Tracking of Ubiquitin Signaling through 3.5 Billion Years of Combinatorial Conjugation.

Ubiquitination is an evolutionary, ancient system of post-translational modification of proteins that occurs through a cascade involving ubiquitin activation, transfer, and conjugation. The maturation of this system has followed two main pathways. The first is the conservation of a universal structural fold of ubiquitin and ubiquitin-like proteins, which are present in both Archaea and Bacteria, as well as in multicellular Eukaryotes. The second is the rise of the complexity of the superfamily of ligases, which conjugate ubiquitin-like proteins to substrates, in terms of an increase in the number of enzyme variants, greater variation in structural organization, and the diversification of their catalytic domains. Here, we examine the diversity of the ubiquitination system among different organisms, assessing the variety and conservation of the key domains of the ubiquitination enzymes and ubiquitin itself. Our data show that E2 ubiquitin-conjugating enzymes of metazoan phyla are highly conservative, whereas the homology of E3 ubiquitin ligases with human orthologues gradually decreases depending on "molecular clock" timing and evolutionary distance. Surprisingly, Chordata and Echinodermata, which diverged over 0.5 billion years ago during the Cambrian explosion, share almost the same homology with humans in the amino acid sequences of E3 ligases but not in their adaptor proteins. These observations may suggest that, firstly, the E2 superfamily already existed in its current form in the last common metazoan ancestor and was generally not affected by purifying selection in metazoans. Secondly, it may indicate convergent evolution of the ubiquitination system and highlight E3 adaptor proteins as the "upper deck" of the ubiquitination system, which plays a crucial role in chordate evolution.

Synthetic Amphipathic Helical Peptide L-37pA Ameliorates the Development of Acute Respiratory Distress Syndrome (ARDS) and ARDS-Induced Pulmonary Fibrosis in Mice.

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 Attenuates Furin-Mediated Bri2 Cleavage and Postpones Its Membrane Trafficking.

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.

Topology of Ubiquitin Chains in the Chromatosomal Environment of the E3 Ubiquitin Ligase RNF168.

Genome stability is critical for normal functioning of cells, it depends on accuracy of DNA replication, chromosome segregation, and DNA repair. Cellular defense mechanisms against DNA damage are important for preventing cancer development and aging. The E3 ubiquitin ligase RNF168 of the RING superfamily is an essential component of the complex responsible for ubiquitination of the H2A/H2A.X histones near DNA double-strand breaks, which is a key step in attracting repair factors to the damage site. In this study, we unequivocally showed that RNF168 does not have the ability to directly distinguish architecture of polyubiquitin chains, except for the tropism of its two ubiquitin-binding domains UDM1/2 to K63 ubiquitin chains. Analysis of intracellular chromatosomal environment of the full-length RNF168 and its domains using the ligand-induced bioluminescence resonance energy transfer (BRET) revealed that the C-terminal part of UDM1 is associated with the K63 ubiquitin chains; RING and the N-terminal part of UDM2 are sterically close to the K63- and K48-ubiquitin chains, while the C-terminal part of UDM1 is co-localized with all possible ubiquitin variants. Our observations together with the available structural data suggest that the C-terminal part of UDM1 binds the K63 polyubiquitin chains on the linker histone H1; RING and the N-terminal part of UDM2 are located in the central part of nucleosome and sterically close to H1 and K48-ubiquitinated alternative substrates of RNF168, such as JMJD2A/B demethylases, while the C-terminal part of UDM1 is in the region of activated ubiquitin residue associated with E2 ubiquitin ligase, engaged by RNF168.

Markers of NETosis in Patients with Systemic Lupus Erythematosus and Antiphospholipid Syndrome.

