Membrane proteins are of outstanding importance in biology, drug discovery and vaccination. A common limiting factor in research and applications involving membrane proteins is the ability to solubilize and stabilize membrane proteins. Although detergents represent the major means for solubilizing membrane proteins, they are often associated with protein instability and poor applicability in structural and biophysical studies. Here, we present a novel lipoprotein nanoparticle system that allows for the reconstitution of membrane proteins into a lipid environment that is stabilized by a scaffold of Saposin proteins. We showcase the applicability of the method on two purified membrane protein complexes as well as the direct solubilization and nanoparticle-incorporation of a viral membrane protein complex from the virus membrane. We also demonstrate that this lipid nanoparticle methodology facilitates high-resolution structural studies of membrane proteins in a lipid environment by single-particle electron cryo-microscopy (cryo-EM) and allows for the stabilization of the HIV-envelope glycoprotein in a functional state.

Prostaglandins (PG) are bioactive lipids produced from arachidonic acid via the action of cyclooxygenases and terminal PG synthases. Microsomal prostaglandin E synthase 1 (MPGES1) constitutes an inducible glutathione-dependent integral membrane protein that catalyzes the oxidoreduction of cyclooxygenase derived PGH 2 into PGE2. MPGES1 has been implicated in a number of human diseases or pathological conditions, such as rheumatoid arthritis, fever, and pain, and is therefore regarded as a primary target for development of novel antiinflammatory drugs. To provide a structural basis for insight in the catalytic mechanism, we determined the structure of MPGES1 in complex with glutathione by electron crystallography from 2D crystals induced in the presence of phospholipids. Together with results from site-directed mutagenesis and activity measurements, we can thereby demonstrate the role of specific amino acid residues. Glutathione is found to bind in a U-shaped conformation at the interface between subunits in the protein trimer. It is exposed to a site facing the lipid bilayer, which forms the specific environment for the oxidoreduction of PGH 2 to PGE 2 after displacement of the cytoplasmic half of the N-terminal transmembrane helix. Hence, insight into the dynamic behavior of MPGES1 and homologous membrane proteins in inflammation and detoxification is provided.electron crystallography ͉ inflammation ͉ MAPEG ͉ membrane protein M icrosomal prostaglandin E synthase 1 (MPGES1) is the key enzyme in pathology related production of PGE 2 from cyclooxygenase (Cox) derived PGH 2 (1). The protein is a member of the MAPEG protein family, which includes 5-lipoxygenase activating protein (FLAP), leukotriene C 4 synthase (LTC4S), microsomal glutathione transferase (MGST)1, MGST2, and MGST3 (2, 3). MPGES1 is the most efficient PGES known and catalyzes the oxidoreduction of prostaglandin endoperoxide H 2 into PGE 2 with an apparent k cat /K m of 310 mM Ϫ1 s Ϫ1 [supporting information (SI) Fig. S1]. The enzyme equally well catalyses the oxidoreduction of endocannabinoids into prostaglandin glycerol esters (4) and PGG 2 into 15-hydroperoxy-PGE 2 (5). In addition, the enzyme confers low glutathione transferase and glutathione-dependent peroxidase activities (5). The biological significance of the latter activities remains unclear but is thought to reflect the close evolutionary distance to MGST1.MPGES1 protein expression levels are in most cases low, and proinflammatory stimuli induce its cellular expression and activity, which is prevented by corticosteroids (1, 6-8). The predominant source of PGH 2 seems derived from Cox-2, although Cox-1 may also contribute (9). Studies, mainly from disruption of the MPGES1 gene in mice, indicate key roles for MPGES1-generated PGE 2 in pathological conditions such as chronic inflammation, pain, fever, anorexia, atherosclerosis, stroke and tumorigenesis (10). Recently, a role for MPGES1 in regulating neonatal respiration was described in ref. 11. MPGES1 has been shown to be overexpressed in rheu...

