Proteasomal Degradation In Response Research Articles

Deacetylation of MTHFD2 by SIRT4 senses stress signal to inhibit cancer cell growth by remodeling folate metabolism.

Folate metabolism plays an essential role in tumor development. Various cancers display therapeutic response to reagents targeting key enzymes of the folate cycle, but obtain chemoresistance later. Therefore, novel targets in folate metabolism are highly demanded. Methylenetetrahydrofolate dehydrogenase/methylenetetrahydrofolate cyclohydrolase 2 (MTHFD2) is one of the key enzymes in folate metabolism and its expression is highly increased in multiple human cancers. However, the underlying mechanism that regulates MTHFD2 expression remains unknown. Here, we elucidate that SIRT4 deacetylates the conserved lysine 50 (K50) residue in MTHFD2. K50 deacetylation destabilizes MTHFD2 by elevating cullin 3 E3 ligase-mediated proteasomal degradation in response to stressful stimuli of folate deprivation, leading to suppression of nicotinamide adenine dinucleotide phosphate production in tumor cells and accumulation of intracellular reactive oxygen species, which in turn inhibits the growth of breast cancer cells. Collectively, our study reveals that SIRT4 senses folate availability to control MTHFD2 K50 acetylation and its protein stability, bridging nutrient/folate stress and cellular redox to act on cancer cell growth.

Evidence that the transcriptional repressor ICER is regulated via the N-end rule for ubiquitination

ICER is a transcriptional repressor that is mono- or poly-ubiquitinated. This either causes ICER to be translocated from the nucleus, or degraded via the proteasome, respectively. In order to further studies the proteins involved in ICER regulation mass spectrometry analysis was performed to identify potential candidates. We identified twenty eight ICER-interacting proteins in human melanoma cells, Sk-Mel-24. In this study we focus on two proteins with potential roles in ICER proteasomal degradation in response to the N-end rule for ubiquitination: the N-alpha-acetyltransferase 15 (NAA15) and the E3 ubiquitin-protein ligase UBR4. Using an HA-tag on the N- or C-terminus of ICER (NHAICER or ICERCHA) it was found that the N-terminus of ICER is important for its interaction to UBR4, whereas NARG1 interaction is independent of HA-tag position. Silencing RNA experiments show that both NAA15 and UBR4 up-regulates ICER levels and that ICER's N-terminus is important for this regulation. The N-terminus of ICER was found to have dire consequences on its regulation by ubiquitination and cellular functions. The half-life of NHAICER was found to be about twice as long as ICERCHA. Polyubiquitination of ICER was found to be dependent on its N-terminus and mediated by UBR4. This data strongly suggests that ICER is ubiquitinated as a response to the N-end rule that governs protein degradation rate through recognition of the N-terminal residue of proteins. Furthermore, we found that NHAICER inhibits transcription two times more efficiently than ICERCHA, and causes apoptosis 5 times more efficiently than ICERCHA. As forced expression of ICER has been shown before to block cells in mitosis, our data represent a potentially novel mechanism for apoptosis of cells in mitotic arrest.

Tyrosine Nitration Contributes to Nitric Oxide-Stimulated Degradation of CYP2B6.

Human cytochrome P450 (P450) CYP2B6 undergoes nitric oxide (NO)-dependent proteasomal degradation in response to the NO donor dipropylenetriamine NONOate (DPTA) and biologic NO in HeLa and HuH7 cell lines. CYP2B6 is also downregulated by NO in primary human hepatocytes. We hypothesized that NO or derivative reactive nitrogen species may generate adducts of tyrosine and/or cysteine residues, causing CYP2B6 downregulation, and selected Tyr and Cys residues for mutation based on predicted solvent accessibility. CYP2B6V5-Y317A, -Y380A, and -Y190A mutant proteins expressed in HuH7 cells were less sensitive than wild-type (WT) enzyme to degradation evoked by DPTA, suggesting that these tyrosines are targets for NO-dependent downregulation. The Y317A or Y380A mutants did not show increases in high molecular mass (HMM) species after treatment with DPTA or bortezomib + DPTA, in contrast to the WT enzyme. Carbon monoxide-releasing molecule 2 treatment caused rapid suppression of 2B6 enzyme activity, significant HMM species generation, and ubiquitination of CYP2B6 protein but did not stimulate CYP2B6 degradation. The CYP2B6 inhibitor 4-(4-chlorophenyl)imidazole blocked NO-dependent CYP2B6 degradation, suggesting that NO access to the active site is important. Molecular dynamics simulations predicted that tyrosine nitrations of CYP2B6 would cause significant destabilizing perturbations of secondary structure and remove correlated motions likely required for enzyme function. We propose that cumulative nitrations of Y190, Y317, and Y380 by reactive nitrogen species cause destabilization of CYP2B6, which may act synergistically with heme nitrosylation to target the enzyme for degradation. SIGNIFICANCE STATEMENT: This work provides novel insight into the mechanisms by which nitric oxide, which is produced in hepatocytes in response to inflammation, triggers the ubiquitin-dependent proteasomal degradation of the cytochrome P450 (P450) enzyme CYP2B6. Our data demonstrate that both nitration of specific tyrosine residues and interaction of nitric oxide (NO) with the P450 heme are necessary for NO to trigger ubiquitination and protein degradation.

