Two subforms of eukaryotic topoisomerase I Purification and structure-function relationships

AbstractA new method for isolation of eukaryotic topoisomerase 1 from calf thymus and from Jurkat-1 cells using HPLC has been developed. The method allows quantitative purification of high molecular weight topo I and of two low molecular weight fractions differing by their isoelectric points. It has been suggested that these fractions be characterized as two subforms of the enzyme possessing structural and functional differences. The differences in their specific activities, sensitivity to camptothecin and in their proteolytic digestion maps have been demonstrated for the two enzymes

CCR5/CXCR3 antagonist TAK-779 prevents diffuse alveolar damage of the lung in the murine model of the acute respiratory distress syndrome

Introduction: The acute respiratory distress syndrome (ARDS), secondary to viral pneumonitis, is one of the main causes of high mortality in patients with COVID-19 (novel coronavirus disease 2019)—ongoing SARS-CoV-2 infection— reached more than 0.7 billion registered cases.Methods: Recently, we elaborated a non-surgical and reproducible method of the unilateral total diffuse alveolar damage (DAD) of the left lung in ICR mice–a publicly available imitation of the ARDS caused by SARS-CoV-2. Our data read that two C–C chemokine receptor 5 (CCR5) ligands, macrophage inflammatory proteins (MIPs) MIP-1α/CCL3 and MIP-1β/CCL4, are upregulated in this DAD model up to three orders of magnitude compared to the background level.Results: Here, we showed that a nonpeptide compound TAK-779, an antagonist of CCR5/CXCR3, readily prevents DAD in the lung with a single injection of 2.5 mg/kg. Histological analysis revealed reduced peribronchial and perivascular mononuclear infiltration in the lung and mononuclear infiltration of the wall and lumen of the alveoli in the TAK-779-treated animals. Administration of TAK-779 decreased the 3–5-fold level of serum cytokines and chemokines in animals with DAD, including CCR5 ligands MIP-1α/β, MCP-1, and CCL5. Computed tomography revealed rapid recovery of the density and volume of the affected lung in TAK-779-treated animals.Discussion: Our pre-clinical data suggest that TAK-779 is more effective than the administration of dexamethasone or the anti-IL6R therapeutic antibody tocilizumab, which brings novel therapeutic modality to TAK-779 and other CCR5 inhibitors for the treatment of virus-induced hyperinflammation syndromes, including COVID-19

Genetically engineered CD80–pMHC-harboring extracellular vesicles for antigen-specific CD4+ T-cell engagement

The identification of low-frequency antigen-specific CD4+ T cells is crucial for effective immunomonitoring across various diseases. However, this task still encounters experimental challenges necessitating the implementation of enrichment procedures. While existing antigen-specific expansion technologies predominantly concentrate on the enrichment of CD8+ T cells, advancements in methods targeting CD4+ T cells have been limited. In this study, we report a technique that harnesses antigen-presenting extracellular vesicles (EVs) for stimulation and expansion of antigen-specific CD4+ T cells. EVs are derived from a genetically modified HeLa cell line designed to emulate professional antigen-presenting cells (APCs) by expressing key costimulatory molecules CD80 and specific peptide–MHC-II complexes (pMHCs). Our results demonstrate the beneficial potent stimulatory capacity of EVs in activating both immortalized and isolated human CD4+ T cells from peripheral blood mononuclear cells (PBMCs). Our technique successfully expands low-frequency influenza-specific CD4+ T cells from healthy individuals. In summary, the elaborated methodology represents a streamlined and efficient approach for the detection and expansion of antigen-specific CD4+ T cells, presenting a valuable alternative to existing antigen-specific T-cell expansion protocols

MHC Class II Presentation in Autoimmunity

Antigen presentation by major histocompatibility complex class II (MHC-II) molecules is crucial for eliciting an efficient immune response by CD4+ T cells and maintaining self-antigen tolerance. Some MHC-II alleles are known to be positively or negatively associated with the risk of the development of different autoimmune diseases (ADs), including those characterized by the emergence of autoreactive T cells. Apparently, the MHC-II presentation of self-antigens contributes to the autoimmune T cell response, initiated through a breakdown of central tolerance to self-antigens in the thymus. The appearance of autoreactive T cell might be the result of (i) the unusual interaction between T cell receptors (TCRs) and self-antigens presented on MHC-II; (ii) the posttranslational modifications (PTMs) of self-antigens; (iii) direct loading of the self-antigen to classical MHC-II without additional nonclassical MHC assistance; (iv) the proinflammatory environment effect on MHC-II expression and antigen presentation; and (v) molecular mimicry between foreign and self-antigens. The peculiarities of the processes involved in the MHC-II-mediated presentation may have crucial importance in the elucidation of the mechanisms of triggering and developing ADs as well as for clarification on the protective effect of MHC-II alleles that are negatively associated with ADs

