Die Quartärstruktur von Rhodopsin und ihre funktionellen Implikationen

The present work focuses on the quaternary structure of rhodopsin and its possible implications for the function of the receptor as a light transducer. Rhodopsin is a prototypical G protein- coupled receptor (GPCR) that is found in high concentrations in the discs of the outer segment of rod photoreceptor cells. Its physiological function is the transduction of light into a biological relevant signal under dim light conditions. There is growing evidence that GPCRs form and might even function as oligomers in membranes (Milligan, Ramsay et al. 2003; Milligan 2006). Oligomers were also reported for rhodopsin by atomic force microscopy (Fotiadis, Liang et al. 2003), chemical cross-linking (Jastrzebska, Maeda et al. 2004; Medina, Perdomo et al. 2004; Jastrzebska, Fotiadis et al. 2006), blue native electrophoresis (Jastrzebska, Maeda et al. 2004) and FRET studies (Kota, Reeves et al. 2006; Mansoor, Palczewski et al. 2006). This view is challenged by early biophysical and biochemical studies suggesting that rhodopsin is monomeric (Cone 1972; Poo and Cone 1974; Chabre 1975; Chabre and le Maire 2005). However, it remains to be elucidated whether its quaternary structure is of any physiological significance for visual signal transduction. In the present thesis, I investigated rhodopsin’s propensity to oligomerize in the plasma membrane of HEK293 and COS-1 cells using bimolecular fluorescence complementation (BiFC) and fluorescence resonance energy transfer (FRET) as techniques. As possible interaction domains for rhodopsin oligomers, helices IV and V (Liang, Fotiadis et al. 2003) as well as helices I, II and VIII (Salom, Lodowski et al. 2006) have been proposed so far. In my thesis, I also tried to verify possible interaction domains using FRET competition experiments. Furthermore, I was interested in investigating whether a change in rhodopsin’s quaternary structure alters its ability of binding or activating its G protein transducin (Gt). For this question I used purified, solubilized rhodopsin and rhodopsin fusion proteins in 0.01% DM to measure FRET as well as Gt activation rates. I found that BiFC yields fluorescing cells upon coexpression of several unrelated membrane and non-membrane proteins with opsin. This suggests that it is not a suitable test for specific membrane protein interaction. Furthermore, I found that opsin shows very high FRET efficiency in the plasma membranes of HEK293 and COS-1 cells. The FRET competition data confirms the idea of helix IV/V as part of the oligomerization interface. When detergent is added to purified membranes as well as to HEK293 cells in vivo, FRET efficiency decreases significantly. In purified, solubilized samples (0.01% DM), no FRET could be measured at all. Under the chosen experimental conditions, solubilized rhodopsin therefore appears to be present as a monomer. Nevertheless, measurements of Gt activation revealed that monomeric rhodopsin efficiently activates its cognate G protein at high rates (Vmax of 40 Gt/s, KM of 3.3 µM). Monomeric rhodopsin therefore works with a specificity constant of 1.3∙10-7M-1s-1, which is close to the diffusion limit (Berg and von Hippel 1985) and can thus be called a ‘perfect enzyme’.In der hier vorliegenden Doktorarbeit wurde die Quartärstruktur von Rhodopsin und ihre Rolle für die Weiterleitung von Lichtsignalen an das G-Protein Transducin untersucht. Rhodopsin ist ein prototypischer G-Protein-gekoppelter Rezeptor (GPCR), der in hohen Konzentrationen in den Membranen der Disks der Stäbchenaußensegmente vorkommt. Seine physiologische