Elena A. Romanova

Peptidoglycan Recognition Protein Tag7 Forms a Cytotoxic Complex with Heat Shock Protein 70 in Solution and in Lymphocytes

The peptidoglycan recognition protein Tag7 is shown to form a stable 1:1 complex with the major stress protein Hsp70. Neither protein is cytotoxic by itself, but their complex induces apoptotic death in several tumor-derived cell lines even at subnanomolar concentrations. The minimal part of Hsp70 needed to evoke cytotoxicity is residues 450–463 of its peptide-binding domain, but full cytotoxicity requires its ATPase activity; remarkably, Tag7 liberated from the complex at high ATP is not cytotoxic. The Tag7-Hsp70 complex is produced by tag7-transfected cells and by lymphokine-activated killers, being assembled within the cell and released into the medium through the Golgi apparatus by a mechanism different from the commonly known granule exocytosis. Thus, we demonstrate how a heat shock protein may perform functions clearly distinct from chaperoning or cell rescue and how peptidoglycan recognition proteins may be involved in innate immunity and anti-cancer defense.

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+ (≈0.5% of total lymphocytes in culture). Remarkably, similar effectors with at least the same activity are often found in fresh donor blood samples (≈104 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.

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

Background: A complex containing an innate immunity protein Tag7, and Hsp70 kills various cancer cells. Results: Tag7 and its complex with Hsp70 bind to the TNFR1 receptor, but only the Tag7-Hsp70 complex induces a cytotoxic effect via apoptosis and necroptosis. Conclusion: A new ligand has been found for the TNFR1 death receptor. Significance: Tag7 may be used as an inhibitor of the TNF-α-induced cytotoxicity. 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.

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.

Comparative analysis of secretion of S100A4 metastatic marker by immune and tumor cells

S100A4 protein is present in low concentrations (2.1–15.7 ng/106 cells) in lymphocyte and neutrophil culture medium. Addition of stimulants to the cells did not lead to an appreciable increase in the content of this protein. The initial content of S100A4 is significantly higher (92–447 ng/106 cells) in culture media of highly metastatic KSML-100 adenocarcinoma and M3 and B16 melanoma cells. The release of S100A4 by these cells significantly increased after addition of lymphocytes and Tag7/Hsp70 cytotoxic complex. Repeated injection of antibodies to S100A4 to mice with transplanted M3 melanoma inhibited tumor growth.

A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7–Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4+ and CD8+ lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.

Innate Immunity Protein Tag7 Induces 3 Distinct Populations of Cytotoxic Cells That Use Different Mechanisms to Exhibit Their Antitumor Activity on Human Leukocyte Antigen-Deficient Cancer Cells

The search for new immune response mechanisms capable of controlling immune-evasive tumor cells devoid of the MHC antigen is a challenging task for immunologists. In this study, we found that the treatment of human peripheral blood lymphocytes with the innate immunity protein Tag7 (PGRP-S, PGLYRP1) induces differentiation of the populations of NK (natural killer) cells and CD8+ and CD4+ T lymphocytes that are cytotoxic for human leukocyte antigen-negative tumor cells. These populations employ different mechanisms of tumor cell lysis (based on the release of granzymes in the case of NK cells and on the FasL-Fas interaction in the case of CD8+ and CD4+ T lymphocytes) and induce different death pathways (apoptosis or necroptosis) in tumor cells. An analysis of genes activated in leukocyte populations after Tag7 treatment and experiments with specific inhibitors have shown that the TREM-1 receptor expressed on the monocyte cell surface is essential for activation of cytotoxic activity. Overall, the results of this study provide evidence for a novel role of the Tag7 protein in the immune response.

The Tag7–Hsp70 cytotoxic complex induces tumor cell necroptosis via permeabilisation of lysosomes and mitochondria

Tag7 (PGRP-S, or PGLYRP1), an innate immunity protein, plays an important role in the immune defense system. It forms a stable cytotoxic complex with the heat shock protein Hsp70. This complex can induce an apoptotic or necroptotic tumor cell death by interacting with the TNFR1 receptor. In this study, we analyzed molecular events involved in the process of the Tag7-Hsp70-induced necroptosis. We found that Tag7 can bind to sTNFR1, a soluble fragment of the TNFR1 receptor, leading to an inhibition of the RIP1 dependent necroptosis. A major role in the downstream phases of the Tag7-Hsp70 induced necroptosis was played by an interaction between lysosomes and mitochondria. The interaction of Tag7-Hsp70 with the TNFR1 receptor triggered a certain sequence of events: at first, it activated RIP1 kinase, and later on, increased intracellular concentration of Са(2+) ions and an activation of calpains, which led to the permeabilization of the lysosomal membranes. The consequent release of the lysosomal enzymes, including cathepsins B and D, resulted in the depolarization of the mitochondrial membrane, ROS production, and eventual cell death.

Cell death of L-929 cells induced by cytotoxic complex Tag7-Hsp70 is analogous to the death of the same cells induced by TNF-α

The identification and studying the molecular bases of functioning of new cytotoxic agents finds an important implication in developing drugs for fighting with tumors. While investigating the cytotoxic action of protein complex Tag7-Hsp70 which was opened in our laboratory previously we found that Tag7-Hsp70 demonstrated the same specificity in regard to different tumor target cells as it was for classical cytokine TNF-α. L-929 cells and Jurkat cells appeared to be good targets representing up to 30% of dead cells within a population and HeLa cells-bad targets representing less than 5% of dead cells after 20 h of incubation with either of the cytotoxic agents. While investigating the action of either TNF-α or Tag7-Hsp70 on L-929 cells we detected two peaks of death: after 3 h and after 20 h. For both cytotoxic agents we observed the first, smaller (13–15%), peak to be eliminated after the addition of caspase inhibitor YVAD-CHO and the second, greater (25–30%), peak to become even bigger in presence of caspase inhibitor. Probably, protein complex Tag7-Hsp70 interacts like TNF-α with a receptor on the surface of tumor cells that results in triggering two alternative mechanisms of programmed cell death: apoptosis and necroptosis.

Interactions and possible functional characteristics of Tag7-S100A4 protein complex.

Peptidoglycane-recognizing protein Tag7 formed a complex with S100A4 (a representative of S100 protein family), the apparent dissociation constants in the absence and presence of Ca2+ were 2×10−8 M and 10−9 M, respectively. Analysis of fluorescence spectra of hydrophobic fluorescent probe 2-toluidinyl naphthalene-6-sulfonate in the presence of S100A4 and Tag7 proteins showed that extensive area or several sites are involved into the complex formation between these proteins. The formation of Tag7-S100A4 complex had virtually no effect on the role of S100A4 in the regulation of intracellular Ca2+ metabolism. Removal of not only Tag7, but also S100A4 from neutrophil conditioned medium reduced lysis of E. coli cell, while addition of the Tag7-S100A4 complex to the medium restored antibacterial activity.