathway to environmental stresses in the vertebrate model system. AREs regulate the transcription of a repertoire of phase II detoxifying and/or oxidative-stress responsive genes, offering protection against toxic chemicals, carcinogens, and xenobiotics. In order to identify and analyze AREs in zebrafish, a pattern search algorithm was developed to identify AREs and computational tools available online were utilized to analyze the identified AREs in zebrafish. This study identified the AREs within 30 kb upstream from the transcription start site of antioxidant genes and mitochondrial genes. We report for the first time the AREs of all the known protein coding genes in the zebrafish genome. Western blotting, RT2 profiler array PCR, and qRT-PCR were performed to test whether AREs influence the Nrf2 target genes expression in the zebrafish larvae using sulforaphane. This study reveals unique AREs that have not been previously reported in the cytoprotective genes. Nine TGAG/CNNNTC and six TGAG/CNNNGC AREs were observed significantly. Our findings suggest that AREs drive the dynamic transcriptional events of Nrf2 target genes in the zebrafish larvae on exposure to sulforaphane. The identified abundant putative AREs will define the Keap1-Nrf2-ARE network and elucidate the precise regulation of Nrf2-ARE pathway in not only diseases but also in embryonic development, inflammation, and aerobic respiration. Our results help to understand the dynamic complexity of the Nrf2-ARE system in zebrafish.

Identification of compounds that inhibit the binding of Keap1a/Keap1b Kelch DGR domain with Nrf2 ETGE/DLG motifs in zebrafish.

The Keap1-Nrf2-ARE system serves as a premier defence mechanism to curb oxidative stress, which remains as one of the major causes of ageing and pathogenesis in various diseases. Nrf2 is the principal master regulator of the cellular defence system, and its activation remains the prospective therapeutic approach against chronic diseases. One of the recent strategies is to disrupt Keap1-Nrf2 protein-protein interaction (PPI) that alters the docking of Keap1 with Nrf2 by compounds occupying a position in the Keap1 blocking the interface with Nrf2. In this study, we made an attempt to identify the compounds with anticancer, antioxidant and anti-inflammatory properties to disrupt Keap1a/b-Nrf2 PPI through in silico molecular docking in zebrafish. The phylogenetic analysis of Keap1 proteins revealed the existence of orthologous Keap1-Nrf2-ARE system in lower vertebrates that includes zebrafish. The DGR domains of zebrafish Keap1a and Keap1b were modelled with Modeller 9.19 using Keap1 of Mus musculus (PDB ID:5CGJ) as template. Based on the docking calculations, top hit compounds were identified to disrupt both Keap1a and Keap1b interaction with Nrf2 which include quercetin 3,4'-diglucoside, flavin adenine dinucleotide disodium salt hydrate, salvianolic acid A, tunicamycin and esculin. The LC50 of esculin in 3 dpf zebrafish larvae is 5 mmol/L, and the qRT-PCR results showed that esculin significantly increased the transcription of Nrf2 target genes-Gstpi, Nqo1, Hmox1a and Prdx1 in 3 dpf zebrafish larvae. These potential hits could serve as safer Nrf2 activators due to their non-covalent disruption of Keap1-Nrf2 PPI and be developed into efficacious preventive/therapeutic agents for various diseases.

Antioxidant response elements: Discovery, classes, regulation and potential applications.

Exposure to antioxidants and xenobiotics triggers the expression of a myriad of genes encoding antioxidant proteins, detoxifying enzymes, and xenobiotic transporters to offer protection against oxidative stress. This articulated universal mechanism is regulated through the cis-acting elements in an array of Nrf2 target genes called antioxidant response elements (AREs), which play a critical role in redox homeostasis. Though the Keap1/Nrf2/ARE system involves many players, AREs hold the key in transcriptional regulation of cytoprotective genes. ARE-mediated reporter constructs have been widely used, including xenobiotics profiling and Nrf2 activator screening. The complexity of AREs is brought by the presence of other regulatory elements within the AREs. The diversity in the ARE sequences not only bring regulatory selectivity of diverse transcription factors, but also confer functional complexity in the Keap1/Nrf2/ARE pathway. The different transcription factors either homodimerize or heterodimerize to bind the AREs. Depending on the nature of partners, they may activate or suppress the transcription. Attention is required for deeper mechanistic understanding of ARE-mediated gene regulation. The computational methods of identification and analysis of AREs are still in their infancy. Investigations are required to know whether epigenetics mechanism plays a role in the regulation of genes mediated through AREs. The polymorphisms in the AREs leading to oxidative stress related diseases are warranted. A thorough understanding of AREs will pave the way for the development of therapeutic agents against cancer, neurodegenerative, cardiovascular, metabolic and other diseases with oxidative stress.

Structure based approach for understanding organism specific recognition of protein-RNA complexes.

