Stress-activated Mitogen-activated Protein Kinase Research Articles

RNA sequencing analysis shows that titanium dioxide nanoparticles induce endoplasmic reticulum stress, which has a central role in mediating plasma glucose in mice

Titanium dioxide nanoparticles (TiO2 NPs) constitute the top five NPs in use today. In this study, oral administration of 50, 100, and 200 mg/kg body weight (b.w.) TiO2 NPs increases plasma glucose in mice, whereas 10 and 20 mg/kg b.w. TiO2 NPs did not. RNA sequencing (RNA-seq) technology was used to investigate genome-wide effects of TiO2 NPs. Clustering analysis of the RNA-seq data showed the most significantly enriched gene ontology terms and KEGG pathways related to the endoplasmic reticulum (ER) and ER stress. Molecular biology verification showed that 50 mg/kg b.w. and higher doses TiO2 NPs activated a xenobiotic biodegradation response and increased expression of cytochrome P450 family genes in mouse livers, thus inducing ER stress in mice. ER stress-activated MAPK and NF-κB pathways and induced an inflammation response, resulting in phosphorylation of the insulin receptor substrate 1 and, consequently, insulin resistance. This was the main mechanism by which TiO2 NPs increased plasma glucose in mice. Meanwhile, ER stress disturbed the monooxygenase system, and thus generated reactive oxygen species (ROS). Relief of ER stress with 4-phenylbutyric acid inhibited all the above effects of TiO2 NPs, including the generation of ROS. Therefore, TiO2 NP-induced ER stress was a decisive factor with a central role in plasma glucose disturbance in mice.

ETAS®50 Attenuates Ultraviolet-B-Induced Interleukin-6 Expression by Suppressing Akt Phosphorylation in Normal Human Dermal Fibroblasts.

We recently reported that ETAS 50, a standardized extract from the Asparagus officinalis stem, exerted anti-inflammatory effects on ultraviolet-B- (UV-B-) irradiated normal human dermal fibroblasts (NHDFs) by inhibiting nuclear factor-κB p65 nuclear import and the resulting interleukin-1β (IL-1β) expression. To further elucidate the antiphotoaging potency of ETAS 50, we examined the anti-inflammatory effects on UV-B-irradiated NHDFs by focusing on the stress-activated mitogen-activated protein kinase (MAPK) and Akt signaling pathways. NHDFs were treated with 1 mg/mL of ETAS 50 or dextrin (vehicle control) after UV-B irradiation (20 mJ/cm2) for different time periods. Phosphorylation levels of c-Jun N-terminal kinase (JNK), p38 MAPK, and Akt were analyzed by western blotting. IL-6 mRNA levels were analyzed by real-time polymerase chain reaction. UV-B-irradiated NHDFs showed increased phosphorylation levels of JNK, p38 MAPK, and Akt, as well as increased mRNA levels of IL-6. ETAS 50 treatment after UV-B irradiation suppressed the increased phosphorylation levels of Akt without affecting those of JNK and p38 MAPK. ETAS 50 as well as Akt inhibitor Perifosine repressed UV-B irradiation-induced IL-6 mRNA expression. These results suggest that ETAS 50 treatment represses UV-B irradiation-induced IL-6 expression by suppressing Akt phosphorylation. The present findings demonstrate the potential of ETAS 50 to prevent photoaging by attenuating UV-B irradiation-induced proinflammatory responses in skin fibroblasts.

A genome-wide screen for FTY720-sensitive mutants reveals genes required for ROS homeostasis.