Neutrophil Extracellular Traps (NETs) have been implicated in systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) pathogenesis. The myeloperoxidase-deoxyribonucleic acid (MPO-DNA) complex and nucleosomes are serum markers of NETosis. The aim of this study was to assess these NETosis parameters as markers for SLE and APS diagnosis and their association with clinical features and disease activity. A total of 138 people were included in the cross-sectional study: 30 with SLE without APS, 47 with SLE and APS, 41 patients with primary antiphospholipid syndrome (PAPS), and 20 seemingly healthy individuals. Serum MPO-DNA complex and nucleosome levels were determined via an enzyme-linked immunosorbent assay (ELISA). Informed consent was obtained from all subjects involved in the study. The Ethics Committee of the V.A. Nasonova Research Institute of Rheumatology (Protocol No. 25 dated 23 December 2021) approved the study. In patients with SLE without APS, the levels of the MPO-DNA complex were significantly higher compared to patients with SLE with APS, with PAPS, and healthy controls (p < 0.0001). Among patients with a reliable diagnosis of SLE, 30 had positive values of the MPO-DNA complex, of whom 18 had SLE without APS, and 12 had SLE with APS. Patients with SLE and positive MPO-DNA complex levels were significantly more likely to have high SLE activity (χ2 = 5.25, p = 0.037), lupus glomerulonephritis (χ2 = 6.82, p = 0.009), positive antibodies to dsDNA (χ2 = 4.82, p = 0.036), and hypocomplementemia (χ2 = 6.72, p = 0.01). Elevated MPO-DNA levels were observed in 22 patients with APS: 12 with SLE with APS and 10 with PAPS. There were no significant associations between positive levels of the MPO-DNA complex and clinical and laboratory manifestations of APS. The concentration of nucleosomes was significantly lower in the group of SLE patients (±APS) compared to controls and PAPS (p < 0.0001). In SLE patients, the frequency of low nucleosome levels was associated with high SLE activity (χ2 = 13.4, p < 0.0001), lupus nephritis (χ2 = 4.1, p = 0.043), and arthritis (χ2 = 3.89, p = 0.048). An increase in the specific marker of NETosis, the MPO-DNA complex, was found in the blood serum of SLE patients without APS. Elevated levels of the MPO-DNA complex can be regarded as a promising biomarker of lupus nephritis, disease activity, and immunological disorders in SLE patients. Lower levels of nucleosomes were significantly associated with SLE (±APS). Low nucleosome levels were more common in patients with high SLE activity, lupus nephritis, and arthritis.

Myelin Basic Protein Fragmentation by Engineered Human Proteasomes with Different Catalytic Phenotypes Revealed Direct Peptide Ligands of MS-Associated and Protective HLA Class I Molecules.

Proteasomes exist in mammalian cells in multiple combinatorial variants due to the diverse regulatory particles and exchange of catalytic subunits. Here, using biotin carboxyl carrier domain of transcarboxylase from Propionibacterium shermanii fused with different proteasome subunits of catalytic and regulatory particles, we report comprehensive characterization of highly homogenous one-step purified human constitutive and immune 20S and 26S/30S proteasomes. Hydrolysis of a multiple sclerosis (MS) autoantigen, myelin basic protein (MBP), by engineered human proteasomes with different catalytic phenotypes, revealed that peptides which may be directly loaded on the HLA class I molecules are produced mainly by immunoproteasomes. We detected at least five MBP immunodominant core regions, namely, LPRHRDTGIL, SLPQKSHGR, QDENPVVHFF, KGRGLSLSRF and GYGGRASDY. All peptides, except QDENPVVHFF, which originates from the encephalitogenic MBP part, were associated with HLA I alleles considered to increase MS risk. Prediction of the affinity of HLA class I to this peptide demonstrated that MS-protective HLA-A*44 and -B*35 molecules are high-affinity binders, whereas MS-associated HLA-A*23, -A*24, -A*26 and -B*51 molecules tend to have moderate to low affinity. The HLA-A*44 molecules may bind QDENPVVHFF and its deamidated form in several registers with unprecedently high affinity, probably linking its distinct protective phenotype with thymic depletion of the repertoire of autoreactive cytotoxic T cells or induction of CD8+ regulatory T cells, specific to the encephalitogenic MBP peptide.

Control of Genome through Variative Nature of Histone-Modifying Ubiquitin Ligases.

