Cellular senescence induced by cathepsin X downregulation

Cellular senescence represents a powerful tumor suppressor mechanism to prevent proliferation and invasion of malignant cells. Since tumor cells as well as primary fibroblasts lacking the lysosomal cysteine-type carboxypeptidase cathepsin X exhibit a reduced invasive capacity, we hypothesized that the underlying reason may be the induction of cellular senescence. To investigate the cellular and molecular mechanisms leading to diminished migration/invasion of cathepsin X-deficient cells, we have analyzed murine embryonic fibroblasts (MEF) derived from cathepsin X-deficient mice and neonatal human dermal fibroblasts (NHDF) transfected with siRNAs targeting cathepsin X. Remarkably, both cell types exhibited a flattened and enlarged cell body, a characteristic phenotype of senescent cells. Additional evidence for accelerated senescence was obtained by detection of the common senescence marker β-galactosidase. Further examination revealed increased expression levels of senescence-associated genes such as p16, p21, p53, and caveolin in these cells along with a reduced proliferation rate. The accelerated cellular senescence induced by cathepsin X deficiency was rescued by simultaneous expression of exogenous cathepsin X. Finally, cell cycle analysis confirmed a marked reduction of the synthesis rate and prolongation of the S-phase, while susceptibility to apoptosis of cathepsin X-deficient cells remained unchanged. In conclusion, cathepsin X deficiency leads to accelerated cellular senescence and consequently to diminished cellular proliferation and migration/invasion implying a potential role of cathepsin X in bypassing cellular senescence.     

Carboxy-monopeptidase substrate specificity of human cathepsin X

Cathepsin X is a papain-like cysteine protease with restricted positional specificity, acting primarily as a carboxy-monopeptidase. We mapped the specificities at the S2, S1, and S1' subsites of human cathepsin X by systematically and independently substituting the P2, P1, and P1' positions of the carboxy-monopeptidase substrate Abz-FRF(4NO(2)) with natural amino acids. Human cathepsin X has broad S2, S1, and S1' specificities within two orders of magnitude in k(cat)/K(M), excluding proline that is not tolerated at these subsites. Glycine is not favored in S2, but is among the preferred residues in S1 and S1', which highlights S2 as the affinity-determinant subsite. The presence of peculiar residues at several binding site positions (Asp76, His234, Asn75, and Glu72) does not translate into a markedly different sequence specificity profile relative to other human cathepsins. These findings suggest that a specific function of human cathepsin X is unlikely to result from sequence specificity, but rather from a combination of its unique positional specificity and the co-localization of enzyme and substrate in a specific cellular environment.     