Fingolimod hydrochloride (FTY720), a sphingosine-1-phosphate (S1P) analogue, is an approved immune modulator for the treatment of multiple sclerosis (MS). Notably, in addition to its well-known mode of action as an S1P modulator, accumulating evidence suggests that FTY720 induces apoptosis in various cancer cells via reactive oxygen species (ROS) generation. Although the involvement of multiple signaling molecules, such as JNK (Jun N-terminal kinase), Akt (alpha serine/threonine-protein kinase) and Sphk has been reported, the exact mechanisms how FTY720 induces cell growth inhibition and the functional relationship between FTY720 and these signaling pathways remain elusive. Our previous reports using the fission yeast Schizosaccharomyces pombe as a model system to elucidate FTY720-mediated signaling pathways revealed that FTY720 induces an increase in intracellular Ca2+ concentrations and ROS generation, which resulted in the activation of the transcriptional responses downstream of Ca2+/calcineurin signaling and stress-activated MAPK signaling, respectively. Here, we performed a genome-wide screening for genes whose deletion induces FTY720-sensitive growth in S. pombe and identified 49 genes. These gene products are related to the biological processes involved in metabolic processes, transport, transcription, translation, chromatin organization, cytoskeleton organization and intracellular signal transduction. Notably, most of the FTY720-sensitive deletion cells exhibited NAC-remedial FTY720 sensitivities and dysregulated ROS homeostasis. Our results revealed a novel gene network involving ROS homeostasis and the possible mechanisms of the FTY720 toxicity.

Therapeutic application of apoptosis signal-regulating kinase 1 inhibitors.

Apoptosis signal-regulating kinase 1 (ASK1) is a member of the stress-activated mitogen-activated protein kinase kinase kinase (MAP3K) family. ASK1 is an attractive drug target, owing to its essential role in a wide variety of human diseases including neurodegenerative disorders, inflammatory diseases and cancer. Recent studies have suggested that pharmacological manipulations using small molecule ASK1 inhibitors may be beneficial in experimental human disease models. In this review, we highlight the current understanding of ASK1 inhibitors as a potential therapy for human diseases.

MAPK signalling pathway in cancers: Olive products as cancer preventive and therapeutic agents.

The mitogen-activated protein kinases (MAPKs) are fundamental in inflammation and cancer control, through the crosstalk between the redox regulated nuclear factor E2-related factor 2 (Nrf2) and nuclear factor-kB (NFκB) gene expression. MAPKs regulate various cellular activities involved in cancer progression, including proliferation, apoptosis and immune escape and blockade of upstream kinases is a current therapeutic strategy. However, these therapies are associated with some adverse effects and with the paradoxical activation of the MAPKs pathway. In the context of cancer prevention and treatment, it has been suggested that dietary factors are able to modulate cancer initiation and progression by interacting with the MAPKs. Within these dietary factors, virgin olive oil (VOO) is of particular interest due to its content in squalene, already used as drug delivery system in cancer therapy. The aim of this review is to discuss the studies pointing to the effects of olive-derived foodstuff and nutraceuticals on MAPKs signalling cascades. The reviewed experimental studies suggest that the stress-activated JNK and p38 MAPKs could be targets of olive-derived nutraceuticals. The latter, including phytochemicals from olive cultivation and processing wastes, could be adjuvants in chemotherapies, whereas VOO could be considered a "natural delivery system" of bioactive phytochemicals due to its high content in squalene.

Regulation of cotton (Gossypiumhirsutum) drought responses by mitogen-activated protein (MAP) kinase cascade-mediated phosphorylation of GhWRKY59.

Drought is a key limiting factor for cotton (Gossypium spp.) production, as more than half of the global cotton supply is grown in regions with high water shortage. However, the underlying mechanism of the response of cotton to drought stress remains elusive. By combining genome-wide transcriptome profiling and a loss-of-function screen using virus-induced gene silencing, we identified Gossypiumhirsutum GhWRKY59 as an important transcription factor that regulates the drought stress response in cotton. Biochemical and genetic analyses revealed a drought stress-activated mitogen-activated protein (MAP) kinase cascade consisting of GhMAP3K15-Mitogen-activated Protein Kinase Kinase 4 (GhMKK4)-Mitogen-activated Protein Kinase 6 (GhMPK6) that directly phosphorylates GhWRKY59 at residue serine 221. Interestingly, GhWRKY59 is required for dehydration-induced expression of GhMAPK3K15, constituting a positive feedback loop of GhWRKY59-regulated MAP kinase activation in response to drought stress. Moreover, GhWRKY59 directly binds to the W-boxes of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 2 (GhDREB2), which encodes a dehydration-inducible transcription factor regulating the plant hormone abscisic acid (ABA)-independent drought response. Our study identified a complete MAP kinase cascade that phosphorylates and activates a key WRKY transcription factor, and elucidated a regulatory module, consisting of GhMAP3K15-GhMKK4-GhMPK6-GhWRKY59-GhDREB2, that is involved in controlling the cotton drought response.

