Biomedical subjects

Pere Garriga

Publications and source records attributed to Pere Garriga.

12 recordsLinked to original sources

PubMed2006

A methyl group at C7 of 11-cis-retinal allows chromophore formation but affects rhodopsin activation.

The newly synthesized 11-cis-7-methylretinal can form an artificial visual pigment with kinetic and spectroscopic properties similar to the native pigment in the dark-state. However, its photobleaching behavior is altered, showing a Meta I-like photoproduct. This behavior reflects a steric constraint imposed by the 7-methyl group that affects the conformational change in the binding pocket as a result of retinal photoisomerization. Transducin activation is reduced, when compared to the native pigment with 11-cis-retinal. Molecular dynamics simulations suggest coupling of the C7 methyl group and the beta-ionone ring with Met207 in transmembrane helix 5 in agreement with recent experimental results.

Animals↗

PubMed2006

Ca2+/recoverin dependent regulation of phosphorylation of the rhodopsin mutant R135L associated with retinitis pigmentosa.

No single molecular mechanism accounts for the effect of mutations in rhodopsin associated with retinitis pigmentosa. Here we report on the specific effect of a Ca2+/recoverin upon phosphorylation of the autosomal dominant retinitis pigmentosa R135L rhodopsin mutant. This mutant shows specific features like impaired G-protein signaling but enhanced phosphorylation in the shut-off process. We now report that R135L hyperphosphorylation by rhodopsin kinase is less efficiently inhibited by Ca2+/recoverin than wild-type rhodopsin. This suggests an involvement of Ca2+/recoverin into the molecular pathogenic effect of the mutation in retinitis pigmentosa which is the cause of rod photoreceptor cell degeneration. This new proposed role of Ca2+/recoverin may be one of the specific features of the proposed new Type III class or rhodopsin mutations associated with retinitis pigmentosa.

Animals↗

PubMed2005

Light-driven activation of beta 2-adrenergic receptor signaling by a chimeric rhodopsin containing the beta 2-adrenergic receptor cytoplasmic loops.

Structure-function studies of rhodopsin indicate that both intradiscal and transmembrane (TM) domains are required for retinal binding and subsequent light-induced structural changes in the cytoplasmic domain. Further, a hypothesis involving a common mechanism for activation of G-protein-coupled receptor (GPCR) has been proposed. To test this hypothesis, chimeric receptors were required in which the cytoplasmic domains of rhodopsin were replaced with those of the beta(2)-adrenergic receptor (beta(2)-AR). Their preparation required identification of the boundaries between the TM domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR necessary for formation of the rhodopsin chromophore and its activation by light and subsequent optimal activation of beta(2)-AR signaling. Chimeric receptors were constructed in which the cytoplasmic loops of rhodopsin were replaced one at a time and in combination. In these replacements, size of the third cytoplasmic (EF) loop critically determined the extent of chromophore formation, its stability, and subsequent signal transduction specificity. All the EF loop replacements showed significant decreases in transducin activation, while only minor effects were observed by replacements of the CD and AB loops. Light-dependent activation of beta(2)-AR leading to Galphas signaling was observed only for the EF2 chimera, and its activation was further enhanced by replacements of the other loops. The results demonstrate coupling between light-induced conformational changes occurring in the transmembrane domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR.

Amino Acid Sequence↗

PubMed2004

Patterns of retinal light absorption related to retinitis pigmentosa mutants from in silico model structures of rhodopsin.

Changes induced by mutations in rhodopsin that are associated with the degenerative visual disease retinitis pigmentosa result in an altered pattern of light absorption according to quantum mechanical simulations and reference experimental works. Eleven single-point mutations associated with retinitis pigmentosa at and in the proximity to the retinal binding pocket of rhodopsin have been modeled in silico and their spectra calculated with the NDOL (Neglect of Differential Overlap accounting L azimuthal quantum number) a priori method. The altered pattern of absorption found would lead to cumulative consequences in energy dissipation with aging. Different energy balances in the case of mutants at the very molecular level, compared to native nonmutated rhodopsin, can cause permanent cellular stress and would play a role in the progression of the retine degenerative process. It could explain the worsening of the pathological condition mostly in adults and suggests the probable beneficial effects of using quenching drugs and protection devices against excess of light in the early stages of life for avoiding or reducing potential damage.

Absorption↗

PubMed2003

[Rhodopsin structure: some light into the shadows of retinal degenerations].

