Role of Tim4 in the regulation of ABCA1+ adipose tissue macrophages and post-prandial cholesterol levels.

Dyslipidemia is a main driver of cardiovascular diseases. The ability of macrophages to scavenge excess lipids implicate them as mediators in this process and understanding the mechanisms underlying macrophage lipid metabolism is key to the development of new treatments. Here, we investigated how adipose tissue macrophages regulate post-prandial cholesterol transport. Single-cell RNA sequencing and protected bone marrow chimeras demonstrated that ingestion of lipids led to specific transcriptional activation of a population of resident macrophages expressing Lyve1, Tim4, and ABCA1. Blocking the phosphatidylserine receptor Tim4 inhibited lysosomal activation and the release of post-prandial high density lipoprotein cholesterol following a high fat meal. Both effects were recapitulated by chloroquine, an inhibitor of lysosomal function. Moreover, clodronate-mediated cell-depletion implicated Tim4+ resident adipose tissue macrophages in this process. Thus, these data indicate that Tim4 is a key regulator of post-prandial cholesterol transport and adipose tissue macrophage function and may represent a novel pathway to treat dyslipidemia.

Specific inhibition of ectodomain shedding of glycoprotein Ibα by targeting its juxtamembrane shedding cleavage site.

BACKGROUND: Ectodomain shedding of glycoprotein Ibα (GPIbα), a proteolytic event in which metalloprotease ADAM17 cleaves the Gly464-Val465 bond and releases glycocalicin to the plasma, is considered a critical step in mediating clearance of stored platelets. Supporting evidence has largely come from studies using ADAM17 inhibitors. However, the definitive proof is lacking due to the broad substrate specificity of ADAM17. AIM: To achieve substrate-specific inhibition of GPIbα shedding. METHODS: Development of monoclonal antibodies that directly bind the sequence around the GPIbα shedding cleavage site and inhibit GPIbα shedding by blocking ADAM17 access to the cleavage site. RESULTS: Six anti-GPIbα monoclonal antibodies with varying binding affinities were obtained. The prototypic clone, designated 5G6, and its monomeric Fab fragment bind specifically purified GPIb-IX complex, human platelets, and transgenic murine platelets expressing human GPIbα. The clone 5G6 showed similar inhibitory potency as a widely used shedding inhibitor GM6001 in both constitutive and induced GPIbα shedding in human platelets. It does not recognize mouse GPIbα or inhibit shedding of other platelet receptors. Finally, 5G6 binding displays no detectable effect on platelet activation and aggregation. CONCLUSIONS: The clone 5G6 specifically inhibits GPIbα shedding with no detectable effect on platelet functions. The method of substrate-specific shedding inhibition by macromolecular binding of the shedding cleavage site can be applicable to many other transmembrane receptors undergoing ectodomain shedding.

Endogenous cytokinins in Cocos nucifera L. in vitro cultures obtained from plumular explants.

Auxin induces in vitro somatic embryogenesis in coconut plumular explants through callus formation. Embryogenic calli and non-embryogenic calli can be formed from the initial calli. Analysis of endogenous cytokinins showed the occurrence of cytokinins with aromatic and aliphatic side chains. Fourteen aliphatic cytokinins and four aromatic cytokinins were analysed in the three types of calli and all the cytokinins were found in each type, although some in larger proportions than others. The most abundant cytokinins in each type of callus were isopentenyladenine-9-glucoside, zeatin-9-glucoside, zeatin riboside, isopentenyladenine riboside, dihydrozeatin and dihydrozeatin riboside in decreasing order. Total cytokinin content was compared between the three types of calli, and it was found to be lower in embryogenic calli compared to non-embryogenic calli or initial calli. The same pattern was observed for individual cytokinins. When explants were cultured in media containing exogenously added cytokinins, the formation of embryogenic calli in the explants was reduced. When 8-azaadenine (an anticytokinin) was added the formation of embryogenic calli and somatic embryos was increased. These results suggest that the difference in somatic embryo formation capacity observed between embryogenic calli and non-embryogenic calli is related to their endogenous cytokinin contents.

A preliminary time-of-flight neutron diffraction study on amicyanin from Paracoccus denitrificans.