Nitric oxide-regulated proteolysis of human CYP2B6 via the ubiquitin-proteasome system

We showed previously that rat cytochrome P450 CYP2B1 undergoes NO-dependent proteasomal degradation in response to inflammatory stimuli, and that the related human enzyme CYP2B6 is also down-regulated by NO in primary human hepatocytes. To investigate the mechanism of CYP2B6 down-regulation, we made several cell lines (HeLa and HuH7 cells) in which native CYP2B6 or CYP2B6 with a C-terminal V5 tag (CYP2B6V5) are expressed from a lentiviral vector with a cytomegalovirus promoter. Native CYP2B6 protein was rapidly down-regulated in HeLa cells within 3h of treatment with the NO donor (Z)−1-[2-(2-Aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate, while its mRNA level was not down-regulated. Treatment of the cells with the NO donor (Z)−1-[N-(3-aminopropyl)-N-(3-ammoniopropyl)amino]diazen-1-ium-1,2-diolate also resulted in rapid down-regulation of CYP2B6 activity, measured as the formation of 7-hydroxy-4-trifluoromethylcoumarin, as well as 2B6 protein in the CYP2B6 HeLa cell line. CYP2B6V5 was also down-regulated by NO donors in HuH7 cells. Down-regulation was observed in the presence of cycloheximide, demonstrating that this occurs via a post-translational mechanism. We generated a HeLa cell line expressing both CYP2B6V5 and human nitric oxide synthase 2 (NOS2), the latter under positive control by tetracycline. The cellular NO produced by doxycycline treatment also effectively down-regulated CYP2B6 protein, which was blocked by the co-treatment with the NOS2 competitive inhibitor L-NG-nitroarginine methyl ester (L-NAME). We next investigated the proteolytic enzymes responsible for NO-dependent CYP2B6 degradation. Neither calpain inhibitors (N-Acetyl-L-leucyl-L-leucyl-L-norleucinal, carbobenzoxy-valinyl-phenylalaninal), nor lysosomal protease inhibitors (3-methyladenine and chloroquine) inhibited the NO dependent CYP2B6V5 down-regulation. The proteasome inhibitors MG132 and bortezomib attenuated, but did not completely block the NO-induced down-regulation in the HuH7 cell line. However, when cells were co-treated with NO donor and proteasome inhibitors, high molecular mass species could be detected on native CYP2B6 as well as CYP2B6V5 Western blots. Further investigation demonstrated that CYP2B6 protein was polyubiquitinated and this was dramatically enhanced by co-treatment with NO donor and bortezomib. Taken together, our data demonstrate that CYP2B6 is down-regulated in an NO-dependent manner via ubiquitination and proteasomal degradation.

