ASK1 and its role in cardiovascular and other disorders: available treatments and future prospects

ABSTRACT

Introduction: Apoptosis signal-regulating kinase 1 (ASK1), also known as MAP3K5, is a member of mitogen-activated protein kinase kinase kinase (MAP3K) family and is well reported as crucial in the regulation of the JNK and P38 pathways. ASK1 is activated in response to a diverse array of stresses such as endoplasmic reticulum stress, lipopolysaccharides, tumor necrosis factor alpha, and reactive oxygen species. The activation of ASK1 induces various stress responses.

Areas covered: Considering ASK1 as an important therapeutic drug target, here we have discussed the role of ASK1 in the progression of various diseases. We have also provided an overview of the available inhibitors for ASK1. The success of computational-based approaches toward ASK1 inhibitor design has also been discussed.

Expert opinion: A number of reports have outlined the prominent role of ASK1 in the pathogenesis of several diseases. The discovery of novel ASK1 inhibitors would have a wide range of applications in medical science. In-silico techniques have been successfully used in the design of some novel ASK1 inhibitors. The use of machine learning-based approaches in combination with structure-based virtual screening (SBVS) and ligand-based virtual screening (LBVS) will be helpful toward the development of potent ASK1 inhibitors.     

Silencing of the integrin-linked kinase gene induces the apoptosis in ovarian carcinoma

Context: Integrin-linked kinase (ILK), a multidomain focal adhesion protein serine/threonine kinase, plays an essential role in ovarian carcinoma. There are reports that the expression and activity of ILK are increased in ovarian cancer.

Objective: To test the hypothesis that ILK pathway mediates the apoptosis of ovarian carcinoma SKOV3 cell influencing the cell survival, we performed these studies.

Materials and methods: We applied lentivirus transfection, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT), apoptotic proteins expressions assay, and Hoechst to study our hypothesis.

Results: We found that silencing of the ILK increases the cell cytotoxic, growth inhibition, and apoptosis. Moreover, after blocking the activation of ILK with ILK shRNA, up-regulation of pro-apoptotic bax expression and down-regulation of the anti-apoptotic bcl-2 expression were found in ovarian cancer SKOV3 cell line. These were associated with an increasing cleaved caspase-3 activity and chromatin condensation of cell nuclear. Furthermore, the expressions of fas and fas ligand (fasL), belonging to the tumor necrosis factor family and controlling the cell apoptosis, were also enhanced.

Conclusions: Thus, these findings indicate that both the intrinsic pathway and the extrinsic death receptor pathway are involved in the process that silencing of the ILK gene induces the apoptosis in ovarian carcinoma SKOV3 cell.     

Plant 5-methylthioribose kinase: properties of the partially purified enzyme from yellow lupin (lupinus luteus L.) seeds

Activity of 5-methylthioribose kinase, the enzyme which catalyzes the ATP-dependent formation of 1-phospho-5-methylthioribose, has been revealed in the extracts from various higher plant species. Almost 2,000-fold-purified enzyme has been obtained from yellow lupin (Lupinus luteus L. cv Topaz) seed extract. Molecular weight of the native enzyme is 70,000 as judged by gel filtration. The lupin 5-methylthioribose kinase exhibits a strict requirement for divalent metal ions. Among the ions tested, only Mg²⁺ and Mn²⁺ acted as cofactors. The curve of kinase initial velocity versus pH reaches plateau at pH 10 to 10.5. The K_m values calculated for 5-methylthioribose and ATP are 4.3 and 8.3 micromolar, respectively.

Among nucleoside triphosphates tested as potential phosphate donors, only dATP could substitute in the reaction for ATP. 5-Isobutylthioribose, an analog of 5-methylthioribose, proved to be the γ-ATP-phosphate acceptor, too. The compound inhibits competitively synthesis of 1-phospho-5-methylthioribose (K_i = 1.4 micromolar). Lupin 5-methylthioribose kinase is completely and irreversibly inhibited by the antisulfhydryl reagent, p-hydroxymercuribenzoate. As in bacteria (Ferro, Barrett, Shapiro 1978 J Biol Chem 253: 6021-6025), the enzyme may be involved in a new, alternative pathway of methionine synthesis in plant tissues.