The role of cathepsin X in cell signaling

Cathepsin X is a lysosomal cysteine protease, found predominantly in cells of monocyte/macrophage lineage. It acts as a monocarboxypepidase and has a strict positional and narrower substrate specificity relative to the other human cathepsins. In our recent studies we identified—β₂ subunit of integrin receptors and α and γ enolase as possible substrates for cathepsin X carboxypeptidase activity. In both cases cathepsin X is capable to cleave regulatory motifs at C-terminus affecting the function of targeted molecules. We demonstrated that via activation of β₂ integrin receptor Mac-1 (CD11b/CD18) active cathepsin X enhances adhesion of monocytes/macrophages to fibrinogen and regulates the phagocytosis. By activation of Mac-1 receptor cathepsin X may regulate also the maturation of dendritic cells, a process, which is crucial in the initiation of adaptive immunity. Cathepsin X activates also the other β₂ integrin receptor, LFA-1 (CD11a/CD18) which is involved in the proliferation of T lymphocytes. By modulating the activity of LFA-1 cathepsin X causes cytoskeletal rearrangements and morphological changes of T lymphocytes enhancing ameboid-like migration in 2-D and 3-D barriers and increasing homotypic aggregation. The cleavage of C-terminal amino acids of α and γ enolase by cathepsin X abolishes their neurotrophic activity affecting neuronal cell survival and neuritogenesis.Key words: cathepsin X, integrin, enolase, T lymphocyte, macrophage, dendritic cell, adhesion, migration, neuritogenesisProteases comprise a group of enzymes that catalyse the cleavage of a peptide bond in a polypeptide chain by nucleophilic attack on the carbonyl carbon. The proteases are either exopeptidases cleaving one or a few amino acids at the N- or C-terminus of polypeptide chain or endopeptidases that cleave the peptide bond internally. According to the catalytic mechanism the endopeptidases are divided into aspartic, cysteine, serine, threonine and metallo endoproteases—see MEROPS database.¹ To date, 561 genes encoding for proteases have been identified in human genome. Among them 148 genes encode for cysteine proteases including a group of eleven lysosomal cysteine proteases (members of C1 family) also called cathepsins. They exhibit different expression patterns, levels and specificities, all of which contribute to their differential physiological roles. Some of them, like cathepsins B, H, L and C are ubiquitously present in tissues, whereas others (cathepsins S, V, X, O, K, F and W) are expressed by specific cell types. Cysteine cathepsins were long believed to be responsible for the terminal protein degradation in the lysosomes, however, this view has changed dramatically when they have been found to be involved in a number of important cellular processes and pathologies.^2,3In contrast to other cathepsins, cathepsin X was discovered only recently. Its gene,^4,5 structure^6,7 and activity properties^8,9 show several unique features that distinguish it clearly from other human cysteine proteases. It has a very short pro-region⁷ and a three residue insertion motif which forms a characteristic “mini loop.”⁶ Cathepsin X exhibits carboxypeptidase activity⁶ and, in contrast to cathepsin B, the other carboxypeptidase, it does not act as an endopeptidase. Contrary to the first reports,⁴ cathepsin X is not widely expressed in cells and tissues, but is restricted to the cells of the immune system, predominantly monocytes, macrophages and dendritic cells.¹⁰ Higher levels of cathepsin X were also found in tumor and immune cells of prostate¹¹ and gastric¹² carcinomas and in macrophages of gastric mucosa, especially after infection by Helicobacter pylori.¹³ Recently it was shown that cathepsin X is abundantly expressed in mouse brain cells, in particular glial cells. Its upregulation was also detected in the brains of patients with Alzheimer disease.¹⁴The involvement of cathepsin X in signal transduction is implied by the integrin-binding motifs, present in its pro-form (RGD: Arg-Gly-Asp) and mature form (ECD: Glu-Cys-Asp).^4,5 Moreover, cathepsin X binds cell surface heparan sulfate proteoglycans¹⁵ which are also involved in integrin regulation. A strong co-localization of pro-cathepsin X with β₃ integrin subunit was demonstrated in our study in pro-monocytic U-937 cells.¹⁶ Further, it was reported that the pro-form of cathepsin X interacts with α_vβ₃ integrin through the RGD motif in lamellipodia of human umbilical vein endothelial cells (HUVECs).¹⁷ However, we showed that the active form of cathepsin X co-localized predominantly with β₂ integrin subunit in various cells of monocytes/macrophage lineage. Active cathepsin X was shown to regulate β₂ integrin-dependent adhesion, phagocytosis and T lymphocyte activation by interaction with macrophage antigen-1 (CD11b/CD18, Mac-1). We showed that inhibitors and monoclonal antibodies, capable to impair cathepsin X enzymatic activity, reduced the binding of differentiated U-937 cells to fibrinogen and polystyrene surface in a dose dependent manner. The co-localization of active cathepsin X with β₂ integrin chain was particularly enhanced in interactions of monocyte/macrophages with endothelial and tumor cells.Besides in monocytes and macrophages the active cathepsin X plays a role in β₂ integrin activation also in dendritic cells (DC), which are crucial for effective antigen presentation and initiation of T cell dependent immune response. Maturation of dendritic cells is accompanied by a range of morphological and cytoskeleton structure changes. In response to maturation stimuli in vitro, DCs rapidly adhere, develop polarity and assemble actin rich structures at the leading edge, known as podosomes.