Revealing complexity and specificity in the activation of lipase-mediated oxylipin biosynthesis: a specific role of the Nicotiana attenuata GLA1 lipase in the activation of jasmonic acid biosynthesis in leaves and roots

The activation of enzymatic oxylipin biosynthesis upon wounding, herbivory and pathogen attack depends on the biochemical activation of lipases that make polyunsaturated fatty acids (PUFAs) available to lipoxygenases (LOXs). The identity and number of the lipases involved in this process remain controversial and they probably differ among plant species. Analysis of transgenic Nicotiana attenuata plants (ir-gla1) stably reduced in the expression of the NaGLA1 gene showed that this plastidial glycerolipase is a major supplier of trienoic fatty acids for jasmonic acid (JA) biosynthesis in leaves and roots after wounding and simulated herbivory, but not during infection with the oomycete Phytophthora parasitica (var. nicotianae). NaGLA1 was not essential for the developmental control of JA biosynthesis in flowers and for the biosynthesis of C(6) volatiles by the hydroperoxide lyase (HPL) pathway; however, it affected the metabolism of divinyl ethers (DVEs) early during infection with P. parasitica (var. nicotianae) and the accumulation of NaDES1 and NaLOX1 mRNAs. Profiling of lysolipids by LC-MS/MS was consistent with a rapid activation of NaGLA1 and indicated that this lipase utilizes different lipid classes as substrates. The results revealed the complexity and specificity of the regulation of lipase-mediated oxylipin biosynthesis, highlighting the existence of pathway- and stimulus-specific lipases.     

An evaluation of detergents for NMR structural studies of membrane proteins

Structural information on membrane proteins lags far behind that on soluble proteins, in large part due to difficulties producing homogeneous, stable, structurally relevant samples in a membrane-like environment. In this study 25 membrane mimetics were screened using 2D (1)H-(15)N heteronuclear single quantum correlation NMR experiments to establish sample homogeneity and predict fitness for structure determination. A single detergent, 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] (LPPG), yielded high quality NMR spectra with sample lifetimes greater than one month for the five proteins tested - R. sphaeroides LH1 alpha and beta subunits, E. coli and B. pseudofirmus OF4 ATP synthase c subunits, and S. aureus small multidrug resistance transporter - with 1, 2, or 4 membrane spanning alpha-helices, respectively. Site-specific spin labeling established interhelical distances in the drug transporter and genetically fused dimers of c subunits in LPPG consistent with in vivo distances. Optical spectroscopy showed that LH1 beta subunits form native-like complexes with bacteriochlorophyll a in LPPG. All the protein/micelle complexes were estimated to exceed 100 kDaltons by translational diffusion measurements. However, analysis of (15)N transverse, longitudinal and (15)N[(1)H] nuclear Overhauser effect relaxation measurements yielded overall rotational correlation times of 8 to 12 nsec, similar to a 15-20 kDalton protein tumbling isotropically in solution, and consistent with the high quality NMR data observed.     

Lysophospholipids Facilitate COPII Vesicle Formation

1. Download high-res image (222KB)
2. Download full-size image

     

Induction,inhibition, and incorporation: Different roles for anionic and zwitterionic lysolipids in the fibrillation of the functional amyloid FapC

Amyloid proteins are widespread in nature both as pathological species involved in several diseases and as functional entities that can provide protection and storage for the organism. Lipids have been found in amyloid deposits from various amyloid diseases and have been shown to strongly affect the formation and structure of both pathological and functional amyloid proteins. Here, we investigate how fibrillation of the functional amyloid FapC from Pseudomonas is affected by two lysolipids, the zwitterionic lipid 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine and the anionic lipid 1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) (LPG). Small-angle X-ray scattering, circular dichroism, dynamic light scattering, and thioflavin T fluorescence measurements were performed simultaneously on the same sample to ensure reproducibility and allow a multimethod integrated analysis. We found that LPG strongly induces fibrillation around its critical micelle concentration (cmc) by promoting formation of large structures, which mature via accumulation of intermediate fibril structures with a large cross section. At concentrations above its cmc, LPG strongly inhibits fibrillation by locking FapC in a core–shell complex. In contrast, lipid 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine induces fibrillation at concentrations above its cmc, not via strong interactions with FapC but by being incorporated during fibrillation and likely stabilizing the fibrillation nucleus to reduce the lag phase. Finally, we show that LPG is not incorporated into the fibril during assembly but rather can coat the final fibril. We conclude that lipids affect both the mechanism and outcome of fibrillation of functional amyloid, highlighting a role for lipid concentration and composition in the onset and mechanism of fibrillation in vivo.     

The structural specificity of anti-phospholipid antibodies in autoimmune disease

Antibodies to phospholipids represent a unique set of incompletely characterized antibodies prevalent in patients with systemic lupus erythematosus and related autoimmune disorders. Little is known concerning their induction and pathogenesis. Here we provide an overview of what is currently known about these antibodies and explore the possibility that they interact with and are perhaps specific for unusual lipid structures. Particular attention is given to the physical chemical nature and phase behaviour of associated putative lipid antigens. This analysis suggests that alterations in the phospholipid architecture of cell membranes may play an important role in the immunoregulation of autoimmunity.     

The diversity of FtsY‐lipid interactions

The bacterial signal recognition particle (SRP) receptor FtsY forms a complex with the SRP Ffh to target nascent polypeptide chains to the bacterial inner membrane. How FtsY interacts with lipids and associates to the membrane is unclear. Here, we show that vesicle binding leads to partial protection against proteolytic degradation and a change in secondary structure, which differs depending on whether the lipids are simple mixtures of zwitterionic and anionic lipids, mimics of Escherichia coli lipids, or lysolipids. Lipid binding alters the stability of FtsY. Thermal unfolding of FtsY in buffer shows two transitions, one occurring at ～60°C and the other at ～90°C. The thermal intermediate accumulating between 60 and 90°C has structural features in common with the state induced by binding to E. coli lipids. E. coli lipid extract induces a single transition around 70°C, anionic lipids have no effect while cooperative unfolding is completely removed in lysolipids. Thus, the lipid environment profoundly influences the dynamic properties of FtsY, leading to three different kinds of FtsY‐lipid interactions with different effects on structure, proteolytic protection, and stability, and is driven both by hydrophobic and electrostatic interactions. Trypsin digestion experiments highlight the central role of the N‐domain in lipid contacts, whereas the A‐ and G‐domains appear to play a more minor part. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 595–606, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com