Caspase-mediated cleavage of miRNA processing proteins Drosha, DGCR8, Dicer, and TRBP2 in heat-shocked cells and its inhibition by HSP70 overexpression.

Cells respond to stress through adaptive mechanisms that limit cellular damage and prevent cell death. MicroRNAs act as regulators of stress responses and stress can impact the functioning of miRNA biogenesis pathways. We were interested in the effect that severe proteotoxic stress capable of inducing apoptosis may have on miRNA biogenesis and the impact of the molecular chaperone protein HSP70 under these conditions. We found that the miRNA processing enzymes Drosha and Dicer and their accessory proteins DGCR8 and TRBP2 are cleaved by caspases in apoptotic cells. Overexpression of HSP70 prevented caspase activation and the degradation of these processing proteins. Caspase cleavage of TRBP2 was mapped to amino acid 234 which separates the two dsRNA-binding domains from the C-terminal Dicer interacting domain. Overexpression of TRBP2 was found to increase miRNA maturation, while expression of either of the fragments generated by caspase cleavage impaired maturation. These results indicate that inactivation of miRNA biogenesis is a critical feature of apoptosis and that cleavage of TRBP2, rather than simply a loss of function, serves to create positive acting inhibitors of pre-miRNA maturation.

The dichotomous effects of caffeine on homologous recombination in mammalian cells.

This study was initiated to examine the effects of caffeine on the DNA damage response (DDR) and homologous recombination (HR) in mammalian cells. A 5 mM caffeine treatment caused the cell cycle to stall at G2/M and cells eventually underwent apoptosis. Caffeine exposure also induced a strong DDR along with subsequent activation of wildtype p53 protein. An unexpected observation was the caffeine-induced depletion of Rad51 (and Brca2) proteins. Consequently, caffeine-treated cells were expected to be inefficient in HR. However, a dichotomy in the HR response of cells to caffeine treatment was revealed. Caffeine treatment rendered cells significantly better at performing the nascent DNA synthesis that accompanies the early strand invasion steps of HR. Additionally, caffeine treatment increased chromatin accessibility and elevated the efficiency of illegitimate recombination. Conversely, the increase in nascent DNA synthesis did not translate into a higher number of gene targeting events. Thus, prolonged caffeine exposure stalls the cell cycle, induces a p53-mediated apoptotic response and a down-regulation of critical HR proteins, and for reasons discussed, stimulates early steps of HR, but not the formation of complete recombination products.

Cardiolipin exposure on the outer mitochondrial membrane modulates α-synuclein.

Neuronal loss in Parkinson's disease (PD) is associated with aberrant mitochondrial function and impaired proteostasis. Identifying the mechanisms that link these pathologies is critical to furthering our understanding of PD pathogenesis. Using human pluripotent stem cells (hPSCs) that allow comparison of cells expressing mutant SNCA (encoding α-synuclein (α-syn)) with isogenic controls, or SNCA-transgenic mice, we show that SNCA-mutant neurons display fragmented mitochondria and accumulate α-syn deposits that cluster to mitochondrial membranes in response to exposure of cardiolipin on the mitochondrial surface. Whereas exposed cardiolipin specifically binds to and facilitates refolding of α-syn fibrils, prolonged cardiolipin exposure in SNCA-mutants initiates recruitment of LC3 to the mitochondria and mitophagy. Moreover, we find that co-culture of SNCA-mutant neurons with their isogenic controls results in transmission of α-syn pathology coincident with mitochondrial pathology in control neurons. Transmission of pathology is effectively blocked using an anti-α-syn monoclonal antibody (mAb), consistent with cell-to-cell seeding of α-syn.

An Endophyte Constructs Fungicide-Containing Extracellular Barriers for Its Host Plant.

