CK2β interacts with and regulates p21-activated kinases in Drosophila

The role of CK2β has been defined as the regulatory subunit of protein kinase CK2, which is a heterotetrameric complex composed of two CK2β and two catalytic active CK2α subunits. The identification of other serine/threonine kinases such as A-Raf, Chk1, and c-Mos that interact with and are regulated by CK2β has challenged this view and provided evidence for functions of CK2β outside the CK2 holoenzyme. In this report we describe the first interaction of Drosophila CK2β outside the CK2 holoenzyme with p21-activated kinase (PAK) proteins. This interaction is seen for distinct PAK and CK2β isoforms. In contrast to the CK2α–CK2β interaction, dimer formation of the CK2β subunits is not a prerequisite for binding of PAK proteins. Our results support the idea that CK2β can bind to PAK proteins in a CK2α independent manner and negatively regulates PAK kinase activity.     

RIOK3 interacts with caspase-10 and negatively regulates the NF-κB signaling pathway

RIOK3 was initially characterized as a homolog of Aspergillus nidulans sudD and showed down-regulation at the invasive front of malignant melanomas, but the molecular mechanism remains elusive. Here, we report that overexpression of RIOK3 inhibits TNFα-induced NF-κB activation, but down-regulation of endogenous RIOK3 expression by siRNA potentiates it. A yeast two-hybrid experiment revealed that RIOK3 interacted with caspase-10, and further, a GST pull-down assay and endogenous coimmunoprecipitation validated the interaction. We subsequently showed that the interaction was mediated by the RIO domain of RIOK3 and each death effector domain of caspase-10. Interestingly, our data demonstrated that RIOK3 suppressed caspase-10-mediated NF-κB activation by competing RIP1 and NIK to bind to caspase-10. Importantly, the kinase activity of RIOK3 was confirmed to be relevant to NF-κB signaling. Taken together, our findings strongly suggest that RIOK3 negatively regulates NF-κB signaling pathway activated by TNFα dependent on its kinase activity and NF-κB signaling pathway activated by caspase-10 independent of its kinase activity.     

Changes in HIF-1α protein, pyruvate dehydrogenase phosphorylation, and activity with exercise in acute and chronic hypoxia

Exercise under acute hypoxia elicits a large increase in blood lactate concentration ([La](b)) compared with normoxic exercise. However, several studies in humans show that with the transition to chronic hypoxia, exercise [La](b) returns to normoxic levels. Although extensively examined over the last decades, the muscle-specific mechanisms responsible for this phenomenon remain unknown. To assess the changes in skeletal muscle associated with a transition from acute to chronic hypoxia, CD-1 mice were exposed for 24 h (24H), 1 wk (1WH), or 4 wk (4WH) to hypobaric hypoxia (equivalent to 4,300 m), exercised under 12% O(2), and compared with normoxic mice (N) at 21% O(2). Since the enzyme pyruvate dehydrogenase (PDH) plays a major role in the metabolic fate of pyruvate (oxidation vs. lactate production), we assessed the changes in its activity and regulation. Here we report that when run under hypoxia, 24H mice exhibited the highest blood and intramuscular lactate of all groups, while the 1WH group approached N group values. Concomitantly, the 24H group exhibited the lowest PDH activity, associated with a higher phosphorylation (inactive) state of the Ser(232) residue of PDH, a site specific to PDH kinase-1 (PDK1). Furthermore, protein levels of PDK1 and its regulator, the hypoxia inducible factor-1α (HIF-1α), were both elevated in the 24H group compared with N and 1WH groups. Overall, our results point to a novel mechanism in muscle where the HIF-1α pathway is desensitized in the transition from acute to chronic hypoxia, leading to a reestablishment of PDH activity and a reduction in lactate production by the exercising muscles.     

Mutations in the gene for the E1β subunit: a novel cause of pyruvate dehydrogenase deficiency

We describe two unrelated patients with pyruvate dehydrogenase (PDH) deficiency attributable to mutations in the gene encoding the E1 subunit of the complex. This is a previously unrecognised form of PDH deficiency, which most commonly results from mutations in the X-linked gene for the E1 subunit. Both patients had reduced immunoreactive E1 protein and both had missense mutations in the E1 gene. Activity of the PDH complex was restored in cultured fibroblasts from both patients by transfection and expression of the normal E1 coding sequence.     