Crystals of the blue copper protein amicyanin suitable for neutron diffraction were grown by the sitting-drop method, followed by repeated macroseeding using solutions prepared with D(2)O. Although the crystal sizes were the same, crystals grown using solutions made up in H(2)O in the initial stages of macroseeding and solutions with D(2)O in later stages did not diffract neutrons well. However, when the protein was initially exchanged with buffered D(2)O and then crystallized and also macroseeded using solutions made up in D(2)O throughout, the crystals diffracted neutrons to high resolution. One of those crystals was used to collect a data set to a resolution of 1.9 A.

Active-site residues are critical for the folding and stability of methylamine dehydrogenase.

Site-directed mutagenesis was used to alter active-site residues of methylamine dehydrogenase (MADH) from Paracoccus denitrificans. Four residues of the beta subunit of MADH which are in close proximity to the tryptophan tryptophylquinone (TTQ) prosthetic group were modified. The crystal structure of MADH reveals that each of these residues participates in hydrogen bonding interactions with other active-site residues, TTQ or water. Relatively conservative mutations which removed the potentially reactive oxygens on the side chains of Thr122, Tyr119, Asp76 and Asp32 each resulted in greatly reduced or undetectable levels of MADH production. The reduction of MADH levels was determined by assays of activity and Western blots of crude extracts with antisera specific for the MADH beta subunit. No activity or cross-reactive protein was detected in extracts of cells expressing D76N, T122A and T122C MADH mutants. Very low levels of active MADH were produced by cells expressing D32N, Y119F, Y119E and Y119K MADH mutants. The Y119F and D32N mutants were purified from cell extracts and found to be significantly less stable than wild-type MADH. Only the T122S MADH mutant was produced at near wild-type levels. Possible roles for these amino acid residues in stabilizing unusual structural features of the MADH beta subunit, protein folding and TTQ biosynthesis are discussed.

Antibody catalysis of the oxidation of water.

Recently we reported that antibodies can generate hydrogen peroxide (H2O2) from singlet molecular oxygen (1O2*). We now show that this process is catalytic, and we identify the electron source for a quasi-unlimited generation of H2O2. Antibodies produce up to 500 mole equivalents of H2O2 from 1O2*, without a reduction in rate, and we have excluded metals or Cl- as the electron source. On the basis of isotope incorporation experiments and kinetic data, we propose that antibodies use H2O as an electron source, facilitating its addition to 1O2* to form H2O3 as the first intermediate in a reaction cascade that eventually leads to H2O2. X-ray crystallographic studies with xenon point to putative conserved oxygen binding sites within the antibody fold where this chemistry could be initiated. Our findings suggest a protective function of immunoglobulins against 1O2* and raise the question of whether the need to detoxify 1O2* has played a decisive role in the evolution of the immunoglobulin fold.

The therapeutic potential for catalytic antibodies: from a concept to a promise.

More than ten years have now elapsed since the first reports confirmed that antibodies not only label antigenic targets but can also perform catalytic functions. Much of the initial research in this area focussed on exploring the scope and utility of these biocatalysts both as enzyme mimics and as programmable protein catalysts. However, their potential in the biomedical field has also been probed. This review details the present perspective of catalytic antibodies as new tools for immunotherapy and specifically focuses on their application to prodrug activation and drug inactivation.

Total synthesis of (+)-zaragozic acid C.

A total synthesis of (+)-zaragozic acid C is described. Key features of the synthesis are the use of a double Sharpless asymmetric dihydroxylation reaction of diene 6 to control stereochemistry at four contiguous stereocenters from C3 to C6; the introduction of the C1-side chain by reaction between the anion derived from the dithiane monosulfoxide 27 and the core aldehyde 12; a high yielding, acid-mediated simultaneous acetonide deprotection-dithiane removal-ketalization procedure leading exclusively to the 2, 8-dioxabicyclo[3.2.1]octane core 34; and a novel triple oxidation procedure allowing installation of the tricarboxylic acid.

Antibodies have the intrinsic capacity to destroy antigens.

