Elucidation of the MD-2/TLR4 interface required for signaling by lipid IVa

LPS signals through a membrane bound-complex of the lipid binding protein MD-2 and the receptor TLR4. In this study we identify discrete regions in both MD-2 and TLR4 that are required for signaling by lipid IVa, an LPS derivative that is an agonist in horse but an antagonist in humans. We show that changes in the electrostatic surface potential of both MD-2 and TLR4 are required in order that lipid IVa can induce signaling. In MD-2, replacing horse residues 57-66 and 82-89 with the equivalent human residues confers a level of constitutive activity on horse MD-2, suggesting that conformational switching in this protein is likely to be important in ligand-induced activation of MD-2/TLR4. We identify leucine-rich repeat 14 in the C terminus of TLR4 as essential for lipid IVa activation of MD-2/TLR4. Remarkably, we identify a single residue in the glycan-free flank of the horse TLR4 solenoid that confers the ability to signal in response to lipid IVa. These results suggest a mechanism of signaling that involves crosslinking mediated by both MD-2-receptor and receptor-receptor contacts in a model that shows striking similarities to the recently published structure (Cell 130: 1071-1082) of the ligand-bound TLR1/2 ectodomain heterodimer.     

Mutational analysis of intrinsic regions of presenilin 2 that determine its endoproteolytic cleavage and pathological function

To investigate the significance of endoproteolytic processing of presenilin 2 (PS2) on its pathological function, we constructed PS2 cDNAs causing amino acid substitutions or deletions around the cleavage site. We found that a PS2 mutant (Del3) with a 20-amino acid deletion was not endoproteolytically processed, while other PS2s with amino acid substitutions and short deletions were cleaved. Overproduction of all the mutant proteins led to a compensatory decrease of endogenous PS1 fragments, but did not affect the amyloid beta peptide X-42/Abeta X-40 ratio without the familial Alzheimer's disease mutation. The Del3 mutant did not exhibit significant deficits in gamma-secretase activity. The turnover rate of the Del3 holoprotein was the same as that of full-length PS2. These data suggest that the determinants of the PS2 cleavage site reside within a large region and that the pathological function of PS2 is exerted by familial Alzheimer's disease mutations not related to the cleavage of holoproteins. We also found that PS2 with an 18-amino acid deletion at the C-terminal end was not processed. Its overexpression led neither to diminished accumulation of endogenous PS1 fragments nor to increased production of amyloid beta peptide X-42. The C-terminal end of PS2 seems to possess the signal for entry into the processing pathway.     

Identification of a novel isoform of MD-2 that downregulates lipopolysaccharide signaling

MD-2 is an association molecule of Toll-like receptor 4 and is indispensable for the recognition of lipopolysaccharide. Here we report the identification of mRNA for an alternatively spliced form of MD-2, named MD-2B, which lacks the first 54 bases of exon 3. When overexpressed with MD-2, MD-2B competitively suppressed NF-kappaB activity induced by LPS. Regardless of the truncation, however, MD-2B still bound to TLR4 as efficiently as MD-2. Flow cytometric analyses revealed that MD-2B inhibited TLR4 from being expressed on the cell surface. Our data indicate that MD-2B may compete with MD-2 for binding to TLR4 and decrease the number of TLR4/MD-2 complexes on the cell surface, resulting in the inhibition of LPS signaling.     

Structural characteristics of a lipid peroxidation product, trans-2-nonenal, that favour inhibition of membrane-associated phosphotyrosine phosphatase activity

