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Increased intracellular water content defined as cytotoxic brain tissue edema is a serious secondary clinical complication to traumatic brain injury (TBI) and stroke and without knowledge to the etiology. Recently a hypothesis to the nervous tissue edema was presented suggesting that external dynamic and internal mechanical static impact forces caused protein unfolding resulting in an increased brain tissue water content. The hypothesis was confirmed by computer simulation tests. In this laboratory study we further evaluated the hypothesis by using the mature protein laminin LN521 upon the effects of both dynamic as well as static impact forces, respectively. Laminin was chosen as a representative protein due to it´s general and abundance presence in the cells. The treated laminin solutions were then analyzed with denatured electrophoresis and Electron Microscopy showing aggregation and fragmentation of the laminin structures. The present results confirm earlier hypothesis and computer simulation suggesting for the first time that dynamic impact force in an accident and increased mechanical static force in stroke unfold mature proteins having the potential to increase the intracellular water content defined as cytotoxic brain tissue edema. The clinical condition resembles the phenomenon when elasmobranchs including white sharks prevent their cells from too high hydrostatic pressure in the deep sea. Thus, the present laboratory study results and knowledge from marine physics may be considered to improve the clinical treatment and outcome of TBI and stroke patients.

Development of new products for the prevention of traumatic brain injuries are mostly based on mechanical investigations. However, there is a demand for new and more sophisticated analyzes focusing on molecular levels to improve protective equipment. Recently we evaluated the mature protein laminin LN521 to find out the consequences to dynamic and semi-static impact and found substantial fragmentation and aggregation of the laminin structures. The objective in the present study was to analyze the effect of polymer materials to prevent the cell membrane protein Laminin from being denatured and, hence, reducing impacts to the head evaluated by using two different mechanical methods, denatured electrophoresis and electron microscopy. Thus, following dynamic impacts without and with the polymer materials the results showed a significant reduction of the force as well as the translational acceleration with up to over 50%. Also, in the present laboratory investigation the mature laminin was used following dynamic impact to find out if this molecule can serve as a complementary analyzer to mechanical methods when searching for optimal protective materials. The results showed that the polymer materials had the capacity to save the laminin structures from both fragmentation and aggregation as evaluated with denatured electrophoresis and electron microscopy. Therefore, proteins may complement today's calculation simulations and mechanical investigations in the search for improved protective systems to the skull bone and brain tissue. The present result shows that laminin structures may become a valuable method to further identify new structures on a molecular level in the search for improved protective materials to the brain tissue at physical exercise and at accidents.

Among the first steps in inflammation is the conversion of arachidonic acid (AA) stored in the cell membranes into leukotrienes. This occurs mainly in leukocytes and depends on the interaction of two proteins: 5-lipoxygenase (5LO), stored away from the nuclear membranes until use and 5-lipoxygenase activating protein (FLAP), a transmembrane, homotrimeric protein, constitutively present in nuclear membrane. We could earlier visualize the binding of 5LO to nanodiscs in the presence of Ca2+-ions by the use of transmission electron microscopy (TEM) on samples negatively stained by sodium phosphotungstate. In the absence of Ca2+-ions 5LO did not bind to the membrane. In the present communication, FLAP reconstituted in the nanodiscs which could be purified if the His-tag was located on the FLAP C-terminus but not the N-terminus. Our aim was to find out if 1) 5LO would bind in a Ca2+-dependent manner also when FLAP is present? 2) Would the substrate (AA) have effects on 5LO binding to FLAP-nanodiscs? TEM was used to assess the complex formation between 5LO and FLAP-nanodiscs along with, sucrose gradient purification, gel-electrophoresis and mass spectroscopy. It was found that presence of AA by itself induces complex formation in the absence of added calcium. This finding corroborates that AA is necessary for the complex formation and that a Ca2+-flush is mainly needed for the recruitment of 5LO to the membrane. Our results also showed that the addition of Ca2+-ions promoted binding of 5LO on the FLAP-nanodiscs as was also the case for nanodiscs without FLAP incorporated. In the absence of added substances no 5LO-FLAP complex was formed. Another finding is that the formation of a 5LO-FLAP complex appears to induce fragmentation of 5LOin vitro.

Increased intracellular water content defined as cytotoxic brain tissue edema is a serious secondary clinical complication to traumatic brain injury (TBI) and stroke and without knowledge to the et ...

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca(2+)-induced binding of 5-lipoxygenase on nanodiscs is presented.

Leukotriene A4 hydrolase/aminopeptidase (LTA4H) (EC 3.3.2.6) is a bifunctional zinc metalloenzyme with both an epoxide hydrolase and an aminopeptidase activity. LTA4H from the African claw toad, Xenopus laevis (xlLTA4H) has been shown to, unlike the human enzyme, convert LTA4 to two enzymatic metabolites, LTB4 and another biologically active product Δ(6)-trans-Δ(8)-cis-LTB4 (5(S),12R-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid). In order to study the molecular aspect of the formation of this product we have characterized the structure and function of xlLTA4H. We solved the structure of xlLTA4H to a resolution of 2.3Å. It is a dimeric structure where each monomer has three domains with the active site in between the domains, similar as to the human structure. An important difference between the human and amphibian enzyme is the phenylalanine to tyrosine exchange at position 375. Our studies show that mutating F375 in xlLTA4H to tyrosine abolishes the formation of the LTB4 isomeric product Δ(6)-trans-Δ(8)-cis-LTB4. In an attempt to understand how one amino acid exchange leads to a new product profile as seen in the xlLTA4H, we performed a conformer analysis of the triene part of the substrate LTA4. Our results show that the Boltzmann distribution of substrate conformers correlates with the observed distribution of products. We suggest that the observed difference in product profile between the human and the xlLTA4H arises from different level of discrimination between substrate LTA4 conformers.

Human 5-lipoxygenase (5-LOX) is responsible for the formation of leukotriene (LT)A4, a pivotal intermediate in the biosynthesis of the leukotrienes, a family of proinflammatory lipid mediators. 5-LOX has thus gained attention as a potential drug target. However, details of the kinetic mechanism of 5-LOX are still obscure. In this Letter, we investigated the kinetic isotope effect (KIE) of 5-LOX with its physiological substrate, arachidonic acid (AA). The observed KIE is 20±4 on kcat and 17±2 on kcat/KM at 25°C indicating a non-classical reaction mechanism. The observed rates show slight temperature dependence at ambient temperatures ranging from 4 to 35°C. Also, we observed low Arrhenius prefactor ratio (AH/AD=0.21) and a small change in activation energy (Ea(D)-Ea(H)=3.6J/mol) which suggests that 5-LOX catalysis involves tunneling as a mechanism of H-transfer. The measured KIE for 5-LOX involves a change in regioselectivity in response to deuteration at position C7, resulting in H-abstraction form C10 and formation of 8-HETE. The viscosity experiments influence the (H)kcat, but not (D)kcat. However the overall kcat/KM is not affected for labeled or unlabeled AA, suggesting that either the product release or conformational rearrangement might be involved in dictating kinetics of 5-LOX at saturating conditions. Investigation of available crystal structures suggests the role of active site residues (F421, Q363 and L368) in regulating the donor-acceptor distances, thus affecting H-transfer as well as regiospecificity. In summary, our study shows that that the H-abstraction is the rate limiting step for 5-LOX and that the observed KIE of 5-LOX is masked by a change in regioselectivity.

An important step in the production of inflammatory mediators of the leukotriene family is the Ca2+ mediated recruitment of 5 Lipoxygenase (5LO) to nuclear membranes. To study this reaction in vitro, the natural membrane mimicking environment of nanodiscs was used. Nanodiscs with 10.5 nm inner diameter were made with the lipid POPC and membrane scaffolding protein MSP1E3D1. Monomeric and dimeric 5LO were investigated. Monomeric 5LO mixed with Ca2+ and nanodiscs are shown to form stable complexes that 1) produce the expected leukotriene products from arachidonic acid and 2) can be, for the first time, visualised by native gel electrophoresis and negative stain transmission electron micros-copy and 3) show a highest ratio of two 5LO per nanodisc. We interpret this as one 5LO on each side of the disc. The dimer of 5LO is visualised by negative stain transmission electron microscopy and is shown to not bind to nanodiscs. This study shows the advantages of nanodiscs to obtain basic structural information as well as functional information of a complex between a monotopic membrane protein and the membrane. QC 20160428

Inflammation is one of the innate defense mechanisms exerted by the human body for protection and to initiate the healing process. Prolonged inflammatory reactions can lead to chronic disease conditions like atherosclerosis, asthma and myocardial infarction. Leukotrienes (LTs) are one of several pro-inflammatory lipid mediators involved in such inflammatory diseases and are derived from arachidonic acid (AA). The key enzyme involved in LT biosynthesis is 5 Lipoxygenase (5LO), Five lipoxygenase activating protein (FLAP) an integral membrane protein and Coactosin like protein (CLP) a scaffolding protein. Upon external stimuli, intracellular calcium concentration increases which translocates 5LO from the cytosol to the nuclear membrane and localizes near FLAP. Then 5LO converts the AA to leukotriene A4. The hypothesis is that AA is transferred from the nuclear membrane to 5LO by homo-trimeric FLAP and CLP is also involved with 5LO in this stage. Though this hypothesis has been studied extensively, the association between these proteins in LT biosynthesis is still clouded. To elucidate these assisted interactions, we reconstituted the FLAP into “Nanodisc” a membrane mimicking system. We grouped the project by first analyzing the interaction of 5LO with ND, to simulate and understand the calcium mediated translocation of 5LO to nuclear membrane in this ND system. We then proceeded to use FLAP-containing nanodiscs (FND) and repeated aforementioned analyses. We employed biochemical assays and transmission electron microscopy to characterize the interactions and to create a 3D model of the functional complex of 5LO,CLP and FLAP. Here, we show our results from the above mentioned projects involved in understanding the interaction of proteins involved in the initiation of leukotriene biosynthesis.

Leukotrienes are pro-inflammatory lipid mediators involved in chronic inflammatory diseases like asthma and atherosclerosis. Different enzymes and proteins are involved in the leukotriene biosynthesis pathway that stems from oxygenation of arachidonic acid (AA) by 5LO (5-lipoxygenase). FLAP (5-lipoxygenase activating protein) is an integral membrane protein, belonging to the MAPEG (Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism) family. Till now, there is no prominent evidence for any biochemical or enzymatic activity for FLAP, except involvement as an “activator” in leukotriene biosynthesis. The hypothesis is that, on increase in intracellular calcium concentration, 5LO moves from the cytosol to the nuclear membrane and localizes near FLAP. The AA is then transferred from the nuclear membrane to 5LO by homo-trimeric FLAP. Then 5LO converts the AA to leukotriene A4. This interaction between FLAP and 5LO is ambiguous due to intricate mechanisms which occur at the interface of the nuclear membrane and also between an enzyme and an integral membrane protein with no documented function. To understand the function and involvement of FLAP in leukotriene biosynthesis in vitro, we employ soluble phospholipid bilayers called “Nanodiscs” which mimic a membrane environment. The nanodisc will act as a stable platform for structural and functional characterization of the interaction between 5LO and FLAP. After initial studies of the Ca2+dependent recruitment of 5LO to empty nanodiscs as well as the reconstitution of FLAP into nanodiscs, the entire complex 5LO-FLAP-nanodisc can be targeted. We employ biochemical assays to characterize the interactions and transmission electron microscopy (single particle analysis) to create a 3D model of the functional complex of 5LO and FLAP. Here, we portray our outcomes from the above mentioned subprojects encompassed in understanding the interaction of 5LO and FLAP.