¹⁸ The adhesion of immature DCs to the extracellular matrix, is accompanied by recruitment of Mac-1 integrin receptor, which can be activated by cathepsin X. We have shown that, during maturation, cathepsin X translocates to the plasma membrane of maturing DCs, enabling Mac-1 activation and, consequently, cell adhesion.¹⁹ In mature DCs cathepsin X redistributes from the membrane to the perinuclear region, which coincides with the de-adhesion of DCs, formation of cell clusters and acquisition of the mature phenotype. Again, the inhibition of cathepsin X activity during DC differentiation and maturation reduced the capacity of DCs to stimulate T lymphocytes.β₂ integrin receptors are important also in T lymphocyte functions, such as migration and invasion across the endothelium and tissues. Lymphocyte function-associated antigen-1 (CD11a/CD18, LFA-1), the predominant β₂ integrin receptor in T lymphocytes enables cell-cell interactions and homotypic aggregation via LFA-1-ICAM-1 (intracellular adhesion molecule-1) interactions. LFA-1 can act also as a true signaling receptor, causing F-actin reorganization that leads to cytoskeletal changes of the cell²⁰ and a switch from a spherical to a polarized shape.²¹ Although the concentration of cathepsin X in T lymphocytes is lower compared to monocytes and macrophages, we showed that it interacts with LFA-1 promoting cytoskeleton-dependent morphological changes and migration across 2D and 3D models of ICAM-1 and Matrigel.^22,23 Its co-localization with LFA-1 was particularly evident at the trailing edge protrusion, the uropod, which plays an important role in T lymphocyte migration and cell-cell interactions (Fig. 1). Uropodal active cathepsin X cleaves C-terminal amino acids of β chain in LFA-1 promoting its high affinity conformation and the binding of the cytoskeletal protein talin. This interaction stabilizes the uropod and promotes its elongation (Jevnikar, et al. submitted).Open in a separate windowFigure 1Activation of LFA-1 integrin receptor by cathepsin X at the uropod of T lymphocyte promotes cytoskeleton-dependent morphological changes and cell migration.³⁰We demonstrated that uropods of cathepsin X upregulated T lymphocytes elongate to extreme length and form cell-to-cell connections, the nanotubes (Obermajer, et al. in press). Membrane (or tunneling) nanotubes were recently found as a new principle of cell-to-cell communication enabling transmission of complex and specific messages to distant cells through a physically connected network. Calcium fluxes, vesicles and cell-surface components can all traffic between cells connected by nanotubes. In immune system nanotubes integrate communities of cells for a better coordination of their action in various stages of immune response. We showed that nanotubes of cathepsin X upregulated T lymphocytes could readily transfer cellular organelles such as mitochondria and lysosomes and proposed that nanotube mediated transfer makes possible T lymphocyte activation without the need for direct contact with antigen presenting cells.The exact mechanism of cathepsin X translocation towards plasma membrane and degradation of C-terminal amino acids of β chain remains unclear. In lysosomes cathepsin X can be found as a pro- and active form. After cell activation cathepsin X containing vesicles translocate towards the plasma membrane,¹⁶ as observed also for some other lysosomal proteases.²⁴ During this process it is possible that pro-cathepsin X is activated by the other cysteine protease cathepsin L, as shown already in vitro.⁸ Both proteases were strongly co-localized with β₂ integrin chain at plasma membrane of activated monocytes/macrophages and at uropodes of T lymphocytes. Simultaneous co-localization with the lysosomal markers demonstrates that at least the initial translocation of cathepsin X towards cytoplasmic tail of β₂ integrin chain is vesicular. The interaction of cathepsin X with β₂ integrin subunit was confirmed by immunoprecipitation and FRET.²² According to in vitro experiments we propose that cathepsin X cleaves sequentially C-terminal aminoacids F⁷⁶⁶, A⁷⁶⁷, E⁷⁶⁸ and S⁷⁶⁹ of β₂ integrin subunit (Fig. 2) until reaching proline in penultimate position, confirming previous observation that the proline in S2 position leads to resistance to cathepsin X proteolysis.²⁵ Also, our results are in agreement with the previously mentioned monocarboxypeptidase activity of cathepsin X.^26,27 Since the signaling to and from the integrins is mainly regulated by the short cytoplasmic tail of β₂ subunit,²⁸ cathepsin X mediated β₂ integrin truncation leads to regulation of the receptor signaling. The interaction of cytoplasmic tail with different cytoskeletal and regulatory proteins, such as talin, filamin, radixin and α-actinin is crucial for signal transduction and modulation of cytoskeleton.²⁹Open in a separate windowFigure 2Cathepsin X activates LFA-1 by sequential cleavage of C-terminal amino acids of β₂ integrin subunit.Besides β₂ integrin chain we recently identified isozymes α and γ enolases as another molecular target for cathepsin X carboxypeptidase activity (Obermajer, et al. submitted). We demonstrated that cathepsin X sequentially cleaves C-terminal amino acids of both isozymes, abolishing their neurotrophic activity. On this way the neuronal cell survival and neuritogenesis can be regulated. Inhibition of cathepsin X activity increases the generation of plasmin, essential for neuronal differentiation and changes the length distribution of neurites, especially in the early phase of neurite outgrowth. Moreover, cathepsin X inhibition increases neuronal survival and reduces serum deprivation induced apoptosis, particularly in the absence of nerve growth factor.     