Funktion ist die Übersetzung von Licht in ein biologisch verwertbares Signal unter Dämmerlicht Bedingungen. Es gibt zunehmend Hinweise, dass GPCRs oligomere Strukturen bilden, die möglicherweise auch ihre funktionellen Einheit darstellen (Milligan, Ramsay et al. 2003; Milligan 2006). Mit Hilfe von Techniken wie atomic force microscopy ( Fotiadis, Liang et al. 2003), cross-linking (Jastrzebska, Maeda et al. 2004; Medina, Perdomo et al. 2004; Jastrzebska, Fotiadis et al. 2006), blue native gel electrophoresis (Jastrzebska, Maeda et al. 2004) und fluorescence resonance energy transfer (FRET) (Kota, Reeves et al. 2006; Mansoor, Palczewski et al. 2006) wurden auch für Rhodopsin Oligomere als Quartärstruktur postuliert. Diese Experimente stehen aber im Widerspruch zu Resultaten aus früheren biophysikalischen und biochemischen Experimenten, in denen keinerlei Evidenz für eine dimere/oligomere Quartärstruktur zu finden war (Cone 1972, Poo und Cone 1974; Chabre 1975; Chabre und le Maire 2005). Weiterhin bleibt zudem unklar, ob eine Oligomerisierung relevant für die physiologische Funktion von Rhodopsin als Licht Rezeptor in vivo ist. In der hier präsentierten Arbeit wurde die Dimerisierung von Rhodopsin in der Plasmamembran von HEK293 und COS-1 Zellen mit Hilfe von bimolecular fluorescence complementation assay (BiFC) und FRET untersucht. Als mögliche Interaktionsdomainen der Oligomerbildung von Rhodopsin wurden bisher Helices IV und V (Liang, Fotiadis et al. 2003) als auch Helices I, II, und VII (Salom, Lodowski et al. 2006) postuliert. In der vorliegenden Arbeit wurden mögliche Interaktionsdomainen der Rhodopsin Oligomerbildung mit Hilfe von FRET untersucht. Weiterhin sollte untersucht werden, ob eine Änderung der Quartärstruktur auch zu einer Änderung in der Katalyseeffizienz der G-Protein Aktivierung führt. Für diese Frage wurde solubilisiertes Rhodopsin und entsprechende Rhodopsin-Fluorophor Fusionsproteine in 0,01% DM präpariert, um G-Protein Aktivierungsraten als auch FRET zu messen. Es zeigt sich, dass die Koexpression von komplementären Opsin-BiFC Fusionsproteinen zu einem starken Fluoreszenzsignal in vivo führte. Die Tatsache, das auch die Koexpression von verschiedener anderer -membranständiger als auch zytoplasmatischer- Proteine als BiFC Konstrukte mit einem komplementären Opsin-BiFC Konstrukt Fluoreszenz zeigten, legt allerdings nahe, dass BiFC kein spezifischer Marker für intermolekulare Interaktion von Membranproteinen ist. FRET Experimente mit geeigneten Opsin-Fluorophor Fusionsproteinen ergaben eine hohe FRET Effizienz in der Plasmamembran von transfizierten HEK293 and COS-1 Zellen. Die FRET Kompetitionsexperimente unterstrichen weiterhin die Theorie, dass Helices IV and V eine Rolle bei der Oligomerisierung spielen. Wenn das Detergens Dodecylmaltosid (DM) zu gereinigten COS-1 Membranen oder HEK293 Zellen in vivo gegeben wurde, verringerte sich die FRET Effizienz signifikant. In aufgereinigten, solubilisierten Proben (0,01% DM) konnte überhaupt kein FRET Signal mehr gemessen werden. Daraus kann geschlussfolgert werden, dass Rhodopsin in 0,01% DM als Monomer vorliegt. Unter denselben experimentellen Bedingungen wurde auch die katalytische G-Protein Aktivierungskapazität von gereinigtem Rhodopsin bestimmt. Es zeigte sich, dass monomeres Rhodopsin sehr effizient in der Lage ist Transducin zu aktivieren (Vmax = 40 Gt/s, KM = 3,3 µM). Daraus folgt, dass monomeres Rhodopsin mit einer Spezifizitätskonstante von 1,3•10-7 M-1s-1 nahe am theoretisch möglichen Diffusionslimit arbeitet, (Berg und von Hippel 1985) und somit als so genanntes ‚perfektes Enzym’ beschrieben werden kann