     

Cytidine Kinase: A Novel Pyrimidine Ribonucleoside Phosphorylating Enzyme in Escherzchza Coli

Abstract

A previously undescribed pyrimidine ribonucleoside kinase that preferentially phosphorylates cytidine to yield its 5′-monophosphate form has been purified to near homogeneity from Escherichia coli.The enzyme was distinct from the known pyrimidine nucleoside kinase in the chromatographic profile, substrate specificity and molecular weight.

     

Synthesis and Biological Activity of 5-Fluoro-2-thiocytosine Nucleosides

Abstract

Two pathways are described for the synthesis of the 2′-deoxynucleosides of 2-thiocytosine and 5-fluoro-2-thiocytosine: (a) by nucleoside condensation, (b) by amination of the corresponding nucleosides of 2,4-dithiouracil. Biological activities vs two cell systems are described. The nucleosides are moderate to weak substrates of deoxycytidine kinase and, partly as a result of this, reasonable good inhibitors of the enzyme     

A discrete affinity-driven elevation of ZAP-70 kinase activity initiates negative selection

Context: Although ZAP-70 is required for T-cell development, it’s unclear how this kinase controls both positive and negative selection.

Objective and methods: Using OT-I pre-selection thymocytes and a panel of peptide major histocompatibility complex (pMHC) ligands of defined affinity, the recruitment, phosphorylation and activity of ZAP-70 was determined at the interface with antigen-presenting cells (APCs).

Results: pMHC ligands promoting negative selection induce a discrete elevation of ZAP-70 recruitment, phosphorylation and enzymatic activity in the thymocyte:APCs interface.

Discussion: The quantity of ZAP-70 kinase activity per cell is a key parameter controlling the fate of a developing thymocyte since partial inhibition of ZAP-70 kinase activity converted negative into positive selection. Surprisingly, the amount of ZAP-70 enzymatic activity observed during negative selection is not controlled by differential phosphorylation of the ZAP-70 protein but rather by the total amount of T-cell receptor and co-associated ZAP-70 recruited to the thymocyte:APC interface.

Conclusions: These data provide evidence that a burst of ZAP-70 activity initiates the signaling pathways for negative selection.     

LRRK2 mutations impair depolarization-induced mitophagy through inhibition of mitochondrial accumulation of RAB10

ABSTRACT

Parkinson disease (PD) is a disabling, incurable disorder with increasing prevalence in the western world. In rare cases PD is caused by mutations in the genes for PINK1 (PTEN induced kinase 1) or PRKN (parkin RBR E3 ubiquitin protein ligase), which impair the selective autophagic elimination of damaged mitochondria (mitophagy). Mutations in the gene encoding LRRK2 (leucine rich repeat kinase 2) are the most common monogenic cause of PD. Here, we report that the LRRK2 kinase substrate RAB10 accumulates on depolarized mitochondria in a PINK1- and PRKN-dependent manner. RAB10 binds the autophagy receptor OPTN (optineurin), promotes OPTN accumulation on depolarized mitochondria and facilitates mitophagy. In PD patients with the two most common LRRK2 mutations (G2019S and R1441C), RAB10 phosphorylation at threonine 73 is enhanced, while RAB10 interaction with OPTN, accumulation of RAB10 and OPTN on depolarized mitochondria, depolarization-induced mitophagy and mitochondrial function are all impaired. These defects in LRRK2 mutant patient cells are rescued by LRRK2 knockdown and LRRK2 kinase inhibition. A phosphomimetic RAB10 mutant showed less OPTN interaction and less translocation to depolarized mitochondria than wild-type RAB10, and failed to rescue mitophagy in LRRK2 mutant cells. These data connect LRRK2 with PINK1- and PRKN-mediated mitophagy via its substrate RAB10, and indicate that the pathogenic effects of mutations in LRRK2, PINK1 and PRKN may converge on a common pathway.

