Calpain-1 Cleaves and Activates Caspase-7

Caspase-7 is an executioner caspase that plays a key role in apoptosis, cancer, and a number of neurodegenerative diseases. The mechanism of caspase-7 activation by granzyme B and caspase-3 has been well characterized. However, whether other proteases such as calpains activate or inactivate caspase-7 is not known. Here, we present that recombinant caspase-7 is directly cleaved by calpain-1 within the large subunit of caspase-7 to produce two novel products, large subunit p18 and p17. This new form of caspase-7 has a 6-fold increase in V_max when compared with the previously characterized p20/p12 form. Zymography revealed that the smaller caspase-7 product (p17) is 18-fold more active than either the caspase-3-cleaved product (p20) or the larger calpain-1 product of caspase-7 (p18). Mass spectrometry and site-directed mutagenesis identified the calpain cleavage sites within the caspase-7 large subunit at amino acid 36 and 45/47. These proteolysis events occur in vivo as indicated by the accumulation of caspase-7 p18 and p17 subunits in cortical neurons undergoing Ca²⁺ dysregulation. Further, cleavage at amino acid 45/47 of caspase-7 by calpain results in a reduction in nuclear localization when compared with the caspase-3 cleavage product of caspase-7 (p20). Our studies suggest the calpain-activated form of caspase-7 has unique enzymatic activity, localization, and binding affinity when compared with the caspase-activated form.Apoptosis is a well-defined cellular destruction pathway that primarily utilizes a family of cysteine proteases, the caspases (1, 2). This cell death program can be initiated by cell death receptor activation (extrinsic pathway) or a variety of drugs or cellular stresses (intrinsic pathway) leading to activation of apical caspase-8, -9, and/or -10 (1, 3, 4). These initiator caspases in turn directly activate the executioner caspases, caspase-3 and -7, which through proteolysis of defined substrates are responsible for the dismantling of the cell and subsequent death (3, 4). Granzyme B, released by cytotoxic T lymphocytes to protect the host from pathogens and tumor cells, can also initiate this apoptotic cascade and therefore is considered an apical caspase mimic (5–7). All caspases, as well as granzyme B, preferentially cleave after aspartic acid residues, with many having well-defined consensus sequences, making substrate cleavage sites easy to predict and establish (3, 4, 7, 8).Caspases exist in a latent form prior to activation. Both the initiator and executioner caspases are synthesized as a single chain protein, which require proteolytic cleavage to become active. Procaspase-7 is expressed as a 303-amino acid residue polypeptide chain. The activation and regulation of executioner caspase-7 by caspases and granzyme B has been extensively studied. Caspase-7 requires cleavage by caspase-3 and caspase-8/-10 or granzyme B, for activation (6, 9). Current evidence suggests that caspase-3 initially cleaves off the first 23 amino acids (propeptide, 2 kDa), followed by caspase-8/-10 or granzyme B cleaving between the large (20 kDa) and small (12 kDa) subunit after amino acid 198 to activate the enzyme. The large subunit containing the catalytic His-237 and Cys-285 (caspase-1 numbering convention), and the small subunit are involved in the formation of the substrate-binding region. In vitro, granzyme B can also activate caspase-7 independently of caspase-3, but this does not appear to occur in vivo (5, 6). Currently, there is no evidence that other classes of proteases play a role in activating or modulating caspase-7 activity.Changes in intracellular Ca²⁺ levels influence apoptosis in a number of cell types (10–13). Because in many of these apoptotic cell models the Ca²⁺-dependent cysteine proteases, calpains, are activated upstream of caspases (14–16), it is possible that calpains may activate and/or modulate caspase activity via direct cleavage. Studies directed at understanding calpains with respect to caspase activation are limited. Calpain-2 was shown to cleave procaspase-9, decreasing its activity (17). In the same study, calpain-2 treatment cleaved procaspase-7 to produce a single, novel fragment, but in this case the effect on enzymatic activity was not investigated (17). To improve our understanding of calpains and the role of calcium in cell death, we carried out studies directed at understanding how calpains activate or modulate caspase activity. We found that calpain treatment produced a large increase in caspase-7 activity. Calpain cleaves procaspase-7 to produce two large subunits of 18.5 and 17.2 kDa, the smaller of which has a robust increase in activity relative to the 20-kDa large subunit produced by caspase-3 cleavage of caspase-7. Both calpain cleavage sites in caspase-7 are identified using mass spectrometry. N-methyl-d-aspartate-induced Ca²⁺-dependent cell death in primary cortical neurons produced calpain-derived caspase-7 cleavage products in vivo. Lastly, the strictly cytosolic localization of the smaller calpain fragment confirms that a previously identified nuclear localization signal (18) is involved in caspase-7 cytosolic/nuclear distribution. Our data suggest that increases in Ca²⁺ leading to activation of calpains may significantly modulate caspase-7 activity and thus, apoptosis.     