Covalent attachment of ubiquitin residue is not only the proteasomal degradation signal, but also a widespread posttranslational modification of cellular proteins in eukaryotes. One of the most important targets of the regulatory ubiquitination are histones. Localization of ubiquitin residue in different regions of the nucleosome attracts a strictly determined set of cellular factors with varied functionality. Depending on the type of histone and the particular lysine residue undergoing modification, histone ubiquitination can lead both to transcription activation and to gene repression, as well as contribute to DNA repair via different mechanisms. An extremely interesting feature of the family of RING E3 ubiquitin ligases catalyzing histone ubiquitination is the striking structural diversity of the domains providing high specificity of modification very similar initial targets. It is obvious that further elucidation of peculiarities of the ubiquitination system involved in histone modification, as well as understanding of physiological role of this process in the maintenance of homeostasis of both single cells and the entire organism, will substantially expand the possibilities of treating a number of socially significant diseases.

Protein PGLYRP1/Tag7 Peptides Decrease the Proinflammatory Response in Human Blood Cells and Mouse Model of Diffuse Alveolar Damage of Lung through Blockage of the TREM-1 and TNFR1 Receptors.

Infection caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2) in many cases is accompanied by the release of a large amount of proinflammatory cytokines in an event known as "cytokine storm", which is associated with severe coronavirus disease 2019 (COVID-19) cases and high mortality. The excessive production of proinflammatory cytokines is linked, inter alia, to the enhanced activity of receptors capable of recognizing the conservative regions of pathogens and cell debris, namely TLRs, TREM-1 and TNFR1. Here we report that peptides derived from innate immunity protein Tag7 inhibit activation of TREM-1 and TNFR1 receptors during acute inflammation. Peptides from the N-terminal fragment of Tag7 bind only to TREM-1, while peptides from the C-terminal fragment interact solely with TNFR1. Selected peptides are capable of inhibiting the production of proinflammatory cytokines both in peripheral blood mononuclear cells (PBMCs) from healthy donors and in vivo in the mouse model of acute lung injury (ALI) by diffuse alveolar damage (DAD). Treatment with peptides significantly decreases the infiltration of mononuclear cells to lungs in animals with DAD. Our findings suggest that Tag7-derived peptides might be beneficial in terms of the therapy or prevention of acute lung injury, e.g., for treating COVID-19 patients with severe pulmonary lesions.

Charge-mediated proteasome targeting.

A majority of thousands of intracellular mammalian proteins are recognized by proteasome only being conjugated with ubiquitin (Ub), representing a universal degradation signal operated by the ubiquitination system. Ub-independent proteasome targeting is rationalized by the existence of 2 types of direct proteasome signals (DPSs), specific amino acid sequences or post-translational modifications, which are recognized by proteasome regulatory subunits. Historically, the first type was shown to exist in ornithine decarboxylase, whereas acetylation of core histones recently was reported as a second type of DPS. Here we declare a third type, representing charge-mediated DPS. This discovered DPS may be classified as a monopartite composition- but not sequence-dependent element of ∼70 Å in length enriched in basic and flexible amino acids. This type of degradation signal, which may be provided by cationic chemicals, is most efficiently engaged by proteasomes capped with regulator (REG)α or REGγ in an ATP-independent manner. Taken together, our findings suggest a novel modality of proteasome-substrate interrelation bypassing ubiquitination.-Kudriaeva, A., Kuzina, E. S., Zubenko, O., Smirnov, I. V., Belogurov, A. Charge-mediated proteasome targeting.

Salicylic acid influences the protease activity and posttranslation modifications of the secreted peptides in the moss Physcomitrella patens.

Plant secretome comprises dozens of secreted proteins. However, little is known about the composition of the whole secreted peptide pools and the proteases responsible for the generation of the peptide pools. The majority of studies focus on target detection and characterization of specific plant peptide hormones. In this study, we performed a comprehensive analysis of the whole extracellular peptidome, using moss Physcomitrella patens as a model. Hundreds of modified and unmodified endogenous peptides that originated from functional and nonfunctional protein precursors were identified. The plant proteases responsible for shaping the pool of endogenous peptides were predicted. Salicylic acid (SA) influenced peptide production in the secretome. The proteasome activity was altered upon SA treatment, thereby influencing the composition of the peptide pools. These results shed more light on the role of proteases and posttranslational modification in the "active management" of the extracellular peptide pool in response to stress conditions. It also identifies a list of potential peptide hormones in the moss secretome for further analysis.