Abbreviations : ACTB: actin beta; ATP5F1B: ATP synthase F1 subunit beta; CALCOCO2: calcium binding and coiled-coil domain 2; CCCP: carbonyl cyanide m-chlorophenylhydrazone; Co-IP: co-immunoprecipitation; EBSS: Earle’s balanced salt solution; GFP: green fluorescent protein; HSPD1: heat shock protein family D (Hsp60) member 1; LAMP1: lysosomal associated membrane protein 1; LRRK2: leucine rich repeat kinase 2; IF: immunofluorescence; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MFN2: mitofusin 2; OMM: outer mitochondrial membrane; OPTN: optineurin; PD: Parkinson disease; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RHOT1: ras homolog family member T1; ROS: reactive oxygen species; TBK1: TANK binding kinase 1; WB: western blot.     

Substrate Specificities of Mitochondrial Thymidine Kinase and Cytosolic Deoxycytidine Kinase Against 5-Aryl Substituted Pyrimidine-2′-deoxyribose Analogues

Abstract

Some 5-aryl-2′-deoxyuridine and -deoxycytidine analogues, many with known antiviral activity, were evaluated as substrates for pure deoxycytidine kinase (dCK) and pure mitochondrial thymidine kinase (TK2). Some of the deoxyuridine compounds were also tested with pure cytosolic thymidine kinase (TK1). TK2 showed the highest activity with this type of analogues.     

Herpes Simplex Virus Thymidine Kinase Gene-Transfected Tumor Cells; Sensitivity to Antiherpetic Drugs

Abstract

A series of antiherpetic 5-substituted 2′-deoxyuridine derivatives (i. e. BVDU) and guanine derivatives (i. e. ganciclovir) have been evaluated for their cytostatic activity against murine mammary carcinoma FM3A cell lines that are deficient in cytosol thymidine kinase, but transfected by the herpes simplex virus type 1 (HSV-1)- or type 2 (HSV-2)-specified thymidine kinase gene. Most compounds were endowed with a markedly higher cytostatic activity against the HSV TK gene-transfected tumor cells than against wild-type tumor cells. The principal target for cytostatic activity of the BVDU derivatives proved thymidylate synthase, whereas the guanine derivatives inhibited HSV TK gene-transfected tumor cell proliferation by competing with cellular DNA polymerase(s) and subsequent incorporation into the cellular genome.

     

Targeting Autophagic Regulation of NF-kappaB in HTLV-I Transformed Cells by Geldanamycin: Implications for Therapeutic Interventions

The IκappaB kinase (IKK)/NF-κappaB signaling pathway plays an essential role in the development and survival of many types of cancers including adult T-cell leukemia (ATL) caused by the human T-cell leukemia virus type I (HTLV-I) infection. Accordingly, targeting NF-κappaB provides an attractive strategy for cancer therapy. We recently found that specific inhibition of Hsp90 by geldanamycin (GA) results in autophagic degradation of IKK and NF-κappaB-inducing kinase (NIK), an upstream kinase of IKK, and inactivation of NF-κappaB in various cell lines. Here, we further report that GA inhibition of Hsp90 also led to IKK autophagic degradation and NF-κappaB inhibition in both HTLV-transformed T cells and ATL-derived cell lines. Importantly, GA treatment led to efficient apoptosis of these malignant cells, whereas inhibition of autophagic degradation of IKK significantly ameliorated the cytotoxic effect of GA. These findings thus not only provide mechanistic insights into the tumor suppression function of autophagy and the anti-tumor activity of GA, but also suggest an immediate therapeutic strategy for ATL and other diseases associated with NF-κappaB activation by targeting autophagic degradation of the central NF-kappaB activating kinases.