戊型肝炎病毒感染激活补体系统

为了探究补体系统与戊型肝炎病毒复制的相关性,分别在HEV感染的A549细胞和BALB/c小鼠中检测C3aR、CD55和CD59蛋白的表达.利用RT-qPCR定量检测细胞和组织中补体的表达,采用免疫组化法检测HEV感染BALB/c小鼠中补体CD59及C5b-9的表达,ELISA检测补体相关炎症因子的变化.HEV感染可以激活补体蛋白C3aR、C5b-9、CD55和CD59的表达,引起补体蛋白相关炎症因子IL-10表达水平下降,IL-12和TNF-α的表达水平的上升,从而导致机体的炎症反应,加剧组织损伤.HEV感染激活补体系统并参与早期的抗病毒反应,HEV感染对补体的持续激活导致炎症因子过度表达,加重机体损伤.     

Ryanodine Activates Sea Urchin Eggs

We have studied the effect on sea urchin eggs of ryanodine, a plant alkaloid that causes muscle contraction by opening calcium channels in the sarcoplasmic reticulum terminal cisternae. Ryanodine, although it is less effective that IP₃, produces full or partial activation in 62% of injected sea urchin eggs. In addition ryanodine inhibits in a dose dependant manner ⁴⁵Ca pumping in the isolated egg cortex or in eggs permeabilized with digitonin. Efflux experiments show that in fact ryanodine as IP₃ stimulates the release of calcium sequestered intracellularly. We further show that these effects of ryanodine are inhibited by Mg⁺⁺, ruthenium red and heparin. Our results suggest that ryanodine-sensitive intracellular calcium channels exist in the sea urchin egg.     

Glutaminolysis Activates Rag-mTORC1 Signaling

Amino acids control cell growth via activation of the?highly conserved kinase TORC1. Glutamine is a particularly important amino acid in cell growth control and metabolism. However, the role of glutamine in TORC1 activation remains poorly defined. Glutamine is metabolized through glutaminolysis to?produce α-ketoglutarate. We demonstrate that glutamine in combination with leucine activates mammalian TORC1 (mTORC1) by enhancing glutaminolysis and α-ketoglutarate production. Inhibition of glutaminolysis prevented GTP loading of RagB and lysosomal translocation and subsequent activation of mTORC1. Constitutively active Rag heterodimer activated mTORC1 in the absence of glutaminolysis. Conversely, enhanced glutaminolysis or?a cell-permeable α-ketoglutarate analog stimulated lysosomal translocation and activation of mTORC1. Finally, cell growth and autophagy, two processes controlled by mTORC1, were regulated by glutaminolysis. Thus, mTORC1 senses and is activated by?glutamine and leucine via glutaminolysis and α-ketoglutarate production upstream of Rag. This may provide an explanation for glutamine addiction in cancer cells.     