Surface cracks create sites for pathogen invasion. Yew trees (Taxus) hyperbranch from long-lived buds that lie underneath the bark [1], resulting in persistent bark cracking and deep air pockets, potentially allowing pathogens to enter the nutrient-rich vascular system (vertical phloem and inter-connected radial medullary rays [MR]). Yew is famous as the source of the anti-cancer diterpenoid drug Taxol. A mystery has been why both the tree and its resident non-pathogenic fungi (endophytes) synthesize Taxol, apparently redundantly [2-7]. These endophytes, as well as pure Taxol, suppress fungal pathogens including wood-decaying fungi (WDF) [8-11]. Here we show that a Taxol-producing fungal endophyte, Paraconiothyrium SSM001 [12], migrates to pathogen entry points including branch cracks. The fungus sequesters Taxol in intracellular hydrophobic bodies that are induced by WDF for release by exocytosis, after which the bodies can coalesce to form remarkable extracellular barriers, laced with the fungicide. We propose that microbial construction of fungicide-releasing hydrophobic barriers might be a novel plant defense mechanism. We further propose that the endophyte might be evolutionarily analogous to animal immune cells, in that it might expand plant immunity by acting as an autonomous, anti-pathogen sentinel that monitors the vascular system.

Heat shock inhibition of CDK5 increases NOXA levels through miR-23a repression.

Hyperthermia is a proteotoxic stress that is lethal when exposure is extreme but also cytoprotective in that sublethal exposure leads to the synthesis of heat shock proteins, including HSP70, which are able to inhibit stress-induced apoptosis. CDK5 is an atypical cyclin-dependent kinase family member that regulates many cellular functions including motility and survival. Here we show that exposure of a human lymphoid cell line to hyperthermia causes CDK5 insolubilization and loss of tyrosine-15 phosphorylation, both of which were prevented in cells overexpressing HSP70. Inhibition of CDK5 activity with roscovitine-sensitized cells to heat induced apoptosis indicating a protective role for CDK5 in inhibiting heat-induced apoptosis. Both roscovitine and heat shock treatment caused increased accumulation of NOXA a pro-apoptotic BH3-only member of the BCL2 family. The increased abundance of NOXA by CDK5 inhibition was not a result of changes in NOXA protein turnover. Instead, CDK5 inhibition increased NOXA mRNA and protein levels by decreasing the expression of miR-23a, whereas overexpressing the CDK5 activator p35 attenuated both of these effects on NOXA and miR-23a expression. Lastly, overexpression of miR-23a prevented apoptosis under conditions in which CDK5 activity was inhibited. These results demonstrate that CDK5 activity provides resistance to heat-induced apoptosis through the expression of miR-23a and subsequent suppression of NOXA synthesis. Additionally, they indicate that hyperthermia induces apoptosis through the insolubilization and inhibition of CDK5 activity.

Regulation of apoptosis by heat shock proteins.

Thermotolerance, the acquired resistance of cells to stress, is a well-established phenomenon. Studies of the key mediators of this response, the heat shock proteins (HSPs), have led to the discovery of the important roles played by these proteins in the regulation of apoptotic cell death. Apoptosis is critical for normal tissue homeostasis and is involved in diverse processes including development and immune clearance. Apoptosis is tightly regulated by both proapoptotic and antiapoptotic factors, and dysregulation of apoptosis plays a significant role in the pathophysiology of many diseases. In the recent years, HSPs have been identified as key determinants of cell survival, which can modulate apoptosis by directly interacting with components of the apoptotic machinery. Therefore, manipulation of the HSPs could represent a viable strategy for the treatment of diseases. Here, we review the current knowledge with regard to the mechanisms of HSP-mediated regulation of apoptosis.

Overexpression of HSP70 inhibits cofilin phosphorylation and promotes lymphocyte migration in heat-stressed cells.

Hyperthermia adversely affects cell structure and function, but also induces adaptive responses that allow cells to tolerate these stressful conditions. For example, heat-induced expression of the molecular chaperone protein HSP70 can prevent stress-induced cell death by inhibiting signaling pathways that lead to apoptosis. In this study, we used high-resolution two-dimensional gel electrophoresis and phosphoprotein staining to identify signaling pathways that are altered by hyperthermia and modulated by HSP70 expression. We found that in heat-shocked cells, the actin-severing protein cofilin acquires inhibitory Ser3 phosphorylation, which is associated with an inhibition of chemokine-stimulated cell migration. Cofilin phosphorylation appeared to occur as a result of the heat-induced insolubilization of the cofilin phosphatase slingshot (SSH1-L). Overexpression of HSP70 reduced the extent of SSH1-L insolubilization and accelerated its resolubilization when cells were returned to 37°C after exposure to hyperthermia, resulting in a more rapid dephosphorylation of cofilin. Cells overexpressing HSP70 also had an increased ability to undergo chemotaxis following exposure to hyperthermia. These results identify a critical heat-sensitive target controlling cell migration that is regulated by HSP70 and point to a role for HSP70 in immune cell functions that depend upon the proper control of actin dynamics.

