Cystatins, cysteine peptidase inhibitors, as regulators of immune cell cytotoxicity

Cystatins comprise a superfamily of evolutionarily related proteins, present in all living organisms, from protozoa to mammals. They act as inhibitors of cysteine peptidases although they can also function independently of their inhibitory function. Cysteine cathepsins are implicated in various physiological and pathological processes. In the immune response they are involved in antigen processing and presentation, the cytotoxicity of natural killer (NK) cells and cytotoxic T lymphocytes (CTL), migration and adhesion of immune cells, cytokine and growth factor regulation and toll-like receptor signalling. Cystatins are probably involved in the regulation of all these processes; importantly, cystatin F has a crucial role in the regulation of immune cell cytotoxicity. NK cells and CTLs exploit the granzyme/perforin pathway for target cell killing, with perforin and granzymes as crucial effector molecules. Granzymes are synthesized as inactive pro-granzymes and need to be proteolytically activated by cathepsins C and H. Cystatin F is the main regulator of the activity of cathepsins C and H in cytotoxic cells and, consequently, regulates their cytotoxicity. The role of cystatins and cysteine cathepsins in the immune response is presented, with emphasis on their role in the regulation of cytotoxicity of NK cells and CTLs.</p

Human CD4+ T-cell clone expansion leads to the expression of the cysteine peptidase inhibitor cystatin F

The existence of CD4+ cytotoxic T cells (CTLs) at relatively high levels under different pathological conditions in vivo suggests their role in protective and/or pathogenic immune functions. CD4+ CTLs utilize the fundamental cytotoxic effector mechanisms also utilized by CD8+ CTLs and natural killer cells. During long-term cultivation, CD4+ T cells were also shown to acquire cytotoxic functions. In this study, CD4+ human T-cell clones derived from activated peripheral blood lymphocytes of healthy young adults were examined for the expression of cytotoxic machinery components. Cystatin F is a protein inhibitor of cysteine cathepsins, synthesized by CD8+ CTLs and natural killer cells. Cystatin F affects the cytotoxic efficacy of these cells by inhibiting the major progranzyme convertases cathepsins C and H as well as cathepsin L, which is involved in perforin activation. Here, we show that human CD4+ T-cell clones express the cysteine cathepsins that are involved in the activation of granzymes and perforin. CD4+ T-cell clones contained both the inactive, dimeric form as well as the active, monomeric form of cystatin F. As in CD8+ CTLs, cysteine cathepsins C and H were the major targets of cystatin F in CD4+ T-cell clones. Furthermore, CD4+ T-cell clones expressed the active forms of perforin and granzymes A and B. The levels of the cystatin F decreased with time in culture concomitantly with an increase in the activities of granzymes A and B. Therefore, our results suggest that cystatin F plays a role in regulating CD4+ T cell cytotoxicity. Since cystatin F can be secreted and taken up by bystander cells, our results suggest that CD4+ CTLs may also be involved in regulating immune responses through cystatin F secretion

Cysteine Cathepsins as Therapeutic Targets in Immune Regulation and Immune Disorders

Cysteine cathepsins, as the most abundant proteases found in the lysosomes, play a vital role in several processes&mdash;such as protein degradation, changes in cell signaling, cell morphology, migration and proliferation, and energy metabolism. In addition to their lysosomal function, they are also secreted and may remain functional in the extracellular space. Upregulation of cathepsin expression is associated with several pathological conditions including cancer, neurodegeneration, and immune-system dysregulation. In this review, we present an overview of cysteine-cathepsin involvement and possible targeting options for mitigation of aberrant function in immune disorders such as inflammation, autoimmune diseases, and immune response in cancer

Cysteine Cathepsins as Therapeutic Targets in Immune Regulation and Immune Disorders

Cysteine cathepsins, as the most abundant proteases found in the lysosomes, play a vital role in several processes—such as protein degradation, changes in cell signaling, cell morphology, migration and proliferation, and energy metabolism. In addition to their lysosomal function, they are also secreted and may remain functional in the extracellular space. Upregulation of cathepsin expression is associated with several pathological conditions including cancer, neurodegeneration, and immune-system dysregulation. In this review, we present an overview of cysteine-cathepsin involvement and possible targeting options for mitigation of aberrant function in immune disorders such as inflammation, autoimmune diseases, and immune response in cancer

Extracellular Cystatin F Is Internalised by Cytotoxic T Lymphocytes and Decreases Their Cytotoxicity

Cystatin F is a protein inhibitor of cysteine cathepsins, peptidases involved in the activation of the effector molecules of the perforin/granzyme pathway. Cystatin F was previously shown to regulate natural killer cell cytotoxicity. Here, we show that extracellular cystatin F has a role in regulating the killing efficiency of cytotoxic T lymphocytes (CTLs). Extracellular cystatin F was internalised into TALL-104 cells, a cytotoxic T cell line, and decreased their cathepsin C and H activity. Correspondingly, granzyme A and B activity was also decreased and, most importantly, the killing efficiency of TALL-104 cells as well as primary human CTLs was reduced. The N-terminally truncated form of cystatin F, which can directly inhibit cathepsin C (unlike the full-length form), was more effective than the full-length inhibitor. Furthermore, cystatin F decreased cathepsin L activity, which, however, did not affect perforin processing. Cystatin F derived from K-562 target cells could also decrease the cytotoxicity of TALL-104 cells. These results clearly show that, by inhibiting cysteine cathepsin proteolytic activity, extracellular cystatin F can decrease the cytotoxicity of CTLs and thus compromise their function

Extracellular cystatin F is internalised by cytotoxic T lymphocytes and decreases their cytotoxicity

