3-D structure and dynamics of protein kinase B—new mechanism for the allosteric regulation of an AGC kinase

New developments regarding the structure and in vivo dynamics of protein kinase B (PKB/Akt) have been recently exposed. Here, we specifically review how the use of multi-disciplinary approaches has resulted in reaching the recent progress made to relate the quaternary structure of PKB to its in vivo function. Using X-ray crystallography, the structure of PKB pleckstrin homology (PH) and kinase domains was determined separately. The molecular mechanisms involved in (a) the binding of the phosphoinositides to the PH domain and (b) the activation of the kinase with the rearrangement of the catalytic site and substrate binding were determined. In vitro, nuclear magnetic resonance and circular dychroism studies gave complementary information on the interaction of the PH domain with the phosphoinositides. However, the molecular nature and the function of the interactions between the PKB domains could not be deduced from the X-ray data since the full-length PKB has not been crystallised. In vitro, dynamic information on the inter-domain conformational changes related to PKB activation states emerged with the use of tandem mass spectrometry. Cell imaging and Förster resonance energy transfer provided in vivo dynamics. Molecular modelling and dynamic simulations in conjunction with mutagenesis and biochemical analysis were used to investigate the complex interactions between the PKB domains in vivo and understand at the molecular level how it linked to its activity. The compilation of the information obtained on the 3-D structure and the spatiotemporal dynamics of this widely studied oncogene could be applied to the study of other proteins. This inter-disciplinary approach led to a more profound understanding of PKB complex activation mechanism in vivo that will shed light onto new ideas and possibilities for modulating its activity.     

Strategies and methods for studying the rhizosphere—the plant science toolbox

This review summarizes and discusses methodological approaches for studies on the impact of plant roots on the surrounding rhizosphere and for elucidation of the related mechanisms, covering a range from simple model experiments up to the field scale. A section on rhizosphere sampling describes tools and culture systems employed for analysis of root growth, root morphology, vitality testing and for monitoring of root activity with respect to nutrient uptake, water, ion and carbon flows in the rhizosphere. The second section on rhizosphere probing covers techniques to detect physicochemical changes in the rhizosphere as a consequence of root activity. This comprises compartment systems to obtain rhizosphere samples, visualisation techniques, reporter gene approaches and remote sensing technologies for monitoring the conditions in the rhizosphere. Approaches for the experimental manipulation of the rhizosphere by use of molecular and genetic methods as tools to study rhizosphere processes are discussed in a third section. Finally it is concluded that in spite of a wide array of methodological approaches developed in the recent past for studying processes and interactions in the rhizosphere mainly under simplified conditions in model experiments, there is still an obvious lack of methods to test the relevance of these findings under real field conditions or even on the scale of ecosystems. This also limits reliable data input and validation in current rhizosphere modelling approaches. Possible interactions between different environmental factors or plant-microbial interactions (e.g. mycorrhizae) are frequently not considered in model experiments. Moreover, most of the available knowledge arises from investigations with a very limited number of plant species, mainly crops and studies considering also intraspecific genotypic differences or the variability within wild plant species are just emerging.     

Mechanism of NF-κB signaling pathway and autophagy in the regulation of osteoblast differentiation

Objective: The objective of the present work was to investigate a possible mechanism of NF-κB signaling pathway and autophagy in the regulation of osteoblast differentiation, and provide experimental basis for the study of tooth eruption disorder.

Methods: Mouse osteoblast-like (MC3T3-E1) cells were inoculated with a cell density of 70%. According to the grouping experimental design, Western blot and monodansylcadaverine (MDC) detection were conducted after dosing for 24?h. The cells were divided into the following five groups: blank control group; 6.25?µg/mL SN50 group; 12.5?µg/mL SN50 group; 25?µg/mL SN50 group and 50?µg/mL SN50 group.

Results: Western blot analysis revealed that the expression of LC3 protein was present in the blank control group; 6.25?µg/mL SN50 group; 12.5?µg/mL SN50 group and 50?µg/mL SN50 group, with no significant differences among these groups. However, the expression of LC3 protein was significantly lower in the 25?µg/mL SN50 group. MDC detection showed that, in the blank control group; 6.25?µg/mL SN50 group; 12.5?µg/mL SN50 group and 50?µg/mL SN50 group, there was obvious green fluorescence in the cytoplasm of the osteoblasts. However, in the 25?µg/mL SN50 group, it was found that there were significantly fewer green fluorescent particles.

Conclusion: The osteoblast itself had a strong function of autophagy. The appropriate concentration of SN50 in blocking the NF-κB pathway of the osteoblast was associated with the obvious inhibition of autophagy. However, the relationship between NF-κB signaling pathway and autophagy in the process of tooth eruption requires further study.     