Acute acidification or amiloride treatment suppresses the ability of Hsp70 to inhibit heat-induced apoptosis.

Inhibition of stress-induced apoptosis by the molecular chaperone protein Hsp70 is a contributing factor in tumorigenesis and suppression of this ability could increase the effectiveness of anti-tumor therapy. Tumor cells exist in an acidic environment and acute acidification can sensitize tumor cells to heat-induced cell death. However, the ability of Hsp70 to prevent apoptosis under these conditions has not been examined. The effect of acute acidification on heat-induced apoptosis was examined in a human T-cell line with tetracycline-regulated Hsp70 expression. Apoptosis was inhibited in cells exposed to hyperthermia in acidic media when examined 6 h after the heat stress, but resumed if cells were returned to physiological pH during this recovery period. Long-term proliferation assays showed that acute acidification sensitized cells to heat-induced apoptosis. Hsp70 expressing cells were also sensitized and this was correlated with a reduced ability to suppress the activation of JNK (c-jun N-terminal kinase), Bax and caspase-3. Further sensitization could be achieved with the NHE1 (Na(+)/H(+) exchanger) inhibitor HMA (5-(N, N-hexamethylene) amiloride), which potentiated JNK activation in heat-shocked cells. These results demonstrate that the ability of Hsp70 to suppress apoptosis is compromised when cells are exposed to hyperthermia in an acidic environment, which is correlated with an impaired ability to inhibit JNK activation.

Regulation of CD4 expression via recycling by HRES-1/RAB4 controls susceptibility to HIV infection.

A novel 2986-base transcript encoded by the antisense strand of the HRES-1 human endogenous retrovirus was isolated from peripheral blood lymphocytes. This transcript codes for a 218-amino acid protein, termed HRES-1/Rab4, based on homology to the Rab4 family of small GTPases. Antibody 13407 raised against recombinant HRES-1/Rab4 detected a native protein of identical molecular weight in human T cells. HRES-1 nucleotides 2151-1606, located upstream of HRES-1/Rab4 exon 1, have promoter activity when oriented in the direction of HRES-1/Rab4 transcription. The human immunodeficiency virus, type 1 (HIV-1), tat gene stimulates transcriptional activity of the HRES-1/Rab4 promoter via trans-activation of the HRES-1 long terminal repeat. Transfection of HIV-1 tat into HeLa cells or infection of H9 and Jurkat cells by HIV-1 increased HRES-1/Rab4 protein levels. Overexpression of HRES-1/Rab4 in Jurkat cells abrogated HIV infection, gag p24 production, and apoptosis, whereas dominant-negative HRES-1/Rab4(S27N) had the opposite effects. HRES-1/Rab4 inhibited surface expression of CD4 and targeted it for lysosomal degradation. HRES-1/Rab4(S27N) enhanced surface expression, recycling, and total cellular CD4 content. Infection by HIV elicited a coordinate down-regulation of CD4 and up-regulation of HRES-1/Rab4 in PBL. Moreover, overexpression of HRES-1/Rab4 reduced CD4 expression on peripheral blood CD4+ T cells. Stimulation by HIV-1 of HRES-1/Rab4 expression and its regulation of CD4 recycling reveal novel coordinate interactions between an infectious retrovirus and the human genome.

Distinct hsp70 domains mediate apoptosis-inducing factor release and nuclear accumulation.

Although hsp70 antagonizes apoptosis-inducing factor (AIF)-mediated cell death, the relative importance of preventing its release from mitochondria versus sequestering leaked AIF in the cytosol remains controversial. To dissect these two protective mechanisms, hsp70 deletion mutants lacking either the chaperone function (hsp70-deltaEEVD) or ATPase function (hsp70-deltaATPase) were selectively overexpressed before exposing cells to a metabolic inhibitor, an insult sufficient to cause mitochondrial AIF release, nuclear AIF accumulation, and apoptosis. Compared with empty vector, overexpression of wild type human hsp70 inhibited bax activation and reduced mitochondrial AIF release after injury. In contrast, mutants lacking either the chaperone function (hsp70-deltaEEVD) or the ATP hydrolytic domain (hsp70-deltaATPase) failed to prevent mitochondrial AIF release. Although hsp70-deltaEEVD did not inhibit bax activation or mitochondrial membrane injury after cell stress, this hsp70 mutant co-immunoprecipitated with leaked AIF in injured cells and decreased nuclear AIF accumulation. In contrast, hsp70-deltaATPase did not interact with AIF either in intact cells or in a cell-free system and furthermore, failed to prevent nuclear AIF accumulation. These results demonstrate that mitochondrial protection against bax-mediated injury requires both intact chaperone and ATPase functions, whereas the ATPase domain is critical for sequestering AIF in the cytosol.