Protein-tyrosine phosphatases (PTPs) are very susceptible to oxidation by reactive oxygen species (ROS), which induce the oxidation of catalytic cysteines, thereby inactivating these PTPs. PTPs are also inactivated by treatment with different aldehydes (such as trans-2-nonenal), produced after tissue damage by ROS. However, the molecular mechanisms behind such aldehyde-due inactivation remain unknown. Using commercially available compounds, we examined the structural characteristics of trans-2-nonenal that allow the inhibition of platelet membrane-associated PTP activity, as well as how these compounds affect the dynamics of SH-, CO- and NH2- protein groups on the membranes. PTP was effectively inhibited by physiological amounts of trans-2-nonenal (1-10 microM). Incubation with trans-2-nonene (10 microM) also decreased PTP activity, although to a lower extent. Treatment with nonyl aldehyde almost eliminated PTP inhibition. Decreases in protein thiols were visible after trans-2-nonenal and trans-2-nonene treatments. Both the latter compounds also increased protein carbonyls (although trans-2-nonenal was more effective) and decreased protein amino groups to an equal extent. Collectively, our data indicate that alpha,beta unsaturation (and not a double bond in another position) is the most important structural determinant for PTP inhibition, the alkenal with 9-carbon atoms being the most effective in eliciting such inhibition. The data allow us to predict the modification of sulfhydryls and/or the formation of addition products with lysyl or histidyl residues, and hence the kind of specific antibodies that it would be necessary to generate in order to test such modifications directly.     

Human MD-2 discrimination of meningococcal lipid A structures and activation of TLR4

MD-2, a eukaryotic accessory protein, is an essential component for the molecular pattern recognition of bacterial endotoxins. MD-2 interacts with lipid A of endotoxins [lipopolysaccharide (LPS) or lipooligosaccharide (LOS)] to activate human toll-like receptor (TLR) 4. The structure of lipid A influences the subsequent activation of human TLR4 and the immune response, but the basis for the discrimination of lipid A structures is unclear. A recombinant human MD-2 (rMD-2) protein was produced in the Pichia pastoris yeast expression system. Human embryonic kidney (HEK293) cells were transfected with human TLR4 and were stimulated with highly purified LOS (0.56 pmol) from Neisseria meningitidis or LPS from other structurally defined bacterial endotoxins in the presence or absence of human rMD-2. Human rMD-2 restored, in a dose-dependent manner, interleukin (IL-8) responsiveness to LOS or LPS in TLR4-transfected HEK293 cells. The interaction of endotoxin with human rMD-2 was then assessed by enzyme-linked immunosorbent assays. Wild-type meningococcal LOS (Wt m LOS) bound human rMD-2, and binding was inhibited by an anti-MD-2 antibody to MD-2 dose-dependently (P < 0.005). Wt m LOS or meningococcal KDO(2)-lipid A had the highest binding affinity for human rMD-2; unglycosylated meningococcal lipid A produced by meningococci with defects in the 3-deoxy-d-manno-2-octulosonic acid (KDO) biosynthesis pathway did not appear to bind human rMD-2 (P < 0.005). The affinity of meningococcal LOS with a penta-acylated lipid A for human rMD-2 was significantly less than that for hexa-acylated LOS (P < 0.05). The hierarchy in the binding affinity of different lipid A structures for human rMD-2 was directly correlated with differences in TLR4 pathway activation and cytokine production by human macrophages.     

Defining the regions of Escherichia coli YidC that contribute to activity

The YidC/Oxa1/Alb3 family of proteins catalyzes membrane protein insertion in bacteria, mitochondria, and chloroplasts. In this study, we investigated which regions of the bacterial YidC protein are important for its function in membrane protein biogenesis. In Escherichia coli, YidC spans the membrane six times, with a large 319-residue periplasmic domain following the first transmembrane domain. We found that this large periplasmic domain is not required for YidC function and that the residues in the exposed hydrophilic loops or C-terminal tail are not critical for YidC activity. Rather, the five C-terminal transmembrane segments that contain the three consensus sequences in the YidC/Oxa1/Alb3 family are important for its function. However, by systematically replacing all the residues in transmembrane segment (TM) 2, TM3, and TM6 with serine and by swapping TM4 and TM5 with unrelated transmembrane segments, we show that the precise sequence of these transmembrane regions is not essential for in vivo YidC activity. Single serine mutations in TM2, TM3, and TM6 impaired the membrane insertion of the Sec-independent procoat-leader peptidase protein. We propose that the five C-terminal transmembrane segments of YidC function as a platform for the translocating substrate protein to support its insertion into the membrane.     