Knockdown of apoptosis signal-regulating kinase 1 modulates basal glycogen synthase kinase-3β kinase activity and regulates cell migration

GSK-3β is a basally active kinase. Axin forms a complex with GSK-3β and β-catenin; this complex promotes the GSK-3β-dependent phosphorylation of β-catenin, thereby inducing its degradation. However, the inhibition of GSK-3β provokes cell migration via the dysregulation of β-catenin. In this study, we determined that the level of apoptosis signal-regulating kinase 1 (ASK1) was lower in a metastatic breast cancer cell line, compared to that of non-metastatic cancer cell lines and the knockdown of ASK1 not only induces β-catenin activation via the inhibition of GSK-3β and collapsing the subsequent protein complex by regulating Axin dynamics, but also stimulates cell migration. Together, the blockage of the GSK-3β-β-catenin pathway resulting from the knockdown of ASK1 modulates the migration of breast cancer cells.     

Nmi interacts with Hsp105β and enhances the Hsp105β-mediated Hsp70 expression

The mammalian stress protein Hsp105α is expressed constitutively and is further induced under stress conditions, whereas the alternative spliced form, Hsp105β is only expressed during mild heat shock. We previously reported that Hsp105α is localized mainly in the cytoplasm, whereas Hsp105β is localized in the nucleus. Consistent with the different localization of these proteins, Hsp105β but not Hsp105α induces the expression of the major stress protein Hsp70. We here identified N-myc and Stat interactor (Nmi), as an Hsp105β-binding protein by yeast two-hybrid screening. Immunoprecipitation and pull-down assay showed that Nmi interacts with Hsp105β in vivo and in vitro. Luciferase reporter gene assay and Western blotting showed that Nmi enhanced both the Hsp105β-induced phosphorylation of Stat3 and the Hsp105β-induced activation of the hsp70 promoter in a manner that is dependent on the Stat3-binding site, which results in an increase in Hsp70 protein levels. Most importantly, mild heat shock-induced Hsp70 expression, which is dependent on Hsp105β, is suppressed by knockdown of endogenous Nmi. These results suggest that Nmi has a role as a positive regulator of Hsp105β-mediated hsp70 gene expression along the Stat3 signaling pathway.     

TRIM59 interacts with ECSIT and negatively regulates NF-κB and IRF-3/7-mediated signal pathways

A series of inhibitors of d-amino acid oxidase (DAAO) are specific in blocking chronic pain, including formalin-induced tonic pain, neuropathic pain and bone cancer pain. This study used RNA interference technology to further validate the notion that spinal DAAO mediates formalin-induced pain. To target DAAO, a siRNA/DAAO formulated in polyetherimide (PEI) complexation and a shRNA/DAAO (shDAAO, with the same sequence as siRNA/DAAO after intracellular processing) expressed in recombinant adenoviral vectors were designed. The siRNA/DAAO was effective in blocking DAAO expression in NRK-52E rat kidney tubule epithelial cells, compared to the nonspecific oligonucleotides. Furthermore, multiple-daily intrathecal injections of both siRNA/DAAO and Ad-shDAAO for 7 days significantly inhibited spinal DAAO expression by 50-80% as measured by real-time quantitative PCR and Western blot, and blocked spinal DAAO enzymatic activity by approximately 60%. Meanwhile, both siRNA/DAAO and Ad-shDAAO prevented formalin-induced tonic phase pain by approximately 60%. Multiple-daily intrathecal injections of siRNA/DAAO and Ad-shDAAO also blocked more than 30% spinal expression of GFAP, a biomarker for the activation of astrocytes. These results further suggest that down-regulation of spinal DAAO expression and enzymatic activity leads to analgesia with its mechanism potentially related to activation of astrocytes in the spinal cord.     