Induction of HSP70 expression and recruitment of HSC70 and HSP70 in the nucleus reduce aggregation of a polyalanine expansion mutant of PABPN1 in HeLa cells.

Nuclear inclusions formed by the aggregation of a polyalanine expansion mutant of the nuclear poly(A)-binding protein (PABPN1) is a hallmark of oculopharyngeal muscular dystrophy (OPMD). OPMD is a dominant autosomal disease in which patients exhibit progressive difficulty of swallowing and eyelid elevation, starting around the age of 50. At present, there is no specific treatment to reduce the aggregate burden in patients. However, in cell culture models of OPMD, reduction of protein aggregation can be achieved by ectopic expression of HSP70. As gene transfer may not be the most effective means to elevate HSP70 levels, we tested four pharmacological agents for their ability to induce HSP70, recruit both HSP70 and HSC70 into the cell nucleus and reduce mutant PABPN1 aggregation in a HeLa cell culture model. We show here that exposure to moderate levels of ZnSO4, 8-hydroxyquinoline, ibuprofen and indomethacin produced a robust stress response resulting in the induction of HSP70 in HeLa cells expressing the mutant PABPN1 as a green fluorescent protein (GFP) fusion protein. Both HSP70 and the constitutive chaperone HSC70 localized in the nucleus of cells treated with any one of the four agents. This stress response was similar to what was observed following hyperthermia. All four agents also caused a significant reduction in the cellular burden of protein aggregates, as was judged by confocal microscopy and solubility changes of the aggregates. A concomitant reduction of cell death in drug-treated mutant PABPN1 expressing cells was also observed.

Hsp70 inhibits heat-induced apoptosis upstream of mitochondria by preventing Bax translocation.

Hsp70 overexpression can protect cells from stress-induced apoptosis. Our previous observation that Hsp70 inhibits cytochrome c release in heat-stressed cells led us to examine events occurring upstream of mitochondrial disruption. In this study we examined the effects of heat shock on the proapoptotic Bcl-2 family member Bax because of its central role in regulating cytochrome c release in stressed cells. We found that heat shock caused a conformational change in Bax that leads to its translocation to mitochondria, stable membrane association, and oligomerization. All of these events were inhibited in cells that had elevated levels of Hsp70. Hsp70 did not physically interact with Bax in control or heat-shocked cells, indicating that Hsp70 acts to suppress signals leading to Bax activation. Hsp70 inhibited stress-induced JNK activation and inhibition of JNK with SP600125 or by expression of a dominant negative mutant of JNK-blocked Bax translocation as effectively as Hsp70 overexpression. Hsp70 did not protect cells expressing a mutant form of Bax that has constitutive membrane insertion capability or cells treated with a small molecule activator of apoptosome formation, indicating that it is unable to prevent cell death after mitochondrial disruption and caspase activation have occurred. These results indicate that Hsp70 blocks heat-induced apoptosis primarily by inhibiting Bax activation and thereby preventing the release of proapoptotic factors from mitochondria. Hsp70, therefore, inhibits events leading up to mitochondrial membrane permeabilization in heat-stressed cells and thereby controls the decision to die but does not interfere with cell death after this event has occurred.

Saccharomyces cerevisiae Hsp104 enhances the chaperone capacity of human cells and inhibits heat stress-induced proapoptotic signaling.

