Mts1 (S100A4) and Its Peptide Demonstrate Cytotoxic Activity in Complex with Tag7 (PGLYRP1) Peptide.

Receptors of cytokines are major regulators of the immune response. In this work, we have discovered two new ligands that can activate the TNFR1 (tumor necrosis factor receptor 1) receptor. Earlier, we found that the peptide of the Tag (PGLYRP1) protein designated 17.1 can interact with the TNFR1 receptor. Here, we have found that the Mts1 (S100A4) protein interacts with this peptide with a high affinity (Kd = 1.28 × 10-8 M), and that this complex is cytotoxic to cancer cells that have the TNFR1 receptor on their surface. This complex induces both apoptosis and necroptosis in cancer cells with the involvement of mitochondria and lysosomes in cell death signal transduction. Moreover, we have succeeded in locating the Mts1 fragment that is responsible for protein-peptide interaction, which highly specifically interacts with the Tag7 protein (Kd = 2.96 nM). The isolated Mts1 peptide M7 also forms a complex with 17.1, and this peptide-peptide complex also induces the TNFR1 receptor-dependent cell death. Molecular docking and molecular dynamics experiments show the amino acids involved in peptide binding and that may be used for peptidomimetics' development. Thus, two new cytotoxic complexes were created that were able to induce the death of tumor cells via the TNFR1 receptor. These results may be used in therapy for both cancer and autoimmune diseases.

The Interaction of HMGB1 with the Proinflammatory TREM-1 Receptor Generates Cytotoxic Lymphocytes Active against HLA-Negative Tumor Cells.

High mobility group protein (HMGB1) is secreted by myeloid cells and cells of damaged tissues during inflammation, causing inflammatory reactions through various receptors, including TLRS and RAGE. TREM-1 is considered to be one of the potential HMGB1 receptors. In this work, we have shown that the HMGB1 protein is able to bind to the TREM-1 receptor at high affinity both in solution and on the cell surface. This binding causes lymphocytes to release cytokines IL-2, IL-1b, IL-6, TNF and Ifny into the medium, which leads to the appearance of cytotoxic lymphocytes in PBMC capable of lysing HLA-negative tumor cells. Expanding the spectra of proinflammatory receptor ligands and understanding the mechanisms of their action is essential for the creation of new immunotherapy pathways.

The 12-Membered TNFR1 Peptide, as Well as the 16-Membered and 6-Membered TNF Peptides, Regulate TNFR1-Dependent Cytotoxic Activity of TNF.

Understanding the exact mechanisms of the activation of proinflammatory immune response receptors is very important for the targeted regulation of their functioning. In this work, we were able to identify the sites of the molecules in the proinflammatory cytokine TNF (tumor necrosis factor) and its TNFR1 (tumor necrosis factor receptor 1), which are necessary for the two-stage cytotoxic signal transduction required for tumor cell killing. A 12-membered TNFR1 peptide was identified and synthesized, interacting with the ligands of this receptor protein's TNF and Tag7 and blocking their binding to the receptor. Two TNF cytokine peptides interacting with different sites of TNFR1 receptors were identified and synthesized. It has been demonstrated that the long 16-membered TNF peptide interferes with the binding of TNFR1 ligands to this receptor, and the short 6-membered peptide interacts with the receptor site necessary for the transmission of a cytotoxic signal into the cell after the ligands' interaction with the binding site. This study may help in the development of therapeutic approaches to regulate the activity of the cytokine TNF.

Short Peptides of Innate Immunity Protein Tag7 (PGLYRP1) Selectively Induce Inhibition or Activation of Tumor Cell Death via TNF Receptor.

In this study, we have found two peptides of Tag7 (PGLYRP1) protein-17.1A (HRDVQRT) and 17.1B (RSNYVLKG), that have different affinities to the TNFR1 receptor and the Hsp70 protein. Peptide 17.1A is able to inhibit signal transduction through the TNFR1 receptor, and peptide 17.1B can activate this receptor in a complex with Hsp70. Thus, it is possible to modulate the activity of the TNFR1 receptor and further perform its specific inhibition or activation in the treatment of various autoimmune or oncological diseases.

N-Terminal Peptide of PGLYRP1/Tag7 Is a Novel Ligand for TREM-1 Receptor.