Involvement of MicroRNAs in Probiotics-Induced Reduction of the Cecal Inflammation by Salmonella Typhimurium.

The microRNAs (miRNAs) have been shown to play important roles in the development of the immune system and in regulation of host inflammation responses. Probiotics can effectively alleviate the inflammation caused by Salmonella in chickens. However, whether and how miRNAs are involved in modulation of the inflammation response in the gut of chickens have not been reported. In this study, the impact of a probiotics, Lactobacillus plantarum Z01 (LPZ01), was investigated on the cecal miRNAs and cytokine secretions in Salmonella Typhimurium (S. Typhimurium)-infected chickens at the age of 3 days. Newly hatched chicks were assigned to four groups (1): NC (basal diet) (2): S (basal diet + S. Typhimurium challenged) (3): SP (basal diet + S. Typhimurium challenged + LPZ01) (4): P (basal diet + LPZ01). In comparison with the S group, chicks in the SP group reduced the number of S. Typhimurium and had lower levels of interferon-γ and lipopolysaccharide-induced tumor necrosis factor alpha factor (LITAF) in ceca post challenge. Expression of 14 miRNAs was significantly affected by the presence of S. Typhimurium and/or lactobacillus. Five differential expression miRNAs (gga-miR-215-5p, gga-miR-3525, gga-miR-193a-5p, gga-miR-122-5p, and gga-miR-375) were randomly selected for confirmation by the RT-PCR. Predicted target genes of differentially expressed miRNAs were enriched in regulation of cAMP-dependent protein kinase activity, stress-activated MAPK cascade, immune system development and regulation of immune system process as well as in immune related pathways such as MAPK and Wnt signaling pathways. The relationship between changes of miRNAs and changes of cytokines was explored. Finally, 119 novel miRNAs were identified in 36 libraries totally. Identification of novel miRNAs significantly expanded the repertoire of chicken miRNAs and provided the basis for understanding the function of miRNAs in the host. Our results suggest that the probiotics reduce the inflammation of the S. Typhimurium infection in neonatal broiler chicks, at least partially, through regulation of miRNAs expression.

Fungal Histidine Phosphotransferase Plays a Crucial Role in Photomorphogenesis and Pathogenesis in Magnaporthe oryzae.

Two-component signal transduction (TCST) pathways play crucial roles in many cellular functions such as stress responses, biofilm formation, and sporulation. The histidine phosphotransferase (HPt), which is an intermediate phosphotransfer protein in a two-component system, transfers a phosphate group to a phosphorylatable aspartate residue in the target protein(s), and up-regulates stress-activated MAP kinase cascades. Most fungal genomes carry a single copy of the gene coding for HPt, which are potential antifungal targets. However, unlike the histidine kinases (HK) or the downstream response regulators (RR) in two-component system, the HPts have not been well-studied in phytopathogenic fungi. In this study, we investigated the role of HPt in the model rice-blast fungal pathogen Magnaporthe oryzae. We found that in M. oryzae an additional isoform of the HPT gene YPD1 was expressed specifically in response to light. Further, the expression of light-regulated genes such as those encoding envoy and blue-light-harvesting protein, and PAS domain containing HKs was significantly reduced upon down-regulation of YPD1 in M. oryzae. Importantly, down-regulation of YPD1 led to a significant decrease in the ability to penetrate the host cuticle and in light-dependent conidiation in M. oryzae. Thus, our results indicate that Ypd1 plays an important role in asexual development and host invasion, and suggest that YPD1 isoforms likely have distinct roles to play in the rice-blast pathogen M. oryzae.

