Treatment with polyamines can prevent monosodium glutamate neurotoxicity in the rat retina

It has been previously shown that treatment of newborn rats with the polyamines putrescine, spermidine and spermine can rescue sympathetic neurons from naturally occurring cell death and from induced death after axotomy or immunosympathectomy. The present study demonstrates that polyamine treatment can also prevent the neurodegenerative effects in the retina and the loss of body weight caused by monosodium glutamate. The findings indicate that polyamine treatment may have a rather general beneficial effect on neuron survival.     

In vivo 15N NMR studies of regulation of nitrogen assimilation and amino acid production by Brevibacterium lactofermentum

Glutamic acid producer Brevibacterium lactofermentum intact cells were used to demonstrate the feasibility of in vivo 15N NMR to follow nitrogen assimilation and amino acid production throughout the growth cycle. The induction of glutamic acid production by different growth conditions was studied. Intracellular and extracellular levels of free metabolites were estimated as function of oxygen supply and biotin concentration. 15N NMR enabled us to distinguish two phases during the fermentation. At the early stage of fermentation, glutamic acid was accumulated intracellularly independent of oxygen supply and no product was excreted. In the late growth phase, the permeability of the cells developed and L-glutamic acid was excreted. The effect of aeration and biotin concentration on cellular contents and excretion was also studied by 15N NMR. Glutamate, N-acetylglutamine, and glutamine were the main nitrogenous pools independent of cell culture conditions. Free ammonia was not accumulated intracellularly although glutamic acid fermentation can be characterized as the process of nitrogen assimilation and the uptake of ammonia is the key step. In conclusion, the application of in vivo 15N NMR spectroscopy unraveled various problems of nitrogen metabolism, in a rapid and nondestructive manner.     

DAPK2 is a novel regulator of mTORC1 activity and autophagy

Autophagy is a tightly regulated catabolic process, which is upregulated in cells in response to many different stress signals. Inhibition of mammalian target of rapmaycin complex 1 (mTORC1) is a crucial step in induction of autophagy, yet the mechanisms regulating the fine tuning of its activity are not fully understood. Here we show that death-associated protein kinase 2 (DAPK2), a Ca²⁺-regulated serine/threonine kinase, directly interacts with and phosphorylates mTORC1, and has a part in suppressing mTOR activity to promote autophagy induction. DAPK2 knockdown reduced autophagy triggered either by amino acid deprivation or by increases in intracellular Ca²⁺ levels. At the molecular level, DAPK2 depletion interfered with mTORC1 inhibition caused by these two stresses, as reflected by the phosphorylation status of mTORC1 substrates, ULK1 (unc-51-like kinase 1), p70 ribosomal S6 kinase and eukaryotic initiation factor 4E-binding protein 1. An increase in mTORC1 kinase activity was also apparent in unstressed cells that were depleted of DAPK2. Immunoprecipitated mTORC1 from DAPK2-depleted cells showed increased kinase activity in vitro, an indication that DAPK2 regulation of mTORC1 is inherent to the complex itself. Indeed, we found that DAPK2 associates with components of mTORC1, as demonstrated by co-immunoprecipitation with mTOR and its complex partners, raptor (regulatory-associated protein of mTOR) and ULK1. DAPK2 was also able to interact directly with raptor, as shown by recombinant protein-binding assay. Finally, DAPK2 was shown to phosphorylate raptor in vitro. This phosphorylation was mapped to Ser721, a site located within a highly phosphorylated region of raptor that has previously been shown to regulate mTORC1 activity. Thus, DAPK2 is a novel kinase of mTORC1 and is a potential new member of this multiprotein complex, modulating mTORC1 activity and autophagy levels under stress and steady-state conditions.Macroautophagy (hereafter referred to as autophagy) is a highly regulated intracellular bulk degradation process found ubiquitously in eukaryotes. During autophagy a double-membrane vesicle, termed an autophagosome, engulfs cytoplasmic materials, including whole organelles. The autophagosome is later fused with the lysosome and its content degraded by hydrolases.¹ Basal levels of autophagy are maintained within the cell during steady state, and are involved in cell homeostasis activities such as turnover of long-lived proteins, preventing accumulation of protein aggregates, and removal of damaged cellular structures.² Beyond this homeostatic function, autophagy is stimulated during various stress conditions, such as nutrient deprivation, intracellular Ca²⁺ increase, hypoxia, ER stress and oxidative stress, to ensure continuous cell survival under stress.³A critical step in the induction of autophagy comprises the inactivation of a key negative regulator of the process, the mammalian target of rapamycin (mTOR).⁴ mTOR is a conserved serine/threonine protein kinase that acts as a master regulator in the cell. mTOR forms a rapamycin-sensitive complex named mTORC1 with its binding partner raptor (regulatory-associated protein of mTOR), which mediates mTOR''s substrate presentation.⁵ mTORC1 senses nutrient availability, growth factors and energy levels, and, in response, regulates cell growth, metabolism and protein synthesis, mainly by phosphorylation of substrates involved in protein translation: the p70 ribosomal S6 kinase (p70S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Under nutrient-rich conditions, mTORC1 suppresses autophagy to basal levels by phosphorylating and inhibiting the autophagy proteins ULK1 (unc-51-like kinase 1) and Atg13. Upon autophagic stimulus, mTORC1 activity is inhibited and the ULK1 complex is activated, leading to autophagy induction.⁶ The activity levels of mTORC1 are regulated by several mechanisms, such as interacting proteins, cellular localization and phosphorylation events. Raptor phosphorylation has been suggested as a mechanism by which upstream kinases such as AMPK,⁷ RSK⁸ and ULK1⁹ can regulate mTORC1 activity.Death-associated protein kinase 2 (DAPK2; also named DRP-1) is a 42-kDa Ca²⁺/calmodulin (CaM)-regulated serine/threonine kinase,¹⁰ and a closely related homolog of DAPK, a gene originally discovered in an attempt to find positive regulators of cell death.¹¹ DAPK2 was identified based on homology to the catalytic domain of DAPK. DAPK2 is a soluble cytoplasmatic protein, which triggers massive membrane blebbing and appearance of double-membrane autophagic vesicles upon its overexpression (for a review see Shiloh et al.¹²). DAPK2''s substrates and interacting proteins are mostly unknown, with the exception of the myosin II regulatory light chain, which has been shown to be an in vitro and in vivo substrate.¹³ Although many publications have studied DAPK, its substrates and its role in cell death and autophagy,^{14, 15} very little is known about DAPK2 substrates, cellular functions or the molecular pathways that it regulates.In this work, we studied the involvement of DAPK2 in the autophagic module. We identified DAPK2 as a novel interacting protein of mTORC1, and as a negative regulator of the complex both during steady-state growth conditions and in response to different stress autophagic signals. We identified mTOR''s binding partner, raptor, as a substrate of DAPK2, and found Ser721 as its phosphorylation site.     

