AB047.Membrane binding of S100A10 protein and AHNAK peptide involved in cell membrane repair

Background:The S100A10 protein might be an early biomarker of diabetes development leading to diabetic retinopathy.The protein complex S100A10/annexin A2 allows the recruitment of the C-terminal of AHNAK protein(AHNAK C-ter peptide)to the membrane in presence of calcium,before forming a platform which can initiate membrane repair.However,no molecular data are currently available on membrane binding of the different proteins involved in this complex.We aim to study the membrane binding of S100A10,AHNAK C-ter peptide and their complex to better understand their roles in cell membrane repair process.Methods:Firstly,S100A10 will be overexpressed and purified by affinity chromatography and AHNAK C-ter peptide will be synthesized.Langmuir monolayers membrane model will then be used to characterize the interactions between these proteins and different phospholipids found in membranes.The secondary structure,orientation and membrane organization of these proteins will be studied by Polarization Modulation Infrared Reflection-Absorption Spectroscopy.Their lateral localization will be determined through the influence of these proteins on the physical state of lipids by fluorescence microscopy.Results:The optimization of the overexpression,purification and cleavage of the GST tag procedure to obtain pure S100A10 was completed.Protein identification by mass spectrometry and circular dichroism stability pre-studies were performed.In parallel,AHNAK C-ter peptide was studied by Langmuir monolayer model and the results indicate this peptide prefers lipids with negatively charged polar heads and unsaturated acyl chains.Preliminary solid-state NMR results confirm this phenomenon at 37℃.Conclusions:Our research will complete current knowledge on membrane binding of S100A10 and AHNAK C-ter peptide.We could also identify the conditions leading to modifications of these membrane bindings,and possibly to the loss of protein function.Thus,this project helps to better determine their roles in membrane repair,as well as in other physiological mechanisms in which these proteins are involved.

Slr0151 in Synechocystis sp. PCC 6803 is required for efficient repair of photosystem Ⅱ under high-light condition

Cyanobacteria are ancient photosynthetic prokareyotes that have adapted successfully to adverse environments including high-light irradiation. Although it is known that the repair of photodamaged photosystem Ⅱ（PSⅡ） in the organisms is a highly regulated process, our knowledge of the molecular components that regulate each step of the process is limited.We have previously identified a hypothetical protein Slr0151 in the membrane fractions of cyanobacterium Synechocystis sp.PCC 6803. Here, we report that Slr0151 is involved in PSⅡ repair of the organism. We generated a mutant strain（Dslr0151）lacking the protein Slr0151 and analyzed its characteristics under normal and high-light conditions. Targeted deletion of slr0151 resulted in decreased PSⅡ activity in Synechocystis. Moreover,the mutant exhibited increased photoinhibition due to impairment of PSⅡ repair under high-light condition. Further analysis using in vivo radioactive labeling and 2-D blue native/sodium dodecylsulfate polyacrylamide gel electrophoresis indicated that the PSⅡ repair cycle was hindered at the levels of D1 synthesis and disassembly and/or assembly of PSⅡ in the mutant. Protein interaction assays demonstrated that Slr0151 interacts with D1 and CP43 proteins. Taken together,these results indicate that Slr0151 plays an important role in regulating PSⅡ repair in the organism under high-light stress condition.

Mitochondrial fragmentation and ROS signaling in wound response and repair

Mitochondria are organelles that serve numerous critical cellular functions,including energy production,Ca2+home-ostasis,redox signaling,and metabolism.These functions are intimately linked to mitochondrial morphology,which is highly dynamic and capable of rapid and transient changes to alter cellular functions in response to environmental cues and cellular demands.Mitochondrial morphology and activity are critical for various physiological processes,including wound healing.In mammals,wound healing is a complex process that requires coordinated function of multiple cell types and progresses in partially overlapping but distinct stages:hemostasis and inflammation,cell pro-liferation and migration,and tissue remodeling.The repair process at the single-cell level forms the basis for wound healing and regeneration in tissues.Recent findings reveal that mitochondria fulfill the intensive energy demand for wound repair and aid wound closure by cytoskeleton remodeling via morphological changes and mitochondrial reactive oxygen species(mtROS)signaling.In this review,we will mainly elucidate how wounding induces changes in mitochondrial morphology and activity and how these changes,in turn,contribute to cellular wound response and repair.