An investigation of innate immunity receptors sheds light on the mechanisms of inflammation and associated immune reactions. One of the key immune regulators is the TREM-1 receptor, which is involved in both inflammation and antitumor immune response. In this article, we have obtained a new ligand for the TREM-1 receptor. The peptide, named N3, is a part of the innate immune protein PGLYRP1/Tag7. It is responsible for activating the TREM-1 signaling pathway. Here, we have demonstrated that the N3 peptide acts like other TREM-1 receptor ligands: its binding results in a mild inflammation response and appearance of cytotoxic lymphocytes. We have shown that cytotoxic populations of lymphocytes in N3 peptide-treated PBMCs are similar to those treated with Tag7 or Hsp70. We also determined the part of the N3 peptide responsible for binding to TREM-1. The resulting peptide (N9) consists of nine amino acids and can be considered as a potential peptide that blocks TREM-1 signaling.

Short Peptides of Innate Immunity Protein Tag7 Inhibit the Production of Cytokines in CFA-Induced Arthritis.

The pathogenesis of autoimmune arthritis is a hot topic in current research. The main focus of this work was to study cytokines released in CFA-induced arthritis in ICR mice as well as the regulation of blood levels of cytokines by two peptides of the innate immunity protein Tag7 (PGLYRP1) capable of blocking the activation of the TNFR1 receptor. Arthritis was induced by local periarticular single-dose injections of 40 µL of complete Freund's adjuvant (CFA) into the left ankle joints of mice. The levels of chemokines and cytokines in plasma were measured using a Bio-Plex Pro Mouse Cytokine Kit at 3, 10, and 21 days after arthritis induction. Tag7 peptides were shown to decrease the blood levels of the pro-inflammatory cytokines IL-6, TNF, and IL-1ß. Administration of peptides also decreased the levels of chemokines MGSA/CXCL1, MIP-2α/CXCL2, ENA78/CXCL5, MIG/CXCL9, IP-10/CXCL10, MCP-1/CCL2, and RANTES/CCL5. Furthermore, a decrease in the levels of cytokines IL7, G-CSF, and M-CSF was demonstrated. Addition of the studied peptides strongly affected IFN-γ concentration. We believe that a decrease in the levels of cytokine IFN-γ was associated with a therapeutic effect of Tag7 peptides manifested in alleviation of the destruction of cartilage and bone tissues in the CFA-induced arthritis.

Hsp70 Interacts with the TREM-1 Receptor Expressed on Monocytes and Thereby Stimulates Generation of Cytotoxic Lymphocytes Active against MHC-Negative Tumor Cells.

The search for and analysis of new ligands for innate immunity receptors are of special significance for understanding the regulatory mechanisms of immune response. Here we show that the major heat shock protein 70 (Hsp70) can bind to and activate TREM-1, the innate immunity receptor expressed on monocytes. The Hsp70-TREM-1 interaction activates expression of TNFα and IFNγ mRNAs in monocytes and stimulates IL-2 secretion by PBMCs. Moreover, incubation of PBMCs with Hsp70 leads to an appearance of cytotoxic lymphocyte subpopulations active against the MHC-negative tumor cells. In addition, both the CD4+ T-lymphocytes and CD14+ monocytes are necessary for the Hsp70 signal transduction and a consequent activation of the cytotoxic lymphocytes. We believe that data presented in this study will broaden the views on the involvement of Hsp70 in the antitumor immunity.

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.

Tilorone activates NK cells and cytotoxic lymphocytes that kill HLA-negative tumor cells.

Tilorone hydrochloride, a low-molecular-weight synthetic compound, induces interferon production and has been reported to have both antiviral and antitumor activities. Here, we have demonstrated the ability of tilorone to activate NK cells and specific subpopulations of cytotoxic CD4+ and CD8+ T lymphocytes that recognize immune-evasive tumor cells and kill them via the FasL-Fas interaction. We have also performed a comparative analysis of characteristics between lymphocytes activated in the fraction of human peripheral blood mononuclear cells (PBMCs) upon treatment with different stimulants of the immune response: tilorone, innate immunity protein Tag7, and cytokine IL-2, a regulator of adaptive immunity. The results show that all the three stimulants, regardless of their nature, activate lymphocytes that are identical with respect to the spectrum of target cells, phenotype, and mechanism of cytotoxic action However, these stimulants induce different mechanisms of lymphocyte activation at early stages of the immune response. © 2018 IUBMB Life, 71(3):376-384, 2019.

CD3+ CD8+ NKG2D+ T Lymphocytes Induce Apoptosis and Necroptosis in HLA-Negative Cells via FasL-Fas Interaction.

