Distinct secondary structures of the leucine-rich repeat proteoglycans decorin and biglycan. Glycosylation-dependent conformational stability

Biglycan and decorin have been overexpressed in eukaryotic cells and two major glycoforms isolated under native conditions: a proteoglycan substituted with glycosaminoglycan chains; and a core protein form secreted devoid of glycosaminoglycans (Hocking, A. M., Strugnell, R. A., Ramamurthy, P., and McQuillan, D. J. (1996) J. Biol. Chem. 271, 19571-19577; Ramamurthy, P., Hocking, A. M., and McQuillan, D. J. (1996) J. Biol. Chem. 271, 19578-19584). Far-UV CD spectroscopy of decorin and biglycan proteoglycans indicates that, although they are predominantly beta-sheet, biglycan has a significantly higher content of alpha-helical structure. Decorin proteoglycan and core protein are very similar, whereas the biglycan core protein exhibits closer similarity to the decorin glycoforms than to the biglycan proteoglycan form. However, enzymatic removal of the chondroitin sulfate chains from biglycan proteoglycan does not induce a shift to the core protein structure, suggesting that the final form is influenced by polysaccharide addition only during biosynthesis. Fluorescence emission spectroscopy demonstrated that the single tryptophan residue, which is at a conserved position at the C-terminal domain of both biglycan and decorin, is found in similar microenvironments. This indicates that in this specific domain the different glycoforms do exhibit apparent conservation of structure. Exposure of decorin and biglycan to 10 M urea resulted in an increase in fluorescent intensity, which indicates that the emission from tryptophan in the native state is quenched. Comparison of urea-induced protein unfolding curves provide further evidence that decorin and biglycan assume different structures in solution. Decorin proteoglycan and core protein unfold in a manner similar to a classic two-state model, in which there is a steep transition to an unfolded state between 1 and 2 M urea. The biglycan core protein also shows a similar steep transition. However, biglycan proteoglycan shows a broad unfolding transition between 1 and 6 M urea, probably indicating the presence of stable unfolding intermediates.     

Activity and stability of recombinant bifunctional rearranged and monofunctional domains of ATP-sulfurylase and adenosine 5'-phosphosulfate kinase

Murine adenosine 3'-phosphate 5'-phosphosulfate (PAPS) synthetase consists of a COOH-terminal ATP-sulfurylase domain covalently linked through a nonhomologous intervening sequence to an NH2-terminal adenosine 5'-phosphosulfate (APS) kinase domain forming a bifunctional fused protein. Possible advantages of bifunctionality were probed by separating the domains on the cDNA level and expressing them as monofunctional proteins. Expressed protein generated from the ATP-sulfurylase domain alone was fully active in both the forward and reverse sulfurylase assays. APS kinase-only recombinants exhibited no kinase activity. However, extension of the kinase domain at the COOH terminus by inclusion of the 36 residue linker region restored kinase activity. An equimolar mixture of the two monofunctional enzymes catalyzed the overall reaction (synthesis of PAPS from ATP + SO42-) comparably to the fused bifunctional enzyme. The importance of the domain order and organization was demonstrated by generation of a series of rearranged recombinants in which the order of the two active domains was reversed or altered relative to the linker region. The critical role of the linker region was established by generation of recombinants that had the linker deleted or rearranged relative to the two active domains. The intrinsic stability of the various recombinants was also investigated by measuring enzyme deactivation as a function of time of incubation at 25 or 37 degrees C. The expressed monofunctional ATP-sulfurylase, which was initially fully active, was unstable compared with the fused bifunctional wild type enzyme, decaying with a t1/2 of 10 min at 37 degrees C. Progressive extension by addition of kinase sequence at the NH2-terminal side of the sulfurylase recombinant eventually stabilized sulfurylase activity. Sulfurylase activity was significantly destabilized in a time-dependent manner in the rearranged proteins as well. In contrast, no significant deactivation of any truncated kinase-containing recombinants or misordered kinase recombinants was observed at either temperature. It would therefore appear that fusion of the two enzymes enhances the intrinsic stability of the sulfurylase only.     

Is the titer of adipokinetic peptides in Leptinotarsa decemlineata fed on genetically modified potatoes increased by oxidative stress?

The level of adipokinetic hormones (AKHs) (Peram-CAH-I and II) in the corpora cardiaca and the hemolymph of Leptinotarsa decemlineata enormously increases in the adults fed on genetically modified potatoes containing either GNA lectin or Cry 3Aa toxin concomitant with increased oxidative stress in gut tissues. A similar enhancement of the AKH titer is achieved when the adults are injected with paraquat that evokes oxidative stress. On the other hand, an injection of exogenous AKH reduces oxidative stress biomarkers in the hemolymph by reducing protein carbonyls and enhancing reduced glutathione levels. These facts indicate that there is a feedback regulation between an oxidative stressor action and the level of AKH in the insect body, and that AKHs might be involved in the activation of an antioxidant protection mechanism. These results are to our knowledge, the first evidence for the involvement of AKHs in oxidative stress mitigation, in addition to a plethora of other roles.     

