Archaeal Unfoldase Counteracts Protein Misfolding Retinopathy in Mice.

Deregulation of cellular proteostasis due to the failure of the ubiquitin proteasome system to dispose of misfolded aggregation-prone proteins is a hallmark of various neurodegenerative diseases in humans. Microorganisms have evolved to survive massive protein misfolding and aggregation triggered by heat shock using their protein-unfolding ATPases (unfoldases) from the Hsp100 family. Because the Hsp100 chaperones are absent in homoeothermic mammals, we hypothesized that the vulnerability of mammalian neurons to misfolded proteins could be mitigated by expressing a xenogeneic unfoldase. To test this idea, we expressed proteasome-activating nucleotidase (PAN), a protein-unfolding ATPase from thermophilic Archaea, which is homologous to the 19S eukaryotic proteasome and similar to the Hsp100 family chaperones in rod photoreceptors of mice. We found that PAN had no obvious effect in healthy rods; however, it effectively counteracted protein-misfolding retinopathy in Gγ1 knock-out mice. We conclude that archaeal PAN can rescue a protein-misfolding neurodegenerative disease, likely by recognizing misfolded mammalian proteins.SIGNIFICANCE STATEMENT This study demonstrates successful therapeutic application of an archaeal molecular chaperone in an animal model of neurodegenerative disease. Introducing the archaeal protein-unfolding ATPase proteasome-activating nucleotidase (PAN) into the retinal photoreceptors of mice protected these neurons from the cytotoxic effect of misfolded proteins. We propose that xenogeneic protein-unfolding chaperones could be equally effective against other types of neurodegenerative diseases of protein-misfolding etiology.

G-protein betagamma-complex is crucial for efficient signal amplification in vision.

A fundamental question of cell signaling biology is how faint external signals produce robust physiological responses. One universal mechanism relies on signal amplification via intracellular cascades mediated by heterotrimeric G-proteins. This high amplification system allows retinal rod photoreceptors to detect single photons of light. Although much is now known about the role of the α-subunit of the rod-specific G-protein transducin in phototransduction, the physiological function of the auxiliary ßγ-complex in this process remains a mystery. Here, we show that elimination of the transducin γ-subunit drastically reduces signal amplification in intact mouse rods. The consequence is a striking decline in rod visual sensitivity and severe impairment of nocturnal vision. Our findings demonstrate that transducin ßγ-complex controls signal amplification of the rod phototransduction cascade and is critical for the ability of rod photoreceptors to function in low light conditions.

Regulation of rod photoreceptor function by farnesylated G-protein γ-subunits.

Heterotrimeric G-protein transducin, Gt, is a key signal transducer and amplifier in retinal rod and cone photoreceptor cells. Despite similar subunit composition, close amino acid identity, and identical posttranslational farnesylation of their Gγ subunits, rods and cones rely on unique Gγ1 (Gngt1) and Gγc (Gngt2) isoforms, respectively. The only other farnesylated G-protein γ-subunit, Gγ11 (Gng11), is expressed in multiple tissues but not retina. To determine whether Gγ1 regulates uniquely rod phototransduction, we generated transgenic rods expressing Gγ1, Gγc, or Gγ11 in Gγ1-deficient mice and analyzed their properties. Immunohistochemistry and Western blotting demonstrated the robust expression of each transgenic Gγ in rod cells and restoration of Gαt1 expression, which is greatly reduced in Gγ1-deficient rods. Electroretinography showed restoration of visual function in all three transgenic Gγ1-deficient lines. Recordings from individual transgenic rods showed that photosensitivity impaired in Gγ1-deficient rods was also fully restored. In all dark-adapted transgenic lines, Gαt1 was targeted to the outer segments, reversing its diffuse localization found in Gγ1-deficient rods. Bright illumination triggered Gαt1 translocation from the rod outer to inner segments in all three transgenic strains. However, Gαt1 translocation in Gγ11 transgenic mice occurred at significantly dimmer background light. Consistent with this, transretinal ERG recordings revealed gradual response recovery in moderate background illumination in Gγ11 transgenic mice but not in Gγ1 controls. Thus, while farnesylated Gγ subunits are functionally active and largely interchangeable in supporting rod phototransduction, replacement of retina-specific Gγ isoforms by the ubiquitous Gγ11 affects the ability of rods to adapt to background light.