Hsp104, the most potent thermotolerance factor in Saccharomyces cerevisiae, is an unusual molecular chaperone that is associated with the dispersal of aggregated, non-native proteins in vivo and in vitro. The close cooperation between Hsp100 oligomeric disaggregases and specific Hsp70 chaperone/cochaperone systems to refold and reactivate heat-damaged proteins has been dubbed a "bichaperone network". Interestingly, animal genomes do not encode a Hsp104 ortholog. To investigate the biochemical and biological consequences of introducing into human cells a stress tolerance factor that has protein refolding capabilities distinct from those already present, Hsp104 was expressed as a transgene in a human leukemic T-cell line (PEER). Hsp104 inhibited heat-shock-induced loss of viability in PEER cells, and this action correlated with reduced procaspase-3 cleavage but not with reduced c-Jun N-terminal kinase phosphorylation. Hsp104 cooperated with endogenous human Hsp70 and Hsc70 molecular chaperones and their J-domain-containing cochaperones Hdj1 and Hdj2 to produce a functional hybrid bichaperone network capable of refolding aggregated luciferase. We also established that Hsp104 shuttles across the nuclear envelope and enhances the chaperoning capacity of both the cytoplasm and nucleoplasm of intact cells. Our results establish the fundamental properties of protein disaggregase function in human cells with implications for the use of Hsp104 or related proteins as therapeutic agents in diseases associated with protein aggregation.

Molecular chaperones and the stress of oncogenesis.

Protein-damaging stresses induce the expression of 'heat-shock proteins', which have essential roles in protecting cells from the potentially lethal effects of stress and proteotoxicity. These stress-protective heat-shock proteins are often overexpressed in cells of various cancers and have been suggested to be contributing factors in tumorigenesis. An underlying basis of oncogenesis is the acquisition and accumulation of mutations that provide the transformed cell with the combined characteristics of deregulated cell proliferation and suppressed cell death. Heat-shock proteins with dual roles as regulators of protein conformation and stress sensors may therefore have intriguing and central roles in both cell proliferation and apoptosis. It has been established that heat-shock proteins exhibit specificity to particular classes of polypeptide substrates and client proteins in vivo, and that chaperones can stabilize mutations that affect the folded conformation. Likewise, overexpression of chaperones has also been shown to protect cells against apoptotic cell death. The involvement of chaperones, therefore, in such diverse roles might suggest novel anticancer therapeutic approaches targeting heat-shock protein function for a broad spectrum of tumor types.

HSP72 inhibits apoptosis-inducing factor release in ATP-depleted renal epithelial cells.

Inhibition of the mitochondrial release and nuclear translocation of apoptosis-inducing factor (AIF) by heat stress protein (HSP)72 may ameliorate apoptosis in renal epithelial cells exposed to a metabolic inhibitor. To evaluate this hypothesis, cells were transiently exposed to 5 mM sodium cyanide in the absence of medium glucose, a maneuver known to induce apoptosis. ATP depletion for 1-2 h resulted in the progressive accumulation of mitochondrial AIF in the cytosol of samples obtained by selectively permeabilizing the plasma membrane with digitonin. During recovery from ATP depletion, time-dependent nuclear AIF accumulation (but not cytochrome c, an F0F1 ATP synthase subunit, or talin) was observed in isolated nuclei. Nuclear AIF accumulation was associated with peripheral chromatin condensation and DNA degradation. Prior heat stress (HS) significantly reduced AIF leakage into the cytosol, decreased nuclear accumulation of AIF, and inhibited DNA degradation. HS also increased the interaction between AIF and HSP72 detected by immunoprecipitation. In ATP depleted cells, selective overexpression of human HSP72 reduced the leakage of mitochondrial AIF in a dose-dependent manner (r = 0.997). This study suggests that mitochondrial membrane injury and subsequent AIF release contribute to nuclear injury and apoptosis in ATP-depleted renal cells. HSP72, an antiapoptotic protein, inhibits cell injury in part by preventing mitochondrial AIF release and perhaps by decreasing its nuclear accumulation.

hsp72 inhibits focal adhesion kinase degradation in ATP-depleted renal epithelial cells.