Retinitis pigmentosa is a group of retinal degenerative diseases, within the broad family of hereditary retinopathies, for which there is no cure at present. Mutations in different genes coding for proteins related to the metabolism of photoreceptor cells, and to the visual phototransduction cascade, are the cause of this disease. Rhodopsin, the photoreceptor protein responsible for light absorption--and key in the first stages of vision--is one of the most studied molecules of the retina. Mutations in the opsin gene account for about 25% of all cases of autosomal dominant retinitis pigmentosa. Recent crystallization of this receptor in its inactive dark state has revealed new structural details yielding further insights into the intra and intermolecular mechanismsin which the protein is involved as a result of its activation.Furthermore, the in vitro study of recombinant rhodopsins carrying mutations previously found in retinitis pigmentosa patients (by means of spectroscopic and functional techniques) has shed new light on the structural requirements for its correct function, as well as the molecular defects underlying the mechanism of photoreceptor cell death. In this study, the main findings of the recent investigations carried out in this field are presented. The relevant information obtained at the molecular level is bound to facilitate our understandingof the molecular processes that will allow suitable therapiesfor different retinal degenerative diseases, particularly retinitis pigmentosa, to be proposed.

Humans↗

PubMed2003

Altered functionality in rhodopsin point mutants associated with retinitis pigmentosa.

Point mutations found in rhodopsin associated with the retinal degenerative disease retinitis pigmentosa have been expressed in mammalian COS-1 cells, purified, and characterised. The mutations characterised-most of them for the first time-have been Met44Thr, Gly114Asp, Arg135Leu, Val137Met, and Pro171Leu in the transmembrane domain; Leu328Pro and Ala346Pro in the C-terminal tail of the cytoplasmic domain; and Gly106Trp in the intradiscal domain. Several of these mutations cause misfolding which results in impaired 11-cis-retinal binding. Two of them, Met44Thr and Val137Met, show spectral and structural features similar to those of wild type rhodopsin (Type I mutants) but significantly increased transducin initial activation rates. We propose that, in the case of these mutants, abnormal functioning resulting in faster activation kinetics could also play a role in retinitis pigmentosa by altering the stoichiometric balance of the different proteins involved in the phototransduction biochemical reactions.

Amino Acid Sequence↗

PubMed2003

Structural and functional role of helices I and II in rhodopsin. A novel interplay evidenced by mutations at Gly-51 and Gly-89 in the transmembrane domain.

The naturally occurring mutations G51A and G51V in transmembrane helix I and G89D in the transmembrane helix II of rhodopsin are associated with the retinal degenerative disease autosomal dominant retinitis pigmentosa. To probe the orientation and packing of helices I and II a number of replacements at positions 51 and 89 were prepared by using site-directed mutagenesis, and the corresponding proteins expressed in COS-1 cells were characterized. Mutations at position 51 (G51V and G51L) bound retinal like wild-type rhodopsin but had thermally destabilized structures in the dark, altered photobleaching behavior, destabilized metarhodopsin II active conformations, and were severely defective in signal transduction. The effects observed can be correlated with the size of the mutated side chains that would interfere with specific interhelical interaction with Val-300 in helix VII. Mutations at position 89 had sensitivity to charge, as in G89K and G89D mutants, which showed reduced transducin activation. G89K showed a second absorbing species in the UV region at 350 nm, suggesting a charge effect of the introduced lysine. Increased formation of non-active forms of rhodopsin, like metarhodopsin III, may have some influence in the molecular defect underlying retinitis pigmentosa in the mutants studied. At the structural level, the effect of the mutations analyzed can be rationalized assuming a very specific set of tertiary interactions in the interhelical packing of the transmembrane segments of rhodopsin.

Amino Acid Sequence↗

PubMed2003

Effect of dodecyl maltoside detergent on rhodopsin stability and function.

Detergent-solubilized bovine rhodopsin produces mixed detergent/lipid/protein micelles. The effect of dodecyl maltoside detergent on the thermal stability of dark-state rhodopsin, and upon formation of the different intermediates after rhodopsin photobleaching (metarhodopsin II and metarhodopsin III), and upon transducin activation has been studied. No significant effect is observed for the thermal stability of dark-state rhodopsin in the range of detergent concentrations studied, but a decrease in the stability of metarhodopsin II and an increase in metarhodopsin III formation is observed with decreasing detergent concentrations. The transducin activation process is also affected by the presence of detergent indicating that this process is dependent on the lipid micro-environment and membrane fluidity, and this stresses the importance of the native lipid environment in rhodopsin normal function.

Detergents↗

PubMed2002

Zinc-induced decrease of the thermal stability and regeneration of rhodopsin.

