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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.

Spinal cord injury (SCI) is a medical challenge that results in the formation of a glial scar preventing recovery of axonal conductivity. Cytokines and chemokines significantly affect the pathogenesis of SCI and represent important targets for therapeutic intervention. Here, dozens of cytokines and chemokines are dynamically monitored in plasma, cerebrospinal fluid, and injury site released in response to experimental SCI conducted in two rodent strains and patients undergoing surgical removal of intramedullary tumors. Dataset comprising 6,172 cytokine/chemokine values across 8 time points suggests that SCI in mammals is accompanied by a massive cytokine storm in cerebrospinal fluid, mainly driven by CXCL1, IL‐6, and CCL2‐5. Sub‐acute phase is mostly associated with IL‐2, IL‐7, CCL22 and CX3CL1, whereas TNFα and IL17α permanently persists in CNS for even weeks following SCI. The effects of mogamulizumab and chemical antagonists of C‐C/C‐X‐C chemokine receptors TAK‐799, SB225002, and MK‐7123 on SCI recovery in rodents are further estimated. Here blockade of CCR5 and CXCR1/2 chemokine receptors is shown beneficial for amelioration of acute SCI, whereas anti‐CCR4 antibody mogamulizumab readily prevents secondary inflammation in the injured area. Summarizing, the current report claims for a novel combined time‐resolved therapeutic modality in SCI treatment, which supports feasibility and motivates off‐label clinical evaluation in appropriate cohorts. Spinal cord injury (SCI) in mammals is accompanied by a massive cytokine storm in cerebrospinal fluid, mainly driven by CXCL1, IL‐6, and CCL2‐5. Sub‐acute phase is mostly associated with IL‐2, IL‐7, CCL22, and CX3CL1, whereas TNFα and IL17α permanently persists in CNS even weeks following SCI. Time‐related combined therapy by CXCR1/2 or ‐CCR5 chemical antagonists, mogamulizumab and infliximab, might ameliorate the evolution of SCI sequelae during the acute, sub‐acute, and chronic phases, respectively.

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.

Most intracellular proteins are degraded by the ubiquitin–proteasome system (UPS), with proteasomes directly hydrolyzing protein substrates. Specific forms of proteasomes (non-constitutive proteasomes), implicated in antigen presentation, cellular homeostasis maintenance and stress response have been described. However, proteasomes were also identified outside cells, where their function remains unclear. Proteasome secretion via extracellular vesicles (EVs) have been reported, though the direct transmission of non-constitutive proteasomes between cells has not been shown. Using genetically modified cells, including a human adenocarcinoma cell line SW620B8-mCherry expressing the β5i subunit of non-constitutive proteasomes fused to the mCherry protein, and a number of techniques, such as differential centrifugation, affinity isolation, unspecific precipitation, NTA and microscopy, EVs containing non-constitutive proteasomes were obtained and characterized. Different cell lines were shown to secrete varying amounts of vesicles containing non-constitutive proteasomes. The content of these proteasomes in EVs was increased after the stimulation of cells with IFN-γ. The interaction of vesicles secreted by SW620B8-mCherry cells with recipient cells was demonstrated. The β5i-mCherry chimera was detected in lysates of different recipient cells following incubation with EVs secreted from SW620B8-mCherry cells. The obtained results highlight the transfer of non-constitutive proteasomes from one cell to another via EVs.

In this study, we evaluated the ability of the synthetic amphipathic helical peptide (SAHP), L-37pA, which mediates pathogen recognition and innate immune responses, to treat acute respiratory distress syndrome (ARDS) accompanied by diffuse alveolar damage (DAD) and chronic pulmonary fibrosis (PF). For the modeling of ARDS/DAD, male ICR mice were used. Intrabronchial instillation (IB) of 200 µL of inflammatory agents was performed by an intravenous catheter 20 G into the left lung lobe only, leaving the right lobe unaffected. Intravenous injections (IVs) of L-37pA, dexamethasone (DEX) and physiological saline (saline) were used as therapies for ARDS/DAD. L37pA inhibited the circulating levels of inflammatory cytokines, such as IL-8, TNFα, IL1α, IL4, IL5, IL6, IL9 and IL10, by 75–95%. In all cases, the computed tomography (CT) data indicate that L-37pA reduced lung density faster to −335 ± 23 Hounsfield units (HU) on day 7 than with DEX and saline, to −105 ± 29 HU and −23 ± 11 HU, respectively. The results of functional tests showed that L-37pA treatment 6 h after ARDS/DAD initiation resulted in a more rapid improvement in the physiological respiratory lung by 30–45% functions compared with the comparison drugs. Our data suggest that synthetic amphipathic helical peptide L-37pA blocked a cytokine storm, inhibited acute and chronic pulmonary inflammation, prevented fibrosis development and improved physiological respiratory lung function in the ARDS/DAD mouse model. We concluded that a therapeutic strategy using SAHPs targeting SR-B receptors is a potential novel effective treatment for inflammation-induced ARDS, DAD and lung fibrosis of various etiologies.