Prior heat stress (HS) or the selective overexpression of hsp72 prevents apoptosis caused by exposure to metabolic inhibitors by protecting the mitochondrial membrane and partially reducing caspase-3 activation. Focal adhesion kinase (FAK), a tyrosine kinase, exhibits anti-apoptotic properties and is a potential target for degradation by caspase-3. This study tested the hypothesis that hsp72 interacts with FAK, preventing caspase-3-mediated degradation during ATP depletion. ATP depletion (5 mm NaCN and 5 mm 2-deoxy-d-glucose in the absence of medium glucose) caused FAK degradation within 15 min. FAK degradation was completely prevented by a caspase-3-specific inhibitor. HS induced the accumulation of hsp72, increased the interaction between hsp72 and FAK, and significantly inhibited FAK degradation during ATP depletion. Selective overexpression of wild-type hsp72 (but not hsp72DeltaEEVD) reproduced the protective effects of HS on FAK cleavage. Purified hsp72 prevented the degradation of FAK by caspase-3 in vitro in a dose-dependent manner without affecting caspase-3 activity. Interaction between hsp72 and FAK is critical because both exogenous ATP and deletion of the substrate-binding site decreased protection of FAK by hsp72. These data indicate that FAK is an early target of injury in cells exposed to metabolic inhibitors and demonstrate that hsp72 reduces caspase-3-mediated proteolysis of FAK, an anti-apoptotic protein.

The R1 subunit of herpes simplex virus ribonucleotide reductase protects cells against apoptosis at, or upstream of, caspase-8 activation.

The R1 subunit of herpes simplex virus (HSV) ribonucleotide reductase, which in addition to its C-terminal reductase domain possesses a unique N-terminal domain of about 400 amino acids, is thought to have an additional, as yet unknown, function. Here, we report that the full-length HSV-2 R1 has an anti-apoptotic function able to protect cells against death triggered by expression of R1(Delta2-357), an HSV-2 R1 subunit with its first 357 amino acids deleted. We further substantiate the R1 anti-apoptotic activity by showing that its accumulation at low level could completely block apoptosis induced by TNF-receptor family triggering. Activation of caspase-8 induced either by TNF or by Fas ligand expression was prevented by the R1 protein. As HSV R1 did not inhibit cell death mediated by several agents acting via the mitochondrial pathway (Bax overexpression, etoposide, staurosporine and menadione), it is proposed that it functions to interrupt specifically death receptor-mediated signalling at, or upstream of, caspase-8 activation. The N-terminal domain on its own did not exhibit anti-apoptotic activity, suggesting that both domains of R1 or part(s) of them are necessary for this new function. Evidence for the importance of HSV R1 in protecting HSV-infected cells against cytokine-induced apoptosis was obtained with the HSV-1 R1 deletion mutants ICP6Delta and hrR3. These results show that, in addition to its ribonucleotide reductase function, which is essential for virus reactivation, HSV R1 could contribute to virus propagation by preventing apoptosis induced by the immune system.

Hsp72 and stress kinase c-jun N-terminal kinase regulate the bid-dependent pathway in tumor necrosis factor-induced apoptosis.

The major inducible heat shock protein Hsp72 has been shown to protect cells from certain apoptotic stimuli. Here we investigated the mechanism of Hsp72-mediated protection from tumor necrosis factor (TNF)-induced apoptosis of primary culture of IMR90 human fibroblasts. Hsp72 temporarily blocked apoptosis in response to TNF and permanently protected cells from heat shock. An Hsp72 mutant (Hsp72 Delta EEVD) with a deletion of the four C-terminal amino acids, which are essential for the chaperone function, blocked TNF-induced apoptosis in a manner similar to that of normal Hsp72 but did not inhibit heat shock-induced death. Therefore, the chaperone activity of Hsp72 is dispensable for suppression of TNF-induced apoptosis but is required for protection from heat shock. In fibroblasts derived from Bid knockout mice, similar temporal inhibition of TNF-induced apoptosis was seen. In these cells neither normal Hsp72 nor Hsp72 Delta EEVD conferred additional protection from apoptosis, suggesting that Hsp72 specifically affects Bid-dependent but not Bid-independent apoptotic pathways. Furthermore, both normal Hsp72 and Delta Hsp72EEVD inhibited Bid activation and downstream events, including release of cytochrome c, activation of caspase 3, and cleavage of poly-ADP-ribose polymerase. Both Hsp72 and Delta Hsp72EEVD blocked activation of the stress kinase c-jun N-terminal kinase (JNK) by TNF, and specific inhibition of JNK similarly temporarily blocked Bid activation and the downstream apoptotic events. These data strongly suggest that in TNF-induced apoptosis, Hsp72 specifically interferes with the Bid-dependent apoptotic pathway via inhibition of JNK.