Reichardt's dye, a highly solvatochromic dye, was encapsulated within poly (glycerol succinic acid) ([Gn]-PGLSA-OH) dendrimers to investigate the interior environment of these dendritic macromolecules. The absorption maximum for the encapsulated Reichardt's dye in water was indicative of a relatively high dielectric constant present within the dye/dendrimer complex. (1)H NMR of the encapsulated complex showed the presence of aromatic protons from Reichardt's dye along with the aliphatic protons of the dendrimer. Additionally, there were substantial changes in T(1) and T(2) times of the encapsulated dye when compared with the free dye, and (1)H NOESY spectra for the complex showed a significant number of intermolecular NOE cross-peaks. These data reveal the close through-space proximity of the dye to the dendrimer and the restricted motion of the encapsulated dye. To demonstrate the potential use of these macromolecules as drug delivery vehicles, the poorly water-soluble anticancer drug 10-hydroxycamptothecin (10HCPT) was encapsulated within a carboxylated PGLSA dendrimer ([G4]-PGLSA-COONa). Cytotoxicity assays with human breast cancer cells showed a significant reduction of cell viability, demonstrating that 10HCPT retains activity upon encapsulation.
Light causes massive translocation of G-protein transducin from the light-sensitive outer segment compartment of the rod photoreceptor cell. Remarkably, significant translocation is observed only when the light intensity exceeds a critical threshold level. We addressed the nature of this threshold using a series of mutant mice and found that the threshold can be shifted to either a lower or higher light intensity, dependent on whether the ability of the GTPase-activating complex to inactivate GTP-bound transducin is decreased or increased. We also demonstrated that the threshold is not dependent on cellular signaling downstream from transducin. Finally, we showed that the extent of transducin ␣ subunit translocation is affected by the hydrophobicity of its acyl modification. This implies that interactions with membranes impose a limitation on transducin translocation. Our data suggest that transducin translocation is triggered when the cell exhausts its capacity to activate transducin GTPase, and a portion of transducin remains active for a sufficient time to dissociate from membranes and to escape from the outer segment. Overall, the threshold marks the switch of the rod from the highly light-sensitive mode of operation required under limited lighting conditions to the less-sensitive energy-saving mode beneficial in bright light, when vision is dominated by cones.
Transducin is a prototypic heterotrimeric G-protein mediating visual signaling in vertebrate photoreceptor cells. Despite its central role in phototransduction, little is known about the mechanisms that regulate its expression and maintain approximately stoichiometric levels of the ␣-and ␥-subunits. Here we demonstrate that the knock-out of transducin ␥-subunit leads to a major downregulation of both ␣-and -subunit proteins, despite nearly normal levels of the corresponding transcripts, and fairly rapid photoreceptor degeneration. Significant fractions of the remaining ␣-and -subunits were mislocalized from the light-sensitive outer segment compartment of the rod. Yet, the tiny amount of the ␣-subunit present in the outer segments of knock-out rods was sufficient to support light signaling, although with a markedly reduced sensitivity. These data indicate that the ␥-subunit controls the expression level of the entire transducin heterotrimer and that heterotrimer formation is essential for normal transducin localization. They further suggest that the production of transducin -subunit without its constitutive ␥-subunit partner sufficiently stresses the cellular biosynthetic and/or chaperone machinery to induce cell death.
Inherited retinal degenerations, caused by mutations in over 100 individual genes, affect approximately 2 million people worldwide. Many of the underlying mutations cause protein misfolding or mistargeting in affected photoreceptors. This places an increased burden on the protein folding and degradation machinery, which may trigger cell death. We analyzed how these cellular functions are affected in degenerating rods of the transducin γ-subunit (Gγ 1 ) knockout mouse. These rods produce large amounts of transducin β-subunit (Gβ 1 ), which cannot fold without Gγ 1 and undergoes intracellular proteolysis instead of forming a transducin βγ-subunit complex. Our data revealed that the most critical pathobiological factor leading to photoreceptor cell death in these animals is insufficient capacity of proteasomes to process abnormally large amounts of misfolded protein. A decrease in the Gβ 1 production in Gγ 1 knockout rods resulted in a significant reduction in proteasomal overload and caused a striking reversal of photoreceptor degeneration. We further demonstrated that a similar proteasomal overload takes place in photoreceptors of other mutant mice where retinal degeneration has been ascribed to protein mistargeting or misfolding, but not in mice whose photoreceptor degenerate as a result of abnormal phototransduction. These results establish the prominence of proteasomal insufficiency across multiple degenerative diseases of the retina, thereby positioning proteasomes as a promising therapeutic target for treating these debilitating conditions. neurodegenerative diseases | protein degradation
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