Regulatory interplay of the Sub1A and CIPK15 pathways in the regulation of α-amylase production in flooded rice plants

Rice (Oryza sativa L.) can successfully germinate and grow even when flooded. Rice varieties possessing the submergence 1A (Sub1A) gene display a distinct flooding-tolerant phenotype, associated with lower carbohydrate consumption and restriction of the fast-elongation phenotype typical of flooding-intolerant rice varieties. Calcineurin B-like interacting protein kinase 15 (CIPK15) was recently indicated as a key regulator of α-amylases under oxygen deprivation, linked to both rice germination and flooding tolerance in adult plants. It is still unknown whether the Sub1A- and CIPK15-mediated pathways act as complementary processes for rice survival under O(2) deprivation. In adult plants Sub1A and CIPK15 may perhaps play an antagonistic role in terms of carbohydrate consumption, with Sub1A acting as a starch degradation repressor and CIPK15 as an activator. In this study, we analysed sugar metabolism in the stem of rice plants under water submergence by selecting cultivars with different traits associated with flooding survival. The relation between the Sub1A and the CIPK15 pathways was investigated. The results show that under O(2) deprivation, the CIPK15 pathway is repressed in the tolerant, Sub1A-containing, FR13A variety. CIPK15 is likely to play a role in the up-regulation of Ramy3D in flooding-intolerant rice varieties that display fast elongation under flooding and that do not possess Sub1A.     

Sclerotization of insect cuticle: A new method for studying the ratio of quinone and β-sclerotization

Sclerotized cuticle upon hydrolysis yields conventional amino acids, ketocatechols and/or aryl amino acid adduct(s) depending on the mode of sclerotization. In the absence of structural details of the bridged hydrolysis products, the two modes of sclerotization have been assessed by differential labelling and chromatography. Insect cuticle labelled with either DOPA or tyrosine was hydrolyzed and chromatographed on dihydroxyboryl cellulose and Dowex 50 columns. Dihydroxyboryl cellulose specifically and quantitatively retained all catechols, while Dowex 50 separated basic aryl amino acid adduct(s) from amino acids. Hydrolysates of cuticles labelled with DOPA were resolved into non-catecholic and catecholic fractions; the ratio of radioactivity present in these two fractions reflected the ratio of quinone tanning and β-sclerotization. However, cuticle labelled with tyrosine required additional chromatography of the non-catecholic fraction on Dowex 50 to determine this ratio.     

A virus-specified mechanism for the prevention of multiple infection—T7- and T3-mutual and superinfection exclusion

Summary Co-and superinfection of cells with T3/T7 result in exclusion (mutual or superinfection exclusion). The exclusion mechanism is also directed against homologous (or identical) virus. Exclusion is established after the adsorption but before the genome becomes available for gene expression or replication, that is only one virus per cell develops. The exclusion is triggered by a constituant of the viral particle. An early T7 gene (M gene) (Schweiger et al., 1975) is essential for the formation of exclusion competent virions.     

A role for PPARα in the regulation of arginine metabolism and nitric oxide synthesis

The pleiotropic effects of PPARα may include the regulation of amino acid metabolism. Nitric oxide (NO) is a key player in vascular homeostasis. NO synthesis may be jeopardized by a differential channeling of arginine toward urea (via arginase) versus NO (via NO synthase, NOS). This was studied in wild-type (WT) and PPARα-null (KO) mice fed diets containing either saturated fatty acids (COCO diet) or 18:3 n-3 (LIN diet). Metabolic markers of arginine metabolism were assayed in urine and plasma. mRNA levels of arginases and NOS were determined in liver. Whole-body NO synthesis and the conversion of systemic arginine into urea were assessed by using ¹⁵N₂-guanido-arginine and measuring urinary ¹⁵NO₃ and [¹⁵N]-urea. PPARα deficiency resulted in a markedly lower whole-body NO synthesis, whereas the conversion of systemic arginine into urea remained unaffected. PPARα deficiency also increased plasma arginine and decreased citrulline concentration in plasma. These changes could not be ascribed to a direct effect on hepatic target genes, since NOS mRNA levels were unaffected, and arginase mRNA levels decreased in KO mice. Despite the low level in the diet, the nature of the fatty acids modulated some effects of PPARα deficiency, including plasma arginine and urea, which increased more in KO mice fed the LIN diet than in those fed the COCO diet. In conclusion, PPARα is largely involved in normal whole-body NO synthesis. This warrants further study on the potential of PPARα activation to maintain NO synthesis in the initiation of the metabolic syndrome.     

Autophagy: An overlooked mechanism of HIV-1 pathogenesis and NeuroAIDS?

Human immunodeficiency virus type 1 (HIV-1) establishes a persistent infection characterized by progressive depletion of CD4⁺ lymphocytes and immunosuppression. Although extensive research has examined the importance of apoptosis as a cause of cell death associated with HIV-1 infection, the role of autophagy has been largely ignored. Our laboratory has examined the autophagic process in HIV-1-infected cells. Following infection of human peripheral blood CD4+ T-cells or U937 cells with HIV-1 for 48 hours, the autophagy proteins Beclin 1 and LC3-II were found to be markedly decreased. Beclin 1 mRNA expression and autophagosomes were also reduced in HIV-1 infected cells. Thus, our data indicate that HIV-1 infection inhibits autophagy in infected cells in contrast to the previously described induction of autophagy by gp120 in uninfected bystander cells. It is likely that HIV-1 has evolved this mechanism as part of an elaborate attempt to evade the immune system while promoting its own replication. We believe that autophagy is an overlooked mechanism in HIV-1 pathogenesis and plays a particularly important role in the early cognitive impairment and dementia often associated with advanced AIDS. A model is presented that describes the potential role of autophagy in NeuroAIDS.