Udu Deficiency Activates DNA Damage Checkpoint

Udu has been shown to play an essential role during blood cell development; however, its roles in other cellular processes remain largely unexplored. In addition, ugly duckling (udu) mutants exhibited somite and myotome boundary defects. Our fluorescence-activated cell sorting analysis also showed that the loss of udu function resulted in defective cell cycle progression and comet assay indicated the presence of increased DNA damage in udu^tu24 mutants. We further showed that the extensive p53-dependent apoptosis in udu^tu24 mutants is a consequence of activation in the Atm–Chk2 pathway. Udu seems not to be required for DNA repair, because both wild-type and udu embryos similarly respond to and recover from UV treatment. Yeast two-hybrid and coimmunoprecipitation data demonstrated that PAH-L repeats and SANT-L domain of Udu interacts with MCM3 and MCM4. Furthermore, Udu is colocalized with 5-bromo-2′-deoxyuridine and heterochromatin during DNA replication, suggesting a role in maintaining genome integrity.     

Auditory Attention Activates Peripheral Visual Cortex

Background

Recent neuroimaging studies have revealed that putatively unimodal regions of visual cortex can be activated during auditory tasks in sighted as well as in blind subjects. However, the task determinants and functional significance of auditory occipital activations (AOAs) remains unclear.

Methodology/Principal Findings

We examined AOAs in an intermodal selective attention task to distinguish whether they were stimulus-bound or recruited by higher-level cognitive operations associated with auditory attention. Cortical surface mapping showed that auditory occipital activations were localized to retinotopic visual cortex subserving the far peripheral visual field. AOAs depended strictly on the sustained engagement of auditory attention and were enhanced in more difficult listening conditions. In contrast, unattended sounds produced no AOAs regardless of their intensity, spatial location, or frequency.

Conclusions/Significance

Auditory attention, but not passive exposure to sounds, routinely activated peripheral regions of visual cortex when subjects attended to sound sources outside the visual field. Functional connections between auditory cortex and visual cortex subserving the peripheral visual field appear to underlie the generation of AOAs, which may reflect the priming of visual regions to process soon-to-appear objects associated with unseen sound sources.     