The role of cathepsin X in cell signaling

Cathepsin X is a lysosomal cysteine protease, found predominantly in cells of monocyte/macrophage lineage. It acts as a monocarboxypepidase and has a strict positional and narrower substrate specificity relative to the other human cathepsins. In our recent studies we identified ? β2 subunit of integrin receptors and α and γ enolase as possible substrates for cathepsin X carboxypeptidase activity. In both cases cathepsin X is capable to cleave regulatory motifs at C-terminus affecting the function of targeted molecules. We demonstrated that via activation of β2 integrin receptor Mac-1 (CD11b/CD18) active cathepsin X enhances adhesion of monocytes/macrophages to fibrinogen and regulates the phagocytosis. By activation of Mac-1 receptor cathepsin X may regulate also the maturation of dendritic cells, a process, which is crucial in the initiation of adaptive immunity. Cathepsin X activates also the other β2 integrin receptor, LFA-1 (CD11a/CD18) which is involved in the proliferation of T lymphocytes. By modulating the activity of LFA-1 cathepsin X causes cytoskeletal rearrangements and morphological changes of T lymphocytes enhancing ameboid-like migration in 2-D and 3-D barriers and increasing homotypic aggregation. The cleavage of C-terminal amino acids of α and γ enolase by cathepsin X abolishes their neurotrophic activity affecting neuronal cell survival and neuritogenesis.     

Suppression of LFA-1 Expression by Spermine Is Associated with Enhanced Methylation of ITGAL,the LFA-1 Promoter Area

Spermine and spermidine, natural polyamines, suppress lymphocyte function-associated antigen 1 (LFA-1) expression and its associated cellular functions through mechanisms that remain unknown. Inhibition of ornithine decarboxylase, which is required for polyamine synthesis, in Jurkat cells by 3 mM D,L-alpha-difluoromethylornithine hydrochloride (DFMO) significantly decreased spermine and spermidine concentrations and was associated with decreased DNA methyltransferase (Dnmt) activity, enhanced demethylation of the LFA-1 gene (ITGAL) promoter area, and increased CD11a expression. Supplementation with extracellular spermine (500 µM) of cells pretreated with DFMO significantly increased polyamine concentrations, increased Dnmt activity, enhanced methylation of the ITGAL promoter, and decreased CD11a expression. It has been shown that changes in intracellular polyamine concentrations affect activities of -adenosyl-L-methionine-decaroboxylase, and, as a result, affect concentrations of the methyl group donor, S-adenosylmethionine (SAM), and of the competitive Dnmt inhibitor, decarboxylated SAM. Additional treatments designed to increase the amount of SAM and decrease the amount of decarboxylated SAM–such as treatment with methylglyoxal bis-guanylhydrazone (an inhibitor of S-adenosyl-L-methionine-decaroboxylase) and SAM supplementation–successfully decreased CD11a expression. Western blot analyses revealed that neither DFMO nor spermine supplementation affected the amount of active Ras-proximate-1, a member of the Ras superfamily of small GTPases and a key protein for regulation of CD11a expression. The results of this study suggest that polyamine-induced suppression of LFA-1 expression occurs via enhanced methylation of ITGAL.     