EML1 is essential for retinal photoreceptor migration and survival.

Calcium regulates the response sensitivity, kinetics and adaptation in photoreceptors. In striped bass cones, this calcium feedback includes direct modulation of the transduction cyclic nucleotide-gated (CNG) channels by the calcium-binding protein CNG-modulin. However, the possible role of EML1, the mammalian homolog of CNG-modulin, in modulating phototransduction in mammalian photoreceptors has not been examined. Here, we used mice expressing mutant Eml1 to investigate its role in the development and function of mouse photoreceptors using immunostaining, in-vivo and ex-vivo retinal recordings, and single-cell suction recordings. We found that the mutation of Eml1 causes significant changes in the mouse retinal structure characterized by mislocalization of rods and cones in the inner retina. Consistent with the fraction of mislocalized photoreceptors, rod and cone-driven retina responses were reduced in the mutants. However, the Eml1 mutation had no effect on the dark-adapted responses of rods in the outer nuclear layer. Notably, we observed no changes in the cone sensitivity in the Eml1 mutant animals, either in darkness or during light adaptation, ruling out a role for EML1 in modulating cone CNG channels. Together, our results suggest that EML1 plays an important role in retina development but does not modulate phototransduction in mammalian rods and cones.

Electron paramagnetic resonance studies of functionally active, nitroxide spin-labeled peptide analogues of the C-terminus of a G-protein alpha subunit.

The C-terminal tail of the transducin alpha subunit, Gtalpha(340-350), is known to bind and stabilize the active conformation of rhodopsin upon photoactivation (R*). Five spin-labeled analogues of Gtalpha(340-350) demonstrated native-like activity in their ability to bind and stabilize R*. The spin-label 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) was employed at interior sites within the peptide, whereas a Proxyl (3-carboxyl-2,2,5,5-tetramethyl-pyrrolidinyloxy) spin-label was employed at the amino terminus of the peptide. Upon binding to R*, the electron paramagnetic resonance spectrum of TOAC(343)-Gtalpha(340-350) revealed greater immobilization of the nitroxide when compared to that of the N-terminally modified Proxyl-Gtalpha(340-350) analogue. A doubly labeled Proxyl/TOAC(348)-Gtalpha(340-350) was examined by DEER spectrocopy to determine the distribution of distances between the two nitroxides in the peptides when in solution and when bound to R*. TOAC and Proxyl spin-labels in this GPCR-G-protein alpha-peptide system provide unique biophysical probes that can be used to explore the structure and conformational changes at the rhodopsin-G-protein interface.

G-protein alpha and beta-gamma subunits interact with conformationally distinct signaling states of rhodopsin.

Light activated rhodopsin interacts with domains on all three subunits of transducin. Two of these domains, the C-terminal regions of the alpha and gamma subunits mimic the ability of transducin to stabilize the active conformation of rhodopsin, metarhodopsin II, but display different roles in transducin activation process. Whether the interactions are with the same or different complimentary sites on Meta II is unknown. We have used chemo-selective thioalkylation of rhodopsin and UV/visible spectroscopy to show that interactions with transducin C-terminal domains can be selectively disrupted. These data provide evidence that formal structural determinants on Meta II for these domains of transducin are different. In a set of complimentary experiments we examined the reactivity of Meta II species produced in the presence of the Gtalpha and Gtgamma subunit peptides to hydroxylamine. Analysis of the rates of Meta II decay confirms that the conformational states of Meta II when bound to Gtalpha and Gtbetagamma represent distinct signaling states of rhodopsin.

Rhodopsin controls a conformational switch on the transducin gamma subunit.