VRK1 phosphorylates and protects NBS1 from ubiquitination and proteasomal degradation in response to DNA damage

NBS1 is an early component in DNA-Damage Response (DDR) that participates in the initiation of the responses aiming to repair double-strand breaks caused by different mechanisms. Early steps in DDR have to react to local alterations in chromatin that are induced by DNA damage. NBS1 participates in the early detection of DNA damage and functions as a platform for the recruitment and assembly of components that are sequentially required for the repair process. In this work we have studied whether the VRK1 chromatin kinase can affect the activation of NBS1 in response to DNA damage induced by ionizing radiation. VRK1 is forming a basal preassembled complex with NBS1 in non-damaged cells. Knockdown of VRK1 resulted in the loss of NBS1 foci induced by ionizing radiation, an effect that was also detected in cell-cycle arrested cells and in ATM (−/−) cells. The phosphorylation of NBS1 in Ser343 by VRK1 is induced by either doxorubicin or IR in ATM (−/−) cells. Phosphorylated NBS1 is also complexed with VRK1. NBS1 phosphorylation by VRK1 cooperates with ATM. This phosphorylation of NBS1 by VRK1 contributes to the stability of NBS1 in ATM (−/−) cells, and the consequence of its loss can be prevented by treatment with the MG132 proteasome inhibitor of RNF8. We conclude that VRK1 regulation of NBS1 contributes to the stability of the repair complex and permits the sequential steps in DDR.

The F-box protein COI1 functions upstream of MYB305 to regulate primary carbohydrate metabolism in tobacco (Nicotiana tabacum L. cv. TN90).

Jasmonate (JA) plays an important role in regulating plant male fertility and secondary metabolism, but its role in regulating primary metabolism remains unclear. The F-box protein CORONATINE INSENSITIVE 1 (COI1) is a critical component of the JA receptor, and mediates JA-signalling by targeting JASMONATE ZIM-domain (JAZ) proteins for proteasomal degradation in response to JA perception. Here, we found that RNA interference-mediated knockdown of NtCOI1 in tobacco (Nicotiana tabacum L. cv. TN90) recapitulated many previously observed phenotypes in coi1 mutants, including male sterility, JA insensitivity, and loss of floral anthocyanin production. It also affected starch metabolism in the pollen, anther wall, and floral nectary, leading to pollen abortion and loss of floral nectar. Transcript levels of genes encoding starch metabolism enzymes were significantly altered in the pollen, anther wall, and floral nectary of NtCOI1-silenced tobacco. Changes in leaf primary metabolism were also observed in the NtCOI1-silenced tobacco. The expression of NtMYB305, an orthologue of MYB305 previously identified as a flavonoid metabolic regulator in Antirrhinum majus flowers and as a floral-nectar regulator mediating starch synthesis in ornamental tobacco, was extremely downregulated in NtCOI1-silenced tobacco. These findings suggest that NtCOI1 functions upstream of NtMYB305 and plays a fundamental role in coordinating plant primary carbohydrate metabolism and correlative physiological processes.

Characterization of ML-IAP protein stability and physiological role in vivo

ML-IAP [melanoma IAP (inhibitor of apoptosis)] is an anti-apoptotic protein that is expressed highly in melanomas where it contributes to resistance to apoptotic stimuli. The anti-apoptotic activity and elevated expression of IAP family proteins in many human cancers makes IAP proteins attractive targets for inhibition by cancer therapeutics. Small-molecule IAP antagonists that bind with high affinities to select BIR (baculovirus IAP repeat) domains have been shown to stimulate auto-ubiquitination and rapid proteasomal degradation of c-IAP1 (cellular IAP1) and c-IAP2 (cellular IAP2). In the present paper, we report ML-IAP proteasomal degradation in response to bivalent, but not monovalent, IAP antagonists. This degradation required ML-IAP ubiquitin ligase activity and was independent of c-IAP1 or c-IAP2. Although ML-IAP is best characterized in melanoma cells, we show that ML-IAP expression in normal mammalian tissues is restricted largely to the eye, being most abundant in ciliary body epithelium and retinal pigment epithelium. Surprisingly, given this pattern of expression, gene-targeted mice lacking ML-IAP exhibited normal intraocular pressure as well as normal retinal structure and function. The results of the present study indicate that ML-IAP is dispensable for both normal mouse development and ocular homoeostasis.