An important problem in cellular immunology is to identify new populations of cytotoxic lymphocytes capable of killing tumor cells that have lost classical components of MHC-machinery and to understand mechanisms of the death of these cells. We have previously found that CD4+ CD25+ lymphocytes appear in the lymphokine-activated killer (LAK) cell culture, which carry Tag7 (PGRP-S) and FasL proteins on their surface and can kill Hsp70- and Fas-expressing HLA-negative cells. In this work, we have continued to study the mechanisms of killing of the HLA-negative tumor cells, focusing this time on the CD8+ lymphocytes. We show that after a tumor antigen contact the IL-2 activated CD8+ lymphocytes acquire ability to lyse tumor cells bearing this antigen. However, activation of the CD8+ lymphocytes in the absence of antigen causes appearance of a cytotoxic population of CD8+ NKG2D+ lymphocytes, which are able to lyse HLA-negative cancer cells that have lost the classic mechanism of antigen presentation. These cells recognize the noncanonical MicA antigen on the surface of HLA-negative K562 cells but kill them via the FasL-Fas interaction, as do cytotoxic T lymphocytes. FasL presented on the lymphocyte surface can trigger both apoptosis and necroptosis. Unlike in the case of TNFR1, another cell death receptor, no switching to alternative processes has been observed upon induction of Fas-dependent cell death. It may well be that the apoptotic and necroptotic signals are transduced separately in the latter case, with the ability of FasL+ lymphocytes to induce necroptosis allowing them to kill tumor cells that escape apoptosis. J. Cell. Biochem. 118: 3359-3366, 2017. © 2017 Wiley Periodicals, Inc.

Innate immunity protein Tag7 (PGRP-S) activates lymphocytes capable of Fasl-Fas-dependent contact killing of virus-infected cells.

The innate immunity protein Tag7 (PGRP-S, PGLYRP1) is involved in antimicrobial and antitumor defense. As shown in our previous studies, Tag7 specifically interacts with the major heat shock protein Hsp70 to form a stable Tag7-Hsp70 complex with cytotoxic activity against tumor cells. A stable complex of Tag7 with the calcium-binding protein Mts1 (S100A4) stimulates migration of lymphocytes. Moreover, Tag7 can activate cytotoxic lymphocytes that recognize and kill HLA-negative tumor cells. Here, we have shown that Tag 7 treatment of human peripheral blood mononuclear cells (PBMCs) results in activation of different cytotoxic lymphocyte populations-natural killer (NK) cells and CD8+ NKG2D+ T lymphocytes-that kill Moloney murine leukemia virus (MMLV) infected SC-1 cells using different mechanisms of cell death induction. This mechanism in NK cells is based on the release of granzymes, which activate apoptosis in target cells, while CD8+ NKG2D+ T lymphocytes recognize the noncanonical MicA antigen on the surface of virus-containing cells and kill them via the FasL-Fas interaction, triggering the apoptotic or necroptotic cell death pathway. Preliminary incubation of PBMCs with virus-infected cells and following incubation with Tag7 results in activation of lymphocytes with a different phenotype. These lymphocytes change the spectrum of target cells and the mechanism of cell death induction, and their interaction with target cells is not species-specific. © 2017 IUBMB Life, 69(12):971-977, 2017.

FasL and the NKG2D receptor are required for the secretion of the Tag7/PGRP-S-Hsp70 complex by the cytotoxic CD8+ lymphocytes.

Tag7 (PGRP-S or PGLYRP1), while possessing an antimicrobial activity, also exhibits an antitumor effect when in complex with the major heat shock protein Hsp70. The cytotoxic Tag7-Hsp70 complex is secreted by lymphocytes after interaction with the HLA-negative tumors. Previously, we have shown that IL-2 induces formation of the CD4+ and CD8+ cytotoxic subpopulations of human lymphocytes, which kill tumor cells through the FasL-Fas interaction. Here, we show that only the CD8+ T cells are able to secrete the Tag7-Hsp70 complex. For its secretion the same proteins on the surface of the lymphocytes and target cells, which are involved in the contact lysis, are necessary as well. The interaction of Fas receptor with FasL leads to an activation of the Tag7-Hsp70 complex in the lymphocyte membrane fraction, and here FasL acts as a receptor that induces intracellular signaling in lymphocytes. An interaction of the MicA stress ligand with the NKG2D receptor is necessary for the release of this cytotoxic complex. It is possible, that CD8+ T lymphocytes interacting with a target cell can both carry out the contact killing of these cells and to secrete the cytotoxic factor. © 2016 IUBMB Life, 69(1):30-36, 2017.

Tag7 (PGLYRP1) in Complex with Hsp70 Induces Alternative Cytotoxic Processes in Tumor Cells via TNFR1 Receptor.

