Structural basis of Irgb6 inactivation by Toxoplasma gondii through the phosphorylation of switch I.

Irgb6 is a priming immune-related GTPase (IRG) that counteracts Toxoplasma gondii. It is known to be recruited to the low virulent type II T. gondii parasitophorous vacuole (PV), initiating cell-autonomous immunity. However, the molecular mechanism by which immunity-related GTPases become inactivated after the parasite infection remains obscure. Here, we found that Thr95 of Irgb6 is prominently phosphorylated in response to low virulent type II T. gondii infection. We observed that a phosphomimetic T95D mutation in Irgb6 impaired its localization to the PV and exhibited reduced GTPase activity in vitro. Structural analysis unveiled an atypical conformation of nucleotide-free Irgb6-T95D, resulting from a conformational change in the G-domain that allosterically modified the PV membrane-binding interface. In silico docking corroborated the disruption of the physiological membrane binding site. These findings provide novel insights into a T. gondii-induced allosteric inactivation mechanism of Irgb6.

Regulation of lymphoid-myeloid lineage bias through regnase-1/3-mediated control of Nfkbiz.

ABSTRACT: Regulation of lineage biases in hematopoietic stem and progenitor cells (HSPCs) is pivotal for balanced hematopoietic output. However, little is known about the mechanism behind lineage choice in HSPCs. Here, we show that messenger RNA (mRNA) decay factors regnase-1 (Reg1; Zc3h12a) and regnase-3 (Reg3; Zc3h12c) are essential for determining lymphoid fate and restricting myeloid differentiation in HSPCs. Loss of Reg1 and Reg3 resulted in severe impairment of lymphopoiesis and a mild increase in myelopoiesis in the bone marrow. Single-cell RNA sequencing analysis revealed that Reg1 and Reg3 regulate lineage directions in HSPCs via the control of a set of myeloid-related genes. Reg1- and Reg3-mediated control of mRNA encoding Nfkbiz, a transcriptional and epigenetic regulator, was essential for balancing lymphoid/myeloid lineage output in HSPCs in vivo. Furthermore, single-cell assay for transposase-accessible chromatin sequencing analysis revealed that Reg1 and Reg3 control the epigenetic landscape on myeloid-related gene loci in early stage HSPCs via Nfkbiz. Consistently, an antisense oligonucleotide designed to inhibit Reg1- and Reg3-mediated Nfkbiz mRNA degradation primed hematopoietic stem cells toward myeloid lineages by enhancing Nfkbiz expression. Collectively, the collaboration between posttranscriptional control and chromatin remodeling by the Reg1/Reg3-Nfkbiz axis governs HSPC lineage biases, ultimately dictating the fate of lymphoid vs myeloid differentiation.

Enhancement of SARS-CoV-2 Infection via Crosslinking of Adjacent Spike Proteins by N-Terminal Domain-Targeting Antibodies.

The entry of SARS-CoV-2 into host cells is mediated by the interaction between the spike receptor-binding domain (RBD) and host angiotensin-converting enzyme 2 (ACE2). Certain human antibodies, which target the spike N-terminal domain (NTD) at a distant epitope from the host cell binding surface, have been found to augment ACE2 binding and enhance SARS-CoV-2 infection. Notably, these antibodies exert their effect independently of the antibody fragment crystallizable (Fc) region, distinguishing their mode of action from previously described antibody-dependent infection-enhancing (ADE) mechanisms. Building upon previous hypotheses and experimental evidence, we propose that these NTD-targeting infection-enhancing antibodies (NIEAs) achieve their effect through the crosslinking of neighboring spike proteins. In this study, we present refined structural models of NIEA fragment antigen-binding region (Fab)-NTD complexes, supported by molecular dynamics simulations and hydrogen-deuterium exchange mass spectrometry (HDX-MS). Furthermore, we provide direct evidence confirming the crosslinking of spike NTDs by NIEAs. Collectively, our findings advance our understanding of the molecular mechanisms underlying NIEAs and their impact on SARS-CoV-2 infection.

Deciphering the antigen specificities of antibodies by clustering their complementarity determining region sequences.

IMPORTANCE: Determining antigen and epitope specificity is an essential step in the discovery of therapeutic antibodies as well as in the analysis adaptive immune responses to disease or vaccination. Despite extensive efforts, deciphering antigen specificity solely from BCR amino acid sequence remains a challenging task, requiring a combination of experimental and computational approaches. Here, we describe and experimentally validate a simple and straightforward approach for grouping antibodies that share antigen and epitope specificities based on their CDR sequence similarity. This approach allows us to identify the specificities of a large number of antibodies whose antigen targets are unknown, using a small fraction of antibodies with well-annotated binding specificities.