Addendum to:

Hsp90 Inhibition Results in Autophagy-Mediated Proteasome-Independent Degradation of IκappaB Kinase (IKK)

G. Qing, P. Yan and G. Xiao

Cell Res 2006; 16:895-901

and

Hsp90 Regulates Processing of NF-κappaB2 p100 Involving Protection of NF-κappaB-Inducing Kinase (NIK) from Autophagy-Mediated Degradation

G. Qing, P. Yan, Z. Qu, H. Liu and G. Xiao

Cell Res 2007; 17:520-30     

CDK Inhibitors: Cell Cycle Arrest Versus Apoptosis

No Abstract Available

Key Words:

Cyclin-dependent kinase (Cdk), CDK inhibitors     

Elongation Factor-2 Kinase: Its Role in Protein Synthesis and Autophagy

Elongation factor-2 kinase (eEF-2 kinase; Ca²⁺/calmodulin-dependent kinase III) controls the rate of peptide chain elongation. The activity of eEF-2 kinase is increased in many malignancies, yet its precise function in carcinogenesis remains unknown. Autophagy, a well-defined survival pathway in yeast, may also play an important role in oncogenesis. Furthermore, the autophagic response to nutrient deprivation is regulated by the mammalian target of rapamycin (mTOR). eEF-2 kinase lies downstream of mTOR and is regulated by several kinases in this pathway. Therefore, we studied the role of eEF-2 kinase in autophagy. Knockdown of eEF-2 kinase by RNA interference inhibited autophagy in several cell types as measured by light chain 3 (LC3)-II formation, acidic vesicular organelle staining, and electron microscopy. In contrast, overexpression of eEF-2 kinase increased autophagy. Furthermore, inhibition of autophagy markedly decreased the viability of glioblastoma cells grown under conditions of nutrient depletion, which increased eEF-2 kinase activity and decreased the activity of S6 kinase, suggesting an involvement of mTOR pathway in the eEF-2 kinase-mediated regulation of autophagy. These results suggest that eEF-2 kinase plays a regulatory role in the autophagic process in tumor cells and may promote cancer cell survival under conditions of nutrient deprivation. Therefore, eEF-2 kinase activation may be a part of a survival mechanism in glioblastoma, and targeting this kinase may represent a novel approach to cancer treatment.

Addendum to:

Elongation Factor-2 Kinase Regulates Autophagy in Human Glioblastoma Cells

H. Wu, J.-M. Yang, S. Jin, H. Zhang and W.N. Hait

Cancer Res 2006; 66:3015-23     

Differential effects of Akt pathway inhibitors on IL-1β-induced protein phosphorylation in human fibroblast-like synoviocytes

Context: Interleukin (IL)-1β activates various signal transduction pathways including p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and Akt in human fibroblast-like synoviocytes (HFLS).

Objective: We investigated the effects of an Akt inhibitor, a phosphatidylinositol 3-kinase (PI3K) inhibitor, and Akt RNAi knockdown on IL-1β-induced protein phosphorylation in HFLS to clarify the role of the PI3K/Akt signaling pathway in the phosphorylation of the inhibitor of κB (IκB)α and heat shock protein 27 (HSP27).

Materials and methods: A multiplex suspension array system was used for the detection of phosphorylated proteins.

Results: IL-1β induced biphasic phosphorylation of IκBα, with the first phase occurring 10?min after IL-1β stimulation, and this was augmented by treatment with Akt inhibitor IV. However, this phenomenon was not observed after treatment with LY-294002, a PI3K inhibitor. Furthermore, Akt inhibitor IV suppressed ERK2 phosphorylation, whereas LY-294002 and Akt RNAi had no effect. In contrast, Akt inhibitor IV, LY-294002, and Akt RNAi augmented HSP27 phosphorylation.

Discussion and conclusions: Modulation of different stages of the PI3K/Akt pathway may differentially affect the phosphorylation of IκBα and HSP27 in HFLS.     

mTOR Kinase and the Regulatory Subunit of Protein Kinase A (PRKAR1A) Spatially and Functionally Interact during Autophagosome Maturation

The regulatory subunit 1-alpha (RI-alpha) of protein kinase A (PKA) and the mTOR kinase are involved in a common pathway regulating mammalian autophagy. RI-alpha was found to localize on Rab7-positive late endosomes and on LC3-positive autophagosomal membranes in cultured cells. RI-alpha was also shown to physically interact with mTOR kinase and affect its phosphorylation and activity.1 In this addendum, we further explore the subcellular distribution of mTOR related to RI_ and LC3. We present experiments showing that mTOR colocalizes with RI-alpha-, Rab7- and LC3-positive membranes in cultured cells. Because RIa regulates the phosphorylation and activity of mTOR kinase, which we now show localizes on autophagosomal membranes, the possibility emerges that the RI-alpha-mTOR complex acts at the level of autophagosome maturation.