Quantitative Signal Intensity in Fluid-Attenuated Inversion Recovery and Treatment Effect in the WAKE-UP Trial

International audienceBackground and Purpose— Relative signal intensity of acute ischemic stroke lesions in fluid-attenuated inversion recovery (fluid-attenuated inversion recovery relative signal intensity [FLAIR-rSI]) magnetic resonance imaging is associated with time elapsed since stroke onset with higher intensities signifying longer time intervals. In the randomized controlled WAKE-UP trial (Efficacy and Safety of MRI-Based Thrombolysis in Wake-Up Stroke Trial), intravenous alteplase was effective in patients with unknown onset stroke selected by visual assessment of diffusion weighted imaging fluid-attenuated inversion recovery mismatch, that is, in those with no marked fluid-attenuated inversion recovery hyperintensity in the region of the acute diffusion weighted imaging lesion. In this post hoc analysis, we investigated whether quantitatively measured FLAIR-rSI modifies treatment effect of intravenous alteplase. Methods— FLAIR-rSI of stroke lesions was measured relative to signal intensity in a mirrored region in the contralesional hemisphere. The relationship between FLAIR-rSI and treatment effect on functional outcome assessed by the modified Rankin Scale (mRS) after 90 days was analyzed by binary logistic regression using different end points, that is, favorable outcome defined as mRS score of 0 to 1, independent outcome defined as mRS score of 0 to 2, ordinal analysis of mRS scores (shift analysis). All models were adjusted for National Institutes of Health Stroke Scale at symptom onset and stroke lesion volume. Results— FLAIR-rSI was successfully quantified in stroke lesions in 433 patients (86% of 503 patients included in WAKE-UP). Mean FLAIR-rSI was 1.06 (SD, 0.09). Interaction of FLAIR-rSI and treatment effect was not significant for mRS score of 0 to 1 ( P =0.169) and shift analysis ( P =0.086) but reached significance for mRS score of 0 to 2 ( P =0.004). We observed a smooth continuing trend of decreasing treatment effects in relation to clinical end points with increasing FLAIR-rSI. Conclusions— In patients in whom no marked parenchymal fluid-attenuated inversion recovery hyperintensity was detected by visual judgement in the WAKE-UP trial, higher FLAIR-rSI of diffusion weighted imaging lesions was associated with decreased treatment effects of intravenous thrombolysis. This parallels the known association of treatment effect and elapsing time of stroke onset

Functional Outcome of Intravenous Thrombolysis in Patients With Lacunar Infarcts in the WAKE-UP Trial

Importance: The rationale for intravenous thrombolysis in patients with lacunar infarcts is debated, since it is hypothesized that the microvascular occlusion underlying lacunar infarcts might not be susceptible to pharmacological reperfusion treatment. Objective: To study the efficacy and safety of intravenous thrombolysis among patients with lacunar infarcts. Design, Setting, and Participants: This exploratory secondary post hoc analysis of the WAKE-UP trial included patients who were screened and enrolled between September 2012 and June 2017 (with final follow-up in September 2017). The WAKE-UP trial was a multicenter, double-blind, placebo-controlled randomized clinical trial to study the efficacy and safety of intravenous thrombolysis with alteplase in patients with an acute stroke of unknown onset time, guided by magnetic resonance imaging. All 503 patients randomized in the WAKE-UP trial were reviewed for lacunar infarcts. Diagnosis of lacunar infarcts was based on magnetic resonance imaging and made by consensus of 2 independent investigators blinded to clinical information. Main Outcomes and Measures: The primary efficacy variable was favorable outcome defined by a score of 0 to 1 on the modified Rankin Scale at 90 days after stroke, adjusted for age and severity of symptoms. Results: Of the 503 patients randomized in the WAKE-UP trial, 108 patients (including 74 men [68.5%]) had imaging-defined lacunar infarcts, whereas 395 patients (including 251 men [63.5%]) had nonlacunar infarcts. Patients with lacunar infarcts were younger than patients with nonlacunar infarcts (mean age [SD], 63 [12] years vs 66 [12] years; P = .003). Of patients with lacunar infarcts, 55 (50.9%) were assigned to treatment with alteplase and 53 (49.1%) to receive placebo. Treatment with alteplase was associated with higher odds of favorable outcome, with no heterogeneity of treatment outcome between lacunar and nonlacunar stroke subtypes. In patients with lacunar strokes, a favorable outcome was observed in 31 of 53 patients (59%) in the alteplase group compared with 24 of 52 patients (46%) in the placebo group (adjusted odds ratio [aOR], 1.67 [95% CI, 0.77-3.64]). There was 1 death and 1 symptomatic intracranial hemorrhage according to Safe Implementation of Thrombolysis in Stroke-Monitoring Study criteria in the alteplase group, while no death and no symptomatic intracranial hemorrhage occurred in the placebo group. The distribution of the modified Rankin Scale scores 90 days after stroke also showed a nonsignificant shift toward better outcomes in patients with lacunar infarcts treated with alteplase, with an adjusted common odds ratio of 1.94 (95% CI, 0.95-3.93). Conclusions and Relevance: While the WAKE-UP trial was not powered to demonstrate the efficacy of treatment in subgroups of patients, the results indicate that the association of intravenous alteplase with functional outcome does not differ in patients with imaging-defined lacunar infarcts compared with those experiencing other stroke subtypes.status: publishe