ICAM-3 interacts with LFA-1 and regulates the LFA-1/ICAM-1 cell adhesion pathway

The interaction of lymphocyte function-associated antigen-1 (LFA-1) with its ligands mediates multiple cell adhesion processes of capital importance during immune responses. We have obtained three anti-ICAM-3 mAbs which recognize two different epitopes (A and B) on the intercellular adhesion molecule-3 (ICAM-3) as demonstrated by sequential immunoprecipitation and cross-competitive mAb-binding experiments. Immunoaffinity purified ICAM-3-coated surfaces were able to support T lymphoblast attachment upon cell stimulation with both phorbol esters and cross-linked CD3, as well as by mAb engagement of the LFA-1 molecule with the activating anti-LFA-1 NKI-L16 mAb. T cell adhesion to purified ICAM-3 was completely inhibited by cell pretreatment with mAbs to the LFA-1 alpha (CD11a) or the LFA-beta (CD18) integrin chains. Anti-ICAM-3 mAbs specific for epitope A, but not those specific for epitope B, were able to trigger T lymphoblast homotypic aggregation. ICAM-3-mediated cell aggregation was dependent on the LFA-1/ICAM-1 pathway as demonstrated by blocking experiments with mAbs specific for the LFA-1 and ICAM-1 molecules. Furthermore, immunofluorescence studies on ICAM-3-induced cell aggregates revealed that both LFA-1 and ICAM-1 were mainly located at intercellular boundaries. ICAM-3 was located at cellular uropods, which in small aggregates appeared to be implicated in cell-cell contacts, whereas in large aggregates it appeared to be excluded from cell-cell contact areas. Experiments of T cell adhesion to a chimeric ICAM-1-Fc molecule revealed that the proaggregatory anti-ICAM-3 HP2/19 mAb was able to increase T lymphoblast attachment to ICAM-1, suggesting that T cell aggregation induced by this mAb could be mediated by increasing the avidity of LFA-1 for ICAM-1. Moreover, the HP2/19 mAb was costimulatory with anti-CD3 mAb for T lymphocyte proliferation, indicating that enhancement of T cell activation could be involved in ICAM-3-mediated adhesive phenomena. Altogether, our results indicate that ICAM-3 has a regulatory role on the LFA-1/ICAM-1 pathway of intercellular adhesion.     

Talin1 regulates TCR-mediated LFA-1 function

The leukocyte integrin LFA-1 plays a critical role in T cell trafficking and T cell adhesion to APCs. It is known that integrin-mediated adhesion is regulated by changes in integrin ligand-binding affinity and valency through inside-out signaling. However, the molecular mechanisms involved in TCR-mediated LFA-1 regulation are not well understood. In this study, we show that the cytoskeletal protein talin1 is required for TCR-mediated activation of LFA-1 through regulation of LFA-1 affinity and clustering. Depletion of talin1 from human T cells by small interfering RNAs impairs TCR-induced adhesion to ICAM-1 and T cell-APC conjugation. TCR-induced LFA-1 polarization, but not actin polarization, is defective in talin1-deficient T cells. Although LFA-1 affinity is also reduced in talin1-deficient T cells, rescue of LFA-1 affinity alone is not sufficient to restore LFA-1 adhesive function. Together, our findings indicate that TCR-induced up-regulation of LFA-1-dependent adhesiveness and resulting T cell-APC conjugation require talin1.     