Deep Functional Profiling Facilitates the Evaluation of the Antibacterial Potential of the Antibiotic Amicoumacin

The global spread of antibiotic resistance is forcing the scientific community to find new molecular strategies to counteract it. Deep functional profiling of microbiomes provides an alternative source for the discovery of novel antibiotic producers and probiotics. Recently, we implemented this ultrahigh-throughput screening approach for the isolation of Bacillus pumilus strains efficiently producing the ribosome-targeting antibiotic amicoumacin A (Ami). Proteomics and metabolomics revealed essential insight into the activation of Ami biosynthesis. Here, we applied omics to boost Ami biosynthesis, providing the optimized cultivation conditions for high-scale production of Ami. Ami displayed a pronounced activity against Lactobacillales and Staphylococcaceae, including methicillin-resistant Staphylococcus aureus (MRSA) strains, which was determined using both classical and massive single-cell microfluidic assays. However, the practical application of Ami is limited by its high cytotoxicity and particularly low stability. The former is associated with its self-lactonization, serving as an improvised intermediate state of Ami hydrolysis. This intramolecular reaction decreases Ami half-life at physiological conditions to less than 2 h, which is unprecedented for a terminal amide. While we speculate that the instability of Ami is essential for Bacillus ecology, we believe that its stable analogs represent attractive lead compounds both for antibiotic discovery and for anticancer drug development

Reactibodies generated by kinetic selection couple chemical reactivity with favorable protein dynamics

Igs offer a versatile template for combinatorial and rational design approaches to the de novo creation of catalytically active proteins. We have used a covalent capture selection strategy to identify biocatalysts from within a human semisynthetic antibody variable fragment library that uses a nucleophilic mechanism. Specific phosphonylation at a single tyrosine within the variable light-chain framework was confirmed in a recombinant IgG construct. High-resolution crystallographic structures of unmodified and phosphonylated Fabs display a 15-Å-deep two-chamber cavity at the interface of variable light (VL) and variable heavy (VH) fragments having a nucleophilic tyrosine at the base of the site. The depth and structure of the pocket are atypical of antibodies in general but can be compared qualitatively with the catalytic site of cholinesterases. A structurally disordered heavy chain complementary determining region 3 loop, constituting a wall of the cleft, is stabilized after covalent modification by hydrogen bonding to the phosphonate tropinol moiety. These features and presteady state kinetics analysis indicate that an induced fit mechanism operates in this reaction. Mutations of residues located in this stabilized loop do not interfere with direct contacts to the organophosphate ligand but can interrogate second shell interactions, because the H3 loop has a conformation adjusted for binding. Kinetic and thermodynamic parameters along with computational docking support the active site model, including plasticity and simple catalytic components. Although relatively uncomplicated, this catalytic machinery displays both stereo- and chemical selectivity. The organophosphate pesticide paraoxon is hydrolyzed by covalent catalysis with rate-limiting dephosphorylation. This reactibody is, therefore, a kinetically selected protein template that has enzyme-like catalytic attributes

Design of targeted B cell killing agents.

B cells play an important role in the pathogenesis of both systemic and organ-specific autoimmune diseases. Autoreactive B cells not only produce autoantibodies, but also are capable to efficiently present specific autoantigens to T cells. Furthermore, B cells can secrete proinflammatory cytokines and amplify the vicious process of self-destruction. B cell-directed therapy is a potentially important approach for treatment of various autoimmune diseases. The depletion of B cells by anti-CD20/19 monoclonal antibody Retuximab® used in autoimmune diseases therapy leads to systemic side effects and should be significantly improved. In this study we designed a repertoire of genetically engineered B cell killers that specifically affected one kind of cells carrying a respective B cell receptor. We constructed immunotoxins (ITs), fused with c-myc epitope as a model targeting sequence, based on barnase, Pseudomonas toxin, Shiga-like toxin E.coli and Fc domain of human antibody IgGγ1. C-MYC hybridoma cell line producing anti-c-myc IgG was chosen as a model for targeted cell depletion. C-myc sequence fused with toxins provided addressed delivery of the toxic agent to the target cells. We demonstrated functional activity of designed ITs in vitro and showed recognition of the fusion molecules by antibodies produced by targeted hybridoma. To study specificity of the proposed B cells killing molecules, we tested a set of created ITs ex vivo, using C-MYC and irrelevant hybridoma cell lines. Pseudomonas-containing IT showed one of the highest cytotoxic effects on the model cells, however, possessed promiscuous specificity. Shiga-like toxin construct demonstrated mild both cytotoxicity and specificity. Barnase and Fc-containing ITs revealed excellent balance between their legibility and toxic properties. Moreover, barnase and Fc molecules fused with c-myc epitope were able to selectively deplete c-myc-specific B cells and decrease production of anti-c-myc antibodies in culture of native splenocytes, suggesting their highest therapeutic potential as targeted B cell killing agents