The cytokine midkine and its receptor RPTPζ regulate B cell survival in a pathway induced by CD74

Lasting B cell persistence depends on survival signals that are transduced by cell surface receptors. In this study, we describe a novel biological mechanism essential for survival and homeostasis of normal peripheral mature B cells and chronic lymphocytic leukemia cells, regulated by the heparin-binding cytokine, midkine (MK), and its proteoglycan receptor, the receptor-type tyrosine phosphatase ζ (RPTPζ). We demonstrate that MK initiates a signaling cascade leading to B cell survival by binding to RPTPζ. In mice lacking PTPRZ, the proportion and number of the mature B cell population are reduced. Our results emphasize a unique and critical function for MK signaling in the previously described MIF/CD74-induced survival pathway. Stimulation of CD74 with MIF leads to c-Met activation, resulting in elevation of MK expression in both normal mouse splenic B and chronic lymphocytic leukemia cells. Our results indicate that MK and RPTPζ are important regulators of the B cell repertoire. These findings could pave the way toward understanding the mechanisms shaping B cell survival and suggest novel therapeutic strategies based on the blockade of the MK/RPTPζ-dependent survival pathway.     

Ivermectin is a specific inhibitor of importin α/β-mediated nuclear import able to inhibit replication of HIV-1 and dengue virus

The movement of proteins between the cytoplasm and nucleus mediated by the importin superfamily of proteins is essential to many cellular processes, including differentiation and development, and is critical to disease states such as viral disease and oncogenesis. We recently developed a high-throughput screen to identify specific and general inhibitors of protein nuclear import, from which ivermectin was identified as a potential inhibitor of importin α/β-mediated transport. In the present study, we characterized in detail the nuclear transport inhibitory properties of ivermectin, demonstrating that it is a broad-spectrum inhibitor of importin α/β nuclear import, with no effect on a range of other nuclear import pathways, including that mediated by importin β1 alone. Importantly, we establish for the first time that ivermectin has potent antiviral activity towards both HIV-1 and dengue virus, both of which are strongly reliant on importin α/β nuclear import, with respect to the HIV-1 integrase and NS5 (non-structural protein 5) polymerase proteins respectively. Ivermectin would appear to be an invaluable tool for the study of protein nuclear import, as well as the basis for future development of antiviral agents.     

Ligand-independent regulation of ErbB4 receptor phosphorylation by activated Ras

The ErbB family of receptor tyrosine kinases regulates cell growth, differentiation and survival. Activation of the receptors is induced by specific growth factors in an autocrine, paracrine or juxtacrine manner. The activated ErbB receptors turn on a large variety of signaling cascades, including the prominent Ras-dependent signaling pathways. The activated Ras can induce secretion of growth factors such as EGF and neuregulin, which activate their respective receptors. In the present study, we demonstrate for the first time that activated Ras can activate ErbB4 receptor in a ligand-independent manner. Expression of constitutively active H-Ras(12V), K-Ras(12V) or N-Ras(13V) in PC12-ErbB4 cells induced ErbB4-receptor phosphorylation, indicating that each of the most abundant Ras isoforms can induce receptor activation. NRG-induced phosphorylation of ErbB4 receptor was blocked by the soluble ErbB4 receptor, which had no effect on the Ras-induced receptor phosphorylation. Moreover, conditioned medium from H-Ras(12V)-transfected PC12-ErbB4 cells had no effect on receptor phosphorylation. It thus indicates that Ras induces ErbB4 phosphorylation in a ligand-independent manner. Each of the Ras effector domain mutants, H-Ras(12V)S35, H-Ras(12V)C40, and H-Ras(12V)G37, which respectively activate Raf1, PI3K, and RalGEF, induced a small but significant receptor phosphorylation. The PI3K inhibitor LY294002 and the MEK inhibitor PD98059 caused a partial inhibition of the Ras-induced ErbB4 receptor phosphorylation. Using a mutant ErbB4 receptor, which lacks kinase activity, we demonstrated that the Ras-mediated ErbB4 phosphorylation depends on the kinase activity of the receptor and facilitates ligand-independent neurite outgrowth in PC12-ErbB4 cells. These experiments demonstrate a novel mechanism controlling ErbB receptor activation. Ras induces ErbB4 receptor phosphorylation in a non-autocrine manner and this activation depends on multiple Ras effector pathways and on ErbB4 kinase activity.