Cathepsin B-like and cell death in the unicellular human pathogen Leishmania.

In several studies reporting cell death (CD) in lower eukaryotes and in the human protozoan parasite Leishmania, proteolytic activity was revealed using pan-caspase substrates or inhibitors such as carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). However, most of the lower eukaryotes do not encode caspase(s) but MCA, which differs from caspase(s) in its substrate specificity and cannot be accountable for the recognition of Z-VAD-FMK. In the present study, we were interested in identifying which enzyme was capturing the Z-VAD substrate. We show that heat shock (HS) induces Leishmania CD and leads to the intracellular binding of Z-VAD-FMK. We excluded binding and inhibition of Z-VAD-FMK to Leishmania major metacaspase (LmjMCA), and identified cysteine proteinase C (LmjCPC), a cathepsin B-like (CPC) enzyme, as the Z-VAD-FMK binding enzyme. We confirmed the specific interaction of Z-VAD-FMK with CPC by showing that Z-VAD binding is absent in a Leishmania mexicana strain in which the cpc gene was deleted. We also show that parasites exposed to various stress conditions release CPC into a soluble fraction. Finally, we confirmed the role of CPC in Leishmania CD by showing that, when exposed to the oxidizing agent hydrogen peroxide (H(2)O(2)), cpc knockout parasites survived better than wild-type parasites (WT). In conclusion, this study identified CPC as the substrate of Z-VAD-FMK in Leishmania and as a potential additional executioner protease in the CD cascade of Leishmania and possibly in other lower eukaryotes.

Cathepsin X cleavage of the beta2 integrin regulates talin-binding and LFA-1 affinity in T cells.

T cell migration, essential for immune surveillance and response, is mediated by the integrin LFA-1. CatX, a cysteine carboxypeptidase, is involved in the regulation of T cell migration by interaction with LFA-1. We show that sequential cleavage of C-terminal amino acids from the β(2) cytoplasmic tail of LFA-1, by CatX, enhances binding of the adaptor protein talin to LFA-1 and triggers formation of the latter's high-affinity form. As shown by SPR analysis of peptides constituting the truncated β(2) tail, the cleavage of three C-terminal amino acids by CatX resulted in a 1.6-fold increase of talin binding. Removal of one more amino acid resulted in a 2.5-fold increase over the intact tail. CatX cleavage increased talin-binding affinity to the MD but not the MP talin-binding site on the β(2) tail. This was shown by molecular modeling of the β(2) tail/talin F3 complex to be a result of conformational changes affecting primarily the distal-binding site. Analysis of LFA-1 by conformation-specific mAb showed that CatX modulates LFA-1 affinity, promoting formation of high-affinity from intermediate-affinity LFA-1 but not the initial activation of LFA-1 from a bent to extended form. CatX post-translational modifications may thus represent a mechanism of LFA-1 fine-tuning that enables the trafficking of T cells.

Cysteine cathepsin proteases: regulators of cancer progression and therapeutic response.

Cysteine cathepsin protease activity is frequently dysregulated in the context of neoplastic transformation. Increased activity and aberrant localization of proteases within the tumour microenvironment have a potent role in driving cancer progression, proliferation, invasion and metastasis. Recent studies have also uncovered functions for cathepsins in the suppression of the response to therapeutic intervention in various malignancies. However, cathepsins can be either tumour promoting or tumour suppressive depending on the context, which emphasizes the importance of rigorous in vivo analyses to ascertain function. Here, we review the basic research and clinical findings that underlie the roles of cathepsins in cancer, and provide a roadmap for the rational integration of cathepsin-targeting agents into clinical treatment.

Combined deletion of cathepsin protease family members reveals compensatory mechanisms in cancer.

Proteases are important for regulating multiple tumorigenic processes, including angiogenesis, tumor growth, and invasion. Elevated protease expression is associated with poor patient prognosis across numerous tumor types. Several multigene protease families have been implicated in cancer, including cysteine cathepsins. However, whether individual family members have unique roles or are functionally redundant remains poorly understood. Here we demonstrate stage-dependent effects of simultaneously deleting cathepsin B (CtsB) and CtsS in a murine pancreatic neuroendocrine tumor model. Early in tumorigenesis, the double knockout results in an additive reduction in angiogenic switching, whereas at late stages, several tumorigenic phenotypes are unexpectedly restored to wild-type levels. We identified CtsZ, which is predominantly supplied by tumor-associated macrophages, as the compensatory protease that regulates the acquired tumor-promoting functions of lesions deficient in both CtsB and CtsS. Thus, deletion of multiple cathepsins can lead to stage-dependent, compensatory mechanisms in the tumor microenvironment, which has potential implications for the clinical consideration of selective versus pan-family cathepsin inhibitors in cancer.