Circadian ribosome profiling reveals a role for the Period2 upstream open reading frame in sleep.

Many mammalian proteins have circadian cycles of production and degradation, and many of these rhythms are altered posttranscriptionally. We used ribosome profiling to examine posttranscriptional control of circadian rhythms by quantifying RNA translation in the liver over a 24-h period from circadian-entrained mice transferred to constant darkness conditions and by comparing ribosome binding levels to protein levels for 16 circadian proteins. We observed large differences in ribosome binding levels compared to protein levels, and we observed delays between peak ribosome binding and peak protein abundance. We found extensive binding of ribosomes to upstream open reading frames (uORFs) in circadian mRNAs, including the core clock gene Period2 (Per2). An increase in the number of uORFs in the 5'UTR was associated with a decrease in ribosome binding in the main coding sequence and a reduction in expression of synthetic reporter constructs. Mutation of the Per2 uORF increased luciferase and fluorescence reporter expression in 3T3 cells and increased luciferase expression in PER2:LUC MEF cells. Mutation of the Per2 uORF in mice increased Per2 mRNA expression, enhanced ribosome binding on Per2, and reduced total sleep time compared to that in wild-type mice. These results suggest that uORFs affect mRNA posttranscriptionally, which can impact physiological rhythms and sleep.

Expression of the readthrough transcript CiDRE in alveolar macrophages boosts SARS-CoV-2 susceptibility and promotes COVID-19 severity.

Lung infection during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via the angiotensin-I-converting enzyme 2 (ACE2) receptor induces a cytokine storm. However, the precise mechanisms involved in severe COVID-19 pneumonia are unknown. Here, we showed that interleukin-10 (IL-10) induced the expression of ACE2 in normal alveolar macrophages, causing them to become vectors for SARS-CoV-2. The inhibition of this system in hamster models attenuated SARS-CoV-2 pathogenicity. Genome-wide association and quantitative trait locus analyses identified a IFNAR2-IL10RB readthrough transcript, COVID-19 infectivity-enhancing dual receptor (CiDRE), which was highly expressed in patients harboring COVID-19 risk variants at the IFNAR2 locus. We showed that CiDRE exerted synergistic effects via the IL-10-ACE2 axis in alveolar macrophages and functioned as a decoy receptor for type I interferons. Collectively, our data show that high IL-10 and CiDRE expression are potential risk factors for severe COVID-19. Thus, IL-10R and CiDRE inhibitors might be useful COVID-19 therapies.

Bacterial induction of B cell senescence promotes age-related changes in the gut microbiota.

The elucidation of the mechanisms of ageing and the identification of methods to control it have long been anticipated. Recently, two factors associated with ageing-the accumulation of senescent cells and the change in the composition of gut microbiota-have been shown to play key roles in ageing. However, little is known about how these phenomena occur and are related during ageing. Here we show that the persistent presence of commensal bacteria gradually induces cellular senescence in gut germinal centre B cells. Importantly, this reduces both the production and diversity of immunoglobulin A (IgA) antibodies that target gut bacteria, thereby changing the composition of gut microbiota in aged mice. These results have revealed the existence of IgA-mediated crosstalk between the gut microbiota and cellular senescence and thus extend our understanding of the mechanism of gut microbiota changes with age, opening up possibilities for their control.

The ClusPro AbEMap web server for the prediction of antibody epitopes.

Antibodies play an important role in the immune system by binding to molecules called antigens at their respective epitopes. These interfaces or epitopes are structural entities determined by the interactions between an antibody and an antigen, making them ideal systems to analyze by using docking programs. Since the advent of high-throughput antibody sequencing, the ability to perform epitope mapping using only the sequence of the antibody has become a high priority. ClusPro, a leading protein-protein docking server, together with its template-based modeling version, ClusPro-TBM, have been re-purposed to map epitopes for specific antibody-antigen interactions by using the Antibody Epitope Mapping server (AbEMap). ClusPro-AbEMap offers three different modes for users depending on the information available on the antibody as follows: (i) X-ray structure, (ii) computational/predicted model of the structure or (iii) only the amino acid sequence. The AbEMap server presents a likelihood score for each antigen residue of being part of the epitope. We provide detailed information on the server's capabilities for the three options and discuss how to obtain the best results. In light of the recent introduction of AlphaFold2 (AF2), we also show how one of the modes allows users to use their AF2-generated antibody models as input. The protocol describes the relative advantages of the server compared to other epitope-mapping tools, its limitations and potential areas of improvement. The server may take 45-90 min depending on the size of the proteins.