Temperature dependence of protein-hydration hydrodynamics by molecular dynamics simulations

The dynamics of water molecules near the protein surface are different from those of bulk water and influence the structure and dynamics of the protein itself. To elucidate the temperature dependence hydration dynamics of water molecules, we present results from the molecular dynamic simulation of the water molecules surrounding two proteins (Carboxypeptidase inhibitor and Ovomucoid) at seven different temperatures (T=273 to 303 K, in increments of 5 K). Translational diffusion coefficients of the surface water and bulk water molecules were estimated from 2 ns molecular dynamics simulation trajectories. Temperature dependence of the estimated bulk water diffusion closely reflects the experimental values, while hydration water diffusion is retarded significantly due to the protein. Protein surface induced scaling of translational dynamics of the hydration waters is uniform over the temperature range studied, suggesting the importance protein-water interactions.     

Molecular adaptations underlying susceptibility and resistance to social defeat in brain reward regions

While stressful life events are an important cause of psychopathology, most individuals exposed to adversity maintain normal psychological functioning. The molecular mechanisms underlying such resilience are poorly understood. Here, we demonstrate that an inbred population of mice subjected to social defeat can be separated into susceptible and unsusceptible subpopulations that differ along several behavioral and physiological domains. By a combination of molecular and electrophysiological techniques, we identify signature adaptations within the mesolimbic dopamine circuit that are uniquely associated with vulnerability or insusceptibility. We show that molecular recapitulations of three prototypical adaptations associated with the unsusceptible phenotype are each sufficient to promote resistant behavior. Our results validate a multidisciplinary approach to examine the neurobiological mechanisms of variations in stress resistance, and illustrate the importance of plasticity within the brain's reward circuits in actively maintaining an emotional homeostasis.     

Sphingosine kinase 1 is a critical component of the copper-dependent FGF1 export pathway

Sphingosine kinase 1 catalyzes the formation of sphingosine-1-phosphate, a lipid mediator involved in the regulation of angiogenesis. Sphingosine kinase 1 is constitutively released from cells, even though it lacks a classical signal peptide sequence. Because copper-dependent non-classical stress-induced release of FGF1 also regulates angiogenesis, we questioned whether sphingosine kinase 1 is involved in the FGF1 release pathway. We report that (i) the coexpression of sphingosine kinase 1 with FGF1 inhibited the release of sphingosine kinase 1 at 37 degrees C; (ii) sphingosine kinase 1 was released at 42 degrees C in complex with FGF1; (iii) sphingosine kinase 1 null cells failed to release FGF1 at stress; (iv) sphingosine kinase 1 is a high affinity copper-binding protein which formed a complex with FGF1 in a cell-free system, and (v) sphingosine kinase 1 over expression rescued the release of FGF1 from inhibition by the copper chelator, tetrathiomolybdate. We propose that sphingosine kinase 1 is a component of the copper-dependent FGF1 release pathway.     

Regulation of axon guidance by slit and netrin signaling in the Drosophila ventral nerve cord

Netrin and Slit signaling systems play opposing roles during the positioning of longitudinal tracts along the midline in the ventral nerve cord of Drosophila embryo. It has been hypothesized that a gradient of Slit from the midline interacts with three different Robo receptors to specify the axon tract positioning. However, no such gradient has been detected. Moreover, overexpression of Slit at the midline has no effect on the positioning of these lateral tracts. In this article, we show that Slit is present outside of the midline along the longitudinal and commissural tracts. Sli from the midline, in a Robo-independent manner, is initially taken up by the commissural axon tracts when they cross the midline and is transported along the commissural tracts into the longitudinal connectives. These results are not consistent with a Sli gradient model. We also find that sli mRNA is maternally deposited and embryos that are genetically null for sli can have weaker guidance defects. Moreover, in robo or robo3 mutants, embryos with normal axon tracts are found and such robo embryos reach pupal stages and die, while robo3 mutant embryos develop into normal individuals and produce eggs. Interestingly, embryos from robo3 homozygous individuals fail to develop but have axon tracts ranging from normal to various defects: robo3 phenotype, robo phenotype, and slit-like phenotype, suggesting a more complex functional role for these genes than what has been proposed. Finally, our previous results indicated that netrin phenotype is epistatic to sli or robo phenotypes. However, it seems likely that this previously reported epistatic relationship might be due to the partial penetrance of the sli, robo, robo3 (or robo2) phenotypes. Our results argue that double mutant epistasis is most definitive only if the penetrance of the phenotypes of the mutants involved is complete.     