Zinc is present at high concentrations in the photoreceptor cells of the retina where it has been proposed to play a role in the visual phototransduction process. In order to obtain more information about this role, the study of the effect of zinc on several properties of the visual photoreceptor rhodopsin has been investigated. A specific effect of Zn(2+) on the thermal stability of rhodopsin, obtained from bovine retinas and solubilized in dodecyl maltoside detergent, in the dark is reported. The thermal stability of rhodopsin in its ground state (dark state) is clearly reduced with increasing Zn(2+) concentrations (0-50 microm Zn(2+)). The thermal bleaching process is accelerated in the presence of Zn(2+) with k rate constants, at 55 degrees C, of 0.028 +/- 0.002 min(-1) (0 microm Zn(2+)) and 0.056 +/- 0.003 min(-1) (50 microm Zn(2+)), corresponding to t(12) values of 24.4 +/- 1.6 min and 11.8 +/- 0.1 min, respectively. Thermodynamic parameters derived from Arrhenius plots show a significant E(a) increase at 50 microm Zn(2+) for the process, with deltaG++ decrease and increase in deltaH++ and deltaS++ possibly reflecting conformational rearrangements and reordering of water molecules. The stability of the metarhodopsin II intermediate is also decreased and changes in the metarhodopsin II decay pathway are also detected. The extent of rhodopsin regeneration in vitro is also reduced by zinc. These effects, specific for zinc, are also seen for rhodopsin in native disc membranes, and may be relevant to the suggested role of Zn(2+) in normal and pathological retinal function.

Animals↗

PubMed2002

Unusual thermal and conformational properties of the rhodopsin congenital night blindness mutant Thr-94 --> Ile.

Naturally occurring point mutations in the opsin gene cause the retinal diseases retinitis pigmentosa and congenital night blindness. Although these diseases involve similar mutations in very close locations in rhodopsin, their progression is very different, with retinitis pigmentosa being severe and causing retinal degeneration. We report on the expression and characterization of the recently found T94I mutation associated with congenital night blindness, in the second transmembrane helix or rhodopsin, and mutations at the same site. T94I mutant rhodopsin folded properly and was able to bind 11-cis-retinal to form chromophore, but it showed a blue-shifted visible band at 478 nm and reduced molar extinction coefficient. Furthermore, T94I showed dramatically reduced thermal stability, extremely long lived metarhodopsin II intermediate, and highly increased reactivity toward hydroxylamine in the dark, when compared with wild type rhodopsin. The results are consistent with the location of Thr-94 in close proximity to Glu-113 counterion in the vicinity of the Schiff base linkage and suggest a role for this residue in maintaining the correct dark inactive conformation of the receptor. The reported results, together with previously published data on the other two known congenital night blindness mutants, suggest that the molecular mechanism underlying this disease may not be structural misfolding, as proposed for retinitis pigmentosa mutants, but abnormal functioning of the receptor by decreased thermal stability and/or constitutive activity.

Amino Acid Sequence↗

PubMed2002

Calcium-induced decrease of the thermal stability and chaperone activity of alpha-crystallin.

Alpha-crystallin, one of the major proteins in the vertebrate eye lens, acts as a molecular chaperone, like the small heat-shock proteins, by protecting other proteins from denaturing under stress or high temperature conditions. alpha-Crystallin aggregation is involved in lens opacification, and high [Ca(2+)] has been associated with cataract formation, suggesting a role for this cation in the pathological process. We have investigated the effect of Ca(2+) on the thermal stability of alpha-crystallin by UV and Fourier-transform infrared (FTIR) spectroscopies. In both cases, a Ca(2+)-induced decrease in the midpoint of the thermal transition is detected. The presence of high [Ca(2+)] results also in a marked decrease of its chaperone activity in an insulin-aggregation assay. Furthermore, high Ca(2+) concentration decreases Cys reactivity towards a sulfhydryl reagent. The results obtained from the spectroscopic analysis, and confirmed by circular dichroism (CD) measurements, indicate that Ca(2+) decreases both secondary and tertiary-quaternary structure stability of alpha-crystallin. This process is accompanied by partial unfolding of the protein and a clear decrease in its chaperone activity. It is concluded that Ca(2+) alters the structural stability of alpha-crystallin, resulting in impaired chaperone function and a lower protective ability towards other lens proteins. Thus, alpha-crystallin aggregation facilitated by Ca(2+) would play a role in the progressive loss of transparency of the eye lens in the cataractogenic process.

Animals↗

PubMed2002

The eye photoreceptor protein rhodopsin. Structural implications for retinal disease.

Rhodopsin is the membrane receptor responsible for photoreception in the vertebrate retina. Its characteristic seven-transmembrane helical structural motif is today widely recognised as a paradigm in signal transduction. Rhodopsin and the phototransduction system are frequently used as structural and mechanistic models for the G-protein coupled receptor superfamily. Recent advances in the activation mechanism (as derived from the structural available data) and the implications for normal and pathological - in retinal disorders - visual function will be reviewed.

Amino Acid Sequence↗