Involvement of LFA-1 in lymphoma invasion and metastasis demonstrated with LFA-1-deficient mutants

Lymphocyte function-associated antigen-1 (LFA-1) is a leukocyte and lymphoma cell surface protein that promotes intercellular adhesion. We have previously shown that the invasion of hepatocyte cultures by lymphoma cells is inhibited by anti-LFA-1 antibodies (Roos, E., and F. F. Roossien. 1987. J. Cell Biol. 105:553-559). In addition, we now report that LFA-1 is also involved in invasion of lymphoma cells into fibroblast monolayers. To investigate the role of LFA-1 in metastasis of these lymphoma cells, we have generated mutants that are deficient in LFA-1 cell surface expression because of impaired synthesis of either the alpha or beta subunit precursor of LFA-1. We identified at least three distinct mutant clones. The invasive potential of the mutant cells in vitro, in both hepatocyte and fibroblast cultures, was considerably lower than that of parental cells. The metastatic potential of the mutants was much reduced, indicating that LFA-1 expression is required for efficient metastasis formation by certain lymphoma cells.     

Inactivation of cathepsin B1 by diazomethyl ketones

Benzyloxycarbonyl-phenylalanyl diazomethyl ketone and benzyloxy-carbonyl-phenylalanyl-phenylalanyl diazomethyl ketone, which have been shown to inactivate the thiol protease papain by a mechanism different from that of substrate chloromethyl ketone derivatives, have now been examined as inhibitors of cathepsin B₁ of beef spleen. The dipeptide derivative irreversibly inactivates this protease rapidly, apparently by affinity labeling.     

RhoA is involved in LFA-1 extension triggered by CXCL12 but not in a novel outside-in LFA-1 activation facilitated by CXCL9

Chemokines presented on endothelial tissues instantaneously trigger LFA-1-mediated arrest on ICAM-1 via rapid inside-out and outside-in (ligand-driven) LFA-1 activation. The GTPase RhoA was previously implicated in CCL21-triggered LFA-1 affinity triggering in murine T lymphocytes and in LFA-1-dependent adhesion strengthening to ICAM-1 on Peyer's patch high endothelial venules stabilized over periods of at least 10 s. In this study, we show that a specific RhoA 23/40 effector region is vital for the initial LFA-1-dependent adhesions of lymphocytes on high endothelial venules lasting 1-3 s. Blocking the RhoA 23/40 region in human T lymphocytes in vitro also impaired the subsecond CXCL12-triggered LFA-1-mediated T cell arrest on ICAM-1 by eliminating the rapid induction of an extended LFA-1 conformational state. However, the inflammatory chemokine CXCL9 triggered robust LFA-1-mediated T lymphocyte adhesion to ICAM-1 at subsecond contacts independently of the RhoA 23/40 region. CXCL9 did not induce conformational changes in the LFA-1 ectodomain, suggesting that particular chemokines can activate LFA-1 through outside-in post ligand binding stabilization changes. Like CXCL9, the potent diacylglycerol-dependent protein kinase C agonist PMA was found to trigger LFA-1 adhesiveness to ICAM-1 also without inducing integrin extension or an a priori clustering and independently of the RhoA 23/40 region. Our results collectively suggest that the 23/40 region of RhoA regulates chemokine-induced inside-out LFA-1 extension before ligand binding, but is not required for a variety of chemokine and non-chemokine signals that rapidly strengthen LFA-1-ICAM-1 bonds without an a priori induction of high-affinity extended LFA-1 conformations.     