Intercellular crosstalk in adult dental pulp is mediated by heparin-binding growth factors Pleiotrophin and Midkine.

BACKGROUND: In-depth knowledge of the cellular and molecular composition of dental pulp (DP) and the crosstalk between DP cells that drive tissue homeostasis are not well understood. To address these questions, we performed a comparative analysis of publicly available single-cell transcriptomes of healthy adult human DP to 5 other reference tissues: peripheral blood mononuclear cells, bone marrow, adipose tissue, lung, and skin. RESULTS: Our analysis revealed that DP resident cells have a unique gene expression profile when compared to the reference tissues, and that DP fibroblasts are the main cell type contributing to this expression profile. Genes coding for pleiotrophin (PTN) and midkine (MDK), homologous heparin-binding growth-factors, possessed the highest differential expression levels in DP fibroblasts. In addition, we identified extensive crosstalk between DP fibroblasts and several other DP resident cells, including Schwann cells, mesenchymal stem cells and odontoblasts, mediated by PTN and MDK. CONCLUSIONS: DP fibroblasts emerge as unappreciated players in DP homeostasis, mainly through their crosstalk with glial cells. These findings suggest that fibroblast-derived growth factors possess major regulatory functions and thus have a potential role as dental therapeutic targets.

Celastrol suppresses humoral immune responses and autoimmunity by targeting the COMMD3/8 complex.

Celastrol, a bioactive molecule extracted from the Tripterygium wilfordii plant, has been shown to exhibit anti-inflammatory properties. However, its mechanism of action has not been fully elucidated. Here, we show that celastrol suppresses humoral immune responses and autoimmunity by disabling a protein complex consisting of copper metabolism MURR1 domain-containing (COMMD) 3 and COMMD8 (COMMD3/8 complex), a signaling adaptor for chemoattractant receptors. Having demonstrated the involvement of the COMMD3/8 complex in a mouse model of rheumatoid arthritis, we identified celastrol as a compound that covalently bound to and dissociated the COMMD3/8 complex. Celastrol inhibited B cell migration, reduced antibody responses, and blocked arthritis progression, recapitulating deficiency of the COMMD3/8 complex. These effects of celastrol were abolished in mice expressing a celastrol-resistant mutant of the COMMD3/8 complex. These findings establish that celastrol exerts immunosuppressive activity by targeting the COMMD3/8 complex. Our study suggests that the COMMD3/8 complex is a potentially druggable target in autoimmune diseases and points to celastrol as a lead pharmacologic candidate in this capacity.

Structural Modeling of Adaptive Immune Responses to Infection.

Antibody and TCR modeling are becoming important as more and more sequence data becomes available to the public. One of the pressing questions now is how to use such data to understand adaptive immune responses to disease. Infectious disease is of particular interest because the antigens driving such responses are often known to some extent. Here, we describe tips for gathering data and cleaning it for use in downstream analysis. We present a method for high-throughput structural modeling of antibodies or TCRs using Repertoire Builder and its extensions. AbAdapt is an extension of Repertoire Builder for antibody-antigen docking from antibody and antigen sequences. ImmuneScape is a corresponding extension for TCR-pMHC 3D modeling. Together, these pipelines can help researchers to understand immune responses to infection from a structural point of view.

Construction of a T cell receptor signaling range for spontaneous development of autoimmune disease.

Thymic selection and peripheral activation of conventional T (Tconv) and regulatory T (Treg) cells depend on TCR signaling, whose anomalies are causative of autoimmunity. Here, we expressed in normal mice mutated ZAP-70 molecules with different affinities for the CD3 chains, or wild type ZAP-70 at graded expression levels under tetracycline-inducible control. Both manipulations reduced TCR signaling intensity to various extents and thereby rendered those normally deleted self-reactive thymocytes to become positively selected and form a highly autoimmune TCR repertoire. The signal reduction more profoundly affected Treg development and function because their TCR signaling was further attenuated by Foxp3 that physiologically repressed the expression of TCR-proximal signaling molecules, including ZAP-70, upon TCR stimulation. Consequently, the TCR signaling intensity reduced to a critical range generated pathogenic autoimmune Tconv cells and concurrently impaired Treg development/function, leading to spontaneous occurrence of autoimmune/inflammatory diseases, such as autoimmune arthritis and inflammatory bowel disease. These results provide a general model of how altered TCR signaling evokes autoimmune disease.

Mapping of antibody epitopes based on docking and homology modeling.

Antibodies are key proteins produced by the immune system to target pathogen proteins termed antigens via specific binding to surface regions called epitopes. Given an antigen and the sequence of an antibody the knowledge of the epitope is critical for the discovery and development of antibody based therapeutics. In this work, we present a computational protocol that uses template-based modeling and docking to predict epitope residues. This protocol is implemented in three major steps. First, a template-based modeling approach is used to build the antibody structures. We tested several options, including generation of models using AlphaFold2. Second, each antibody model is docked to the antigen using the fast Fourier transform (FFT) based docking program PIPER. Attention is given to optimally selecting the docking energy parameters depending on the input data. In particular, the van der Waals energy terms are reduced for modeled antibodies relative to x-ray structures. Finally, ranking of antigen surface residues is produced. The ranking relies on the docking results, that is, how often the residue appears in the docking poses' interface, and also on the energy favorability of the docking pose in question. The method, called PIPER-Map, has been tested on a widely used antibody-antigen docking benchmark. The results show that PIPER-Map improves upon the existing epitope prediction methods. An interesting observation is that epitope prediction accuracy starting from antibody sequence alone does not significantly differ from that of starting from unbound (i.e., separately crystallized) antibody structure.

The evolution of structural genomics.

Structural genomics began as a global effort in the 1990s to determine the tertiary structures of all protein families as a response to large-scale genome sequencing projects. The immediate outcome was an influx of tens of thousands of protein structures, many of which had unknown functions. At the time, the value of structural genomics was controversial. However, the structures themselves were only the most obvious output. In addition, these newly solved structures motivated the emergence of huge data science and infrastructure efforts, which, together with advances in Deep Learning, have brought about a revolution in computational molecular biology. Here, we review some of the computational research carried out at the Protein Data Bank Japan (PDBj) during the Protein 3000 project under the leadership of Haruki Nakamura, much of which continues to flourish today.

Advances in antibody discovery from human BCR repertoires.

Antibodies make up an important and growing class of compounds used for the diagnosis or treatment of disease. While traditional antibody discovery utilized immunization of animals to generate lead compounds, technological innovations have made it possible to search for antibodies targeting a given antigen within the repertoires of B cells in humans. Here we group these innovations into four broad categories: cell sorting allows the collection of cells enriched in specificity to one or more antigens; BCR sequencing can be performed on bulk mRNA, genomic DNA or on paired (heavy-light) mRNA; BCR repertoire analysis generally involves clustering BCRs into specificity groups or more in-depth modeling of antibody-antigen interactions, such as antibody-specific epitope predictions; validation of antibody-antigen interactions requires expression of antibodies, followed by antigen binding assays or epitope mapping. Together with innovations in Deep learning these technologies will contribute to the future discovery of diagnostic and therapeutic antibodies directly from humans.

Landscape of infection enhancing antibodies in COVID-19 and healthy donors.

To assess the frequency of SARS-CoV-2 infection in the general population, we searched over 64 million heavy chain antibody sequences from healthy unvaccinated, healthy BNT162b2 vaccinated and COVID-19 patient repertoires for sequences similar to 11 previously reported enhancing antibodies. Although the distribution of sequence identities was similar in all three groups of repertoires, the COVID-19 and healthy vaccinated hits were significantly more clonally expanded than healthy unvaccinated hits. Furthermore, among the tested hits, 17 out of 94 from COVID-19 and 9 out of 59 from healthy vaccinated, compared with only 2 out of 96 from healthy unvaccinated, bound to the enhancing epitope. A total of 9 of the 28 epitope-binding antibodies enhanced ACE2 receptor binding to the spike protein. Together, this study revealed that infection enhancing-like antibodies are far more frequent in COVID-19 patients or healthy vaccinated donors than in healthy unvaccinated donors, but a reservoir of potential enhancing antibodies exists in healthy donors that could potentially mature to actual enhancing antibodies upon infection.

The protein-stabilizing effects of TMAO in aqueous and non-aqueous conditions.

We present a new water-dependent molecular mechanism for the widely-used protein stabilizing osmolyte, trimethylamine N-oxide (TMAO), whose mode of action has remained controversial. Classical interpretations, such as osmolyte exclusion from the vicinity of protein, cannot adequately explain the behavior of this osmolyte and were challenged by recent data showing the direct interactions of TMAO with proteins, mainly via hydrophobic binding. Solvent effect theories also fail to propose a straightforward mechanism. To explore the role of water and the hydrophobic association, we disabled osmolyte-protein hydrophobic interactions by replacing water with hexane and using lipase enzyme as an anhydrous-stable protein. Biocatalysis experiments showed that under this non-aqueous condition, TMAO does not act as a stabilizer, but strongly deactivates the enzyme. Molecular dynamics (MD) simulations reveal that TMAO accumulates near the enzyme and makes many hydrogen bonds with it, like denaturing osmolytes. Some TMAO molecules even reach the active site and interact strongly with the catalystic traid. In aqueous solvent, the enzyme functions well: the extent of TMAO interactions is reduced and can be divided into both polar and non-polar terms. Structural analysis shows that in water, some TMAO molecules bind to the enzyme surface like a surfactant. We show that these interactions limit water-protein hydrogen bonds and unfavorable water-hydrophobic surface contacts. Moreover, a more hydrophobic environment is formed in the solvation layer, which reduces water dynamics and subsequently, rigidifies the backbone in aqueous solution. We show that osmolyte amphiphilicity and protein surface heterogeneity can address the weaknesses of exclusion and solvent effect theories about the TMAO mechanism.

Improved Antibody-Specific Epitope Prediction Using AlphaFold and AbAdapt.

Antibodies recognize their cognate antigens with high affinity and specificity, but the prediction of binding sites on the antigen (epitope) corresponding to a specific antibody remains a challenging problem. To address this problem, we developed AbAdapt, a pipeline that integrates antibody and antigen structural modeling with rigid docking in order to derive antibody-antigen specific features for epitope prediction. In this study, we systematically assessed the impact of integrating the state-of-the-art protein modeling method AlphaFold with the AbAdapt pipeline. By incorporating more accurate antibody models, we observed improvement in docking, paratope prediction, and prediction of antibody-specific epitopes. We further applied AbAdapt-AF in an anti-receptor binding domain (RBD) antibody complex benchmark and found AbAdapt-AF outperformed three alternative docking methods. Also, AbAdapt-AF demonstrated higher epitope prediction accuracy than other tested epitope prediction tools in the anti-RBD antibody complex benchmark. We anticipate that AbAdapt-AF will facilitate prediction of antigen-antibody interactions in a wide range of applications.

Engineering ACE2 decoy receptors to combat viral escapability.

Decoy receptor proteins that trick viruses to bind to them should be resistant to viral escape because viruses that require entry receptors cannot help but bind decoy receptors. Angiotensin-converting enzyme 2 (ACE2) is the major receptor for coronavirus cell entry. Recombinant soluble ACE2 was previously developed as a biologic against acute respiratory distress syndrome (ARDS) and verified to be safe in clinical studies. The emergence of COVID-19 reignited interest in soluble ACE2 as a potential broad-spectrum decoy receptor against coronaviruses. In this review, we summarize recent developments in preclinical studies using various high-affinity mutagenesis and Fc fusion approaches to achieve therapeutic efficacy of recombinant ACE2 decoy receptor against coronaviruses. We also highlight the relevance of stimulating effector immune cells through Fc-receptor engagement and the potential of using liquid aerosol delivery of ACE2 decoy receptors for defense against ACE2-utilizing coronaviruses.

An engineered ACE2 decoy neutralizes the SARS-CoV-2 Omicron variant and confers protection against infection in vivo.

The Omicron (B.1.1.529) SARS-CoV-2 variant contains an unusually high number of mutations in the spike protein, raising concerns of escape from vaccines, convalescent serum, and therapeutic drugs. Here, we analyzed the degree to which Omicron pseudo-virus evades neutralization by serum or therapeutic antibodies. Serum samples obtained 3 months after two doses of BNT162b2 vaccination exhibited 18-fold lower neutralization titers against Omicron than parental virus. Convalescent serum samples from individuals infected with the Alpha and Delta variants allowed similar frequencies of Omicron breakthrough infections. Domain-wise analysis using chimeric spike proteins revealed that this efficient evasion was primarily achieved by mutations clustered in the receptor binding domain but that multiple mutations in the N-terminal domain contributed as well. Omicron escaped a therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective. Angiotensin-converting enzyme 2 (ACE2) decoys are another virus-neutralizing drug modality that are free, at least in theory, from complete escape. Deep mutational analysis demonstrated that an engineered ACE2 molecule prevented escape for each single-residue mutation in the receptor binding domain, similar to immunized serum. Engineered ACE2 neutralized Omicron comparably to the Wuhan strain and also showed a therapeutic effect against Omicron infection in hamsters and human ACE2 transgenic mice. Similar to previous SARS-CoV-2 variants, some sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge.