Relevance of caspase activity during apoptosis in pubertal rat spermatogenesis

Caspases are a family of cysteine-proteases, activated upon several different stimuli, which execute apoptosis in many cell death models. Previous work of our group has shown rats have the highest rate of apoptosis during the first wave of spermatogenesis (between 20 and 25 days after birth), as evaluated by TUNEL and caspase activity. However, the hierarchical order of caspase activation and the relevance of each caspase during germ cell apoptosis are not clear. Thus, the goal of this work is to take a pharmacological approach to dissect the apoptosis pathway of caspase activation. Results showed that intratesticular injection of a caspase-8 inhibitor (z-IETD-fmk), or a pan-caspase inhibitor (z-VAD- fmk), significantly decreased the cleavage of p115 and PARP, two endogenous substrates of caspases, in 22-day-old rats. Additionally, these inhibitors promoted a significant reduction in the number of apoptotic germ cells. On the other hand, intratesticular injection of two different inhibitors of the intrinsic pathway (z-LEHD-fmk and minocycline) did not have any effect upon caspase substrates cleavage (p115 and PARP) or the number of apoptotic germ cells. Therefore, we conclude that the extrinsic pathway of apoptosis plays an important role in physiological germ cell apoptosis during the first round of spermatogenesis in the rat.     

Caspase家族在细胞凋亡中的研究进展

半胱氨酸蛋白酶（caspase）家族成员是近两年来发现的在细胞凋亡过程中起关键作用的酶,对其深入研究有助于揭示细胞凋亡的发生机制,阐明不同疾病的发病机理。本文介绍了Caspase家族及其在细胞凋亡中的研究近况。     

Inactivation of ceramide transfer protein during pro-apoptotic stress by Golgi disassembly and caspase cleavage

The mammalian Golgi apparatus is composed of multiple stacks of cisternal membranes organized laterally into a polarized ribbon. Furthermore, trans-Golgi membranes come in close apposition with ER (endoplasmic reticulum) membranes to form ER-trans-Golgi contact sites, which may facilitate transfer of newly synthesized ceramide from the ER to SM (sphingomyelin) synthase at the trans-Golgi via CERT (ceramide transfer protein). CERT interacts with both ER and Golgi membranes, and together with Golgi morphology contributes to efficient SM synthesis. In the present study, we show that Golgi disassembly during pro-apoptotic stress induced by TNFα (tumour necrosis factor α) and anisomycin results in decreased levels of CERT at the Golgi region. This is accompanied by a caspase-dependent loss of full-length CERT and reduction in de novo SM synthesis. In vitro, CERT is cleaved by caspases 2, 3 and 9. Truncated versions of CERT corresponding to fragments generated by caspase 2 cleavage at Asp213 were mislocalized and did not promote efficient de novo SM synthesis. Thus it is likely that during cellular stress, disassembly of Golgi structure together with inactivation of CERT by caspases causes a reduction in ceramide trafficking and SM synthesis, and could contribute to the cellular response to pro-apoptotic stress.     

Conservation of caspase substrates across metazoans suggests hierarchical importance of signaling pathways over specific targets and cleavage site motifs in apoptosis

Caspases, cysteine proteases with aspartate specificity, are key players in programmed cell death across the metazoan lineage. Hundreds of apoptotic caspase substrates have been identified in human cells. Some have been extensively characterized, revealing key functional nodes for apoptosis signaling and important drug targets in cancer. But the functional significance of most cuts remains mysterious. We set out to better understand the importance of caspase cleavage specificity in apoptosis by asking which cleavage events are conserved across metazoan model species. Using N-terminal labeling followed by mass spectrometry, we identified 257 caspase cleavage sites in mouse, 130 in Drosophila, and 50 in Caenorhabditis elegans. The large majority of the caspase cut sites identified in mouse proteins were found conserved in human orthologs. However, while many of the same proteins targeted in the more distantly related species were cleaved in human orthologs, the exact sites were often different. Furthermore, similar functional pathways are targeted by caspases in all four species. Our data suggest a model for the evolution of apoptotic caspase specificity that highlights the hierarchical importance of functional pathways over specific proteins, and proteins over their specific cleavage site motifs.     

Partial cleavage of A-type lamins concurs with their total disintegration from the nuclear lamina during apoptosis

Although activated caspase 6 is capable of cleaving both A- and B-type lamins during apoptosis, the higher-order structure of the nuclear lamina may cause a differential breakdown of these two types of lamins. In order to obtain a better understanding of the dynamics and the consequences of the rapid, coordinated breakdown of the lamina complex, we applied the green fluorescent protein (GFP) technology in living cells, in which the fate of individual caspase cleavage fragments of A- and B-type lamins was examined. CHO-K1 cells were stably transfected with cDNA constructs encoding N-terminally GFP-labelled hybrids of lamin A, lamin Adelta10, lamin C or lamin B1. The course of the apoptotic process, induced by the kinase inhibitor staurosporine or by the proteasome inhibitor MG132, was monitored by digital imaging microscopy or confocal microscopy. Time-lapse recordings showed that parallel to DNA condensation N-terminally GFP-tagged A-type lamins became diffusely dispersed throughout the nucleoplasm and rapidly translocated to the cytoplasm. In contrast, the majority of GFP-lamin B1 signal remained localised at the nuclear periphery, even after extensive DNA condensation. Comparison of lamin B1-GFP signal with A-type lamin antibody staining in the same apoptotic cells confirmed the temporal differences between A- and B-type lamina dispersal. Immunoblotting revealed only a partial cleavage of A-type lamins and an almost complete cleavage of lamin B1 during apoptosis. In contrast to lamin B1 in normal cells, this cleaved lamin B1, which is apparently still associated with the nuclear membrane, can be completely extracted by methanol or ethanol. Fluorescence loss of intensity after photobleaching experiments showed that in apoptotic cells A-type lamin-GFP molecules diffuse almost freely in both nucleoplasm and cytoplasm, while the lamin B1-GFP fragments remain more stably associated with the nuclear membrane, which is confirmed by co-localisation immunofluorescence studies with a nucleoporin p62 antibody. Our results therefore clearly show a differential behaviour of A- and B-type lamins during apoptosis, suggesting not only distinct differences in the organisation of the lamina filaments, but also that caspase cleavage of only a small fraction of A-type lamins is needed for its complete disintegration.     

Detection of elevated caspase activation and early apoptosis in liver diseases

Apoptosis has been implicated in the pathogenesis of many diseases including various forms of liver failure. The apoptotic process is essentially regulated by intracellular proteases, called caspases, which cleave several vital proteins. Despite the rapid elucidation of apoptotic signaling cascades, however, almost no information exists about the activation of caspases in situ. In the present study, a monoclonal antibody was employed which selectively recognized cleavage site-specific fragments of the caspase substrate cytokeratin-18. We demonstrate that this antibody labeled apoptotic hepatocytes in culture and, in addition, could be used to monitor caspase activation in formalin-fixed tissue biopsies. In liver sections of different liver diseases an increased number of early apoptotic cells was detected which were not found in normal tissue. Our data reveal that hepatobiliary diseases are characterized by elevated caspase activation and apoptosis, which can be specifically detected in situ by a cleavage site-specific antibody against cytokeratin-18.     

Caspase-mediated cleavage of the stacking protein GRASP65 is required for Golgi fragmentation during apoptosis

The mammalian Golgi complex is comprised of a ribbon of stacked cisternal membranes often located in the pericentriolar region of the cell. Here, we report that during apoptosis the Golgi ribbon is fragmented into dispersed clusters of tubulo-vesicular membranes. We have found that fragmentation is caspase dependent and identified GRASP65 (Golgi reassembly and stacking protein of 65 kD) as a novel caspase substrate. GRASP65 is cleaved specifically by caspase-3 at conserved sites in its membrane distal COOH terminus at an early stage of the execution phase. Expression of a caspase-resistant form of GRASP65 partially preserved cisternal stacking and inhibited breakdown of the Golgi ribbon in apoptotic cells. Our results suggest that GRASP65 is an important structural component required for maintenance of Golgi apparatus integrity.     

Caspase-2 is an initiator caspase responsible for pore-forming toxin-mediated apoptosis

Bacterial pathogens modulate host cell apoptosis to establish a successful infection. Pore-forming toxins (PFTs) secreted by pathogenic bacteria are major virulence factors and have been shown to induce various forms of cell death in infected cells. Here we demonstrate that the highly conserved caspase-2 is required for PFT-mediated apoptosis. Despite being the second mammalian caspase to be identified, the role of caspase-2 during apoptosis remains enigmatic. We show that caspase-2 functions as an initiator caspase during Staphylococcus aureus α-toxin- and Aeromonas aerolysin-mediated apoptosis in epithelial cells. Downregulation of caspase-2 leads to a strong inhibition of PFT-mediated apoptosis. Activation of caspase-2 is PIDDosome-independent, and endogenous caspase-2 is recruited to a high-molecular-weight complex in α-toxin-treated cells. Interestingly, prevention of PFT-induced potassium efflux inhibits the formation of caspase-2 complex, leading to its inactivation, thus resisting apoptosis. These results revealed a thus far unknown, obligatory role for caspase-2 as an initiator caspase during PFT-mediated apoptosis.     

Crystal structures of human caspase 6 reveal a new mechanism for intramolecular cleavage self-activation

Dimeric effectors caspase 3 and caspase 7 are activated by initiator caspase processing. In this study, we report the crystal structures of effector caspase 6 (CASP6) zymogen and N-Acetyl-Val-Glu-Ile-Asp-al-inhibited CASP6. Both of these forms of CASP6 have a dimeric structure, and in CASP6 zymogen the intersubunit cleavage site (190)TEVD(193) is well structured and inserts into the active site. This positions residue Asp 193 to be easily attacked by the catalytic residue Cys 163. We demonstrate biochemically that intramolecular cleavage at Asp 193 is a prerequisite for CASP6 self-activation and that this activation mechanism is dependent on the length of the L2 loop. Our results indicate that CASP6 can be activated and regulated through intramolecular self-cleavage.     

Next-generation Interactomics: Considerations for the Use of Co-elution to Measure Protein Interaction Networks

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Highlights

• •Co-elution stands out as a global interactome mapping method.
• •Benefits include all-to-all protein analysis and measurement of interactome perturbations.
• •Different separation, quantification and bioinformatic strategies are available.
• •Design considerations depend largely on system under study.

     

Time-lapse,single cell based confocal imaging analysis of caspase activation and phosphatidylserine flipping during cellular apoptosis

Abstract

Apoptosis is an important phenomenon for investigating the efficacy of anti-cancer drug candidates. The conventional assays for cellular apoptosis, such as enzyme-linked immunosorbent assay, absorbance monitoring for the activity of caspase, and flow cytometric assay, have focused only on biochemical events. We investigated the staurosporine (STS)-induced apoptosis of the murine macrophage RAW-264.7 cell using a cell based bioimaging technique. Using time-lapse confocal microscopy, we monitored caspase-3 activation during apoptosis by imaging the translocation of green fluorescent protein from the cytosol to the nuclei. Five hours after 1 μM STS treatment, caspase-3 was observed to be activated and membrane blebbing was observed simultaneously. Also, the loss of phosphatidylserine (PS) asymmetry in the phospholipid bilayer of plasma membrane during early apoptosis was monitored by imaging annexin-V labeled with fluorescein isocyanate binding to the externalized PS at various concentrations of STS. Moreover, disintegration of the plasma membrane during late apoptosis was confirmed using a nuclear dye, propidium iodide. The single cell based bioimaging data agreed well with those of the biochemical assays for caspase activation and morphological observation for membrane integrity.     

Measurement of apoptosis in cytological specimens by flow cytometry: comparison of Annexin V,caspase cleavage and dUTP incorporation assays

H. P. Dong, A. Holth, M. G. Ruud, E. Emilsen, B. Risberg and B. Davidson Measurement of apoptosis in cytological specimens by flow cytometry: comparison of annexin V, caspase cleavage and dUTP incorporation assays Objective: To compare the performance of different assays for measuring apoptosis in cytological specimens. Methods: Apoptosis was assessed in 27 specimens (22 effusions, five fine needle aspirates; 20 malignant, seven reactive) using flow cytometry, applying assays for the measurement of annexin V expression, caspase‐3 and ‐8 cleavage and deoxynucleotidyl transferase deoxyuridine triphosphates (dUTP) incorporation. Results were studied for differences between reactive and malignant specimens, as well as performance across assays. Results: Wide variation in the degree of apoptosis was observed in both benign and malignant specimens using all assays. However, the percentage of annexin V‐positive cells was higher compared with those showing caspase cleavage or dUTP incorporation in the majority of cases, irrespective of specimen type. Comparative analysis of benign and malignant specimens showed no significant differences in expression of any of the studied parameters. However, tumour cells and reactive mesothelial cells in pleural effusions had a significantly lower level of dUTP incorporation compared with their counterparts in peritoneal specimens (P = 0.001). Conclusions: The present data are in agreement with our previous observation in ovarian carcinoma effusions, that measurement of apoptosis by the annexin V assay provides higher expression values than those obtained by other assays, suggesting that this assay does not accurately reflect the degree of apoptosis in benign or malignant cells in effusions.     

Alix is involved in caspase 9 activation during calcium-induced apoptosis

The cytoplasmic protein Alix/AIP1 (ALG-2 interacting protein X) is involved in cell death through mechanisms which remain unclear but require its binding partner ALG-2 (apoptosis-linked gene-2). The latter was defined as a regulator of calcium-induced apoptosis following endoplasmic reticulum (ER) stress. We show here that Alix is also a critical component of caspase 9 activation and apoptosis triggered by calcium. Indeed, expression of Alix dominant-negative mutants or downregulation of Alix afford significant protection against cytosolic calcium elevation following thapsigargin (Tg) treatment. The function of Alix in this paradigm requires its interaction with ALG-2. In addition, we demonstrate that caspase 9 activation is necessary for apoptosis induced by Tg and that this activation is impaired by knocking down Alix. Altogether, our findings identify, for the first time, Alix as a crucial mediator of Ca²⁺ induced caspase 9 activation.     

Molecular mechanisms of caspase regulation during apoptosis

Caspases, which are the executioners of apoptosis, comprise two distinct classes, the initiators and the effectors. Although general structural features are shared between the initiator and the effector caspases, their activation, inhibition and release of inhibition are differentially regulated. Biochemical and structural studies have led to important advances in understanding the underlying molecular mechanisms of caspase regulation. This article reviews these latest advances and describes our present understanding of caspase regulation during apoptosis.     

Protease cleavage site fingerprinting by label‐free in‐gel degradomics reveals pH‐dependent specificity switch of legumain

Determination of protease specificity is of crucial importance for understanding protease function. We have developed the first gel‐based label‐free proteomic approach (DIPPS—direct in‐gel profiling of protease specificity) that enables quick and reliable determination of protease cleavage specificities under large variety of experimental conditions. The methodology is based on in‐gel digestion of the gel‐separated proteome with the studied protease, enrichment of cleaved peptides by gel extraction, and subsequent mass spectrometry analysis combined with a length‐limited unspecific database search. We applied the methodology to profile ten proteases ranging from highly specific (trypsin, endoproteinase GluC, caspase‐7, and legumain) to broadly specific (matrix‐metalloproteinase‐3, thermolysin, and cathepsins K, L, S, and V). Using DIPPS, we were able to perform specificity profiling of thermolysin at its optimal temperature of 75°C, which confirmed the applicability of the method to extreme experimental conditions. Moreover, DIPPS enabled the first global specificity profiling of legumain at pH as low as 4.0, which revealed a pH‐dependent change in the specificity of this protease, further supporting its broad applicability.     

The caspase 6 derived N-terminal fragment of DJ-1 promotes apoptosis via increased ROS production

In pathological conditions, the amount of DJ-1 determines whether a cell can survive or engage a cell death program. This is exemplified in epithelial cancers, in which DJ-1 expression is increased, while autosomal recessive early onset Parkinson''s disease mutations of DJ-1 generally lead to decreased stability and expression of the protein. We have shown previously that DJ-1 is cleaved by caspase-6 during induction of apoptosis. We demonstrate here that the N-terminal cleaved fragment of DJ-1 (DJ-1 Nt) is specifically expressed in the nucleus and promotes apoptosis in SH-SY5Y neuroblastoma cell lines. In addition, overexpression of DJ-1 Nt in different cell lines leads to a loss of clonogenic potential and sensitizes to staurosporin and 1-methyl-4-phenylpyridinium (MPP+)-mediated caspase activation and apoptosis. Importantly, inhibition of endogenous DJ-1 expression with sh-RNA or DJ-1 deficiency mimics the effect of DJ-1 Nt on cell growth and apoptosis. Moreover, overexpression of DJ-1 Nt increases reactive oxygen species (ROS) production, and sensitizes to MPP+-mediated apoptosis and DJ-1 oxidation. Finally, specific exclusion of DJ-1 Nt from the nucleus abrogates its pro-apoptotic effect. Taken together, our findings identify an original pathway by which generation of a nuclear fragment of DJ-1 through caspase 6-mediated cleavage induces ROS-dependent amplification of apoptosis.     

Beclin 1 cleavage by caspase-3 inactivates autophagy and promotes apoptosis

Autophagy and apoptosis are both highly regulated biological processes that play essential roles in tissue homeostasis, development and diseases. Autophagy is also described as a mechanism of death pathways, however, the precise mechanism of how autophagy links to cell death remains to be fully understood. Beclin 1 is a dual regulator for both autophagy and apoptosis. In this study we found that Beclin 1 was a substrate of caspase-3 with two cleavage sites at positions 124 and 149, respectively. Furthermore, the autophagosome formation occurred, followed by the appearance of morphological hallmarks of apoptosis after staurosporine treatment. The cleavage products of Beclin 1 reduced autophagy and promoted apoptosis in HeLa cells and the cells in which Beclin 1 was stably knocked down by specific shRNA. In addition, the cleavage of Beclin 1 resulted in abrogating the interaction between Bcl-2 with Beclin 1, which could be blocked by z-VAD-fmk. Thus, our results suggest that the cleavage of Beclin 1 by caspase-3 may contribute to inactivate autophagy leading towards augmented apoptosis.