Engineering visual arrestin-1 with special functional characteristics.

Arrestin-1 preferentially binds active phosphorylated rhodopsin. Previously, a mutant with enhanced binding to unphosphorylated active rhodopsin (Rh*) was shown to partially compensate for lack of rhodopsin phosphorylation in vivo. Here we showed that reengineering of the receptor binding surface of arrestin-1 further improves the binding to Rh* while preserving protein stability. In mammals, arrestin-1 readily self-associates at physiological concentrations. The biological role of this phenomenon can only be elucidated by replacing wild type arrestin-1 in living animals with a non-oligomerizing mutant retaining all other functions. We demonstrate that constitutively monomeric forms of arrestin-1 are sufficiently stable for in vivo expression. We also tested the idea that individual functions of arrestin-1 can be independently manipulated to generate mutants with the desired combinations of functional characteristics. Here we showed that this approach is feasible; stable forms of arrestin-1 with high Rh* binding can be generated with or without the ability to self-associate. These novel molecular tools open the possibility of testing of the biological role of arrestin-1 self-association and pave the way to elucidation of full potential of compensational approach to gene therapy of gain-of-function receptor mutations.

Silent scaffolds: inhibition OF c-Jun N-terminal kinase 3 activity in cell by dominant-negative arrestin-3 mutant.

We established a new in vivo arrestin-3-JNK3 interaction assay based on bioluminescence resonance energy transfer (BRET) between JNK3-luciferase and Venus-arrestins. We tested the ability of WT arrestin-3 and its 3A mutant that readily binds ß2-adrenergic receptors as well as two mutants impaired in receptor binding, Δ7 and KNC, to directly bind JNK3 and to promote JNK3 phosphorylation in cells. Both receptor binding-deficient mutants interact with JNK3 significantly better than WT and 3A arrestin-3. WT arrestin-3 and Δ7 mutant robustly promoted JNK3 activation, whereas 3A and KNC mutants did not. Thus, receptor binding, JNK3 interaction, and JNK3 activation are three distinct arrestin functions. We found that the KNC mutant, which tightly binds ASK1, MKK4, and JNK3 without facilitating JNK3 phosphorylation, has a dominant-negative effect, competitively decreasing JNK activation by WT arrestin-3. Thus, KNC is a silent scaffold, a novel type of molecular tool for the suppression of MAPK signaling in living cells.

Rapid degeneration of rod photoreceptors expressing self-association-deficient arrestin-1 mutant.

Arrestin-1 binds light-activated phosphorhodopsin and ensures timely signal shutoff. We show that high transgenic expression of an arrestin-1 mutant with enhanced rhodopsin binding and impaired oligomerization causes apoptotic rod death in mice. Dark rearing does not prevent mutant-induced cell death, ruling out the role of arrestin complexes with light-activated rhodopsin. Similar expression of WT arrestin-1 that robustly oligomerizes, which leads to only modest increase in the monomer concentration, does not affect rod survival. Moreover, WT arrestin-1 co-expressed with the mutant delays retinal degeneration. Thus, arrestin-1 mutant directly affects cell survival via binding partner(s) other than light-activated rhodopsin. Due to impaired self-association of the mutant its high expression dramatically increases the concentration of the monomer. The data suggest that monomeric arrestin-1 is cytotoxic and WT arrestin-1 protects rods by forming mixed oligomers with the mutant and/or competing with it for the binding to non-receptor partners. Thus, arrestin-1 self-association likely serves to keep low concentration of the toxic monomer. The reduction of the concentration of harmful monomer is an earlier unappreciated biological function of protein oligomerization.

Monoterpene hydrocarbons may serve as antipredation defensive compounds in Boisea trivittata, the boxelder bug.

Boxelder bugs, Boisea trivittata, are deterred from predation by green anoles (Anolis carolinensis). Hydrodistillation and GC-MS analysis reveals B. trivittata to contain the volatile monoterpene hydrocarbons beta-pinene (83.9%), limonene (14.7%), myrcene (0.8%), and (E)-beta-ocimene (0.6%). The presence of these antifeedant volatile chemicals may serve to provide some protection of boxelder bugs from predation.

Volatile constituents and antibacterial screening of the essential oil of Chenopodium ambrosioides L. growing in Nigeria.

The essential oil of the aerial parts of Chenopodium ambrosioides L. has been isolated by hydrodistillation and analyzed using GC-MS. The major components were found to be alpha-terpinene (63.1%), p-cymene (26.4%) and ascaridole (3.9%). The oil displayed no antibacterial activity against either Gram-positive bacteria Bacillus cereus or Staphylococcus aureus, or the Gram-negative bacterium Escherichia coli (MIC=1250 microg/mL). A cluster analysis of C. ambrosioides essential oils reveals at least seven distinct chemotypes: ascaridole, alpha-terpinene, alpha-pinene, p-cymene, carvacrol, alpha-terpinyl acetate, and limonene.

Seasonal variation and bioactivity in the leaf oil of Liriodendron tulipifera growing in Huntsville, Alabama.

The seasonal variation in the chemical composition of the leaf essential oil of Liriodendron tulipifera has been analyzed by GC-MS. Two individual trees were sampled five times during the course of the growing season. Twenty components were identified in the leaf oils, which were dominated by sesquiterpene hydrocarbons, principally germacrene D and beta-elemene, in the early part of the season (42-44% and 18-23%, respectively,) but monoterpene hydrocarbons, largely (Z)-beta-ocimene, dominated the later season leaf oils (40-60%). The leaf oils exhibited in-vitro antibacterial activity against Bacillus cereus and Staphylococcus aureus as well as cytotoxic activity on MDA-MB-231 and Hs 578T human breast tumor cells.