Novel benzothiazinones (BTOs) as allosteric modulator or substrate competitive inhibitor of glycogen synthase kinase 3β (GSK-3β) with cellular activity of promoting glucose uptake

Glycogen synthase kinase 3β (GSK-3β) plays a key role in insulin metabolizing pathway and therefore inhibition of the enzyme might provide an important therapeutic approach for treatment of insulin resistance and type 2 diabetes. Recently, discovery of ATP noncompetitive inhibitors is gaining importance not only due to their generally increased selectivity but also for the potentially subtle modulation of the target. These kinds of compounds include allosteric modulators and substrate competitive inhibitors. Here we reported two benzothiazinone compounds (BTO), named BTO-5h (IC₅₀ = 8 μM) and BTO-5s (IC₅₀ = 10 μM) as novel allosteric modulator and substrate competitive inhibitor of GSK-3β, respectively. Their different action modes were proved by kinetic experiments. Furthermore, BTO-5s was selected to check the kinases profile and showed little or even no activity to a panel of ten protein kinases at 100 μM, indicating it has good selectivity. Docking studies were performed to give suggesting binding modes which can well explain their impacts on the enzyme. Moreover, cell experiments displayed both compounds reduced the phosphorylation level of glycogen synthase in an intact cell, and greatly enhanced the glucose uptake in both HpG2 and 3T3-L1 cells. All of these results suggested BTO-5s and BTO-5h maybe have potentially therapeutic value for anti-diabetes. The results also offer a new scaffold for designing and developing selective inhibitors with novel mechanisms of action.     

Inhibitors of type 1 17β-hydroxysteroid dehydrogenase with reduced estrogenic activity: Modifications of the positions 3 and 6 of estradiol

Breast cancer is the second most frequent cancer affecting women. Among all endocrine therapies for the treatment of breast cancer, inhibition of estrogen biosynthesis is becoming an interesting complementary approach to the use of antiestrogens. The enzyme type 1 17β-hydroxysteroid dehydrogenase (17β-HSD) plays a critical role in the biosynthesis of estradiol catalyzing preferentially the reduction of estrone into estradiol, the most active estrogen. Consequently, this enzyme is an interesting biological target for designing drugs for the treatment of estrogen-sensitive diseases such as breast cancer. Our group has reported the synthesis and the biological evaluation of N-methyl, N-butyl 6β-(thiaheptamamide)estradiol as a potent reversible inhibitor of type 1 17β-HSD. Unfortunately, this inhibitor has shown an estrogen effect, thus reducing its possible therapeutic interest. Herein three strategies to modify the biological profile (estrogenicity and inhibitory potency) of the initial lead compound were reported. In a first approach, the thioether bond was replaced with a more stable ether bond. Secondly, the hydroxyl group at position 3, which is responsible for a tight binding with the estrogen receptor, was removed. Finally, the amide group of the side-chain was changed to a methyl group. Moreover, the relationship between the inhibitory potency and the configuration of the side-chain at position 6 was investigated. The present study confirmed that the 6β-configuration of the side chain led to a much better inhibition than the 6α-configuration. The replacement of the 3-OH by a hydrogen atom as well as that of the amide group by a methyl was clearly unfavorable for the inhibition of type 1 17β-HSD. Changing the thioether for an ether bond decreased by 10-fold the estrogenic profile of the lead compound while the inhibitory potency on type 1 17β-HSD was only decreased by 5-fold. This study contributes to the knowledge required for the development of compounds with the desired profile, that is, a potent inhibitor of type 1 l7β-HSD without estrogen-like effects.     