The Transcriptome of Type I Murine Astrocytes under Interferon-Gamma Exposure and Remyelination Stimulus.

Astrocytes are considered to be an important contributor to central nervous system (CNS) disorders, particularly multiple sclerosis. The transcriptome of these cells is greatly affected by cytokines released by lymphocytes, penetrating the blood-brain barrier-in particular, the classical pro-inflammatory cytokine interferon-gamma (IFNγ). We report here the transcriptomal profiling of astrocytes treated using IFNγ and benztropine, a putative remyelinization agent. Our findings indicate that the expression of genes involved in antigen processing and presentation in astrocytes are significantly upregulated upon IFNγ exposure, emphasizing the critical role of this cytokine in the redirection of immune response towards self-antigens. Data reported herein support previous observations that the IFNγ-induced JAK-STAT signaling pathway may be regarded as a valuable target for pharmaceutical interventions.

Ubiquitin-independent proteosomal degradation of myelin basic protein contributes to development of neurodegenerative autoimmunity.

Recent findings indicate that the ubiquitin-proteasome system is involved in the pathogenesis of cancer as well as autoimmune and several neurodegenerative diseases, and is thus a target for novel therapeutics. One disease that is related to aberrant protein degradation is multiple sclerosis, an autoimmune disorder involving the processing and presentation of myelin autoantigens that leads to the destruction of axons. Here, we show that brain-derived proteasomes from SJL mice with experimental autoimmune encephalomyelitis (EAE) in an ubiquitin-independent manner generate significantly increased amounts of myelin basic protein peptides that induces cytotoxic lymphocytes to target mature oligodendrocytes ex vivo. Ten times enhanced release of immunogenic peptides by cerebral proteasomes from EAE-SJL mice is caused by a dramatic shift in the balance between constitutive and ß1i(high) immunoproteasomes in the CNS of SJL mice with EAE. We found that during EAE, ß1i is increased in resident CNS cells, whereas ß5i is imported by infiltrating lymphocytes through the blood-brain barrier. Peptidyl epoxyketone specifically inhibits brain-derived ß1i(high) immunoproteasomes in vitro (kobs/[I] = 240 M(-1)s(-1)), and at a dose of 0.5 mg/kg, it ameliorates ongoing EAE in vivo. Therefore, our findings provide novel insights into myelin metabolism in pathophysiologic conditions and reveal that the ß1i subunit of the immunoproteasome is a potential target to treat autoimmune neurologic diseases.

Multiple sclerosis autoantigen myelin basic protein escapes control by ubiquitination during proteasomal degradation.

The vast majority of cellular proteins are degraded by the 26S proteasome after their ubiquitination. Here, we report that the major component of the myelin multilayered membrane sheath, myelin basic protein (MBP), is hydrolyzed by the 26S proteasome in a ubiquitin-independent manner both in vitro and in mammalian cells. As a proteasomal substrate, MBP reveals a distinct and physiologically relevant concentration range for ubiquitin-independent proteolysis. Enzymatic deimination prevents hydrolysis of MBP by the proteasome, suggesting that an abnormally basic charge contributes to its susceptibility toward proteasome-mediated degradation. To our knowledge, our data reveal the first case of a pathophysiologically important autoantigen as a ubiquitin-independent substrate of the 26S proteasome.

Myelin basic protein antagonizes the SARS-CoV-2 protein ORF3a-induced autophagy inhibition.