Rhodopsin, a prototypical G protein-coupled receptor, catalyzes the activation of a heterotrimeric G protein, transducin, to initiate a visual signaling cascade in photoreceptor cells. The betagamma subunit complex, especially the C-terminal domain of the transducin gamma subunit, Gtgamma(60-71)farnesyl, plays a pivotal role in allosteric regulation of nucleotide exchange on the transducin alpha subunit by light-activated rhodopsin. We report that this domain is unstructured in the presence of an inactive receptor but forms an amphipathic helix upon rhodopsin activation. A K65E/E66K charge reversal mutant of the gamma subunit has diminished interactions with the receptor and fails to adopt the helical conformation. The identification of this conformational switch provides a mechanism for active GPCR utilization of the betagamma complex in signal transfer to G proteins.

The arrestin-bound conformation and dynamics of the phosphorylated carboxy-terminal region of rhodopsin.

Visual arrestin binds to the phosphorylated carboxy-terminal region of rhodopsin to block interactions with transducin and terminate signaling in the rod photoreceptor cells. A synthetic seven-phospho-peptide from the C-terminal region of rhodopsin, Rh(330-348), has been shown to bind arrestin and mimic inhibition of signal transduction. In this study, we examine conformational changes in this synthetic peptide upon binding to arrestin by high-resolution proton nuclear magnetic resonance (NMR). We show that the peptide is completely disordered in solution, but becomes structured upon binding to arrestin. A control, unphosphorylated peptide that fails to bind to arrestin remains highly disordered. Specific NMR distance constraints are used to model the arrestin-bound conformation. The models suggest that the phosphorylated carboxy-terminal region of rhodopsin, Rh(330-348), undergoes significant conformational changes and becomes structured upon binding to arrestin.

Signaling states of rhodopsin in rod disk membranes lacking transducin ßγ-complex.

PURPOSE: To characterize the possible role of transducin Gtßγ-complex in modulating the signaling properties of photoactivated rhodopsin and its lifetime in rod disc membranes and intact rods. METHODS: Rhodopsin photolysis was studied using UV-visible spectroscopy and rapid scanning spectroscopy in the presence of hydroxylamine in highly purified wild-type and Gtγ-deficient mouse rod disc membranes. Complex formation between photoactivated rhodopsin and transducin was measured by extra-metarhodopsin (meta) II assay. Recovery of dark current and flash sensitivity in individual intact wild-type and Gtγ-deficient mouse rods was measured by single-cell suction recordings. RESULTS: Photoconversion of rhodopsin to meta I/meta II equilibrium proceeds normally after elimination of the Gtßγ-complex. The meta I/meta II ratio, the rate of meta II decay, the reactivity of meta II toward hydroxylamine, and the rate of meta III formation in Gtγ-deficient rod disc membranes were identical with those observed in wild-type samples. Under low-intensity illumination, the amount of extra-meta II in Gtγ-deficient discs was significantly reduced. The initial rate of dark current recovery after 12% rhodopsin bleach was three times faster in Gtγ-deficient rods, whereas the rate of the late current recovery was largely unchanged. Mutant rods also exhibited faster postbleach recovery of flash sensitivity. CONCLUSIONS: Photoactivation and thermal decay of rhodopsin proceed similarly in wild-type and Gtγ-deficient mouse rods, but the complex formation between photoactivated rhodopsin and transducin is severely compromised in the absence of Gtßγ. The resultant lower transduction activation contributes to faster photoresponse recovery after a moderate pigment bleach in Gtγ-deficient rods.

Using ligand-based virtual screening to allosterically stabilize the activated state of a GPCR.