Myelin basic protein (MBP) is the second most abundant protein in the central nervous system and is responsible for structural maintenance of the myelin sheath covering axons. Previously, we showed that MBP has a more proactive role in the oligodendrocyte homeostasis, interacting with membrane-associated proteins, including integral membrane protein 2B (ITM2B or Bri2) that is associated with familial dementias. Here, we report that the molecular dynamics of the in silico-generated MBP-Bri2 complex revealed that MBP covers a significant portion of the Bri2 ectodomain, assumingly trapping the furin cleavage site, while the surface of the BRICHOS domain, which is responsible for the multimerization and activation of the Bri2 high-molecular-weight oligomer chaperone function, remains unmasked. These observations were supported by the co-expression of MBP with Bri2, its mature form, and disease-associated mutants, which showed that in mammalian cells, MBP indeed modulates the post-translational processing of Bri2 by restriction of the furin-catalyzed release of its C-terminal peptide. Moreover, we showed that the co-expression of MBP and Bri2 also leads to an altered cellular localization of Bri2, restricting its membrane trafficking independently of the MBP-mediated suppression of the Bri2 C-terminal peptide release. Further investigations should elucidate if these observations have physiological meaning in terms of Bri2 as a MBP chaperone activated by the MBP-dependent postponement of Bri2 membrane trafficking.

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.

Various pathological processes are known to be associated with the production of IgG autoantibodies, which have high affinity for self-antigens and often cause tissue injury and the development of autoimmune diseases. However, the mechanism of their cytotoxic activity is not clearly understood yet. Here, we have shown that the action of these autoantibodies on cells expressing TNFR1 (the cell surface receptor for TNFα) can cause both caspase-dependent apoptosis and necroptosis of these cells, with suppression of apoptosis resulting in switching to RIP1-dependent necroptosis. Analysis of necroptotic mechanisms has shown that a critical point of necroptosis is phosphorylation of RIP1 and RIP3 kinases, which is followed by the involvement of lysosomes and mitochondria in this process. The induction of cytotoxicity is initiated by the interaction of autoantibodies with TNFR1, and autoantibodies can therefore be regarded as a new functional ligand for this receptor. The innate immunity protein Tag7 (PGLYRP1) described in our recent studies is also a ligand for TNFR1 and competes with autoantibodies for binding with it. Supposedly, the cytotoxic effect of autoantibodies is one of the factors responsible for autoimmune diseases that lead to tissue injury.

The creation of effective bioscavengers as a pretreatment for exposure to nerve agents is a challenging medical objective. We report a recombinant method using chemical polysialylation to generate bioscavengers stable in the bloodstream. Development of a CHO-based expression system using genes encoding human butyrylcholinesterase and a proline-rich peptide under elongation factor promoter control resulted in self-assembling, active enzyme multimers. Polysialylation gives bioscavengers with enhanced pharmacokinetics which protect mice against 4.2 LD₅₀ of S-(2-(diethylamino) ethyl) O-isobutyl methanephosphonothioate without perturbation of long-term behavior.

To simulate acute lung injury (ALI) in SD male rats they we administered intratracheally with lipopolysaccharide (LPS) followed by hyperventilation of the lungs (HVL), which lead to functional changes in the respiratory system and an increase in the blood serum concentration of inflammatory cytokines. LPS + HVL after 4 h lead to pronounced histological signs of lung damage. We have studied the effectiveness of Derinat ® when administered intramuscularly at dose of 7.5 mg/kg for 8 days in the ALI model. Derinat ® administration lead to an increase in the concentration of most of the studied cytokines in a day. In the ALI model the administration of Derinat ® returned the concentration of cytokines to its original values already 48 h after LPS + HVL, and also normalized the parameters of pulmonary respiration in comparison with animals without treatment. By the eighth day after LPS + HVL, respiratory parameters and cytokine levels, as well as biochemical and hematological parameters did not differ between groups, while histological signs of residual effects of lung damage were found in all animals, and were more pronounced in Derinat ® group, which may indicate stimulation of the local immune response. Thus, the administration of Derinat ® stimulates the immune response, has a pronounced protective effect against cytokinemia and respiratory failure caused by ALI, has immunomodulatory effect, and also stimulates a local immune response in lung tissues. Thus, Derinat ® is a promising treatment for ALI.