Rap1A regulates generation of T regulatory cells via LFA-1-dependent and LFA-1-independent mechanisms

The small GTPase Rap1A has a critical role in regulating cell-matrix and cell-cell adhesion. In T lymphocytes, Rap1A mediates LFA-1 activation and LFA-1-mediated adhesion. LFA-1 reduces the threshold of TCR signals for low affinity ligands. Previously, we determined that mice expressing constitutively active Rap1A on T cells have increased frequency of CD103⁺ T regulatory cells (Treg). We hypothesized that Rap1A-GTP might affect the differentiation of Treg by regulating LFA-1 activation. Using Foxp3-GFP-KI, LFA-1-KO and Rap1A-GTP-Tg mice we determined that Rap1A has an active role in the development of thymic Treg but LFA-1 is not mandatory for this function. Rap1A is also involved in the generation of peripheral Treg and this effect is mediated via LFA-1-dependent and LFA-1-independent mechanisms. Identification of the signaling pathways via which Rap1-GTP contributes to the differentiation of Treg will provide new insights to the function of Rap1A and to designing targeted approaches for generation of Treg for therapeutic applications.     

Analysis of big endothelin-1 digestion by cathepsin D

Digestion of big endothelin (ET)-1 by cathepsin D, which is the only substantially identified protease showing ET converting enzyme activity, was characterized. Increased doses of cathepsin D showed decrease of immunoreactive (ir-) ET produced from big ET-1. Time course of big ET-1 conversion showed marked increase of ir-ET in a relatively short period, but further incubation resulted in the decrease of ir-ET. Incubation at various pHs with different doses of cathepsin D revealed that low doses of cathepsin D yielded the maximum production of ir-ET at pH 3.5-4.0, but higher doses of cathepsin D showed a bimodal curve of ir-ET production, which may be due to degradation of ir-ET. HPLC analysis revealed that cathepsin D cleaves Asn18-Ile19 bond in addition to Trp21-Val22 bond of big ET-1. These data suggests cathepsin D is not a physiological endothelin converting enzyme.     

Peptide synthesis by recombinant Fasciola hepatica cathepsin L1

Synthesis of the tripeptide Z-Phe-Arg-SerNH2 has been accomplished by a recombinant cysteine protease, cathepsin L1 from liver fluke (Fasciola hepatica), using Z-Phe-Arg-OMe as acyl acceptor and SerNH2 as nucleophile in 0.1 M ammonium acetate pH 9.0-12.5% v/v acetonitrile at 37 degrees C. LC-MS detection indicated tripeptide formation after 10 min, continuing up to 5.5 h. The ester Z-Phe-Arg-OMe was detected throughout the experiment but the hydrolysis product Z-Phe-Arg-OH appeared early and in quite large amounts. We believe that this is the first application of a parasite protease in enzymatic peptide synthesis.     

Occupancy of lymphocyte LFA-1 by surface-immobilized ICAM-1 is critical for TCR- but not for chemokine-triggered LFA-1 conversion to an open headpiece high-affinity state

Lymphocyte arrest and spreading on ICAM-1-expressing APCs require activation of lymphocyte LFA-1 by TCR signals, but the conformational switches of this integrin during these critical processes are still elusive. Using Ab probes that distinguish between different LFA-1 conformations, we found that, unlike strong chemokine signals, potent TCR stimuli were insufficient to trigger LFA-1 extension or headpiece opening in primary human lymphocytes. Nevertheless, LFA-1 in these TCR-stimulated T cells became highly adhesive to both anchored and mobile surface-bound ICAM-1, although it failed to bind soluble ICAM-1 with measurable affinity. Rapid rearrangement of LFA-1 by immobilized ICAM-1 switched the integrin to an open headpiece conformation within numerous scattered submicron focal dots that did not readily collapse into a peripheral LFA-1 ring. Headpiece-activated LFA-1 microclusters were enriched with talin but were devoid of TCR and CD45. Notably, LFA-1 activation by TCR signals as well as subsequent T cell spreading on ICAM-1 took place independently of cytosolic Ca(2+). In contrast to LFA-1-activating chemokine signals, TCR activation of LFA-1 readily took place in the absence of external shear forces. LFA-1 activation by TCR signals also did not require internal myosin II forces but depended on intact actin cytoskeleton. Our results suggest that potent TCR signals fail to trigger LFA-1 headpiece activation unless the integrin first gets stabilized by surface-bound ICAM-1 within evenly scattered actin-dependent LFA-1 focal dots, the quantal units of TCR-stimulated T cell arrest and spreading on ICAM-1.     