G-protein coupled receptors play an essential role in many biological processes. Despite an increase in the number of solved X-ray crystal structures of G-protein coupled receptors, capturing a G-protein coupled receptor in its activated state for structural analysis has proven to be difficult. An unexplored paradigm is stabilization of one or more conformational states of a G-protein coupled receptor via binding a small molecule to the intracellular loops. A short tetrazole peptidomimetic based on the photoactivated state of rhodopsin-bound structure of Gt(alpha)(340-350) was previously designed and shown to stabilize the photoactivated state of rhodopsin, the G-protein coupled receptor involved in vision. A pharmacophore model derived from the designed tetrazole tetrapeptide was used for ligand-based virtual screening to enhance the possible discovery of novel scaffolds. Maybridge Hitfinder and National Cancer Institute diversity libraries were screened for compounds containing the pharmacophore. Forty-seven compounds resulted from virtually screening the Maybridge library, whereas no hits resulted with the National Cancer Institute library. Three of the 47 Maybridge compounds were found to stabilize the MII state. As these compounds did not inhibit binding of transducin to photoactivated state of rhodopsin, they were assumed to be allosteric ligands. These compounds are potentially useful for crystallographic studies where complexes with these compounds might capture rhodopsin in its activated conformational state.

Modulating G-protein coupled receptor/G-protein signal transduction by small molecules suggested by virtual screening.

Modulation of interactions between activated GPCRs (G-protein coupled receptors) and the intracellular (IC) signal transducers, heterotrimeric G-proteins, is an attractive, yet essentially unexplored, paradigm for treatment of certain diseases. Regulating downstream signaling for treatment of congenital diseases due to constitutively active GPCRs, as well as tumors where GPCRs are often overexpressed, requires the development of new methodologies. Modeling, experimental data, docking, scoring, and experimental testing (MEDSET) was developed to discover inhibitors that target the IC loops of activated GPCRs. As proof-of-concept, MEDSET developed and utilized a model of the interface between photoactivated rhodopsin (R*) and transducin (Gt), its G-protein. A National Cancer Institute (NCI) compound library was screened to identify compounds that bound at the interface between R* and its G-protein. High-scoring compounds from this virtual screen were obtained and tested experimentally for their ability to stabilize R* and prevent Gt from binding to R*. Several compounds that modulate signal transduction have been identified.

Rhodopsin-interacting surface of the transducin gamma subunit.

The visual signaling pathway is initiated by photoactivation of the GPCR rhodopsin, which activates nucleotide exchange on the heterotrimeric G-protein transducin (Gt). Domains on both Gtalpha and Gtbetagamma subunits participate in coupling to rhodopsin. Previously, we have shown by high-resolution NMR that the farnesylated C-terminal peptide of Gtgamma(60-71), DKNPFKELKGGC, assumes an amphipathic helical conformation during interaction with metarhodopsin II [Kisselev, O. G., and Downs, M. A. (2003) Structure 11, 367-373]. This conformation was docked to the structure of holo-Gt to create a model of rhodopsin-Gt interaction. Here we test this model by mutational analysis of Gt. To evaluate the contribution of specific amino acids of the Gtgamma C-terminal region involved in binding and GTP-dependent release of transducin from native rhodopsin membranes, we have systematically substituted each of the amino acids in the C-terminal region of Gtgamma for alanine. The mutants were co-expressed with six-histidine-tagged Gtbeta subunits in Sf9 insect cells. The Gtbeta-6-His-gamma mutant proteins were purified and assayed in the presence of Gtalpha for the GTP-dependent interactions with light-activated rhodopsin. Several of the alanine mutants, N62A, P63A, and F64A, exhibited significant functional defects at the level of R*-Gt complex formation. These data show that the conserved N-terminal end of the helical domain in the Gtgamma(60-71) region has the most significant effect on rhodopsin-Gt interactions, which places important constraints on the model of the rhodopsin-Gt complex.

Alternate binding mode of C-terminal phenethylamine analogs of G(t)alpha(340-350) to photoactivated rhodopsin.