Troglitazone activates TRPV1 and causes deacetylation of PPARγ in 3T3-L1 cells

Published research suggests that activation of transient receptor potential vanilloid subfamily 1 (TRPV1) enhances the expression and deacetylation of peroxisome proliferator-activated receptor gamma (PPARγ) to cause browning of white adipose tissue. Here, we show that TRPV1 activation by capsaicin significantly prevents high fat diet-induced obesity in mice. This is associated with an increase in the expression and deacetylation of PPARγ in the epididymal fat of these mice. Consistent with the TRPV1 activation in vivo, overexpression of TRPV1 enhanced the PPARγ and other thermogenic genes in cultured 3T3-L1 preadipocytes. To determine the interaction between TRPV1 and PPARγ signaling, we analyzed the effect of Troglitazone (Trog; a thiazolidinedione derivative and an agonist of PAARγ) treatment on cultured 3T3-L1 cells. Trog enhanced the expression of TRPV1, PPARγ and thermogenic proteins in undifferentiated 3T3-L1 cells but not in differentiated cells. Acute application of Trog stimulated a robust Ca²⁺ influx into 3T3-L1 cells and TRPV1 inhibition by capsazepine prevented this. More interestingly, Trog or capsaicin treatment caused the deacetylation of PPARγ in 3T3-L1 cells and inhibition of TRPV1 or Sirtuin 1 - prevented this. Our data suggest a novel effect of Trog to induce PPARγ deacetylation by activating TRPV1. This research has a significant implication on the role of TRPV1 and PPARγ signaling in the browning of white adipose tissue.     

Classical autophagy proteins LC3B and ATG4B facilitate melanosome movement on cytoskeletal tracks

Macroautophagy/autophagy is a dynamic and inducible catabolic process that responds to a variety of hormonal and environmental cues. Recent studies highlight the interplay of this central pathway in a variety of pathophysiological diseases. Although defective autophagy is implicated in melanocyte proliferation and pigmentary disorders, the mechanistic relationship between the 2 pathways has not been elucidated. In this study, we show that autophagic proteins LC3B and ATG4B mediate melanosome trafficking on cytoskeletal tracks. While studying melanogenesis, we observed spatial segregation of LC3B-labeled melanosomes with preferential absence at the dendritic ends of melanocytes. This LC3B labeling of melanosomes did not impact the steady-state levels of these organelles but instead facilitated their intracellular positioning. Melanosomes primarily traverse on microtubule and actin cytoskeletal tracks and our studies reveal that LC3B enables the assembly of microtubule translocon complex. At the microtubule-actin crossover junction, ATG4B detaches LC3B from melanosomal membranes by enzymatic delipidation. Further, by live-imaging we show that melanosomes transferred to keratinocytes lack melanocyte-specific LC3B. Our study thus elucidates a new role for autophagy proteins in directing melanosome movement and reveal the unconventional use of these proteins in cellular trafficking pathways. Such crosstalk between the central cellular function and housekeeping pathway may be a crucial mechanism to balance melanocyte bioenergetics and homeostasis.     

Genetic analyses using GGE model and a mixed linear model approach,and stability analyses using AMMI bi-plot for late-maturity alpha-amylase activity in bread wheat genotypes

Low falling number and discounting grain when it is downgraded in class are the consequences of excessive late-maturity α-amylase activity (LMAA) in bread wheat (Triticum aestivum L.). Grain expressing high LMAA produces poorer quality bread products. To effectively breed for low LMAA, it is necessary to understand what genes control it and how they are expressed, particularly when genotypes are grown in different environments. In this study, an International Collection (IC) of 18 spring wheat genotypes and another set of 15 spring wheat cultivars adapted to South Dakota (SD), USA were assessed to characterize the genetic component of LMAA over 5 and 13 environments, respectively. The data were analysed using a GGE model with a mixed linear model approach and stability analysis was presented using an AMMI bi-plot on R software. All estimated variance components and their proportions to the total phenotypic variance were highly significant for both sets of genotypes, which were validated by the AMMI model analysis. Broad-sense heritability for LMAA was higher in SD adapted cultivars (53%) compared to that in IC (49%). Significant genetic effects and stability analyses showed some genotypes, e.g. ‘Lancer’, ‘Chester’ and ‘LoSprout’ from IC, and ‘Alsen’, ‘Traverse’ and ‘Forefront’ from SD cultivars could be used as parents to develop new cultivars expressing low levels of LMAA. Stability analysis using an AMMI bi-plot revealed that ‘Chester’, ‘Lancer’ and ‘Advance’ were the most stable across environments, while in contrast, ‘Kinsman’, ‘Lerma52’ and ‘Traverse’ exhibited the lowest stability for LMAA across environments.