Ultracytochemical demonstration and probable localization of 3β-hydroxysteroid dehydrogenase activity with a ferricyanide technique

Summary In order to localize 3-hydroxysteroid dehydrogenase activity on the ultrastructural level, sections of Newt and Rat adrenocortical tissues, fixed in a mixture of glutaraldehyde (0.25%) and formaldehyde (1%), were incubated in a medium containing namely a 3-hydroxysteroid as substrate, NAD, potassium ferricyanide as final electron acceptor, and copper sulfate. In some experiments, phenazine methosulfate (PMS), an electron carrier which can substitute for the activity of the endogenous NADH-diaphorase, is added at various concentrations to the incubation medium.A final precipitate of copper ferrocyanide is observed in the immediate vicinity of the tubules of the smooth endoplasmic reticulum, or in contact with their external faces. The reaction product can also be seen in mitochondrial cristae. The reaction does not take place in incubation media lacking substrate or containing cyanoketone, a specific inhibitor of 3-hydroxysteroid dehydrogenase. The addition of PMS to the incubation medium increases the intensity of the reaction, but does not modify the localization of the precipitate.     

How ubiquitination regulates the TGF-β signalling pathway: new insights and new players: new isoforms of ubiquitin-activating enzymes in the E1-E3 families join the game

Ubiquitination of protein species in regulating signal transduction pathways is universally accepted as of fundamental importance for normal development, and defects in this process have been implicated in the progression of many human diseases. One pathway that has received much attention in this context is transforming growth factor-beta (TGF-β) signalling, particularly during the regulation of epithelial-mesenchymal transition (EMT) and tumour progression. While E3-ubiquitin ligases offer themselves as potential therapeutic targets, much remains to be unveiled regarding mechanisms that culminate in their regulation. With this in mind, the focus of this review highlights the regulation of the ubiquitination pathway and the significance of a recently described group of NEDD4 E3-ubiquitin ligase isoforms in the context of TGF-β pathway regulation. Moreover, we now broaden these observations to incorporate a growing number of protein isoforms within the ubiquitin ligase superfamily as a whole, and discuss their relevance in defining a new 'iso-ubiquitinome'.     

Synthesis and 11β hydroxysteroid dehydrogenase 1 inhibition of thiazolidine derivatives with an adamantyl group

A new series of thiazolidine derivatives with an adamantyl group was synthesized and evaluated for their ability to inhibit 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1). Our initial compound 5a showed a weak inhibitory activity. Significant improvements in potency were achieved by substituent modification. The potent compound 8g (E) showed good in vitro inhibitory activity toward human 11β-HSD1, selectivity toward 11β-HSD2, metabolic stability, pharmacokinetic, and safety profile. Furthermore, this compound significantly inhibited 11β-HSD1 activity in rat and monkey models, and showed improved glycemic control in KKAy mice.     

PI(3,4,5)P3 potentiates phospholipase C-β activity

Phospholipase C-β (PLC-β) isozymes are key effectors in G protein-coupled signaling pathways. Previously, we showed that PLC-β1 and PLC-β3 bound immobilized PIP₃. In this study, PIP₃ was found to potentiate Ca²⁺-stimulated PLC-β activities using an in vitro reconstitution assay. LY294002, a specific PI 3-kinase inhibitor, significantly inhibited 10 min of agonist-stimulated total IP accumulation. Both LY294002 and wortmannin inhibited 90 sec of agonist-stimulated IP₃ accumulation in intact cells. Moreover, transfected p110CAAX, a constitutively activated PI 3-kinase catalytic subunit, increased 90 sec of oxytocin-stimulated IP₃ accumulation. Receptor-ligand binding assays indicated that LY294002 did not affect G protein-coupled receptors directly, suggesting a physiological role for PIP₃ in directly potentiating PLC-β activity. When coexpressed with p110CAAX, fluorescence-tagged PLC-β3 was increasingly localized to the plasma membrane. Additional observations suggest that the PH domain of PLC-β is not important for p110CAAX-induced membrane association.     

The determination by microdensitometry of the initial maximum velocity rate of rat ovarian 3β hydroxysteroid dehydrogenase activity

Summary The determination of the initial maximum velocity rate of 3 hydroxysteroid dehydrogenase activity (3 HSD) of the newest generation of corpora lutea of the adult dioestrous rat ovary is described. Under conditions where the substrate and co-factor concentrations are not rate limiting (4×10^–4 M epiandrosterone; 3×10^–3 M NAD⁺ respectively) the initial maximum reaction rate was maintained for approximately 6 min. NADH production during this period was 4.9±0.2 moles/min/cm³ of corpora lutea.