Intravenous alteplase for stroke with unknown time of onset guided by advanced imaging: systematic review and meta-analysis of individual patient data

Background: Patients who have had a stroke with unknown time of onset have been previously excluded from thrombolysis. We aimed to establish whether intravenous alteplase is safe and effective in such patients when salvageable tissue has been identified with imaging biomarkers. Methods: We did a systematic review and meta-analysis of individual patient data for trials published before Sept 21, 2020. Randomised trials of intravenous alteplase versus standard of care or placebo in adults with stroke with unknown time of onset with perfusion-diffusion MRI, perfusion CT, or MRI with diffusion weighted imaging-fluid attenuated inversion recovery (DWI-FLAIR) mismatch were eligible. The primary outcome was favourable functional outcome (score of 0–1 on the modified Rankin Scale [mRS]) at 90 days indicating no disability using an unconditional mixed-effect logistic-regression model fitted to estimate the treatment effect. Secondary outcomes were mRS shift towards a better functional outcome and independent outcome (mRS 0–2) at 90 days. Safety outcomes included death, severe disability or death (mRS score 4–6), and symptomatic intracranial haemorrhage. This study is registered with PROSPERO, CRD42020166903. Findings: Of 249 identified abstracts, four trials met our eligibility criteria for inclusion: WAKE-UP, EXTEND, THAWS, and ECASS-4. The four trials provided individual patient data for 843 individuals, of whom 429 (51%) were assigned to alteplase and 414 (49%) to placebo or standard care. A favourable outcome occurred in 199 (47%) of 420 patients with alteplase and in 160 (39%) of 409 patients among controls (adjusted odds ratio [OR] 1·49 [95% CI 1·10–2·03]; p=0·011), with low heterogeneity across studies (I 2=27%). Alteplase was associated with a significant shift towards better functional outcome (adjusted common OR 1·38 [95% CI 1·05–1·80]; p=0·019), and a higher odds of independent outcome (adjusted OR 1·50 [1·06–2·12]; p=0·022). In the alteplase group, 90 (21%) patients were severely disabled or died (mRS score 4–6), compared with 102 (25%) patients in the control group (adjusted OR 0·76 [0·52–1·11]; p=0·15). 27 (6%) patients died in the alteplase group and 14 (3%) patients died among controls (adjusted OR 2·06 [1·03–4·09]; p=0·040). The prevalence of symptomatic intracranial haemorrhage was higher in the alteplase group than among controls (11 [3%] vs two [<1%], adjusted OR 5·58 [1·22–25·50]; p=0·024). Interpretation: In patients who have had a stroke with unknown time of onset with a DWI-FLAIR or perfusion mismatch, intravenous alteplase resulted in better functional outcome at 90 days than placebo or standard care. A net benefit was observed for all functional outcomes despite an increased risk of symptomatic intracranial haemorrhage. Although there were more deaths with alteplase than placebo, there were fewer cases of severe disability or death. Funding: None