Structural basis of autoregulatory scaffolding by apoptosis signal-regulating kinase 1

Apoptosis signal-regulating kinases (ASK1-3) are apical kinases of the p38 and JNK MAP kinase pathways. They are activated by diverse stress stimuli, including reactive oxygen species, cytokines, and osmotic stress; however, a molecular understanding of how ASK proteins are controlled remains obscure. Here, we report a biochemical analysis of the ASK1 kinase domain in conjunction with its N-terminal thioredoxin-binding domain, along with a central regulatory region that links the two. We show that in solution the central regulatory region mediates a compact arrangement of the kinase and thioredoxin-binding domains and the central regulatory region actively primes MKK6, a key ASK1 substrate, for phosphorylation. The crystal structure of the central regulatory region reveals an unusually compact tetratricopeptide repeat (TPR) region capped by a cryptic pleckstrin homology domain. Biochemical assays show that both a conserved surface on the pleckstrin homology domain and an intact TPR region are required for ASK1 activity. We propose a model in which the central regulatory region promotes ASK1 activity via its pleckstrin homology domain but also facilitates ASK1 autoinhibition by bringing the thioredoxin-binding and kinase domains into close proximity. Such an architecture provides a mechanism for control of ASK-type kinases by diverse activators and inhibitors and demonstrates an unexpected level of autoregulatory scaffolding in mammalian stress-activated MAP kinase signaling.

Neuronal Cell Death and Degeneration through Increased Nitroxidative Stress and Tau Phosphorylation in HIV-1 Transgenic Rats.

The underlying mechanisms for increased neurodegeneration and neurocognitive deficits in HIV-infected people are unclear. Therefore, this study was aimed to investigate the mechanisms of increased neurodegeneration in 5-month old male HIV-1 Transgenic (Tg) rats compared to the age- and gender-matched wild-type (WT) by evaluating histological changes and biochemical parameters of the key proteins involved in the cell death signaling and apoptosis. Histological and immunohistochemical analyses revealed decreased neuronal cells with elevated astrogliosis in HIV-1 Tg rats compared to WT. Mechanistic studies revealed that increased levels of nitroxidative stress marker proteins such as NADPH-oxidase, cytochrome P450-2E1 (CYP2E1), inducible nitric oxide synthase (iNOS), the stress-activated mitogen-activated protein kinases such as JNK and p38K, activated cell-cycle dependent CDK5, hypoxia-inducible protein-1α, nitrated proteins, hyperphosphorylated tau, and amyloid plaques in HIV-Tg rats were consistently observed in HIV-1 Tg rats. Confocal microscopy and cell viability analyses showed that treatment with an antioxidant N-acetylcysteine or a specific inhibitor of iNOS 1400W significantly prevented the increased apoptosis of neuro-2A cells by HIV-1 Tat or gp120 protein, demonstrating the causal role of HIV-1 mediated nitroxidative stress and protein nitration in promoting neuronal cell death. Immunoprecipitation and immunoblot analysis confirmed nitration of Hsp90, evaluated as an example of nitrated proteins, suggesting possible involvement of nitrated proteins in neuronal damage. Further, activated p-JNK directly binds tau and phosphorylates multiple amino acids, suggesting an important role of p-JNK in tau hyperphosphorylation and tauopathy. These changes were accompanied with elevated levels of many apoptosis-related proteins Bax and cleaved (activated) caspase-3 as well as proinflammatory cytokines including TNF-α, IL-6 and MCP-1. Collectively, these results indicate that raised nitroxidative stress accompanied by elevated inflammation, cell death signaling pathway including activated p-JNK, C-terminal C99 amyloid fragment formation and tau hyperphosphorylation are responsible for increased apoptosis of neuronal cells and neurodegeneration in 5-month old HIV-Tg rats.

The Complexity of Fungal Vision.

Life, as we know it, would not be possible without light. Light is not only a primary source of energy, but also an important source of information for many organisms. To sense light, only a few photoreceptor systems have developed during evolution. They are all based on an organic molecule with conjugated double bonds that allows energy transfer from visible (or UV) light to its cognate protein to translate the primary physical photoresponse to cell-biological actions. The three main classes of receptors are flavin-based blue-light, retinal-based green-light (such as rhodopsin), and linear tetrapyrrole-based red-light sensors. Light not only controls the behavior of motile organisms, but is also important for many sessile microorganisms including fungi. In fungi, light controls developmental decisions and physiological adaptations as well as the circadian clock. Although all major classes of photoreceptors are found in fungi, a good level of understanding of the signaling processes at the molecular level is limited to some model fungi. However, current knowledge suggests a complex interplay between light perception systems, which goes far beyond the simple sensing of light and dark. In this article we focus on recent results in several fungi, which suggest a strong link between light-sensing and stress-activated mitogen-activated protein kinases.