The C-terminus of the Galpha-subunit of transducin plays an important role in receptor recognition. Synthetic peptides corresponding to the last 11 residues of the subunit have been shown to stabilize the photoactivated form of rhodopsin, Rh*. The Rh*-bound structure of the G(t)alpha(340-350) peptide has been determined using transferred nuclear overhauser effect NMR. In that structure, we observed two interactions between Lys341 and Phe350, a cation-pi interaction between the epsilon-amine and the aromatic ring of Phe350 and a salt-bridge between the epsilon-amine and the C-terminal carboxylate. A series of C-terminal phenethylamine analogs of the G(t)alpha(340-350) peptide were synthesized, lacking the C-terminal carboxylate group, to investigate the forces that contribute to the stability of the Rh*-bound conformation of the peptide. Rh*-stabilization assay data suggest that the C-terminal carboxylate is not necessary to maintain binding affinity. Transferred nuclear overhauser effect NMR experiments reveal that these C-terminal phenethylamine peptides adopt an Rh*-bound structure that is similar overall, but lacking some of the intramolecular interactions observed in the native Rh*-bound G(t)alpha(340-350) structure. These studies suggest that the binding site for G(t)alpha(340-350) on Rh* is adaptable, and we propose that the charged carboxylate of Phe350 does not play a significant role in the interaction with Rh*, but helps stabilize the Rh*-bound confirmation of the native peptide.

Relative strength of cation-pi vs salt-bridge interactions: the Gtalpha(340-350) peptide/rhodopsin system.

Interactions between cationic and aromatic side chains of amino acid residues, the so-called cation-pi interaction, are thought to contribute to the overall stability of the folded structure of peptides and proteins. The transferred NOE NMR structure of the G(t)alpha(340-350) peptide bound to photoactivated rhodopsin (R*) geometrically suggests a cation-pi interaction stabilizing the structure between the epsilon-amine of Lys341 and the aromatic ring of the C-terminal residue, Phe350. This interaction has been explored by varying substituents on the phenyl ring to alter the electron density of the aromatic ring of Phe350 and observing the impact on binding of the peptide to R*. The results suggest that while a cation-pi interaction geometrically exists in the G(t)alpha(340-350) peptide when bound to R*, its energetic contribution to the stability of the receptor-bound structure is relatively insignificant, as it was not observed experimentally. The presence of an adjacent and competing salt-bridge interaction between the epsilon-amine of Lys341 and the C-terminal carboxylate of Phe350 effectively shields the charge of the ammonium group. Experimental data supporting a significant cation-pi interaction can be regained through a series of Phe350 analogues where the C-terminal carboxyl has been converted to the neutral carboxamide, thus eliminating the shielding salt-bridge. TrNOE NMR experiments confirmed the existence of the cation-pi interaction in the carboxamide analogues. Various literature estimates of the strength of cation-pi interactions, including some that estimate strengths in excess of salt-bridges, are compromised by omission of the relevant anion in the calculations.

Conformational changes in the phosphorylated C-terminal domain of rhodopsin during rhodopsin arrestin interactions.

Phosphorylation of activated G-protein-coupled receptors and the subsequent binding of arrestin mark major molecular events of homologous desensitization. In the visual system, interactions between arrestin and the phosphorylated rhodopsin are pivotal for proper termination of visual signals. By using high resolution proton nuclear magnetic resonance spectroscopy of the phosphorylated C terminus of rhodopsin, represented by a synthetic 7-phosphopolypeptide, we show that the arrestin-bound conformation is a well ordered helix-loop structure connected to rhodopsin via a flexible linker. In a model of the rhodopsin-arrestin complex, the phosphates point in the direction of arrestin and form a continuous negatively charged surface, which is stabilized by a number of positively charged lysine and arginine residues of arrestin. Opposite to the mostly extended structure of the unphosphorylated C-terminal domain of rhodopsin, the arrestin-bound C-terminal helix is a compact domain that occupies a central position between the cytoplasmic loops and occludes the key binding sites of transducin. In conjunction with other binding sites, the helix-loop structure provides a mechanism of shielding phosphates in the center of the rhodopsin-arrestin complex and appears critical in guiding arrestin for high affinity binding with rhodopsin.