Cystatin F is a protein inhibitor of cysteine cathepsins, peptidases involved in the activation of the effector molecules of the perforin/granzyme pathway. Cystatin F was previously shown to regulate natural killer cell cytotoxicity. Here, we show that extracellular cystatin F has a role in regulating the killing efficiency of cytotoxic T lymphocytes (CTLs). Extracellular cystatin F was internalised into TALL-104 cells, a cytotoxic T cell line, and decreased their cathepsin C and H activity. Correspondingly, granzyme A and B activity was also decreased and, most importantly, the killing efficiency of TALL-104 cells as well as primary human CTLs was reduced. The N-terminally truncated form of cystatin F, which can directly inhibit cathepsin C (unlike the full-length form), was more effective than the full-length inhibitor. Furthermore, cystatin F decreased cathepsin L activity, which, however, did not affect perforin processing. Cystatin F derived from K-562 target cells could also decrease the cytotoxicity of TALL-104 cells. These results clearly show that, by inhibiting cysteine cathepsin proteolytic activity, extracellular cystatin F can decrease the cytotoxicity of CTLs and thus compromise their function

Reshaping of T-lymphocyte compartment in adult prepubertaly ovariectomised rats: A putative role for progesterone deficiency

This study explores the role of ovarian hormones in the phenotypic shaping of peripheral T-cell pool over the reproductive lifespan of rats. For this purpose, 2-month-old prepubertally ovariectomised (Ox) rats, showing oestrogen and progesterone deficiency, and 11-month-old Ox rats, exhibiting only progesterone deficiency, were examined for thymus output, and cellularity and composition of major TCR alpha beta+ peripheral blood lymphocyte (PBL) and splenocyte subsets. Although ovariectomy increased thymic output in both 2- and 11-month-old rats, the count of both CD4+ and CD8+ PBLs and splenocytes increased only in the former. In the blood and spleen of 11-month-old Ox rats only the count of CD8+ cells increased. Although ovariectomy affected the total CD4+ count in none of the examined compartments from the 11-month-old rats, it increased CD4+FoxP3+ PBL and splenocyte relative proportions over those in the age-matched controls. The age-related differences in the cellularity and the major subset composition in Ox rats were linked to the differences in the ovarian steroid hormone levels registered in 2- and 11-month-old rats. The administration of progesterone to Ox rats during the seven days before the sacrificing confirmed contribution of this hormone deficiency to the ovariectomy-induced changes in the TCR alpha beta+ PBL and splenocyte pool from 11-month-old rats. The expansion of the CD8+ splenocyte subset in the 11-month-old Ox rats reflected increases in cellularity of memory and, particularly, nave cells. This was due to greater thymic output of CD8+ cells and homeostatic proliferation than apoptosis in 11-month-old Ox rats when compared with age-matched sham-Ox control rats. The homeostatic changes within CD8+ splenocyte pool from 11-month-old Ox rats, most likely, reflected the enhanced splenic IL-7 and TGF-beta mRNA expression. Overall, in adult female rats, circulating oestrogen and progesterone provide maintenance of T-cell counts, a diversity of T-cell repertoire, and the main T-cell subset composition in the periphery. Progesterone deficiency affects mainly the CD8+ lymphocyte compartment through increasing thymic CD8+ cell export and upsetting homeostatic regulation within the CD8+ splenocyte pool. These alterations were reversible through progesterone supplementation. (C) 2013 Elsevier GmbH, All rights reserved

Cocaprins, β-Trefoil Fold Inhibitors of Cysteine and Aspartic Proteases from Coprinopsis cinerea.

We introduce a new family of fungal protease inhibitors with β-trefoil fold from the mushroom Coprinopsis cinerea, named cocaprins, which inhibit both cysteine and aspartic proteases. Two cocaprin-encoding genes are differentially expressed in fungal tissues. One is highly transcribed in vegetative mycelium and the other in the stipes of mature fruiting bodies. Cocaprins are small proteins (15 kDa) with acidic isoelectric points that form dimers. The three-dimensional structure of cocaprin 1 showed similarity to fungal β-trefoil lectins. Cocaprins inhibit plant C1 family cysteine proteases with Ki in the micromolar range, but do not inhibit the C13 family protease legumain, which distinguishes them from mycocypins. Cocaprins also inhibit the aspartic protease pepsin with Ki in the low micromolar range. Mutagenesis revealed that the β2-β3 loop is involved in the inhibition of cysteine proteases and that the inhibitory reactive sites for aspartic and cysteine proteases are located at different positions on the protein. Their biological function is thought to be the regulation of endogenous proteolytic activities or in defense against fungal antagonists. Cocaprins are the first characterized aspartic protease inhibitors with β-trefoil fold from fungi, and demonstrate the incredible plasticity of loop functionalization in fungal proteins with β-trefoil fold

Development of Recombinant <i>Lactococcus lactis</i> Displaying Albumin-Binding Domain Variants against Shiga Toxin 1 B Subunit - Fig 7

<p>(A) SDS PAGE analysis of lysates of <i>L</i>. <i>lacti</i>s cells expressing S1B22, S1B26, ABDwt and H6-ABDwt (ABDwtH), all in fusion with Usp45 secretion signal and the LysM-containing cA domain, and stained with Coomassie brilliant blue. ABD fusion proteins are high-lighted with arrows. (B) Flow cytometric analysis of ABD surface display, detection with FITC-conjugated human serum albumin. The MFI value of ABDwt was compared with that of the control using Student’s t test. *** p<0.001. Cont.: control containing empty plasmid pNZ8148.</p