Addendum to: Zhou D, Spector SA. Human immunodeficiency virus type-1 infection inhibits autophagy. Aids 2008;22:695-9.     

Autophagy: an overlooked mechanism of HIV-1 pathogenesis and neuroAIDS?

Human immunodeficiency virus type 1 (HIV-1) establishes a persistent infection characterized by progressive depletion of CD4(+) lymphocytes and immunosuppression. Although extensive research has examined the importance of apoptosis as a cause of cell death associated with HIV-1 infection, the role of autophagy has been largely ignored. Our laboratory has examined the autophagic process in HIV-1-infected cells. Following infection of human peripheral blood CD4(+) T-cells or U937 cells with HIV-1 for 48 hours, the autophagy proteins Beclin 1 and LC3-II were found to be markedly decreased. Beclin 1 mRNA expression and autophagosomes were also reduced in HIV-1 infected cells. Thus, our data indicate that HIV-1 infection inhibits autophagy in infected cells in contrast to the previously described induction of autophagy by gp120 in uninfected bystander cells. It is likely that HIV-1 has evolved this mechanism as part of an elaborate attempt to evade the immune system while promoting its own replication. We believe that autophagy is an overlooked mechanism in HIV-1 pathogenesis and plays a particularly important role in the early cognitive impairment and dementia often associated with advanced AIDS. A model is presented that describes the potential role of autophagy in NeuroAIDS.     

Autophagy and Vacuole Homeostasis: A Case for Self-Degradation?

The vacuole of yeast plays an important role in pH- and ion-homeostasis. Another important function of the vacuole, especially during nutrient deprivation, is the degradation of proteins, other macromolecules and organelles. To deliver these components into the vacuolar lumen, specific and sophisticated transport pathways such as autophagy have evolved. This review will first look at autophagy and its relationship to vacuole homeostasis, then move to the topic of vacuole self-degradation and possible reasons for its existence, and close by pointing very briefly to some areas for further research in these topics.     

The NAD World: A New Systemic Regulatory Network for Metabolism and Aging—Sirt1, Systemic NAD Biosynthesis,and Their Importance

For the past several years, it has been demonstrated that the NAD-dependent protein deacetylase Sirt1 and nicotinamide phosphoribosyltransferase (Nampt)-mediated systemic NAD biosynthesis together play a critical role in the regulation of metabolism and possibly aging in mammals. Based on our recent studies on these two critical components, we have developed a hypothesis of a novel systemic regulatory network, named “NAD World”, for mammalian aging. Conceptually, in the NAD World, systemic NAD biosynthesis mediated by intra- and extracellular Nampt functions as a driver that keeps up the pace of metabolism in multiple tissues/organs, and the NAD-dependent deacetylase Sirt1 serves as a universal mediator that executes metabolic effects in a tissue-dependent manner in response to changes in systemic NAD biosynthesis. This new concept of the NAD World provides important insights into a systemic regulatory mechanism that fundamentally connects metabolism and aging and also conveys the ideas of functional hierarchy and frailty for the regulation of metabolic robustness and aging in mammals.     

Time-resolved FRET reveals the structural mechanism of SERCA–PLB regulation

We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to characterize the interaction between phospholamban (PLB) and the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA) under conditions that relieve SERCA inhibition. Unphosphorylated PLB inhibits SERCA in cardiac SR, but inhibition is relieved by either micromolar Ca²⁺ or PLB phosphorylation. In both cases, it has been proposed that inhibition is relieved by dissociation of the complex. To test this hypothesis, we attached fluorophores to the cytoplasmic domains of SERCA and PLB, and reconstituted them functionally in lipid bilayers. TR-FRET, which permitted simultaneous measurement of SERCA–PLB binding and structure, was measured as a function of PLB phosphorylation and [Ca²⁺]. In all cases, two structural states of the SERCA–PLB complex were resolved, probably corresponding to the previously described T and R structural states of the PLB cytoplasmic domain. Phosphorylation of PLB at S16 completely relieved inhibition, partially dissociated the SERCA–PLB complex, and shifted the T/R equilibrium within the bound complex toward the R state. Since the PLB concentration in cardiac SR is at least 10 times that in our FRET measurements, we calculate that most of SERCA contains bound phosphorylated PLB in cardiac SR, even after complete phosphorylation. 4 μM Ca²⁺ completely relieved inhibition but did not induce a detectable change in SERCA–PLB binding or cytoplasmic domain structure, suggesting a mechanism involving structural changes in SERCA’s transmembrane domain. We conclude that Ca²⁺ and PLB phosphorylation relieve SERCA–PLB inhibition by distinct mechanisms, but both are achieved primarily by structural changes within the SERCA–PLB complex, not by dissociation of that complex.