Tag7 (also known as peptidoglycan recognition protein PGRP-S, PGLYRP1), an innate immunity protein, interacts with Hsp70 to form a stable Tag7-Hsp70 complex with cytotoxic activity against some tumor cell lines. In this study, we have analyzed the programmed cell death mechanisms that are induced when cells interact with the Tag7-Hsp70 complex, which was previously shown to be released by human lymphocytes and is cytotoxic to cancer cells. We show that this complex induces both apoptotic and necroptotic processes in the cells. Apoptosis follows the classic caspase-8 and caspase-3 activation pathway. Inhibition of apoptosis leads to a switch to the RIP1-dependent necroptosis. Both of these cytotoxic processes are initiated by the involvement of TNFR1, a receptor for TNF-α. Our results suggest that the Tag7-Hsp70 complex is a novel ligand for this receptor. One of its components, the innate immunity protein Tag7, can bind to the TNFR1 receptor, thereby inhibiting the cytotoxic actions of the Tag7-Hsp70 complex and TNF-α, an acquired immunity cytokine.

Liposome-encapsulated peptides protect against experimental allergic encephalitis.

Multiple sclerosis (MS) is a severe inflammatory and neurodegenerative disease with an autoimmune background. Despite the variety of therapeutics available against MS, the development of novel approaches to its treatment is of high importance in modern pharmaceutics. In this study, experimental autoimmune encephalomyelitis (EAE) in Dark Agouti rats has been treated with immunodominant peptides of the myelin basic protein (MBP) encapsulated in mannosylated small unilamellar vesicles. The results show that liposome-encapsulated MBP(46-62) is the most effective in reducing maximal disease score during the first attack, while MBP(124-139) and MBP(147-170) can completely prevent the development of the exacerbation stage. Both mannosylation of liposomes and encapsulation of peptides are critical for the therapeutic effect, since neither naked peptides nor nonmannosylated liposomes, loaded or empty, have proved effective. The liposome-mediated synergistic effect of the mixture of 3 MBP peptides significantly suppresses the progression of protracted EAE, with the median cumulative disease score being reduced from 22 to 14 points, compared to the placebo group; prevents the production of circulating autoantibodies; down-regulates the synthesis of Th1 cytokines; and induces the production of brain-derived neurotrophic factor in the central nervous system. Thus, the proposed formulation ameliorates EAE, providing for a less severe first attack and rapid recovery from exacerbation, and offers a promising therapeutic modality in MS treatment.

The heat shock-binding protein (HspBP1) protects cells against the cytotoxic action of the Tag7-Hsp70 complex.

Heat shock-binding protein HspBP1 is a member of the Hsp70 co-chaperone family. The interaction between HspBP1 and the ATPase domain of the major heat shock protein Hsp70 up-regulates nucleotide exchange and reduces the affinity between Hsp70 and the peptide in its peptide-binding site. Previously we have shown that Tag7 (also known as peptidoglycan recognition protein PGRP-S), an innate immunity protein, interacts with Hsp70 to form a stable Tag7-Hsp70 complex with cytotoxic activity against some tumor cell lines. This complex can be produced in cytotoxic lymphocytes and released during interaction with tumor cells. Here the effect of HspBP1 on the cytotoxic activity of the Tag7-Hsp70 complex was examined. HspBP1 could bind not only to Hsp70, but also to Tag7. This interaction eliminated the cytotoxic activity of Tag7-Hsp70 complex and decreased the ATP concentration required to dissociate Tag7 from the peptide-binding site of Hsp70. Moreover, HspBP1 inhibited the cytotoxic activity of the Tag7-Hsp70 complex secreted by lymphocytes. HspBP1 was detected in cytotoxic CD8+ lymphocytes. This protein was released simultaneously with Tag7-Hsp70 during interaction of these lymphocytes with tumor cells. The simultaneous secretion of the cytotoxic complex with its inhibitor could be a mechanism protecting normal cells from the cytotoxic effect of this complex.

Opposite roles of metastasin (S100A4) in two potentially tumoricidal mechanisms involving human lymphocyte protein Tag7 and Hsp70.