The Pannexin 1 Channel Activates the Inflammasome in Neurons and Astrocytes

The inflammasome is a multiprotein complex involved in innate immunity. Activation of the inflammasome causes the processing and release of the cytokines interleukins 1β and 18. In primary macrophages, potassium ion flux and the membrane channel pannexin 1 have been suggested to play roles in inflammasome activation. However, the molecular mechanism(s) governing inflammasome signaling remains poorly defined, and it is undetermined whether these mechanisms apply to the central nervous system. Here we show that high extracellular potassium opens pannexin channels leading to caspase-1 activation in primary neurons and astrocytes. The effect of K⁺ on pannexin 1 channels was independent of membrane potential, suggesting that stimulation of inflammasome signaling was mediated by an allosteric effect. The activation of the inflammasome by K⁺ was inhibited by the pannexin 1 channel blocker probenecid, supporting a role of pannexin 1 in inflammasome activation. Co-immunoprecipitation of neuronal lysates indicates that pannexin 1 associates with components of the multiprotein inflammasome complex, including the P2X7 receptor and caspase-1. Moreover antibody neutralization of the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) blocked ATP-induced cell death in oocytes co-expressing P2X7 receptor and pannexin 1. Thus, in contrast to macrophages and monocytes in which low intracellular K⁺ has been suggested to trigger inflammasome activation, in neural cells, high extracellular K⁺ activates caspase-1 probably through pannexin 1.Pannexin 1 is a vertebrate ortholog of the invertebrate innexin gap junction proteins (1), but it does not appear to form functional gap junctions in vivo. Instead pannexin 1 acts as a membrane channel that carries ions and signaling molecules between the cytoplasm and the extracellular space (2, 3). As such, it is a candidate ATP release channel in various cell types, including erythrocytes, astrocytes, bronchial epithelial cells, and taste cells. Various functional roles have been ascribed to pannexin 1 including local vascular perfusion control and propagation of intercellular calcium waves (4–6). Recently pannexin 1 was also shown to form the large pore of the P2X7 purinergic receptor (7, 8). P2X7 plays a major role in inflammation, and its activation by extracellular ATP results in release of interleukin (IL)²-1β from macrophages, probably involving pannexin 1 as a signaling molecule (7).IL-1β production and maturation are tightly regulated by caspase-1 incorporated into large protein complexes termed inflammasomes (9–11). The molecular composition of the inflammasome depends on the identity of the NOD-like receptor (NLR) family member serving as a scaffold protein in the complex (12). The members of the cytosolic NLR family appear to recognize conserved microbial and viral components termed pathogen-associated molecular patterns in intracellular compartments (13). The bipartite adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) bridges the interaction between NLR proteins and inflammatory caspases and plays a central role in the assembly of inflammasomes and the activation of caspase-1 in response to a broad range of pathogen-associated molecular patterns and intracellular pathogens (14). In addition, the inflammasome can be activated by danger-associated molecular patterns, molecules endogenous to the organism that signal stress or injury, including extracellular ATP acting at ionotropic P2X7 receptors, fibronectin, or monosodium urate crystals (15, 16). Moreover it has been suggested that a rapid K⁺ efflux through ATP-activated P2X7 receptors induces inflammasome assembly (17–20).Despite the recent advances in the understanding of accessory proteins required for full activation of caspase-1, little is known about the signaling pathways that trigger inflammasome activation, particularly in the central nervous system (CNS). Recently we reported that spinal cord neurons contain the NLRP1/NALP1 inflammasome consisting of NLRP1, ASC, caspase-1, caspase-11, and the X-linked inhibitor of apoptosis protein (XIAP) and that spinal cord injury induces rapid activation of the inflammasome, causing processing and secretion of IL-1β and IL-18. Moreover antibody neutralization of ASC reduces caspase-1 activation and IL-1 cytokine processing, leading to significant tissue sparing and functional improvement (21). In this study, we focused on signaling events coupling pannexin 1 and P2X7 receptors to rapid caspase-1 activation in primary neurons and astrocytes. We provide compelling evidence that high extracellular K⁺ opens the pannexin 1 channel and activates inflammasomes in neurons and astrocytes, but not THP-1 cells, thus leading to caspase-1 activation. This signaling pathway in neurons is mediated through protein interactions between pannexin 1 and inflammasome proteins. We also provide evidence that ATP acting on P2X7 induces rapid cell death and that antibody neutralization of ASC blocks ATP-induced cell death. Thus, contrary to the widely accepted view in macrophages and monocytes that low intracellular K⁺ triggers inflammasome activation, high extracellular K⁺ surrounding cells such as neurons and astrocytes opens pannexin 1 channels and induces processing of caspase-1.     