Structural basis for the activity of drugs that inhibit phosphodiesterases

Phosphodiesterases (PDEs) comprise a large family of enzymes that catalyze the hydrolysis of cAMP or cGMP and are implicated in various diseases. We describe the high-resolution crystal structures of the catalytic domains of PDE4B, PDE4D, and PDE5A with ten different inhibitors, including the drug candidates cilomilast and roflumilast, for respiratory diseases. These cocrystal structures reveal a common scheme of inhibitor binding to the PDEs: (i) a hydrophobic clamp formed by highly conserved hydrophobic residues that sandwich the inhibitor in the active site; (ii) hydrogen bonding to an invariant glutamine that controls the orientation of inhibitor binding. A scaffold can be readily identified for any given inhibitor based on the formation of these two types of conserved interactions. These structural insights will enable the design of isoform-selective inhibitors with improved binding affinity and should facilitate the discovery of more potent and selective PDE inhibitors for the treatment of a variety of diseases.     

Structural heterogeneity regarding local Shwartzman activity of lipid A

The relation of chemical structure to local Shwartzman activity of lipid A preparations purified by thin-layer chromatography from five bacterial strains was examined. Two lipid A fractions from E. coli F515--Ec-A2 and Ec-A3--exhibited strong activity, similar to that of previous synthetic E. coli-type lipid A (compound 506 or LA-15-PP). The Ec-A3 fraction contained a component that appeared to be structurally identical to compound 506, and the main component of Ec-A2 fraction was structurally similar to compound 506 except that it carried a 3-hydroxytetradecanoyl group at the C-3' position of the backbone in place of a 3-tetradecanoyloxytetradecanoyl group. Free lipid A (12 C) and purified lipid A fractions, Ec-A2 (12 C) and Ec-A3 (12 C), respectively, obtained from bacteria grown at 12 C, exhibited activity comparable to Ec-A2 or Ec-A3. In these preparations, a large part of the 3-dodecanoyloxytetradecanoyl group might be replaced by 3-hexadecenoyloxytetradecanoyl group. Salmonella minnesota R595 free lipid A also contained at least two active lipid A components as seen in E. coli lipid A, but the third component corresponding to the synthetic Salmonella-type lipid A (compound 516 or LA-16-PP) exhibited low activity. A lipid A fraction, Cv-A4 from Chromobacterium violaceum IFO 12614, which was proposed to have two acyloxyacyl groups at the C-2 and C-2' positions with other acyl groups, exhibited weaker activity than the free lipid A or LPS. The purified lipid A fractions from Pseudomonas diminuta JCM 2788 and Pseudomonas vesicularis JCM 1477 contained an unusual backbone with 2,3-diamino-2,3-dideoxy-D-glucose disaccharide phosphomonoester, and these lipid A (Pd-A3 and Pv-A3) exhibited strong activity comparable to the E. coli lipid A. Thus, the present results show that the local Shwartzman reaction can be expressed by partly different lipid A structures in both hydrophilic backbone and fatty acyl residues; when they have the same backbone the potency varies markedly depending on the structure of the acyl residues.     

Identification of the individual residues that determine human CD59 species selective activity

Formation of the cytolytic membrane attack complex of complement on host cells is inhibited by the membrane-bound glycoprotein, CD59. The inhibitory activity of CD59 is species restricted, and human CD59 is not effective against rat complement. Previous functional analysis of chimeric human/rat CD59 proteins indicated that the residues responsible for the species selective function of human CD59 map to a region contained between positions 40 and 66 in the primary structure. By comparative analysis of rat and human CD59 models and by mutational analysis of candidate residues, we now identify the individual residues within the 40-66 region that confer species selective function on human CD59. All nonconserved residues within the 40-66 sequence were substituted from human to rat residues in a series of chimeric human/rat CD59 mutant proteins. Functional analysis revealed that the individual human to rat residue substitutions F47A, T51L, R55E, and K65Q each produced a mutant human CD59 protein with enhanced rat complement inhibitory activity with the single F47A substitution having the most significant effect. Interestingly, the side chains of the residues at positions 47, 51, and 55 are all located on the short single helix (residues 47-55) of CD59 and form an exposed continuous strip parallel to the helix axis. A single human CD59 mutant protein containing rat residue substitutions at all three helix residues produced a protein with species selective activity comparable to that of rat CD59. We further found that synthetic peptides spanning the human CD59 helix sequence were able to inhibit the binding of human CD59 to human C8, but had little effect on the binding of rat CD59 to rat C8.     

Identification of functionally distinct regions that mediate biological activity of the protein kinase a homolog Tpk2

Kinases regulate key signaling processes that are increasingly implicated in development and disease. Kinase modulators have become important therapeutic tools and often target catalytic domains that are among the most structurally and functionally conserved regions of these enzymes. Such therapies lose efficacy as mutations conferring resistance arise. Because interactions between distinct and often distant regions of kinases can be critical, we took an unbiased genetic approach to identify sites within the protein kinase A homolog Tpk2 that contribute to its biological activity. Because many of these map outside the conserved core, this approach should be broadly useful in identifying new, more kinase-specific therapeutic targets.     

Measurement of doxorubicin-induced lipid peroxidation under the conditions that determine cytotoxicity in cultured tumor cells.

We have investigated doxorubicin-induced lipid peroxidation by the measure of malondialdehyde (MDA) formation in rat glioblastoma cells and human breast carcinoma cells in culture. There was a significant production of MDA when the cells were incubated with pharmacologically relevant doxorubicin concentrations, i.e., concentrations that produce a significant cytotoxicity (0.1 micrograms/ml). At equitoxic doses, vincristine provided no lipid peroxidation, indicating that MDA formation is not a consequence of cell death. Doxorubicin-induced lipid peroxidation was maximal 24 h after incubation of the cells with doxorubicin, indicating that a delay was necessary for the free radical-mediated membrane damage induced by doxorubicin. In the presence of alpha-tocopherol in the culture medium, the doxorubicin-induced MDA formation was inhibited. The development of this method will help in defining the role of free radicals and lipid peroxidation in the cytotoxicity of doxorubicin.     

Ornithine-containing lipid of Bordetella pertussis that carries hemagglutinating activity

The proposed structure of the ornithine-containing lipid of Bordetella pertussis is 3-hydroxyhexadecanoic acid amide-linked to ornithine and esterified to the second hexadecanoic acid. The aminolipid strongly agglutinates type A and B human erythrocytes.     

Structural determinants that target the hepatitis C virus core protein to lipid droplets

Hepatitis C virus core protein is targeted to lipid droplets, which serve as intracellular storage organelles, by its C-terminal domain, termed D2. From circular dichroism and nuclear magnetic resonance analyses, we demonstrate that the major structural elements within D2 consist of two amphipathic alpha-helices (Helix I and Helix II) separated by a hydrophobic loop. Both helices require a hydrophobic environment for folding, indicating that lipid interactions contribute to their structural integrity. Mutational studies revealed that a combination of Helix I, the hydrophobic loop, and Helix II is essential for efficient lipid droplet association and pointed to an in-plane membrane interaction of the two helices at the phospholipid layer interface. Aside from lipid droplet association, membrane interaction of D2 is necessary for folding and stability of core following maturation at the endoplasmic reticulum membrane by signal peptide peptidase. These studies identify critical determinants within a targeting domain that enable trafficking and attachment of a viral protein to lipid droplets. They also serve as a unique model for elucidating the specificity of protein-lipid interactions between two membrane-bound organelles.     

Requirement of the adenovirus IVa2 protein for virus assembly

The adenovirus L1 52/55-kDa protein is required for viral DNA packaging and interacts with the viral IVa2 protein, which binds to the viral packaging sequence. Previous reports suggest that the IVa2 protein plays a role in viral DNA packaging and that this function of the IVa2 protein is serotype specific. To further examine the function of the IVa2 protein in viral DNA packaging, a mutant virus that does not express the IVa2 protein was constructed by introducing two stop codons at the beginning of the IVa2 open reading frame in a full-length bacterial clone of adenovirus type 5. The mutant virus, pm8002, was defective for growth in 293 cells, although it replicated its DNA and produced early and late viral proteins. Electron microscopic and gradient analyses revealed that the mutant virus did not assemble any viral particles in 293 cells. In 293-IVa2 cells, which express the IVa2 protein, infectious viruses were produced, although the titer of the mutant virus was lower than that of the wild-type virus, indicating that these cells may not fully complement the mutation. The mutant viral particles produced in 293-IVa2 cells were heterogeneous in size and shape, less stable, and did not traffic efficiently to the nucleus. Marker rescue experiments with a wild-type IVa2 DNA fragment confirmed that the only mutations present in pm8002 were in the IVa2 gene. The results indicate that the IVa2 protein is required for adenovirus assembly and suggest that virus particles may be assembled around the DNA rather than DNA being packaged into preformed capsids.     

Two regions in the isolated brainstem of the frog that modulate respiratory-related activity

Using microinjection techniques, we have explored the isolated, complete midline sectioned brainstem of the frog (Rana catesbeiana) to identify regions that influence the endogenous respiratory-related motor activity. Ten-nanoliter injections of lidocaine (1%), GABA (100 mM) and glutamate (10 and 100 mM) into discrete regions of the rostral and the caudal brainstem produced different effects on the phasic neural discharge. In the rostral site lidocaine, GABA and glutamate injections altered neural burst frequency with little or no effect on burst amplitude. In the caudal site, responses to lidocaine and GABA injections consisted primarily of decreases in neural burst amplitude, often, but not always associated with minor decreases in burst frequency. In this same region, the response to glutamate was characterized by a temporary interruption of the rhythmic neural burst activity. The largest responses to substance injection in both regions were obtained at sites ranging between 200 and 500 m from the ventral surface, in the ventral medullary reticular formation. The results reveal the existence of two areas in the frog brainstem that influence respiratory motor output, one related to the respiratory burst frequency and the other related to the amplitude of the motor output.Abbreviations V trigeminal nerve - VI abducens nerve - VII facial nerve - VIII auditory nerve - X vagal nerve - H hypoglossal nerve - VRG ventral respiratory group - NTS nucleus of the solitary tract     

Identification of regions in alleles of the flax rust resistance gene L that determine differences in gene-for-gene specificity

Thirteen alleles (L, L1 to L11, and LH) from the flax L locus, which encode Toll/interleukin-1 receptor homology-nucleotide binding site-leucine-rich repeat (TIR-NBS-LRR) rust resistance proteins, were sequenced and compared to provide insight into their evolution and into the determinants of gene-for-gene resistance specificity. The predicted L6 and L11 proteins differ solely in the LRR region, whereas L6 and L7 differ solely in the TIR region. Thus, specificity differences between alleles can be determined by both the LRR and TIR regions. Functional analysis in transgenic plants of recombinant alleles constructed in vitro provided further information: L10-L2 and L6-L2 recombinants, encoding the LRR of L2, conferred L2 resistance specificity, and an L2-L10 recombinant, encoding the LRR of L10, conferred a novel specificity. The sequence comparisons also indicate that the evolution of L alleles has probably involved reassortment of variation, resulting from accumulated point mutations, by intragenic recombination. In addition, large deletion events have occurred in the LRR-encoding regions of L1 and L8, and duplication events have occurred in the LRR-encoding region of L2.     

Structural features of 5,10-dideaza-5,6,7,8-tetrahydrofolate that determine inhibition of mammalian glycinamide ribonucleotide formyltransferase

We have investigated the structural features of 5,10-dideaza-5,6,7,8-tetrahydrofolate (DDATHF) that determine the activity of this compound as an inhibitor of glycinamide ribonucleotide formyltransferase (GARFT) purified from mouse L1210 cells. 5-Deazatetrahydrofolate was as good an inhibitor of GARFT as DDATHF, indicating that isosteric replacement of nitrogen by carbon at the 5-position of tetrahydrofolate is sufficient for inhibition of GARFT. 5,10-Dideazafolic acid, 5,8,10-trideazatetrahydrofolate, and 2-desamino-5,10-dideazatetrahydrofolate were poor inhibitors of GARFT, indicating that a reduced pyridopyrimidine ring, N-8, and the 2-amino group of DDATHF, respectively, play an important role in the binding of tetrahydrofolate analogues to this enzyme. DDATHF analogues in which the phenyl ring was replaced either by a cyclohexyl ring or by methylene groups retained activity as inhibitors. 5,10-Dideazatetrahydrohomofolate was about 6 times more potent as an inhibitor of GARFT than DDATHF, but 5,10-dideazatetrahydronorfolate had about one-fifth of the activity of DDATHF. An analogue of DDATHF in which the glutamic acid side chain was replaced by aspartic acid (which was not a substrate for polyglutamation and was only weakly cytotoxic) was equiactive with DDATHF as an inhibitor of purified GARFT. Surprisingly, 5,10-dideazatetrahydropteroic acid was about as active as DDATHF as an inhibitor of GARFT, an indication that the glutamic acid in the side chain of DDATHF does not play a role in this ligand-enzyme interaction. The polyglutamate derivatives of DDATHF bound up to 100 times tighter to GARFT than DDATHF itself; longer chain polyglutamates conformed to Goldstein's zone B behavior under experimental conditions and were projected to be in zone C, i.e., stoichiometric inhibition, in vivo. We conclude that the presence of carbon at the 5-position of tetrahydrofolate analogues is sufficient for inhibition of GARFT, that N-8 and the 2-amino group are involved in binding of DDATHF to GARFT, probably through hydrogen bonds, and that the structures of the phenyl ring and amino acid side chain of DDATHF analogues are not primary determinants of GARFT inhibition by monoglutamate forms of these compounds. We also conclude that polyglutamation plays a major role in the potent cytotoxicity of DDATHF.     

Structural features of galectin-9 and galectin-1 that determine distinct T cell death pathways

The galectin family of lectins regulates multiple biologic functions, such as development, inflammation, immunity, and cancer. One common function of several galectins is the ability to trigger T cell death. However, differences among the death pathways triggered by various galectins with regard to glycoprotein receptors, intracellular death pathways, and target cell specificity are not well understood. Specifically, galectin-9 and galectin-1 both kill thymocytes, peripheral T cells, and T cell lines; however, we have found that galectin-9 and galectin-1 require different glycan ligands and glycoprotein receptors to trigger T cell death. The two galectins also utilize different intracellular death pathways, as galectin-9, but not galectin-1, T cell death was blocked by intracellular Bcl-2, whereas galectin-1, but not galectin-9, T cell death was blocked by intracellular galectin-3. Target cell susceptibility also differed between the two galectins, as galectin-9 and galectin-1 killed different subsets of murine thymocytes. To define structural features responsible for distinct activities of the tandem repeat galectin-9 and dimeric galectin-1, we created a series of bivalent constructs with galectin-9 and galectin-1 carbohydrate recognition domains connected by different peptide linkers. We found that the N-terminal carbohydrate recognition domain and linker peptide contributed to the potency of these constructs. However, we found that the C-terminal carbohydrate recognition domain was the primary determinant of receptor recognition, death pathway signaling, and target cell susceptibility. Thus, carbohydrate recognition domain specificity, presentation, and valency make distinct contributions to the specific effects of different galectins in initiating T cell death.     

Mechanical properties that influence antimicrobial peptide activity in lipid membranes

Antimicrobial peptides are small amphiphilic proteins found in animals and plants as essential components of the innate immune system and whose function is to control bacterial infectious activity. In order to accomplish their function, antimicrobial peptides use different mechanisms of action which have been deeply studied in view of their potential exploitation to treat antibiotic-resistant bacterial infections. One of the main mechanisms of action of these peptides is the disruption of the bacterial membrane through pore formation, which, in some cases, takes place via a monomer to oligomer cooperative transition. Previous studies have shown that lipid composition, and the presence of exogenous components, such as cholesterol in model membranes or carotenoids in bacteria, can affect the potency of distinct antimicrobial peptides. At the same time, considering the membrane as a two-dimensional material, it has been shown that membrane composition defines its mechanical properties which might be relevant in many membrane-related processes. Nevertheless, the correlation between the mechanical properties of the membrane and antimicrobial peptide potency has not been considered according to the importance it deserves. The relevance of these mechanical properties in membrane deformation due to peptide insertion is reviewed here for different types of pores in order to elucidate if indeed membrane composition affects antimicrobial peptide activity by modulation of the mechanical properties of the membrane. This would also provide a better understanding of the mechanisms used by bacteria to overcome antimicrobial peptide activity.