Inhibition of autophagy is one of the hallmarks of the SARS-CoV-2 infection. Recently it was reported that SARS-CoV-2 protein ORF3a inhibits fusion of autophagosomes with lysosomes via interaction with VPS39 thus preventing binding of homotypic fusion and protein sorting (HOPS) complex to RAB7 GTPase. Here we report that myelin basic protein (MBP), a major structural component of the myelin sheath, binds ORF3a and is colocalized with it in mammalian cells. Co-expression of MBP with ORF3a restores autophagy in mammalian cells, inhibited by viral protein. Our data suggest that basic charge of MBP drives suppression of ORF3a-induced autophagy inhibition as its deaminated variants lost ability to bind ORF3a and counteract autophagy blockade. These results together with our recent findings, indicating that MBP interacts with structural components of the vesicle transport machinery-synaptosomal-associated protein 23 (SNAP23), vesicle-associated membrane protein 3 (VAMP3) and Sec1/Munc18-1 family members, may suggest protective role of the MBP in terms of the maintaining of protein traffic and autophagosome-lysosome fusion machinery in oligodendrocytes during SARS-CoV-2 infection. Finally, our data may indicate that deimination of MBP observed in the patients with multiple sclerosis (MS) may contribute to the previously reported worser outcomes of COVID-19 and increase of post-COVID-19 neurologic symptoms in patients with MS.

Asymmetric T-cell division: insights from cutting-edge experimental techniques and implications for immunotherapy.

Asymmetric cell division is a fundamental process conserved throughout evolution, employed by both prokaryotic and eukaryotic organisms. Its significance lies in its ability to govern cell fate and facilitate the generation of diverse cell types. Therefore, attaining a detailed mechanistic understanding of asymmetric cell division becomes essential for unraveling the complexities of cell fate determination in both healthy and pathological conditions. However, the role of asymmetric division in T-cell biology has only recently been unveiled. Here, we provide an overview of the T-cell asymmetric division field with the particular emphasis on experimental methods and models with the aim to guide the researchers in the selection of appropriate in vitro/in vivo models to study asymmetric division in T cells. We present a comprehensive investigation into the mechanisms governing the asymmetric division in various T-cell subsets underscoring the importance of the asymmetry in fate-determining factor segregation and transcriptional and epigenetic regulation. Furthermore, the intricate interplay of T-cell receptor signaling and the asymmetric division geometry are explored, shedding light on the spatial organization and the impact on cellular fate.

Anticancer therapeutic strategies for targeting mutant p53-Y220C.

The tumor suppressor p53 is a transcription factor with a powerful antitumor activity that is controlled by its negative regulator murine double minute 2 (MDM2, also termed HDM2 in humans) through a feedback mechanism. At the same time, TP53 is the most frequently mutated gene in human cancers. Mutant p53 proteins lose wild-type p53 tumor suppression functions but acquire new oncogenic properties, among which are deregulating cell proliferation, increasing chemoresistance, disrupting tissue architecture, and promoting migration, invasion and metastasis as well as several other pro-oncogenic activities. The oncogenic p53 mutation Y220C creates an extended surface crevice in the DNA-binding domain destabilizing p53 and causing its denaturation and aggregation. This cavity accommodates stabilizing small molecules that have therapeutic values. The development of suitable small-molecule stabilizers is one of the therapeutic strategies for reactivating the Y220C mutant protein. In this review, we summarize approaches that target p53-Y220C, including reactivating this mutation with small molecules that bind Y220C to the hydrophobic pocket and developing immunotherapies as the goal for the near future, which target tumor cells that express the p53-Y220C neoantigen.

Systemic lupus erythematosus therapeutic strategy: From immunotherapy to gut microbiota modulation.

Systemic lupus erythematosus (SLE) is characterized by a systemic dysfunction of the innate and adaptive immune systems, leading to an attack on healthy tissues of the body. During the development of SLE, pathogenic features, such as the formation of autoantibodies to self-nuclear antigens, caused tissue damage including necrosis and fibrosis, with an increased expression of type Ⅰ interferon (IFN) regulated genes. Treatment of lupus with immunosuppressants and glucocorticoids, which are used as the standard therapy, is not effective enough and causes side effects. As an alternative, more effective immunotherapies have been developed, including monoclonal and bispecific antibodies that target B cells, T cells, co-stimulatory molecules, cytokines or their receptors, and signaling molecules. Encouraging results have been observed in clinical trials with some of these therapies. Furthermore, a chimeric antigen receptor T cells (CAR-T) therapy has emerged as the most effective, safe, and promising treatment option for SLE, as demonstrated by successful pilot studies. Additionally, emerging evidence suggests that gut microbiota dysbiosis may play a significant role in the severity of SLE, and the use of methods to normalize the gut microbiota, particularly fecal microbiota transplantation (FMT), opens up new opportunities for effective treatment of SLE.