Aurora-A Phosphorylates,Activates, and Relocalizes the Small GTPase RalA

The small GTPase Ras, which transmits extracellular signals to the cell, and the kinase Aurora-A, which promotes proper mitosis, can both be inappropriately activated in human tumors. Here, we show that Aurora-A in conjunction with oncogenic Ras enhances transformed cell growth. Furthermore, such transformation and in some cases also tumorigenesis depend upon S194 of RalA, a known Aurora-A phosphorylation site. Aurora-A promotes not only RalA activation but also translocation from the plasma membrane and activation of the effector protein RalBP1. Taken together, these data suggest that Aurora-A may converge upon oncogenic Ras signaling through RalA.Ras small GTPases (H-, N-, and K-Ras) function as regulated binary switches, typically at the plasma membrane, whereby extracellular signal-stimulated cell surface receptors stimulate guanine nucleotide exchange factors (GEFs) to promote GDP/GTP exchange to favor the formation of active, GTP-bound Ras. This, in turn, induces a conformational change in the effector binding domain in Ras, permitting the binding and activation of effector proteins, such as Raf proteins, phosphatidylinositol 3-kinase (PI3K), and RalGEF proteins, that mediate Ras signaling (19, 53). One-third of human cancers harbor point mutations in Ras that render the protein in a constitutively active GTP-bound state, promoting a host of cancer cell phenotypes (40).Aurora-A belongs to a family of three related serine/threonine mitotic kinases critical for many stages of mitosis. Studies of a number of model systems indicate that Aurora-A phosphorylates a growing number of proteins in a spatially and temporally restricted manner to ensure proper centrosomal maturation and separation, mitotic entry, mitotic spindle assembly, chromosome alignment and separation, and subsequent cytokinesis (18, 42). Overexpression of Aurora-A is seen in human cancers (22, 31, 32) and can cause growth transformation of Rat1 and NIH 3T3 rodent fibroblast cell lines (5). That it can cause tumor formation in the mammary epithelia of mice only after a long latency (60, 68) and that alone it does not transform primary rodent cells (1) or induce pancreatic cancer formation in mice (61) argue that Aurora-A acts in concert with other changes to promote a transformed state.Although the mechanism by which Aurora-A promotes oncogenesis remains to be understood, emerging evidence suggests that Aurora-A may cooperate with the Ras oncoprotein. First, activating mutations in KRAS occur in nearly all pancreatic cancers (28), and Aurora-A has been found to be overexpressed in this tumor type by gene amplification (21) or by elevated levels of mRNA or protein (21, 35). This overexpression likely fosters tumor growth, as suppression of Aurora-A expression by interfering RNA or treatment with Aurora-A inhibitors also impairs pancreatic cancer cell growth (26, 48). Second, overexpression of Aurora-A enhances Ras-induced transformation of murine 3T3A31-1 fibroblasts (59). Third, Aurora kinases physically interact with RasGAP in vitro (23), and inhibition of Aurora-B binding to RasGAP causes apoptosis (49). Fourth, two components of the RalGEF-Ral effector pathway of Ras, which is known to promote Ras oncogenesis (38), are substrates for Aurora-A (66). Specifically, active Ras binds to RalGEF proteins, a family of guanine nucleotide exchange factors (GEFs) and activators of the related small GTPases RalA and RalB. Both the RalGEF protein RalGDS and RalA were shown to be phosphorylated by Aurora-A. With regard to the latter, RalA is phosphorylated at S194 in its C-terminal membrane binding domain, leading to elevated levels of activated RalA-GTP. Constitutively active RalA (G23V) also cooperated with ectopically expressed Aurora-A to promote anchorage-independent growth of MDCK epithelial cells, whereas a RalA mutant that could not be phosphorylated by Aurora-A (RalA^G23V,S194A) was impaired in this activity (66). Moreover, overexpression of both Aurora-A and RalA mRNA is associated with advanced human bladder cancer (58). Finally, as indirect evidence for the importance of the Aurora-A phosphorylation site in RalA, S194 and S183 were identified as sites of dephosphorylation by the phosphatase PP2A. Short hairpin RNA (shRNA) silencing of PP2A expression increased phosphorylation of S194 and S183 and formation of RalA-GTP, whereas replacing endogenous RalA with S194A or S183A mutants resulted in a loss of tumorigenic growth of human embryonic kidney (HEK) cells expressing oncogenic H-Ras, hTERT, and the early region of simian virus 40 (SV40) (56). Given these observations, we explored the molecular connection between Aurora-A and the Ras-RalGEF-RalA pathway.     