Inhibition kinetics and regulation of sphingosine kinase 1 expression in prostate cancer cells: Functional differences between sphingosine kinase 1a and 1b

Sphingosine kinase 1 catalyses the formation of the bioactive lipid, sphingosine 1-phosphate and is a target for anti-cancer agents. We demonstrate here that 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole (SKi, also referred to as SKI-II), FTY720 (Fingolimod), and (S)-FTY720 vinylphosphonate inhibit sphingosine kinase 1 activity with distinct kinetics, indicating that these compounds exhibit different binding modalities with sphingosine kinase 1. Thus, SKi is a mixed inhibitor of sphingosine and ATP binding, whereas FTY720 is competitive with sphingosine and uncompetitive with ATP, and (S)-FTY720 vinylphosphonate is uncompetitive with sphingosine and is a mixed inhibitor with respect to ATP. A novel ‘see-saw’ model is proposed for the binding of inhibitor to catalytic and allosteric sites, the latter dependent on substrate binding, that provides an explanation for the different inhibitor kinetics. In addition, we demonstrate that the expression level and properties unique to an N-terminal 86 amino-acid isoform variant of sphingosine kinase 1 (SK1b) in prostate cancer cells reduce its sensitivity to SKi-induced proteasomal degradation in comparison to SK1a, i.e. these two N-terminal variants of sphingosine kinase 1 (SK1a and SK1b) have different properties. The reduced sensitivity of SK1b to proteasomal degradation in response to SKi is translated into specific changes in ceramide and S1P levels that leads to apoptosis of androgen-sensitive but not androgen-independent LNCaP prostate cancer cells. Therefore, our proposed ‘see-saw’ model might be usefully employed in the design of sphingosine kinase inhibitors to promote apoptosis of chemotherapeutic resistant cancer cells.

DNA polymerase eta is targeted by Mdm2 for polyubiquitination and proteasomal degradation in response to ultraviolet irradiation

DNA polymerase eta (PolH), the product of the xeroderma pigmentosum variant (XPV) gene and a Y-family DNA polymerase, plays a pivotal role in translesion DNA synthesis. Loss of PolH leads to early onset of malignant skin cancer in XPV patients and increases UV-induced carcinogenesis. Thus, the pathways by which PolH expression and activity are controlled may be explored as a strategy to prevent UV-induced cancer. In this study, we found that Mdm2, a RING finger E3 ligase, promotes PolH degradation. Specifically, we showed that knockdown of Mdm2 increases PolH expression in both p53-proficient and -deficient cells. In addition, we showed that UV-induced PolH degradation is attenuated by Mdm2 knockdown. In contrast, ectopically expression of Mdm2 decreases PolH expression, which can be abrogated by the proteasome inhibitor MG132. Moreover, we showed that Mdm2 physically associates with PolH and promotes PolH polyubiquitination in vivo and in vitro. Finally, we showed that knockdown of Mdm2 increases the formation of PolH replication foci and decreases the sensitivity of cells to UV-induced lesions in a PolH-dependent manner. Taken together, we uncovered that Mdm2 serves as an E3 ligase for PolH polyubiquitination and proteasomal degradation in cells under the basal condition and in response to UV irradiation.

The fission yeast rDNA-binding protein Reb1 regulates G1 phase under nutritional stress

Yeast Reb1 and its mammalian ortholog TTF1 are conserved Myb-type DNA-binding proteins that bind to specific sites near the 3'-end of rRNA genes (rDNA). Here, they participate in the termination of transcription driven by RNA polymerase I and block DNA replication forks approaching in the opposite direction. We found that Schizosaccharomyces pombe Reb1 also upregulates transcription of the ste9(+) gene that is required for nitrogen-starvation-induced growth arrest with a G1 DNA content and sexual differentiation. Ste9 activates the anaphase-promoting complex or cyclosome ('APC/C') in G1, targeting B-cyclin for proteasomal degradation in response to nutritional stress. Reb1 binds in vivo and in vitro to a specific DNA sequence at the promoter of ste9(+), similar to the sequence recognized in the rDNA, and this binding is required for ste9(+) transcriptional activation and G1 arrest. This suggests that Reb1 acts as a link between rDNA metabolism and cell cycle control in response to nutritional stress. In agreement with this new role for Reb1 in the regulation of the G1-S transition, reb1Δ and wee1(ts) mutations are synthetically lethal owing to the inability of these cells to lengthen G1 before entering S phase. Similarly, reb1Δ cdc10(ts) cells are unable to arrest in G1 and die at the semi-permissive temperature.