Research throughout the last century has led to a consensus as to the strategy of the humoral component of the immune system. The essence is that, for killing, the antibody molecule activates additional systems that respond to antibody-antigen union. We now report that the immune system seems to have a previously unrecognized chemical potential intrinsic to the antibody molecule itself. All antibodies studied, regardless of source or antigenic specificity, can convert molecular oxygen into hydrogen peroxide, thereby potentially aligning recognition and killing within the same molecule. Aside from pointing to a new chemical arm for the immune system, these results may be important to the understanding of how antibodies evolved and what role they may play in human diseases.

Molecular basis for complex formation between methylamine dehydrogenase and amicyanin revealed by inverse mutagenesis of an interprotein salt bridge.

Methylamine dehydrogenase (MADH) and amicyanin form a physiologic complex which is required for interprotein electron transfer. The crystal structure of this protein complex is known, and the importance of certain residues on amicyanin in its interaction with MADH has been demonstrated by site-directed mutagenesis. In this study, site-directed mutagenesis of MADH, kinetic data, and thermodynamic analysis are used to probe the molecular basis for stabilization of the protein complex by an interprotein salt bridge between Arg99 of amicyanin and Asp180 of the alpha subunit of MADH. This paper reports the first site-directed mutagenesis of MADH, as well as the construction, heterologous expression, and characterization of a six-His-tagged MADH. alpha Asp180 of MADH was converted to arginine to examine the effect on complex formation with native and mutant amicyanins. This mutation had no effect on the parameters for methylamine oxidation by MADH, but significantly affected its interaction with amicyanin. Of the native and mutant proteins that were studied, their observed order of affinity for each other was as follows: native MADH and native amicyanin > native MADH and R99D amicyanin > alpha D180R MADH and native amicyanin > alpha D180R MADH and R99D amicyanin, and alpha D180R MADH and R99L amicyanin. The alpha D180R mutation also eliminated the ionic strength dependence of the reaction of MADH with amicyanin that is observed with wild-type MADH. Interestingly, the inverse mutation pair of alpha D180R MADH and R99D amicyanin did not restore the favorable salt bridge, but instead disrupted complex formation much more severely than did either individual mutation. These results are explained using molecular modeling and thermodynamic analysis of the kinetic data to correlate the energy contributions of specific stabilizing and destabilizing interactions that are present in the wild-type and mutant complexes. A model is also proposed to describe the sequence of events that leads to stable complex formation between MADH and amicyanin.

Tyr(30) of amicyanin is not critical for electron transfer to cytochrome c-551i: implications for predicting electron transfer pathways.

A Pathways analysis of the methylamine dehydrogenase-amicyanin-cytochrome c-551i protein electron transfer (ET) complex predicts two sets of ET pathways of comparable efficiency from the type I copper of amicyanin to the heme of cytochrome c-551i. In one pathway, the electron exits copper via the Cys(92) copper ligand, and in the other, it exits via the Met(98) copper ligand. If the Pathways algorithm is modified to include contributions from the anisotropy of metal-ligand coupling, independent of differences in copper-ligand bond length, then the pathways via Cys(92) are predicted to be at least 100-fold more strongly coupled than the pathways via any of the other copper ligands. All of the favored pathways via Cys(92) include a through-space jump from Cys(92) to the side chain of Tyr(30). To determine whether or not the pathways via Cys(92) are preferentially used for ET, Tyr(30) was changed to other amino acid residues by site-directed mutagenesis. Some mutant proteins were very unstable suggesting a role for Tyr(30) in stabilizing the protein structure. Y30F and Y30I mutant amicyanins could be isolated and analyzed. For the Y30I mutant, the modified Pathways analysis which favors ET via Cys(92) predicts a decrease in ET rate of at least two orders of magnitude, whereas the standard Pathways analysis predicts no change in ET rate since ET via Met(98) is not affected. Experimentally, the ET rates of the Y30I and Y30F mutants were indistinguishable from that of wild-type amicyanin. Likely explanations for these observations are discussed as are their implications for predicting pathways for ET reactions of metalloproteins.

Heterologous expression of correctly assembled methylamine dehydrogenase in Rhodobacter sphaeroides.