Role of the Talaromyces marneffei (Penicillium marneffei) sakA gene in nitrosative stress response, conidiation and red pigment production.

Stress-activated MAPK pathways are systems used to regulate the stress adaptation of most fungi. It has been shown that in Talaromyces marneffei (Penicillium marneffei), a pathogenic dimorphic fungus, the sakA gene is involved, not only in tolerance against oxidative and heat stresses, but also in playing a role in asexual development, yeast cell generation in vitro and survival inside macrophage cell lines. In this study, the role of the T. marneffei sakA gene on the nitrosative stress response and the regulation of gene expression were investigated. The susceptibility of the sakA mutant to NaNO2 was investigated using drop dilution assay and the expression of genes of interest were determined by RT-PCR, comparing them to the wild-type and complemented strains. The results demonstrated that the T. marneffei sakA gene played a role in the stress response to NaNO2, the expression of genes functioning in conidial development (brlA, abaA and wetA) and red pigment biosynthesis (pks3, rp1, rp2 and rp3). These findings suggest that T. marneffei sakA is broadly involved in a wide variety of cell activities, including stress response, cell morphogenesis, asexual development and pigmentation.

FOS-1 functions as a transcriptional activator downstream of the C. elegans JNK homolog KGB-1

JNK proteins are conserved stress-activated MAP kinases. In C. elegans, the JNK-homolog KGB-1 plays essential roles in protection from heavy metals and protein folding stress. However, the contributions of KGB-1 are age-dependent, providing protection in larvae, but reducing stress resistance and shortening lifespan in adults. Attenuation of DAF-16 was linked to the detrimental contributions of KGB-1 in adults, but its involvement in KGB-1-dependent protection in larvae remains unclear. To characterize age-dependent contributions of KGB-1, we used microarray analysis to measure gene expression following KGB-1 activation either in developing larvae or in adults, achieved by knocking down its negative phosphatase regulator vhp-1. This revealed a robust KGB-1 regulon, most of which consisting of genes induced following KGB-1 activation regardless of age; a smaller number of genes was regulated in an age-dependent manner. We found that the bZIP transcription factor FOS-1 was essential for age-invariant KGB-1-dependent gene induction, but not for age-dependent expression. The latter was more affected by DAF-16, which was further found to be required for KGB-1-dependent cadmium resistance in larvae. Our results identify FOS-1 as a transcriptional activator mediating age-invariant contributions of KGB-1, including a regulatory loop of KGB-1 signaling, but also stress the importance of DAF-16 as a mediator of age-dependent contributions.

Effect of D609 on the expression of GADD45β protein: Potential inhibitory role in the growth of glioblastoma cancer stem like cells

GADD45β (Growth Arrest and DNA Damage inducible protein) is a stress activated protein which plays an important role in regulating apoptosis, proliferation, DNA repair and potentially may have a role in cancer. In this study we examined the role of anti-oxidative stress on the expression of GADD45β in glioma stem-like cells (GSC). We show that patient derived GSCs have high survival in the absence of exogenous growth factors. Addition of D609 (Tricyclodecan-9-yl-xanthogenate), a known anti-oxidative compound, to GSCs reduced the cellular ATP content with significant effects observed when GSCs were cultured in growth factor free medium. D609 exposure also resulted in a decrease in the protein and an increase in mRNA of GADD45β with a concomitant decline in the survival of cells. However, under similar conditions the phosphorylation of p38 MAP kinase (stress activated MAP kinase), a downstream target of GADD45β, was significantly enhanced in response to D609. Therefore it appears that GADD45β might play a role in glioma stem cell survival and that p38 MAP kinase may not be directly activated by GADD45β. Together these observations suggest that anti-oxidative compounds like D609 can target GADD45β which may be one strategy to curtail the growth of glioma stem like cells.

Gene expression profiling analysis of ovarian cancer.