We compare the physical and functional interactions between three widespread multifunctional proteins [metastasin (Mts1/S100A4), innate immunity-related Tag7/PGRP-S, and Hsp70] in two experimental models relevant to host-tumor relationships on humoral and cellular levels. (i) Tag7 and Hsp70 in solution or in a lymphocyte make a stable binary complex that is highly cytotoxic for some tumor cells. Here, we show that Mts1 prevents Tag7.Hsp70 assembly in solution, and an excess of Mts1 disrupts the existing Tag7.Hsp70 complex; accordingly, Tag7.Hsp70 cytotoxicity (exemplified with L929 cells) is diminished in the presence of excess Mts1. (ii) Tag7 exposed on a specialized subset of lymphokine-activated killer cells makes specific contact with Hsp70 exposed on some HLA-negative tumor cells, thus enabling FasL/Fas-mediated induction of apoptosis. Here, we show that some CD4(+)CD25(+) cells coexpose Mts1 with Tag7 and FasL, that Mts1 and Tag7 closely contact the same Hsp70 molecule on the target K562 cell (as evidenced by cross-linking), and that killing of such targets is abolished by Mts1-specific antibodies (or selective removal of Mts1-exposing lymphocytes). Thus, this phenotype active against immunoevasive cancerous cells is defined as CD4(+)CD25(+), FasL(+), Tag7(+)Mts1(+) (approximately 0.5% of total lymphocytes in culture). Remarkably, similar effectors with at least the same activity are often found in fresh donor blood samples (approximately 10(4) effectors/mL). Thus, our models suggest that interactions between the three proteins in different situations may have opposite functional outcomes as regards antitumor defense, immune escape, and metastasis.

A 8-mer Peptide of PGLYRP1/Tag7 Innate Immunity Protein Binds to TNFR1 Receptor and Inhibits TNFα-Induced Cytotoxic Effect and Inflammation.

Search for novel regulatory protein fragments with potential functional roles is required both for understanding the immune response mechanisms and the development of targeted immunotherapy. Earlier we demonstrated that the PGLYRP1/Tag7 innate immunity protein can be regarded as an inhibitor of TNFα cytotoxic activity via the interaction with its TNF receptor 1 (TNFR1). A C-terminal peptide fragment 17.1 of the molecule is responsible for this function. In this study we have identified a minimal 8-mer region of this peptide (hereinafter - 17.1A) capable to bind to TNFR1. As a result of such interaction, the cytotoxic signals induced by this receptor are blocked. Also, this peptide demonstrates an anti-inflammatory activity in vivo in the complete Freund's adjuvant (CFA)-induced arthritis model in laboratory mice. Peptide 17.1A is capable to reduce periarticular inflammation, inhibit the development of synovitis and exhibit a protective effect on cartilage and bone tissues. This peptide can turn out to be a promising medicinal agent for autoimmune arthritis and other diseases.

Cytokines TNFα, IFNγ and IL-2 Are Responsible for Signal Transmission from the Innate Immunity Protein Tag7 (PGLYRP1) to Cytotoxic Effector Lymphocytes.

Studies on the mechanisms of activation of cytotoxic lymphocyte subpopulations are an important research direction in modern immunology. This study provides a detailed analysis of the effect of Tag7 (PGRP-S, PGLYRP1) on the development of lymphocyte subpopulations cytotoxic against MHC-negative tumor cells in a pool of peripheral blood mononuclear cells (PBMCs). The results show that Tag7 can bind to the TREM-1 receptor on the surfaces of monocytes, thereby triggering the expression of mRNA TNFα and IFNγ. The appearance of these cytokines in conditioned medium leads to IL-2 cytokine secretion by CD3+CD4+ lymphocytes. In turn, IL-2 facilitates unspecific activation of three cytotoxic cell subpopulations in the PBMC pool: NK (CD16+CD56+), CD3+CD4+ and CD3+CD8+. These subpopulations appear after a certain period of incubation with Tag7 and show toxicity against tumor cells.

Autoantibodies from SLE patients induce programmed cell death in murine fibroblast cells through interaction with TNFR1 receptor.

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.

A 12-mer Peptide of Tag7 (PGLYRP1) Forms a Cytotoxic Complex with Hsp70 and Inhibits TNF-Alpha Induced Cell Death.

Investigation of interactions between a pro-inflammatory cytokine tumor necrosis factor (TNFα) and its receptor is required for the development of new treatments for autoimmune diseases associated with the adverse effects of TNFα. Earlier, we demonstrated that the innate immunity protein Tag7 (PGRP-S, PGLYRP1) can interact with the TNFα receptor, TNFR1, and block the transduction of apoptotic signals through this receptor. A complex formed between the Tag7 protein and the major heat shock protein Hsp70 can activate TNFR1 receptor and induce tumor cell death via either apoptotic or necroptotic pathway. In this study, we show that a 12-mer peptide, designated 17.1, which was derived from the Tag7 protein, can be regarded as a novel TNFα inhibitor, also is able to form a cytotoxic complex with the heat shock protein Hsp70. This finding demonstrates a new role for Hsp70 protein in the immune response. Also, this new inhibitory 17.1 peptide demonstrates an anti-inflammatory activity in the complete Freund's adjuvant (CFA)-induced autoimmune arthritis model in laboratory mice. It appears that the 17.1 peptide could potentially be used as an anti-inflammatory agent.