The biosynthesis of methylamine dehydrogenase (MADH) from Paracoccus denitrificans requires four genes in addition to those that encode the two structural protein subunits, mauB and mauA. The accessory gene products appear to be required for proper export of the protein to the periplasm, synthesis of the tryptophan tryptophylquinone (TTQ) prosthetic group, and formation of several structural disulfide bonds. To accomplish the heterologous expression of correctly assembled MADH, eight genes from the methylamine utilization gene cluster of P. denitrificans, mauFBEDACJG, were placed under the regulatory control of the coxII promoter of Rhodobacter sphaeroides and introduced into R. sphaeroides by using a broad-host-range vector. The heterologous expression of MADH was constitutive with respect to carbon source, whereas the native mau promoter allows induction only when cells are grown in the presence of methylamine as a sole carbon source and is repressed by other carbon sources. The recombinant MADH was localized exclusively in the periplasm, and its physical, spectroscopic, kinetic and redox properties were indistinguishable from those of the enzyme isolated from P. denitrificans. These results indicate that mauM and mauN are not required for MADH or TTQ biosynthesis and that mauFBEDACJG are sufficient for TTQ biosynthesis, since R. sphaeroides cannot synthesize TTQ. A similar construct introduced into Escherichia coli did not produce detectable MADH activity or accumulation of the mauB and mauA gene products but did lead to synthesizes of amicyanin, the mauC gene product. This finding suggests that active recombinant MADH is not expressed in E. coli because one of the accessory gene products is not functionally expressed. This study illustrates the potential utility of R. sphaeroides and the coxII promoter for heterologous expression of complex enzymes such as MADH which cannot be expressed in E. coli. These results also provide the foundation for future studies on the molecular mechanisms of MADH and TTQ biosynthesis, as well as a system for performing site-directed mutagenesis of the MADH gene and other mau genes.

Cleavage of the cyclohexyl-subunit of rapamycin results in loss of immunosuppressive activity.

The cyclohexyl-subunit of rapamycin was cleaved by a sequence involving a Baeyer-Villiger reaction and acid hydrolysis of the resulting lactone-acetal as key steps. Binding of this new rapamycin derivative to FKBP12 was only slightly reduced by this modification, whereas the loss of antiproliferative and immunosuppressive activity was dramatic. These findings indicate that part of the cyclohexyl-subunit of rapamycin could belong to its effector domain.

Site-directed mutagenesis of Phe 97 to Glu in amicyanin alters the electronic coupling for interprotein electron transfer from quinol methylamine dehydrogenase.

Conversion by site-directed mutagenesis of Phe 97 of amicyanin to Glu significantly decreases the rate constant for the electron-transfer reaction from the quinol form of methylamine dehydrogenase to amicyanin. It is shown that the DeltaG degrees and reorganizational energy (lambda) associated with the electron-transfer reaction are unaffected by the mutation and that the decrease in the electron-transfer rate is attributable completely to a decrease in the electronic coupling matrix element (HAB). Phe 97 is not a part of the predicted pathway for electron-transfer from the tryptophan tryptophylquinone cofactor of MADH to the copper of amicyanin. The most likely explanation for these results is that the mutation of this residue at the protein-protein interface causes an increase in the interprotein distance within the protein complex. The change in distance necessary to cause the observed reduction of HAB is calculated assuming a range of beta values, and assuming either solely a direct distance dependence or a pathway dependence, for the long-range electron-transfer reaction. Thermodynamic analysis of the association constants for complex formation reveal that the reaction with the mutant amicyanin exhibits a large positive change in heat capacity whereas this is not observed in the reaction with the wild-type. This may be explained by the replacement of a hydrophobic residue with a polar residue at what is normally a hydrophobic protein-protein interface. The impact of these results on possible explanations for the relatively large reorganizational energy associated with this interprotein electron-transfer reaction is also discussed.

Factors which stabilize the methylamine dehydrogenase-amicyanin electron transfer protein complex revealed by site-directed mutagenesis.

Methylamine dehydrogenase (MADH) and amicyanin form a physiologic complex within which electrons are transferred from the tryptophan tryptophylquinone (TTQ) cofactor of MADH to the type 1 copper of amicyanin. Interactions responsible for complex formation may be inferred from the crystal structures of complexes of these proteins. Site-directed mutagenesis has been performed to probe the roles of specific amino acid residues of amicyanin in stabilizing the MADH-amicyanin complex and determining the observed ionic strength dependence of complex formation. Conversion of Phe97 to Glu severely disrupted binding, establishing the importance of hydrophobic interactions involving this residue. Conversion of Arg99 to either Asp or to Leu increased the Kd for complex formation by 2 orders of magnitude at low ionic strength, establishing the importance of ionic interactions which were inferred from the crystal structure involving Arg99. Conversion of Lys68 to Ala did not disrupt binding at low ionic strength, but it did greatly diminish the observed ionic strength dependence of complex formation that is seen with wild-type amicyanin. These results demonstrate that the physiologic interaction between MADH and amicyanin is stabilized by a combination of ionic and van der Waals interactions and that individual amino acid residues on the protein surface are able to dictate specific interactions between these soluble redox proteins. These results also indicate that the orientation of MADH and amicyanin when they react with each other in solution is the same as the orientation of the proteins which is seen in the structure of the crystallized protein complex.

Electron transfer from copper to heme within the methylamine dehydrogenase--amicyanin--cytochrome c-551i complex.

Methylamine dehydrogenase (MADH), amicyanin, and cytochrome c-551i are soluble redox proteins that form a complex in solution [Chen, L., Durley, R., Mathews, F. S., & Davidson, V. L. (1994) Science 264, 86-90] which is required for the physiologic electron transfer from the tryptophan tryptophylquinone cofactor of MADH to heme via the copper center of amicyanin. The electron transfer reaction from copper to heme within the protein complex has been characterized by transient kinetic and thermodynamic analysis. The rate of this electron transfer reaction is 87 s-1 at 30 degrees C, and it varied with temperature. The reaction exhibited a reorganizational energy (lambda) of 1.1 eV and an electronic coupling (H(AB)) of 0.3 cm-1. The results of these analyses also predict an electron transfer distance, depending upon the value of beta which is used, of 13-24 A. The larger value approximates the direct copper to heme distance observed in the crystal structure of the complex. The most efficient pathways for electron transfer were predicted from the crystal structure using the Greenpath program, and these predictions were correlated with the results of the solution studies of the electron transfer reaction. It is concluded that electron transfer is, in fact, rate limiting for the observed electron transfer reaction in solution and that the two redox centers are strongly coupled, given the distance which separates them.

Assessment of pelvic floor function: a series of simple tests in nulliparous women.

The aim of the study was to assess normal ranges, variations, test-retest reliability and correlations between tests for a battery of simple, minimally invasive tests of pelvic floor function in nulliparous asymptomatic women. Women were recruited by advertisement and underwent dipstick urinalysis, simple cystometrics, provocative tests for incontinence, Q-tip test and surface electromyography with acrylic plug electrodes. Subjects were retested at a later date. Nineteen subjects underwent initial testing and 12 were retested. A wide range of normal values was obtained. Cystometric measures of first and strong urge to void the maximum bladder capacity, Q-tip test and rectal perineometry had significant correlations between the two visits. There were no significant between-test correlation coefficients. It was concluded that the simple tests used demonstrate a mixed ability to follow pelvic floor function longitudinally.

Complex formation with methylamine dehydrogenase affects the pathway of electron transfer from amicyanin to cytochrome c-551i.

Methylamine dehydrogenase (MADH), amicyanin, and cytochrome c-551i are soluble redox proteins that form a complex in solution (Chen, L., Durley, R., Mathews, F. S., and Davidson, V. L. (1994) Science 264, 86-90), which is required for the physiologic electron transfer from the tryptophan tryptophylquinone cofactor of MADH to heme via the copper center of amicyanin. The reduction of cytochrome by amicyanin within the complex in solution has been demonstrated using rapid scanning stopped-flow spectroscopy. Electron transfer from free, uncomplexed, amicyanin to cytochrome c-551i occurs much more rapidly but only to a very small extent because the reaction is thermodynamically much less favorable when amicyanin is not associated with MADH (Gray, K. A., Davidson, V. L., and Knaff, D. B. (1988) J. Biol. Chem. 263, 13987-13990). These kinetic data suggest that amicyanin binding to cytochrome c-551i occurs at different sites when amicyanin is free and when it is in complex with MADH. A model for the interactions of these proteins is presented.