As a gynecological oncology, ovarian cancer has high incidence and mortality. To study the mechanisms of ovarian cancer, the present study analyzed the GSE37582 microarray. GSE37582 was downloaded from Gene Expression Omnibus and included data from 74 ovarian cancer cases and 47 healthy controls. The differentially-expressed genes (DEGs) were screened using linear models for microarray data package in R and were further screened for functional annotation. Next, Gene Ontology and pathway enrichment analysis of the DEGs was conducted. The interaction associations of the proteins encoded by the DEGs were searched using the Search Tool for the Retrieval of Interacting Genes, and the protein-protein interaction (PPI) network was visualized by Cytoscape. Moreover, module analysis of the PPI network was performed using the BioNet analysis tool in R. A total of 284 DEGs were screened, consisting of 145 upregulated genes and 139 downregulated genes. In particular, downregulated FBJ murine osteosarcoma viral oncogene homolog (FOS) was an oncogene, while downregulated cyclin-dependent kinase inhibitor 1A (CDKN1A) was a tumor suppressor gene and upregulated cluster of differentiation 44 (CD44) was classed as an ‘other’ gene. The enriched functions included collagen catabolic process, stress-activated mitogen-activated protein kinases cascade and insulin receptor signaling pathway. Meanwhile, FOS (degree, 15), CD44 (degree, 9), B-cell CLL/lymphoma 2 (BCL2; degree, 7), CDKN1A (degree, 7) and matrix metallopeptidase 3 (MMP3; degree, 6) had higher connectivity degrees in the PPI network for the DEGs. These genes may be involved in ovarian cancer by interacting with other genes in the module of the PPI network (e.g., BCL2-FOS, BCL2-CDKN1A, FOS-CDKN1A, FOS-CD44, MMP3-MMP7 and MMP7-CD44). Overall, BCL2, FOS, CDKN1A, CD44, MMP3 and MMP7 may be correlated with ovarian cancer.

The Centrosome Undergoes Plk1-Independent Interphase Maturation during Inflammation and Mediates Cytokine Release

Cytokine production is a necessary event in the immune response during inflammation and is associated with mortality during sepsis, autoimmune disorders, cancer, and diabetes. Stress-activated MAP kinase signaling cascades that mediate cytokine synthesis are well established. However, the downstream fate of cytokines before they are secreted remains elusive. We report that pro-inflammatory stimuli lead to recruitment of pericentriolar material, specifically pericentrin and γ-tubulin, to the centrosome. This is accompanied by enhanced microtubule nucleation and enrichment of the recycling endosome component FIP3, all of which are hallmarks of centrosome maturation during mitosis. Intriguingly, centrosome maturation occurs during interphase in an MLK-dependent manner, independent of the classic mitotic kinase, Plk1. Centrosome disruption by chemical prevention of centriole assembly or genetic ablation of pericentrin attenuated interleukin-6, interleukin-10, and MCP1 secretion, suggesting that the centrosome is critical for cytokine production. Our results reveal a function of the centrosome in innate immunity.

A Trichoderma atroviride stress-activated MAPK pathway integrates stress and light signals.

Cells possess stress-activated protein kinase (SAPK) signalling pathways, which are activated practically in response to any cellular insult, regulating responses for survival and adaptation to harmful environmental changes. To understand the function of SAPK pathways in T. atroviride, mutants lacking the MAPKK Pbs2 and the MAPK Tmk3 were analysed under several cellular stresses, and in their response to light. All mutants were highly sensitive to cellular insults such as osmotic and oxidative stress, cell wall damage, high temperature, cadmium, and UV irradiation. Under oxidative stress, the Tmk3 pathway showed specific roles during development, which in conidia are essential for tolerance to oxidant agents and appear to play a minor role in mycelia. The function of this pathway was more evident in Δpbs2 and Δtmk3 mutant strains when combining oxidative stress or cell wall damage with light. Light stimulates tolerance to osmotic stress through Tmk3 independently of the photoreceptor Blr1. Strikingly, photoconidiation and expression of blue light regulated genes was severally affected in Δtmk3 and Δpbs2 strains, indicating that this pathway regulates light responses. Furthermore, Tmk3 was rapidly phosphorylated upon light exposure. Thus, our data indicate that Tmk3 signalling cooperates with the Blr photoreceptor complex in the activation of gene expression.