Activating Ly-49 receptors regulate LFA-1-mediated adhesion by NK cells

NK cells are important for innate resistance to tumors and viruses. Engagement of activating Ly-49 receptors expressed by NK cells leads to rapid NK cell activation resulting in target cell lysis and cytokine production. The ITAM-containing DAP12 adapter protein stably associates with activating Ly-49 receptors, and couples receptor recognition with generation of NK responses. Activating Ly-49s are potent stimulators of murine NK cell functions, yet how they mediate such activities is not well understood. We demonstrate that these receptors trigger LFA-1-dependent tight conjugation between NK cells and target cells. Furthermore, we show that activating Ly-49 receptor engagement leads to rapid DAP12-dependent up-regulation of NK cell LFA-1 adhesiveness to ICAM-1 that is also dependent on tyrosine kinases of the Syk and Src families. These results indicate for the first time that activating Ly-49s control adhesive properties of LFA-1, and by DAP12-dependent inside-out signaling. Ly-49-driven mobilization of LFA-1 adhesive function may represent a fundamental proximal event during NK cell interactions with target cells involving activating Ly-49 receptors, leading to target cell death.     

Activation of LFA-1 by ionomycin is independent of calpain-mediated talin cleavage

Activation of calpains by calcium flux leading to talin cleavage is thought to be an important process of LFA-1 activation by inside-out signalling. Here, we tested the effects of the calcium ionophore ionomycin and calpain inhibitor calpeptin on LFA-1-mediated adhesion of a T cell hybridoma line, cytotoxic T cells and primary resting T cells. Ionomycin activated LFA-1-mediated adhesion of all three types of T cells, and calpeptin inhibited the effects of ionomycin. However, calpeptin also inhibited activation of LFA-1 by PMA, which did not induce calcium flux. Cleavage of talin was undetectable in ionomycin-treated T cells. Furthermore, treatment with ionomycin and calpeptin induced apoptosis of T cells. Inhibitors of phosphatidyl Inositol-3 kinase inhibited activation of LFA-1 by ionomycin, but not by PMA, whereas the protein kinase C inhibitor inhibited the effects of PMA, but not ionomycin. Thus, activation of LFA-1 by ionomycin is independent of calpain-mediated talin cleavage.     

A fine-tuning mechanism underlying self-control for autophagy: deSUMOylation of BECN1 by SENP3

ABSTRACT

The roles of SUMOylation and the related enzymes in autophagic regulation are unclear. Based on our previous studies that identified the SUMO2/3-specific peptidase SENP3 as an oxidative stress-responsive molecule, we investigated the correlation between SUMOylation and macroautophagy/autophagy. We found that Senp3^± mice showed increased autophagy in the liver under basal and fasting conditions, compared to Senp3^+/+ mice. We constructed a liver-specific senp3 knockout mouse; these Senp3-deficient liver tissues showed increased autophagy as well. Autophagic flux was accelerated in hepatic and other cell lines following knockdown of SENP3, both before and after the cells underwent starvation in the form of the serum and amino acid deprivation. We demonstrated that BECN1/beclin 1, the core molecule of the BECN1-PIK3C3 complex, could be SUMO3-conjugated by PIAS3 predominantly at K380 and deSUMOylated by SENP3. The basal SUMOylation of BECN1 was increased upon cellular starvation, which enhanced autophagosome formation by facilitating BECN1 interaction with other complex components UVRAG, PIK3C3 and ATG14, thus promoting PIK3C3 activity. In contrast, SENP3 deSUMOylated BECN1, which impaired BECN1-PIK3C3 complex formation or stability to suppress the PIK3C3 activity. DeSUMOylation of BECN1 restrained autophagy induction under basal conditions and especially upon starvation when SENP3 had accumulated in response to the increased generation of reactive oxygen species. Thus, while reversible SUMOylation regulated the degree of autophagy, SENP3 provided an intrinsic overflow valve for fine-tuning autophagy induction.