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Short‐chain peptides are transported across membranes through promiscuous proton‐dependent oligopeptide transporters (POTs)—a subfamily of the major facilitator superfamily (MFS). The human POTs, PEPT1 and PEPT2, are also involved in the absorption of various drugs in the gut as well as transport to target cells. Here, we present a structure of an oligomeric POT transporter from Shewanella oneidensis (PepTSo2), which was crystallized in the inward open conformation in complex with the peptidomimetic alafosfalin. All ligand‐binding residues are highly conserved and the structural insights presented here are therefore likely to also apply to human POTs.

EPR spectroscopy was applied to investigate the inhibition of electron transport in photosystem II by Cu2+ ions. Our results show that Cu2+ has inhibitory effects on both the donor and the acceptor side of photosystem II. In the presence of Cu2+, neither EPR signal IIvery fast nor signal IIfast, which both reflect oxidation of tyrosinez, could be induced by illumination. This shows that Cu2+ inhibits electron transfer from tyrosinez to the oxidized primary donor P680+. Instead of tyrosinez oxidation, illumination results in the formation of a new radical with g = 2.0028 +/- 0.0002 and a spectral width of 9.5 +/- 0.3 G. At room temperature, this radical amounts to one spin per PS II reaction center. Incubation of photosystem II membranes with cupric ions also results in release of the 16 kDa extrinsic subunit and conversion of cytochrome b559 to the low-potential form. On the acceptor side, QA can still be reduced by illumination or chemical reduction with dithionite. However, incubation with Cu2+ results in loss of the normal EPR signal from QA- which is coupled to the non-heme Fe2+ on the acceptor side (the QA(-)-Fe2+ EPR signal). Instead, reduction of QA results in the formation of a free radical spectrum which is 9.5 G wide and centered at g = 2.0044. This signal is attributed to QA- which is magnetically decoupled from the non-heme iron. This suggests that Cu2+ displaces the Fe2+ or severely alters its binding properties. The inhibition of tyrosinez is reversible upon removal of the copper ions with EDTA while the modification of QA was found to be irreversible.

Tumour hypoxia contributes to poor treatment outcome in locally advanced rectal cancer (LARC) and circulating extracellular vesicles (EVs) as potential biomarkers of tumour hypoxia and adverse prognosis have not been fully explored. We examined EV miRNAs from hypoxic colorectal cancer cell lines as template for relevant miRNAs in LARC patients participating in a prospective biomarker study (NCT01816607). Five cell lines were cultured under normoxia (21% O2) or hypoxia (0.2% O2) for 24 h, and exosomes were isolated by differential ultracentrifugation. Using a commercial kit, exosomes were precipitated from 24 patient plasma samples collected at the time of diagnosis. Exosome size distribution and protein cargo were determined by cryo-electron microscopy, nanoparticle tracking analysis, immunoblotting and flow cytometry. The vesicles harboured strong cell line-specific miRNA profiles with 35 unique miRNAs differentially expressed between hypoxic and normoxic cells. Six of these miRNAs were considered candidate-circulating markers of tumour hypoxia in the patients based on the frequency or magnitude of variance in hypoxic versus normoxic cell line experiments and prevalence in patient plasma. Of these, low plasma levels of exosomal miR-486-5p and miR-181a-5p were associated with organ-invasive primary tumour (p = 0.029) and lymph node metastases (p = 0.024), respectively, both attributes of adverse LARC prognosis. In line with this, the plasma level of exosomal miR-30d-5p was elevated in patients who experienced metastatic progression (p = 0.036). Our strategy confirmed that EVs from colorectal cancer cell lines were exosomes containing the oxygen-sensitive miRNAs 486-5p, 181a-5p and 30d-5p, which were retrieved as circulating markers of high-risk LARC.

Human, microsomal, and glutathione-dependent prostaglandin (PG) E synthase-1 (mPGES-1) was expressed with a histidine tag in Escherichia coli. mPGES-1 was purified to apparent homogeneity from Triton X-100-solubilized bacterial extracts by a combination of hydroxyapatite and immobilized metal affinity chromatography. The purified enzyme displayed rapid glutathionedependent conversion of PGH 2 to PGE 2 (V max ; 170 mol min ؊1 mg ؊1 ) and high k cat /K m (310 mM ؊1 s ؊1 ). Purified mPGES-1 also catalyzed glutathione-dependent conversion of PGG 2 to 15-hydroperoxy-PGE 2 (V max ; 250 mol min ؊1 mg ؊1 ). The formation of 15-hydroperoxy-PGE 2 represents an alternative pathway for the synthesis of PGE 2 , which requires further investigation. Purified mPGES-1 also catalyzed glutathione-dependent peroxidase activity toward cumene hydroperoxide (0.17 mol min ؊1 mg ؊1 ), 5-hydroperoxyeicosatetraenoic acid (0.043 mol min ؊1 mg ؊1 ), and 15-hydroperoxy-PGE 2 (0.04 mol min ؊1 mg ؊1 ). In addition, purified mPGES-1 catalyzed slow but significant conjugation of 1-chloro-2,4-dinitrobenzene to glutathione (0.8 mol min ؊1 mg ؊1 ). These activities likely represent the evolutionary relationship to microsomal glutathione transferases. Two-dimensional crystals of purified mPGES-1 were prepared, and the projection map determined by electron crystallography demonstrated that microsomal PGES-1 constitutes a trimer in the crystal, i.e. an organization similar to the microsomal glutathione transferase 1. Hydrodynamic studies of the mPGES-1-Triton X-100 complex demonstrated a sedimentation coefficient of 4.1 S, a partial specific volume of 0.891 cm 3 /g, and a Stokes radius of 5.09 nm corresponding to a calculated molecular weight of 215,000. This molecular weight, including bound Triton X-100 (2.8 g/g protein), is fully consistent with a trimeric organization of mPGES-1.Prostaglandin (PG) 1 E 2 is a prostanoid with potent biological functions; among those functions, its role as a mediator of pain and fever in inflammatory reactions is considered of major importance (1-4). The biosynthesis of PGE 2 from arachidonic acid is catalyzed in a sequential action by PGH synthase (PGHS) forming first the endoperoxide PGG 2 and then PGH 2 by reduction. Subsequently, PGE synthase (PGES) (EC 5.3.99.3) converts PGH 2 into PGE 2 (5). Two forms of PGHS exist, PGHS-1 and PGHS-2, with similar enzymatic properties but distinctly different biological functions. PGHS-1 is constitutively expressed in many cells and organs and takes part in housekeeping functions such as the regulation of vascular homeostasis. PGHS-2, in contrast, is strongly induced in response to proinflammatory stimuli and takes part in various pathophysiological events (6, 7). PGES activity, in most cases glutathione (GSH)-dependent, has been detected both in microsomal and cytosolic fractions of various cells, and apparently, more than one form of PGES exist (8 -12). Microsomal, inducible PGES-1 (mPGES-1) is a member of the membrane-associated proteins in eicosanoid and glutathione metabo...

Microsomal prostaglandin E synthase-1 (MPGES1) is induced during an inflammatory reaction from low basal levels by pro-inflammatory cytokines and subsequently involved in the production of the important mediator of inflammation, prostaglandin E2. Nonsteroidal anti-inflammatory drugs prevent prostaglandin E2 production by inhibiting the upstream enzymes cyclooxygenases 1 and 2. In contrast to these conventional drugs, a new generation of NSAIDs targets the terminal enzyme MPGES1. Some of these compounds potently inhibit human MPGES1 but do not have an effect on the rat orthologue. We investigated this interspecies difference in a rat/human chimeric form of the enzyme as well as in several mutants and identified key residues Thr-131, Leu-135, and Ala-138 in human MPGES1, which play a crucial role as gate keepers for the active site of MPGES1. These residues are situated in transmembrane helix 4, lining the entrance to the cleft between two subunits in the protein trimer, and regulate access of the inhibitor in the rat enzyme. Exchange toward the human residues in rat MPGES1 was accompanied with a gain of inhibitor activity, whereas exchange in human MPGES1 toward the residues found in rat abrogated inhibitor activity. Our data give evidence for the location of the active site at the interface between subunits in the homotrimeric enzyme and suggest a model of how the natural substrate PGH2, or competitive inhibitors of MPGES1, enter the active site via the phospholipid bilayer of the membrane.