Workshops & seminars

"Enzymatic activity, structure and membrane binding of lecithin retinol acyltransferase which is involved in the retinoids visual cycle"
Dr. Christian Salesse (Laval)

DATE & TIME
Friday, January 29, 2016
2:30 p.m. – 3:30 p.m.
SPEAKER(S)

Dr. Christian Salesse

COST

This event is free

Website

CONTACT

Dajana Vuckovic

WHERE

Richard J. Renaud Science Complex
7141 Sherbrooke W.
Room SP-S110

WHEEL CHAIR ACCESSIBLE

Yes

The visual pigment of retinal photoreceptors is responsible for light absorption. It is made of a protein (opsin) and of a chromophore (a derivative of vitamin A, 11-cis-retinal). Upon light absorption by the visual pigment of rod photoreceptors (rhodopsin), 11-cis-retinal is isomerised to all-trans-retinal. As a result, all-trans-retinal dissociates from opsin and is conveyed to the retinal pigment epithelium (RPE) where it is recycled to 11-cis-retinal through the « retinoids visual cycle ». One of the key enzymes of the visual cycle in the RPE, lecithin retinol acyltransferase (LRAT), is responsible for the conversion of all-trans-retinol to all-trans-retinyl ester. LRAT is a membrane-associated protein. We have thus expressed a truncated LRAT (tLRAT, amino acids 31-196) without its N- and C-terminal α-helices. All essential amino acids for the catalytic activity of LRAT are present in tLRAT. The enzymatic reaction catalyzed by LRAT is postulated to take place in two steps. 1) Non-acylated LRAT first hydrolyzes the sn-1 fatty acyl chain of phospholipids, which results in the production of acylated LRAT. 2) Acylated LRAT then transfers its acyl chain to retinol, which produces a retinyl ester. We have recently developed the first reliable method to quantitatively determine the enzymatic activity of tLRAT, which allowed characterizing its biochemical properties in details. We found that tLRAT enzymatic activity is more than 55,000-fold larger than the highest activity reported for this enzyme. Then, the C161S/C168S-tLRAT mutant was prepared to eliminate acylation heterogeneity. We also prepared a uniformly 15N,13C-labeled C161S/C168S-tLRAT sample, and succeeded to assign 100% of backbone amides and 100% of the 13C’, 13C𝛂 and 13C𝛃 by Nuclear Magnetic Resonance (NMR). We derived the secondary structure of tLRAT based on the assigned chemical shifts. Moreover, several mutations of LRAT (Y61A-, A106T-, R109L-, P173L- and S175R) are leading to a complete loss of vision. These mutants have very little or no enzymatic activity. In addition, the comparison between our 15N-Heteronuclear Single Quantum Coherence (HSQC) NMR spectra of tLRAT and those of the P173L-, Y61A-, A106T- and S175R-tLRAT mutants allowed to suggest that these mutations result in local structural changes in the protein and that its global folding remains unchanged. The N- and C-terminal segments of LRAT have also been studied to determine their respective contribution to the membrane anchoring of LRAT. Furthermore, our membrane binding experiments with tLRAT suggested that it has a strong affinity for membranes despite the absence of its N- and C-terminal hydrophobic segments. Other regions of LRAT must be involved in its membrane anchoring such as an α-helical internal segment that we have identified from our NMR characterization.

Christian Salesse graduated in biophysics from the UQTR. He was then a postdoctoral fellow at Mainz university in Germany. He was then professor at the UQTR for 14 years until he moved to Université Laval in 2002. He was a FRQS fellow from the Junior 1 to the National level. He is also adjunct professor at Jilin University in China. He was an invited professor at several universities in France. He is the head of the Vision research unit at the Centre de recherche du CHU of Quebec and of the retina division of the FRQS Vision research network.

He is the guest of Dr.Christine DeWolf.

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