Proteasomal Degradation of Indoleamine 2,3-Dioxygenase in CD8+ Dendritic Cells is Mediated by Suppressor of Cytokine Signaling 3 (SOCS3)

Indoleamine 2,3-dioxygenase (IDO) catalyzes the initial and rate-limiting step of tryptophan catabolism in a specific pathway, resulting in a series of extracellular messengers collectively known as kynurenines. IDO has been recognized as an authentic regulator of immunity not only in mammalian pregnancy, but also in infection, autoimmunity, inflammation, allergy, transplantation, and neoplasia. Its suppressive effects are mostly mediated by dendritic cells (DCs) and involve tryptophan deprivation and/or production of kynurenines, which act on IDO-negative DCs as well as CD4(+) and CD8(+) T cells. We have found that mouse IDO contains two tyrosine residues within two distinct putative immunoreceptor tyrosine-based inhibitory motifs, VPY(115)CEL and LLY(253)EGV. We have also found that Suppressor of Cytokine Signaling 3 (SOCS3)-known to interact with phosphotyrosine-containing peptides and be selectively induced by interleukin 6 (IL-6)-binds mouse IDO, recruits the ECS (Elongin-Cullin-SOCS) E3 ligase, and targets the IDO/SOCS3 complex for proteasomal degradation. This event underlies the ability of IL-6 to convert otherwise tolerogenic, IDO-competent DCs into immunogenic cells. Thus onset of immunity in response to antigen within an early inflammatory context demands that IDO be degraded in tolerogenic DCs. These studies support the finding that IDO is regulated by proteasomal degradation in response to immunogenic and inflammatory stimuli.

Development of a High-Throughput Cell-Based Reporter Assay to Identify Stabilizers of Tumor Suppressor Pdcd4

The novel tumor suppressor Pdcd4 affects tumorigenesis by inhibiting translation. Pdcd4 is phosphorylated and subsequently lost by proteasomal degradation in response to tumor-promoting conditions. Here, the authors describe the development of a reporter cell system to monitor the stability of Pdcd4. The phosphorylation-dependent degradation domain ("target") or an adjacent ("off-target") region of Pdcd4 was cloned into a luciferase expression system. The target constructs were responsive to Pdcd4 degrading conditions (e.g., TPA, p70(S6K1) overactivation), whereas the off-target constructs remained stable. The system was optimized for and shown to be reliable in a high-throughput compatible 384-well format. Screening of 15,275 pure compounds resulted in a hit rate of 0.30% (>50% inhibition of TPA-induced loss of signal, confirmed by reassay). Among the hits were inhibitors of previously identified critical signaling events for TPA-induced Pdcd4 degradation. One compound was identified to be nonspecific using the off-target control cell line. Screening of 135,678 natural product extracts yielded 42 confirmed, specific hits. Z' averaged 0.58 across 446 plates. Further characterization of active natural products and synthetic compounds is expected to identify novel Pdcd4 stabilizers that may be useful in targeting translation to prevent or treat cancers.

MKK4/SEK1 Is Negatively Regulated through a Feedback Loop Involving the E3 Ubiquitin Ligase Itch

Cells exposed to environmental stress rapidly activate the MAPK cascade (MKKK/MKK/MAPK). The transient nature of stress signaling is a consequence of negative feedback signals that lead to kinase dephosphorylation, degradation, and sequestration, which have not been fully elucidated for MKK family members. Here, we investigated the signals that negatively regulate MKK4/SEK1, an upstream activator of the MAPKs JNK and p38/HOG1. Following exposure of cells to sorbitol, MKK4 underwent ubiquitination and degradation in a proteasome-dependent manner. MKK4 ubiquitination required JNK kinase activity. The JNK substrate Itch (a HECT domain-containing Nedd4-like ubiquitin protein ligase) bound to MKK4, ubiquitinated lysines 140 and 143, and promoted MKK4 degradation. Other E3 ligases within the MAPK modular complex did not ubiquitinate MKK4. These data suggest that MKK4 is negatively regulated through a feedback loop involving the E3 ubiquitin ligase Itch, which has a fundamental role in the mechanism that controls MKK4 protein levels.