BACKGROUND: Protein-RNA interactions perform diverse functions within the cell. Understanding the recognition mechanism of protein-RNA complexes has been a challenging task in molecular and computational biology. In earlier works, the recognition mechanisms have been studied for a specific complex or using a set of non-redundant complexes. In this work, we have constructed 18 sets of same protein-RNA complexes belonging to different organisms from Protein Data Bank (PDB). The similarities and differences in each set of complexes have been revealed in terms of various sequence and structure based features such as root mean square deviation, sequence homology, propensity of binding site residues, variance, conservation at binding sites, binding segments, binding motifs of amino acid residues and nucleotides, preferred amino acid-nucleotide pairs and influence of neighboring residues for binding. RESULTS: We found that the proteins of mesophilic organisms have more number of binding sites than thermophiles and the binding propensities of amino acid residues are distinct in E. coli, H. sapiens, S. cerevisiae, thermophiles and archaea. Proteins prefer to bind with RNA using a single residue segment in all the organisms while RNA prefers to use a stretch of up to six nucleotides for binding with proteins. We have developed amino acid residue-nucleotide pair potentials for different organisms, which could be used for predicting the binding specificity. Further, molecular dynamics simulation studies on aspartyl tRNA synthetase complexed with aspartyl tRNA showed specific modes of recognition in E. coli, T. thermophilus and S. cerevisiae. CONCLUSION: Based on structural analysis and molecular dynamics simulations we suggest that the mode of recognition depends on the type of the organism in a protein-RNA complex.

Do we see what we should see? Describing non-covalent interactions in protein structures including precision.

The power of X-ray crystal structure analysis as a technique is to 'see where the atoms are'. The results are extensively used by a wide variety of research communities. However, this 'seeing where the atoms are' can give a false sense of security unless the precision of the placement of the atoms has been taken into account. Indeed, the presentation of bond distances and angles to a false precision (i.e. to too many decimal places) is commonplace. This article has three themes. Firstly, a basis for a proper representation of protein crystal structure results is detailed and demonstrated with respect to analyses of Protein Data Bank entries. The basis for establishing the precision of placement of each atom in a protein crystal structure is non-trivial. Secondly, a knowledge base harnessing such a descriptor of precision is presented. It is applied here to the case of salt bridges, i.e. ion pairs, in protein structures; this is the most fundamental place to start with such structure-precision representations since salt bridges are one of the tenets of protein structure stability. Ion pairs also play a central role in protein oligomerization, molecular recognition of ligands and substrates, allosteric regulation, domain motion and α-helix capping. A new knowledge base, SBPS (Salt Bridges in Protein Structures), takes these structural precisions into account and is the first of its kind. The third theme of the article is to indicate natural extensions of the need for such a description of precision, such as those involving metalloproteins and the determination of the protonation states of ionizable amino acids. Overall, it is also noted that this work and these examples are also relevant to protein three-dimensional structure molecular graphics software.

Single-Cell Profiling of the Antigen-Specific Response to BNT162b2 SARS-CoV-2 RNA Vaccine

RNA-based vaccines against SARS-CoV-2 are critical to limiting COVID-19 severity and spread. Cellular mechanisms driving antigen-specific responses to these vaccines, however, remain uncertain. We used single-cell technologies to identify and characterized antigen-specific cells and antibody responses to the RNA vaccine BNT162b2 in longitudinal samples from a cohort of healthy donors. Mass cytometry and machine learning pinpointed a novel expanding, population of antigen-specific non-canonical memory CD4+ and CD8+ T cells. B cell sequencing suggested progression from IgM, with apparent cross-reactivity to endemic coronaviruses, to SARS-CoV-2-specific IgA and IgG memory B cells and plasmablasts. Responding lymphocyte populations correlated with eventual SARS-CoV-2 IgG and a donor lacking these cell populations failed to sustain SARS-CoV-2-specific antibodies and experienced breakthrough infection. These integrated proteomic and genomic platforms reveal an antigen-specific cellular basis of RNA vaccine-based immunity. ONE SENTENCE SUMMARYSingle-cell profiling reveals the cellular basis of the antigen-specific response to the BNT162b2 SARS-CoV-2 RNA vaccine.

Efficient discovery of potently neutralizing SARS-CoV-2 antibodies using LIBRA-seq with ligand blocking

SARS-CoV-2 therapeutic antibody discovery efforts have met with notable success but have been associated with a generally inefficient process, requiring the production and characterization of exceptionally large numbers of candidates for the identification of a small set of leads. Here, we show that incorporating antibody-ligand blocking as part of LIBRA-seq, the high-throughput sequencing platform for antibody discovery, results in efficient identification of ultra-potent neutralizing antibodies against SARS-CoV-2. LIBRA-seq with ligand blocking is a general platform for functional antibody discovery targeting the disruption of antigen-ligand interactions.

Potent neutralization of SARS-CoV-2 variants of concern by an antibody with a unique genetic signature and structural mode of spike recognition

The emergence of novel SARS-CoV-2 lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of COVID-19. Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the LIBRA-seq technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 showed potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryo-EM structure of 54042-4 in complex with the SARS-CoV-2 spike revealed an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses unique genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings motivate 54042-4 as a lead candidate for clinical development to counteract current and future SARS-CoV-2 VOCs.