Incautious design of shRNAs for stable overexpression of miRNAs could result in generation of undesired isomiRs.

shRNA-mediated strategy of miRNA overexpression based on RNA Polymerase III (Pol III) expression cassettes is widely used for miRNA functional studies. For some miRNAs, e.g., encoded in the genome as a part of a polycistronic miRNA cluster, it is most likely the only way for their individual stable overexpression. Here we have revealed that expression of miRNAs longer than 19 nt (e.g. 23 nt in length hsa-miR-93-5p) using such approach could be accompanied by undesired predominant generation of 5' end miRNA isoforms (5'-isomiRs). Extra U residues (up to five) added by Pol III at the 3' end of the transcribed shRNA during transcription termination could cause a shift in the Dicer cleavage position of the shRNA. This results in the formation of 5'-isomiRs, which have a significantly altered seed region compared to the initially encoded canonical hsa-miR-93-5p. We demonstrated that the commonly used qPCR method is insensitive to the formation of 5'-isomiRs and cannot be used to confirm miRNA overexpression. However, the predominant expression of 5'-isomiRs without three or four first nucleotides instead of the canonical isoform could be disclosed based on miRNA-Seq analysis. Moreover, mRNA sequencing data showed that the 5'-isomiRs of hsa-miR-93-5p presumably regulate their own mRNA targets. Thus, omitting miRNA-Seq analysis may lead to erroneous conclusions regarding revealed mRNA targets and possible molecular mechanisms in which studied miRNA is involved. Overall, the presented results show that structures of shRNAs for stable overexpression of miRNAs requires careful design to avoid generation of undesired 5'-isomiRs.

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-1ß/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/ß, 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.

Identification of Myelin Basic Protein Proximity Interactome Using TurboID Labeling Proteomics.

Myelin basic protein (MBP) is one of the key structural elements of the myelin sheath and has autoantigenic properties in multiple sclerosis (MS). Its intracellular interaction network is still partially deconvoluted due to the unfolded structure, abnormally basic charge, and specific cellular localization. Here we used the fusion protein of MBP with TurboID, an engineered biotin ligase that uses ATP to convert biotin to reactive biotin-AMP that covalently attaches to nearby proteins, to determine MBP interactome. Despite evident benefits, the proximity labeling proteomics technique generates high background noise, especially in the case of proteins tending to semi-specific interactions. In order to recognize unique MBP partners, we additionally mapped protein interaction networks for deaminated MBP variant and cyclin-dependent kinase inhibitor 1 (p21), mimicking MBP in terms of natively unfolded state, size and basic amino acid clusters. We found that in the plasma membrane region, MBP is colocalized with adhesion proteins occludin and myelin protein zero-like protein 1, solute carrier family transporters ZIP6 and SNAT1, Eph receptors ligand Ephrin-B1, and structural components of the vesicle transport machinery-synaptosomal-associated protein 23 (SNAP23), vesicle-associated membrane protein 3 (VAMP3), protein transport protein hSec23B and cytoplasmic dynein 1 heavy chain 1. We also detected that MBP potentially interacts with proteins involved in Fe2+ and lipid metabolism, namely, ganglioside GM2 activator protein, long-chain-fatty-acid-CoA ligase 4 (ACSL4), NADH-cytochrome b5 reductase 1 (CYB5R1) and metalloreductase STEAP3. Assuming the emerging role of ferroptosis and vesicle cargo docking in the development of autoimmune neurodegeneration, MBP may recruit and regulate the activity of these processes, thus, having a more inclusive role in the integrity of the myelin sheath.