Probing the Cell Cycle Significance of Cdt1 Phosphorylation at Novel Sites

The cell cycle is regulated by proteins as it goes through the phases of G1, S, G2, and mitosis. DNA replication should occur once and only once in S phase. When re‐replication occurs erroneously it can lead to genomic instability such as cancer. Cdt1 is a protein that is highly regulated in the cell cycle and Cdt1 is degraded during S phase so that DNA cannot be re‐replicated. It plays a role in recruiting DNA replication MCM helicases to the origin recognition complex (ORC) in the G1 phase of the cell cycle. Cyclin dependent kinases (CDK) are regulatory proteins that phosphorylate Cdt1 for degradation by proteasomes in the S phase. Previous studies have investigated stress activated MAP kinase phosphorylation sites within Cdt1. Findings demonstrate that when those sites are mutated, such that they can no longer be phosphorylated, changes can occur in stability and function. Cdt1 is phosphorylated at two additional sites, Serine 93 and Threonine 152, but the functions of these phosphorylations are unknown. Our goal is to mutate those two sites to alanine, which cannot be phosphorylated, express the altered Cdt1 genes in human cells, and test for changes in Cdt1 behavior in a cell cycle dependent manner. We successfully created the S93A, T152A and S93AT152A (double mutant) Cdt1 expression plasmids by first, using PCR to create our inserts, then combining each mutant with a pENTR vector by Gibson assembly. The mutant Cdt1 constructs were transferred from the pENTR plasmids to pBabe‐puro plasmids by LR reaction for gene expression. For our transient transfection we used 293T cells to express our gene. Our stable Hela cell line was produced by retroviral transfection of 293T cells, then extracting the virus for transduction of Hela cells. Western blot analysis of 293T cells transiently transfected with these plasmids did not yield conclusive results with regard to the precise nature of the expression of the Cdt1 mutants. However, we are in the process of creating stable Hela cell lines using these plasmids. Our T152A and S93AT152A Hela cell line did not become stable as opposed to our S93A Hela cell line. We therefore suspect that our T152A mutant might have a negative effect on cell viability.Support or Funding InformationNIH/NIGMS Grant #R25GM076162NSF Grant #DBI‐1038160

APOL1 kidney disease risk variants cause cytotoxicity by depleting cellular potassium and inducing stress-activated protein kinases

Two specific genetic variants of the apolipoprotein L1 (APOL1) gene are responsible for the high rate of kidney disease in people of recent African ancestry. Expression in cultured cells of these APOL1 risk variants, commonly referred to as G1 and G2, results in significant cytotoxicity. The underlying mechanism of this cytotoxicity is poorly understood. We hypothesized that this cytotoxicity is mediated by APOL1 risk variant-induced dysregulation of intracellular signaling relevant for cell survival. To test this hypothesis, we conditionally expressed WT human APOL1 (G0), the APOL1 G1 variant, or the APOL1 G2 variant in human embryonic kidney cells (T-REx-293) using a tetracycline-mediated (Tet-On) system. We found that expression of either G1 or G2 APOL1 variants increased apparent cell swelling and cell death compared with G0-expressing cells. These manifestations of cytotoxicity were preceded by G1 or G2 APOL1-induced net efflux of intracellular potassium as measured by X-ray fluorescence, resulting in the activation of stress-activated protein kinases (SAPKs), p38 MAPK, and JNK. Prevention of net K(+) efflux inhibited activation of these SAPKs by APOL1 G1 or G2. Furthermore, inhibition of SAPK signaling and inhibition of net K(+) efflux abrogated cytotoxicity associated with expression of APOL1 risk variants. These findings in cell culture raise the possibility that nephrotoxicity of APOL1 risk variants may be mediated by APOL1 risk variant-induced net loss of intracellular K(+) and subsequent induction of stress-activated protein kinase pathways.

MytiLec, a Mussel R-Type Lectin, Interacts with Surface Glycan Gb3 on Burkitt's Lymphoma Cells to Trigger Apoptosis through Multiple Pathways.

MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galβ1-4Glc). MytiLec revealed β-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt's lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt’s lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface.