B cell receptor-induced protein dynamics and the emerging role of SUMOylation revealed by proximity proteomics.

Successful B cell activation, which is critical for high-affinity antibody production, is controlled by the B cell antigen receptor (BCR). However, we still lack a comprehensive protein-level view of the very dynamic multi-branched cellular events triggered by antigen binding. Here, we employed APEX2 proximity biotinylation to study antigen-induced changes, 5-15 min after receptor activation, at the vicinity of the plasma membrane lipid rafts, wherein BCR enriches upon activation. The data reveals dynamics of signaling proteins, as well as various players linked to the subsequent processes, such as actin cytoskeleton remodeling and endocytosis. Interestingly, our differential expression analysis identified dynamic responses in various proteins previously not linked to early B cell activation. We demonstrate active SUMOylation at the sites of BCR activation in various conditions and report its functional role in BCR signaling through the AKT and ERK1/2 axes.

AutoCoEv-A High-Throughput In Silico Pipeline for Predicting Inter-Protein Coevolution.

Protein-protein interactions govern cellular processes via complex regulatory networks, which are still far from being understood. Thus, identifying and understanding connections between proteins can significantly facilitate our comprehension of the mechanistic principles of protein functions. Coevolution between proteins is a sign of functional communication and, as such, provides a powerful approach to search for novel direct or indirect molecular partners. However, an evolutionary analysis of large arrays of proteins in silico is a highly time-consuming effort that has limited the usage of this method for protein pairs or small protein groups. Here, we developed AutoCoEv, a user-friendly, open source, computational pipeline for the search of coevolution between a large number of proteins. By driving 15 individual programs, culminating in CAPS2 as the software for detecting coevolution, AutoCoEv achieves a seamless automation and parallelization of the workflow. Importantly, we provide a patch to the CAPS2 source code to strengthen its statistical output, allowing for multiple comparison corrections and an enhanced analysis of the results. We apply the pipeline to inspect coevolution among 324 proteins identified to be located at the vicinity of the lipid rafts of B lymphocytes. We successfully detected multiple coevolutionary relations between the proteins, predicting many novel partners and previously unidentified clusters of functionally related molecules. We conclude that AutoCoEv, can be used to predict functional interactions from large datasets in a time- and cost-efficient manner.

Humoral immunological kinetics of severe acute respiratory syndrome coronavirus 2 infection and diagnostic performance of serological assays for coronavirus disease 2019: an analysis of global reports.

As the coronavirus disease 2019 (COVID-19) pandemic continues to rise and second waves are reported in some countries, serological test kits and strips are being considered to scale up an adequate laboratory response. This study provides an update on the kinetics of humoral immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and performance characteristics of serological protocols (lateral flow assay [LFA], chemiluminescence immunoassay [CLIA] and ELISA) used for evaluations of recent and past SARS-CoV-2 infection. A thorough and comprehensive review of suitable and eligible full-text articles was performed on PubMed, Scopus, Web of Science, Wordometer and medRxiv from 10 January to 16 July 2020. These articles were searched using the Medical Subject Headings terms 'COVID-19', 'Serological assay', 'Laboratory Diagnosis', 'Performance characteristics', 'POCT', 'LFA', 'CLIA', 'ELISA' and 'SARS-CoV-2'. Data from original research articles on SARS-CoV-2 antibody detection ≥second day postinfection were included in this study. In total, there were 7938 published articles on humoral immune response and laboratory diagnosis of COVID-19. Of these, 74 were included in this study. The detection, peak and decline period of blood anti-SARS-CoV-2 IgM, IgG and total antibodies for point-of-care testing (POCT), ELISA and CLIA vary widely. The most promising of these assays for POCT detected anti-SARS-CoV-2 at day 3 postinfection and peaked on the 15th day; ELISA products detected anti-SARS-CoV-2 IgM and IgG at days 2 and 6 then peaked on the eighth day; and the most promising CLIA product detected anti-SARS-CoV-2 at day 1 and peaked on the 30th day. The most promising LFA, ELISA and CLIA that had the best performance characteristics were those targeting total SARS-CoV-2 antibodies followed by those targeting anti-SARS-CoV-2 IgG then IgM. Essentially, the CLIA-based SARS-CoV-2 tests had the best performance characteristics, followed by ELISA then POCT. Given the varied performance characteristics of all the serological assays, there is a need to continuously improve their detection thresholds, as well as to monitor and re-evaluate their performances to assure their significance and applicability for COVID-19 clinical and epidemiological purposes.

B cells rapidly target antigen and surface-derived MHCII into peripheral degradative compartments.

In order to mount high-affinity antibody responses, B cells internalise specific antigens and process them into peptides loaded onto MHCII for presentation to T helper cells (TH cells). While the biochemical principles of antigen processing and MHCII loading have been well dissected, how the endosomal vesicle system is wired to enable these specific functions remains much less studied. Here, we performed a systematic microscopy-based analysis of antigen trafficking in B cells to reveal its route to the MHCII peptide-loading compartment (MIIC). Surprisingly, we detected fast targeting of internalised antigen into peripheral acidic compartments that possessed the hallmarks of the MIIC and also showed degradative capacity. In these vesicles, internalised antigen converged rapidly with membrane-derived MHCII and partially overlapped with cathepsin-S and H2-M, both required for peptide loading. These early compartments appeared heterogenous and atypical as they contained a mixture of both early and late endosomal markers, indicating a specialized endosomal route. Together, our data suggest that, in addition to in the previously reported perinuclear late endosomal MIICs, antigen processing and peptide loading could have already started in these specialized early peripheral acidic vesicles (eMIIC) to support fast peptide-MHCII presentation.

Insights into mitochondrial fatty acid synthesis from the structure of heterotetrameric 3-ketoacyl-ACP reductase/3R-hydroxyacyl-CoA dehydrogenase.

Mitochondrial fatty acid synthesis (mtFAS) is essential for respiratory growth in yeast and mammalian embryonic survival. The human 3-ketoacyl-acyl carrier protein (ACP) reductase (KAR) of mtFAS is a heterotetrameric α2ß2-assembly composed of 17ß-hydroxysteroid dehydrogenase type-8 (HSD17B8, α-subunit) and carbonyl reductase type-4 (CBR4, ß-subunit). Here we provide a structural explanation for the stability of the heterotetramer from the crystal structure with NAD(+) and NADP(+) bound to the HSD17B8 and CBR4 subunits, respectively, and show that the catalytic activity of the NADPH- and ACP-dependent CBR4 subunit is crucial for a functional HsKAR. Therefore, mtFAS is NADPH- and ACP dependent, employing the 3R-hydroxyacyl-ACP intermediate. HSD17B8 assists in the formation of the competent HsKAR assembly. The intrinsic NAD(+)- and CoA-dependent activity of the HSD17B8 subunit on the 3R-hydroxyacyl-CoA intermediates may indicate a role for this subunit in routing 3R-hydroxyacyl-CoA esters, potentially arising from the metabolism of unsaturated fatty acids, into the mitochondrial ß-oxidation pathway.