Enhancement of autophagic flux after neonatal cerebral hypoxia-ischemia and its region-specific relationship to apoptotic mechanisms.

The multiplicity of cell death mechanisms induced by neonatal hypoxia-ischemia makes neuroprotective treatment against neonatal asphyxia more difficult to achieve. Whereas the roles of apoptosis and necrosis in such conditions have been studied intensively, the implication of autophagic cell death has only recently been considered. Here, we used the most clinically relevant rodent model of perinatal asphyxia to investigate the involvement of autophagy in hypoxic-ischemic brain injury. Seven-day-old rats underwent permanent ligation of the right common carotid artery, followed by 2 hours of hypoxia. This condition not only increased autophagosomal abundance (increase in microtubule-associated protein 1 light chain 3-11 level and punctuate labeling) but also lysosomal activities (cathepsin D, acid phosphatase, and beta-N-acetylhexosaminidase) in cortical and hippocampal CA3-damaged neurons at 6 and 24 hours, demonstrating an increase in the autophagic flux. In the cortex, this enhanced autophagy may be related to apoptosis since some neurons presenting a high level of autophagy also expressed apoptotic features, including cleaved caspase-3. On the other hand, enhanced autophagy in CA3 was associated with a more purely autophagic cell death phenotype. In striking contrast to CA3 neurons, those in CA1 presented only a minimal increase in autophagy but strong apoptotic characteristics. These results suggest a role of enhanced autophagy in delayed neuronal death after severe hypoxia-ischemia that is differentially linked to apoptosis according to the cerebral region.

Colitis induced in mice with dextran sulfate sodium (DSS) is mediated by the NLRP3 inflammasome.

BACKGROUND: The proinflammatory cytokines interleukin 1beta (IL-1beta) and IL-18 are central players in the pathogenesis of inflammatory bowel disease (IBD). In response to a variety of microbial components and crystalline substances, both cytokines are processed via the caspase-1-activating multiprotein complex, the NLRP3 inflammasome. Here, the role of the NLRP3 inflammasome in experimental colitis induced by dextran sodium sulfate (DSS) was examined. METHODS: IL-1beta production in response to DSS was studied in macrophages of wild-type, caspase-1(-/-), NLRP3(-/-), ASC(-/-), cathepsin B(-/-) or cathepsin L(-/-) mice. Colitis was induced in C57BL/6 and NLRP3(-/-) mice by oral DSS administration. A clinical disease activity score was evaluated daily. Histological colitis severity and expression of cytokines were determined in colonic tissue. RESULTS: Macrophages incubated with DSS in vitro secreted high levels of IL-1beta in a caspase-1-dependent manner. IL-1beta secretion was abrogated in macrophages lacking NLRP3, ASC or caspase-1, indicating that DSS activates caspase-1 via the NLRP3 inflammasome. Moreover, IL-1beta secretion was dependent on phagocytosis, lysosomal maturation, cathepsin B and L, and reactive oxygen species (ROS). After oral administration of DSS, NLRP3(-/-) mice developed a less severe colitis than wild-type mice and produced lower levels of proinflammatory cytokines in colonic tissue. Pharmacological inhibition of caspase-1 with pralnacasan achieved a level of mucosal protection comparable with NLRP3 deficiency. CONCLUSIONS: The NLRP3 inflammasome was identified as a critical mechanism of intestinal inflammation in the DSS colitis model. The NLRP3 inflammasome may serve as a potential target for the development of novel therapeutics for patients with IBD.

Angiotensin II is an endogenous neurotransmitter for rat and human mesenteric resistance blood vessels

Angiotensin II (Ang II) is one of the most potent vasoconstrictors. We document here the innervation of rat and human mesenteric resistance arteries (MRA) by angiotensinergic neurons of the rat and human sympathetic coeliac ganglia. Angiotensinogen (Ang-N)-mRNA and angiotensin converting enzyme-mRNA but no renin-mRNA were detected by using quantitative real time polymerase chain reaction in total RNA extracts of rat coeliac ganglia. In the same extracts, cathepsin D-mRNA was detected: This protease also cleaves Ang I from Ang-N and could therefore account for the generation of neuronal Ang peptides in the absence of renin. In situ hybridization confirmed the presence of Ang-N-mRNA in the cytoplasm of rat coeliac ganglia. By using solid-phase extraction, high performance liquid chromatography and subsequent radioimmunoassay, Ang II and its metabolites were detected in rat and also in human coeliac ganglia. Immunoreactivity for Ang II was demonstrated in rat and human coeliac ganglia neurons and their projections innervating MRA. In addition, segmental angiotensinergic innervation of MRA was also observed. By means of confocal laser scanning microscopy we were able to demonstrate the presence of angiotensinergic synapses en passant along side of vascular smooth muscle cells. Our findings could indicate that Ang II is synthesized inside the neurons of sympathetic coeliac ganglia and may act as an endogenous neurotransmitter locally in MRA.

Beclin 1-independent autophagy contributes to apoptosis in cortical neurons.

Neuronal autophagy is enhanced in many neurological conditions, such as cerebral ischemia and traumatic brain injury, but its role in associated neuronal death is controversial, especially under conditions of apoptosis. We therefore investigated the role of autophagy in the apoptosis of primary cortical neurons treated with the widely used and potent pro-apoptotic agent, staurosporine (STS). Even before apoptosis, STS enhanced autophagic flux, as shown by increases in autophagosomal (LC3-II level, LC3 punctate labeling) and lysosomal (cathepsin D, LAMP1, acid phosphatase, β-hexasominidase) markers. Inhibition of autophagy by 3-methyladenine, or by lentivirally-delivered shRNAs against Atg5 and Atg7, strongly reduced the STS-induced activation of caspase-3 and nuclear translocation of AIF, and gave partial protection against neuronal death. Pan-caspase inhibition with Q-VD-OPH likewise protected partially against neuronal death, but failed to affect autophagy. Combined inhibition of both autophagy and caspases gave strong synergistic neuroprotection. The autophagy contributing to apoptosis was Beclin 1-independent, as shown by the fact that Beclin 1 knockdown failed to reduce it but efficiently reduced rapamycin-induced autophagy. Moreover the Beclin 1 knockdown sensitized neurons to STS-induced apoptosis, indicating a cytoprotective role of Beclin 1 in cortical neurons. Caspase-3 activation and pyknosis induced by two other pro-apoptotic stimuli, MK801 and etoposide, were likewise found to be associated with Beclin 1-independent autophagy and reduced by the knockdown of Atg7 but not Beclin 1. In conclusion, Beclin 1-independent autophagy is an important contributor to both the caspase-dependent and -independent components of neuronal apoptosis and may be considered as an important therapeutic target in neural conditions involving apoptosis.

Postischemic treatment of neonatal cerebral ischemia should target autophagy.

OBJECTIVE: To evaluate the contributions of autophagic, necrotic, and apoptotic cell death mechanisms after neonatal cerebral ischemia and hence define the most appropriate neuroprotective approach for postischemic therapy. METHODS: Rats were exposed to transient focal cerebral ischemia on postnatal day 12. Some rats were treated by postischemic administration of pan-caspase or autophagy inhibitors. The ischemic brain tissue was studied histologically, biochemically, and ultrastructurally for autophagic, apoptotic, and necrotic markers. RESULTS: Lysosomal and autophagic activities were increased in neurons in the ischemic area from 6 to 24 hours postinjury, as shown by immunohistochemistry against lysosomal-associated membrane protein 1 and cathepsin D, by acid phosphatase histochemistry, by increased expression of autophagosome-specific LC3-II and by punctate LC3 staining. Electron microscopy confirmed the presence of large autolysosomes and putative autophagosomes in neurons. The increases in lysosomal activity and autophagosome formation together demonstrate increased autophagy, which occurred mainly in the border of the lesion, suggesting its involvement in delayed cell death. We also provide evidence for necrosis near the center of the lesion and apoptotic-like cell death in its border, but in nonautophagic cells. Postischemic intracerebroventricular injections of autophagy inhibitor 3-methyladenine strongly reduced the lesion volume (by 46%) even when given >4 hours after the beginning of the ischemia, whereas pan-caspase inhibitors, carbobenzoxy-valyl-alanyl-aspartyl(OMe)-fluoromethylketone and quinoline-val-asp(OMe)-Ch2-O-phenoxy, provided no protection. INTERPRETATION: The prominence of autophagic neuronal death in the ischemic penumbra and the neuroprotective efficacy of postischemic autophagy inhibition indicate that autophagy should be a primary target in the treatment of neonatal cerebral ischemia.

Interaction of cationic ultrasmall superparamagnetic iron oxide nanoparticles with human melanoma cells.

Ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) are currently under development for the intracellular delivery of therapeutics. However, the mechanisms of cellular uptake and the cellular reaction to this uptake, independent of therapeutics, are not well defined. The interactions of biocompatible cationic aminoUSPIONs with human cells was studied in 2D and 3D cultures using biochemical and electron microscopy techniques. AminoUSPIONs were internalized by human melanoma cells in 2D and 3D cultures. Uptake was clathrin mediated and the particles localized in lysosomes, inducing activation of the lysosomal cathepsin D and decreasing the expression of the transferrin receptor in human melanoma cells and/or skin fibroblasts. AminoUSPIONs deeply invaded 3D spheroids of human melanoma cells. Thus, aminoUSPIONs can invade tumors and their uptake by human cells induces cell reaction.

Identification of Leishmania major cysteine proteinases as targets of the immune response in humans.

In this study, we report the identification of two parasite polypeptides recognized by human sera of patients infected with Leishmania major. Isolation and sequencing of the two genes encoding these polypeptides revealed that one of the genes is similar to the L. major cathepsin L-like gene family CPB, whereas the other gene codes for the L. major homologue of the cysteine proteinase a (CPA) of L. mexicana. By restriction enzyme digestion of genomic DNA, we show that the CPB gene is present in multiple copies in contrast to the cysteine proteinase CPA gene which could be unique. Specific antibodies directed against the mature regions of both types expressed in Escherichia coli were used to analyze the expression of these polypeptides in different stages of the parasite's life cycle. Polypeptides of 27 and 40 kDa in size, corresponding to CPA and CPB respectively, were detected at higher level in amastigotes than in stationary phase promastigotes. Purified recombinant CPs were also used to examine the presence of specific antibodies in sera from either recovered or active cases of cutaneous leishmaniasis patients. Unlike sera from healthy uninfected controls, all the sera reacted with recombinant CPA and CPB. This finding indicates that individuals having recovered from cutaneous leishmaniasis or with clinically apparent disease have humoral responses to cysteine proteinases demonstrating the importance of these proteinases as targets of the immune response and also their potential use for serodiagnosis.

Endogenous angiotensinergic system in neurons of rat and human trigeminal ganglia.

To clarify the role of Angiotensin II (Ang II) in the sensory system and especially in the trigeminal ganglia, we studied the expression of angiotensinogen (Ang-N)-, renin-, angiotensin converting enzyme (ACE)- and cathepsin D-mRNA, and the presence of Ang II and substance P in the rat and human trigeminal ganglia. The rat trigeminal ganglia expressed substantial amounts of Ang-N- and ACE mRNA as determined by quantitative real time PCR. Renin mRNA was untraceable in rat samples. Cathepsin D was detected in the rat trigeminal ganglia indicating the possibility of existence of pathways alternative to renin for Ang I formation. In situ hybridization in rat trigeminal ganglia revealed expression of Ang-N mRNA in the cytoplasm of numerous neurons. By using immunocytochemistry, a number of neurons and their processes in both the rat and human trigeminal ganglia were stained for Ang II. Post in situ hybridization immunocytochemistry reveals that in the rat trigeminal ganglia some, but not all Ang-N mRNA-positive neurons marked for Ang II. In some neurons Substance P was found colocalized with Ang II. Angiotensins from rat trigeminal ganglia were quantitated by radioimmunoassay with and without prior separation by high performance liquid chromatography. Immunoreactive angiotensin II (ir-Ang II) was consistently present and the sum of true Ang II (1-8) octapeptide and its specifically measured metabolites were found to account for it. Radioimmunological and immunocytochemical evidence of ir-Ang II in neuronal tissue is compatible with Ang II as a neurotransmitter. In conclusion, these results suggest that Ang II could be produced locally in the neurons of rat trigeminal ganglia. The localization and colocalization of neuronal Ang II with Substance P in the trigeminal ganglia neurons may be the basis for a participation and function of Ang II in the regulation of nociception and migraine pathology.

The role of LEKTI in fate determination of keratinocyte stem cells

SPINK5 (serine protease inhibitor Kazal-type 5) encodes the putative proteinase inhibitor LEKTI (lympho-epithelial Kazal-type related inhibitor). In skin, LEKTI expression is restricted to the stratum granulosum of the epidermis and the inner root sheath of hair follicles. Mutations that create premature termination codons in SPINK5 have been reported as the cause of Netherton syndrome (NS), a human autosomal recessive disorder characterized by congenital ichthyosis with defective cornification, a specific hair shaft defect known as trichorrexis invaginata or 'bamboo hair', and severe atopic manifestations, including atopic dermatitis and hayfever. Althought recombinant human LEKTI inhibits a battery of serine proteases including plasmin, trypsin, subtilisin A, cathepsin G, and elastase, the precise role of LEKTI in the physiopathology of NS remains unclear. Spink5−/− mice display a NS-like phenotype. Surprisingly, a psoriasis-like hyperplasia, basement membrane breakdown followed by evagination of spindle-shaped epidermal cells into the dermal compartment, and the presence of numerous sweat gland-like structures were also observed when the skin of Spink5−/− newborn mice, which die at birth, was transplanted onto the back of nude mice. Collectively, these observations suggest that LEKTI may play a role on cell proliferation and stem cell fate. Our current work aims at elucidating the mechanisms by which LEKTI impact these biological processes. Using keratinocyte stem cells obtained from NS patients, we have identified LEKTI as a regulator node in several signaling pathways involved in stem cell behavior.

Uptake of particulate vaccine adjuvants by dendritic cells activates the NALP3 inflammasome.

Many currently used and candidate vaccine adjuvants are particulate in nature, but their mechanism of action is not well understood. Here, we show that particulate adjuvants, including biodegradable poly(lactide-co-glycolide) (PLG) and polystyrene microparticles, dramatically enhance secretion of interleukin-1beta (IL-1beta) by dendritic cells (DCs). The ability of particulates to promote IL-1beta secretion and caspase 1 activation required particle uptake by DCs and NALP3. Uptake of microparticles induced lysosomal damage, whereas particle-mediated enhancement of IL-1beta secretion required phagosomal acidification and the lysosomal cysteine protease cathepsin B, suggesting a role for lysosomal damage in inflammasome activation. Although the presence of a Toll-like receptor (TLR) agonist was required to induce IL-1beta production in vitro, injection of the adjuvants in the absence of TLR agonists induced IL-1beta production at the injection site, indicating that endogenous factors can synergize with particulates to promote inflammasome activation. The enhancement of antigen-specific antibody production by PLG microparticles was independent of NALP3. However, the ability of PLG microparticles to promote antigen-specific IL-6 production by T cells and the recruitment and activation of a population of CD11b(+)Gr1(-) cells required NALP3. Our data demonstrate that uptake of microparticulate adjuvants by DCs activates the NALP3 inflammasome, and this contributes to their enhancing effects on innate and antigen-specific cellular immunity.

Early apoptosis of rod photoreceptors in Rpe65(-/-) mice is associated with the upregulated expression of lysosomal-mediated autophagic genes.

RPE65-related Leber's congenital amaurosis (LCA) is a rod-cone dystrophy whose clinical outcome is mainly attributed to the loss of rod photoreceptors followed by cone degeneration. Pathogenesis in Rpe65(-/-) mice is characterized by a slow and progressive degeneration of rods dependent on the constitutive activation of unliganded opsin. We previously reported that this opsin-mediated apoptosis of rods was dependent on Bcl-2-apoptotic pathway and Bax-induced pro-death activity. In this study, we report early initial apoptosis in the newly differentiated retina of Rpe65(-/-) mice. Apoptotic photoreceptors were identified as rods and resulted from pathological phototransduction signaling. This wave of early apoptosis triggered Bcl-2-related pathway and Bax apoptotic activity, while activation of the caspases was not induced. Following cellular stress, multiple signaling pathways are initiated which either commit cells to death or trigger pro-survival responses including autophagy. We report that Bcl-2-related early rod apoptosis was associated with the upregulation of autophagy markers including chaperone-mediated autophagy (CMA) substrate receptor LAMP-2 and lysosomal hydrolases Cathepsin S and Lysozyme. This suggests that lysosomal-mediated autophagy may be triggered in response to early rod apoptosis in Rpe65-LCA disease. These results highlight that Rpe65-related primary stress induces early signaling events, which trigger Bax-induced-apoptotic pathway and autophagy-mediated cellular response. These events may determine retinal cell fate, progression and severity of the disease.