Thiazolidinediones Mimic Glucose Starvation in Facilitating Sp1 Degradation through the Up-Regulation of β-Transducin Repeat-Containing Protein

This study investigated the mechanism by which the transcription factor Sp1 is degraded in prostate cancer cells. We recently developed a thiazolidinedione derivative, (Z)-5-(4-hydroxy-3-trifluoromethylbenzylidene)-3-(1-methylcyclohexyl)-thiazolidine-2,4-dione (OSU-CG12), that induces Sp1 degradation in a manner paralleling that of glucose starvation. Based on our finding that thiazolidinediones suppress beta-catenin and cyclin D1 by up-regulating the E3 ligase SCF(beta-TrCP), we hypothesized that beta-transducin repeat-containing protein (beta-TrCP) targets Sp1 for proteasomal degradation in response to glucose starvation or OSU-CG12. Here we show that either treatment of LNCaP cells increased specific binding of Sp1 with beta-TrCP. This direct binding was confirmed by in vitro pull-down analysis with bacterially expressed beta-TrCP. Although ectopic expression of beta-TrCP enhanced the ability of OSU-CG12 to facilitate Sp1 degradation, suppression of endogenous beta-TrCP function by a dominant-negative mutant or small interfering RNA-mediated knockdown blocked OSU-CG12-facilitated Sp1 ubiquitination and/or degradation. Sp1 contains a C-terminal conventional DSG destruction box ((727)DSGAGS(732)) that mediates beta-TrCP recognition and encompasses a glycogen synthase kinase 3beta (GSK3beta) phosphorylation motif (SXXXS). Pharmacological and molecular genetic approaches and mutational analyses indicate that extracellular signal-regulated kinase-mediated phosphorylation of Thr739 and GSK3beta-mediated phosphorylation of Ser728 and Ser732 were critical for Sp1 degradation. The ability of OSU-CG12 to mimic glucose starvation to activate beta-TrCP-mediated Sp1 degradation has translational potential to foster novel strategies for cancer therapy.

Functional Interaction between Peroxisome Proliferator-Activated Receptor γ and β-Catenin

Studies have demonstrated cross talk between beta-catenin and peroxisome proliferator-activated receptor gamma (PPARgamma) signaling pathways. Specifically, activation of PPARgamma induces the proteasomal degradation of beta-catenin in cells that express an adenomatous polyposis coli-containing destruction complex. In contrast, oncogenic beta-catenin is resistant to such degradation and inhibits the expression of PPARgamma target genes. In the present studies, we demonstrate a functional interaction between beta-catenin and PPARgamma that involves the T-cell factor (TCF)/lymphocyte enhancer factor (LEF) binding domain of beta-catenin and a catenin binding domain (CBD) within PPARgamma. Mutation of K312 and K435 in the TCF/LEF binding domain of an oncogenic beta-catenin (S37A) significantly reduces its ability to interact with and inhibit the activity of PPARgamma. Furthermore, these mutations render S37A beta-catenin susceptible to proteasomal degradation in response to activation of PPARgamma. Mutation of F372 within the CBD (helices 7 and 8) of PPARgamma disrupts its binding to beta-catenin and significantly reduces the ability of PPARgamma to induce the proteasomal degradation of beta-catenin. We suggest that in normal cells, PPARgamma can function to suppress tumorigenesis and/or Wnt signaling by targeting phosphorylated beta-catenin to the proteasome through a process involving its CBD. In contrast, oncogenic beta-catenin resists proteasomal degradation by inhibiting PPARgamma activity, which requires its TCF/LEF binding domain.

The DDB2 nucleotide excision repair gene product p48 enhances global genomic repair in p53 deficient human fibroblasts

The tumor suppressor protein p53 functions in many cellular responses to UV-induced DNA damage, including activating the global nucleotide excision repair (NER) pathway. A potential mechanism for the effect on NER is through the ability of p53 to transcriptionally regulate genes that are directly involved in NER. DDB2 is one such gene that is regulated by p53 at both the basal and UV inducible levels. In order to further understand p53’s role in NER, we transfected and selected clones that stably overexpress DDB2 in a human p53 deficient cell line. Global genomic repair (GGR) of cyclobutane pyrimidine dimers was significantly increased in the DDB2 expressing cells in comparison to controls, demonstrating that p53 wt protein itself is not directly required for efficient GGR. The protein product of DDB2, p48, is also post-translationally regulated by proteasomal degradation in response to UV irradiation. The regulation of p48 at both the transcriptional level by p53, and post-translationally by the proteasome suggests that p48 may be a rate limiting component of NER.