Addendum to:

Depletion of Type IA Regulatory Subunit (RI-alpha) of Protein Kinase A (PKA) in Mammalian Cells and Tissues Activates mTOR and Causes Autophagic Deficiency

M. Mavrakis, J. Lippincott-Schwartz, C.A. Stratakis and I. Bossis

Hum Mol Genet 2006; 15:2962-71     

Erythrocytenenzyme der Primaten

Zusammenfassung Die Adenylatkinasen der Primaten zeigen eine genetisch determinierte Variabilität. Bei der Untersuchung von 334 subhumanen Primaten (289 Simiae der Alten Welt, 45 Prosimiae) konnten wir vier Adenylatkinase-Varianten nachweisen: die AK 1, die bei allen Menschenpopulationen eine hohe Frequenz besitzt und bei den Pongidae ausschließlich vorkommt, wurde nur bei Cercopithecus aethiops und Lemur catta angetroffen; die AK 3, eine beim Menschen sehr selten vorkommende Variante, besitzt bei den Simiae der Alten Welt eine auffallend hohe Frequenz und kommt auch bei einigen Vertretern der Prosimiae vor; die AK 5 und AK 6, weit anodisch wandernde Varianten, die bislang beim Menschen nicht nachgewiesen wurden, fanden wir bei Vertretern der Prosimiae.-Die Varianten AK 2 und AK 4 wurden bei den subhumanen Primaten nicht vorgefunden.

---

Red cell enzymes of primatesAdenylate kinase; EC: 2.7.4.3

Summary The polymorphism of adenylate kinase has been investigated in 334 subhuman Primates (289 Old World Monkeys and 45 Prosimians). Four adenylate kinase variants were found to be present: AK 1, most frequent in human populations and exclusively present in the Pongidae, only occurs in the African Cercopithecus aethiops and the Madagasian Lemur catta; AK 3, extremely rare in human populations, is a common phenotype of Old World Monkeys and also present in some Prosimians; AK 5 and AK 6, faster anodal migrating adenylate kinase variants not present in human populations, were found in other Prosimians.-AK 2 and AK 4, probably specific for humans, were not detected in subhuman Primates.

---

---

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Epidermal growth factor receptor (EGFR) mutations as biomarker for head and neck squamous cell carcinomas (HNSCC)

Abstract

Context: Mutations in tyrosine kinase domain (TK) of epidermal growth factor receptor (EGFR) lead to signalling interruptions in several cancers.

Objective: To understand EGFR mutations in head and neck squamous cell carcinomas (HNSCC), and their role as biomarkers.

Methods: Screened 129 HNSCC patients and 150 controls for mutations in the TK domain using polymerase chain reaction (PCR), single strand confirmatory polymorphism (SSCP) and sequencing.

Results: 81.39% of HNSCC had four mutations: G2155C, G2176A, C2188G and G2471A among these two mutations were also reported in other cancers where as two novel mutations are being reported for the first time in HNSCC. Mutational frequency was significantly associated with an advanced stage of HNSCC, habits of tobacco/alcohol and ages above 49 years.

Conclusion: EGFR single nucleotide polymorphisms could be useful biomarkers of HNSCC.     

Interspecific variability of red cell phosphoglycerate kinase in primates

Summary The genetic polymorphism of red cell phosphoglycerate kinase was investigated in primates. Five variants of the enzyme were detected.

---

Zusammenfassung Für die erythrocytäre Phosphoglyceratkinase der Primaten konnte eine genetisch determinierte Variabilität (5 Enzymvarianten) nachgewiesen werden.

---

---

Supported by the Deutsche Forschungsgemeinschaft.