SUMO Chain-Induced Dimerization Activates RNF4

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Equid Herpesvirus Type 1 Activates Platelets

Equid herpesvirus type 1 (EHV-1) causes outbreaks of abortion and neurological disease in horses. One of the main causes of these clinical syndromes is thrombosis in placental and spinal cord vessels, however the mechanism for thrombus formation is unknown. Platelets form part of the thrombus and amplify and propagate thrombin generation. Here, we tested the hypothesis that EHV-1 activates platelets. We found that two EHV-1 strains, RacL11 and Ab4 at 0.5 or higher plaque forming unit/cell, activate platelets within 10 minutes, causing α-granule secretion (surface P-selectin expression) and platelet microvesiculation (increased small events double positive for CD41 and Annexin V). Microvesiculation was more pronounced with the RacL11 strain. Virus-induced P-selectin expression required plasma and 1.0 mM exogenous calcium. P-selectin expression was abolished and microvesiculation was significantly reduced in factor VII- or X-deficient human plasma. Both P-selectin expression and microvesiculation were re-established in factor VII-deficient human plasma with added purified human factor VIIa (1 nM). A glycoprotein C-deficient mutant of the Ab4 strain activated platelets as effectively as non-mutated Ab4. P-selectin expression was abolished and microvesiculation was significantly reduced by preincubation of virus with a goat polyclonal anti-rabbit tissue factor antibody. Infectious virus could be retrieved from washed EHV-1-exposed platelets, suggesting a direct platelet-virus interaction. Our results indicate that EHV-1 activates equine platelets and that α-granule secretion is a consequence of virus-associated tissue factor triggering factor X activation and thrombin generation. Microvesiculation was only partly tissue factor and thrombin-dependent, suggesting the virus causes microvesiculation through other mechanisms, potentially through direct binding. These findings suggest that EHV-1-induced platelet activation could contribute to the thrombosis that occurs in clinically infected horses and provides a new mechanism by which viruses activate hemostasis.     

Malarial Hemozoin Activates the NLRP3 Inflammasome through Lyn and Syk Kinases

The intraerythrocytic parasite Plasmodium—the causative agent of malaria—produces an inorganic crystal called hemozoin (Hz) during the heme detoxification process, which is released into the circulation during erythrocyte lysis. Hz is rapidly ingested by phagocytes and induces the production of several pro-inflammatory mediators such as interleukin-1β (IL-1β). However, the mechanism regulating Hz recognition and IL-1β maturation has not been identified. Here, we show that Hz induces IL-1β production. Using knockout mice, we showed that Hz-induced IL-1β and inflammation are dependent on NOD-like receptor containing pyrin domain 3 (NLRP3), ASC and caspase-1, but not NLRC4 (NLR containing CARD domain). Furthermore, the absence of NLRP3 or IL-1β augmented survival to malaria caused by P. chabaudi adami DS. Although much has been discovered regarding the NLRP3 inflammasome induction, the mechanism whereby this intracellular multimolecular complex is activated remains unclear. We further demonstrate, using pharmacological and genetic intervention, that the tyrosine kinases Syk and Lyn play a critical role in activation of this inflammasome. These findings not only identify one way by which the immune system is alerted to malarial infection but also are one of the first to suggest a role for tyrosine kinase signaling pathways in regulation of the NLRP3 inflammasome.     

Turning the Tables: Myc Activates Wnt in Breast Cancer

Previous molecular and genetic data implicate the c-myc gene as a critical downstream effector of the Wnt/TCF pathway in colon cancer. However, the involvement of c-myc in mammary epithelial cell transformation had not been explored. We recently showed that c-Myc induces a profound morphological transformation in human mammary epithelial cells accompanied by anchorage-independent growth. The mechanism of c-Myc transformation was revealed in part through the finding that, in contrast to colon cancer, c-Myc activates the Wnt pathway and endogenous TCF activity by suppressing the Wnt inhibitors DKK1 and SFRP1. Notably, DKK1 and SFRP1 were found to be strongly suppressed in human breast cancer cell lines and their re-expression inhibited the transformed phenotype. We demonstrated that breast cancer cells become dependent on repression of the Wnt inhibitors for cell proliferation, i.e. they have acquired an “oncogene addiction”, suggesting that the Myc-Wnt pathway is an attractive therapeutic target. We propose that a positive feedback loop of c-myc and Wnt signaling operates in breast cancer.