Biosynthetic thiolase catalyzes the formation of acetoacetyl-CoA from two molecules of acetyl-CoA. This is a key step in the synthesis of many biological compounds, including steroid hormones and ketone bodies. The thiolase reaction involves two chemically distinct steps; during acyl transfer, an acetyl group is transferred from acetyl-CoA to Cys89, and in the Claisen condensation step, this acetyl group is further transferred to a second molecule of acetyl-CoA, generating acetoacetyl-CoA. Here, new crystallographic data for Zoogloea ramigera biosynthetic thiolase are presented, covering all intermediates of the thiolase catalytic cycle. The high-resolution structures indicate that the acetyl group goes through four conformations while being transferred from acetyl-CoA via the acetylated enzyme to acetoacetyl-CoA. This transfer is catalyzed in a rigid cavity lined by mostly hydrophobic side chains, in addition to the catalytic residues Cys89, His348, and Cys378. The structures highlight the importance of an oxyanion hole formed by a water molecule and His348 in stabilizing the negative charge on the thioester oxygen atom of acetyl-CoA at two different steps of the reaction cycle. Another oxyanion hole, composed of the main chain nitrogen atoms of Cys89 and Gly380, complements a negative charge of the thioester oxygen anion of the acetylated intermediate, stabilizing the tetrahedral transition state of the Claisen condensation step. The reactivity of the active site may be modulated by hydrogen bonding networks extending from the active site toward the back of the molecule.

MARCO is a trimeric class A scavenger receptor of macrophages and dendritic cells that recognizes polyanionic particles and pathogens. The distal, scavenger receptor cysteine-rich (SRCR) domain of the extracellular part of this receptor has been implicated in ligand binding. To provide a structural basis for understanding the ligand-binding mechanisms of MARCO, we have determined the crystal structure of the mouse MARCO SRCR domain. The recombinant SRCR domain purified as monomeric and dimeric forms, and their structures were determined at 1.78 and 1.77 Å resolution, respectively. The monomer has a compact globular fold with a twisted five-stranded antiparallel ␤-sheet and a long loop covering a single ␣-helix, whereas the dimer is formed via ␤-strand swapping of two monomers, thus containing a large eight-stranded ␤-sheet. Calculation of the surface electrostatic potential revealed that the ␤-sheet region with several arginines forms a basic cluster. Unexpectedly, an acidic cluster was found in the long loop region. In the monomer, the acidic cluster is involved in metal ion binding. Studies with cells expressing various SRCR domain mutants showed that all of the arginines of the basic cluster are involved in ligand binding, suggesting a cooperative binding mechanism. Ligand binding is also dependent on the acidic cluster and Ca 2؉ ions whose depletion appears to affect ligand binding at least by modulating the electrostatic potential or relative domain orientation. We propose that the SRCR domain dimerization can contribute to the recognition of large ligands by providing a means for the MARCO receptor oligomerization.MARCO belongs to a diverse group of scavenger receptors (SRs) 3 expressed by macrophages, dendritic cells, and certain endothelial cells (1). These germ line-encoded receptors, also known as pattern recognition receptors due to their ability to recognize conserved pathogen-associated molecular patterns, are considered as an important part of innate immunity, the evolutionarily conserved, first line host defense mechanism. In addition to pathogen-associated molecular patterns, a long list of SR ligands, often polyanionic in nature, includes pollution particles, polyribonucleotides, bacterial lipopolysaccharides, modified host molecules such as oxidized low density lipoprotein (LDL), and unmodified endogenous proteins (1, 2). The SRs are classified into several subgroups, of which class A SRs have primarily been associated with innate immunity. This class consists of five members: SR-A (SR-AI, -II, and -III/ SCARA1) (3, 4), MARCO (macrophage receptor with collagenous domain)/SCARA2 (5), CSR1 (cellular stress response 1) and CSR2/SCARA3 (6), SRCL (scavenger receptor with C-type lectin) I and II/SCARA4 (7, 8), and Tesr (testis-expressed scavenger receptor)/SCARA5 (class A scavenger receptor 5) (9, 10). All of these are trimeric type II membrane proteins with a similar predicted tertiary structure consisting of a short intracellular domain, a transmembrane domain, and a large extracellular domain with...

The majority of macromolecular crystal structures are determined using the method of molecular replacement, in which known related structures are rotated and translated to provide an initial atomic model for the new structure. A theoretical understanding of the signal-to-noise ratio in likelihood-based molecular replacement searches has been developed to account for the influence of model quality and completeness, as well as the resolution of the diffraction data. Here we show that, contrary to current belief, molecular replacement need not be restricted to the use of models comprising a substantial fraction of the unknown structure. Instead, likelihood-based methods allow a continuum of applications depending predictably on the quality of the model and the resolution of the data. Unexpectedly, our understanding of the signal-to-noise ratio in molecular replacement leads to the finding that, with data to sufficiently high resolution, fragments as small as single atoms of elements usually found in proteins can yield ab initio solutions of macromolecular structures, including some that elude traditional direct methods.O ver the past century, determination of novel crystal structures has evolved from an exercise in logic identifying the locations of single atoms by inspecting diffraction patterns (1) or vector maps (2), through the development of direct methods for small molecules (3) and of isomorphous replacement (4, 5) or anomalous diffraction (6, 7) phasing for molecules as large as proteins.Currently, about 80% of protein structures are solved by the method of molecular replacement (8), exploiting prior structural knowledge of related proteins. In principle, molecular replacement (MR) involves rotational and translational searches over many possible placements of a molecular model within the unit cell of an unknown structure. The most sensitive method of evaluating the fit to the observed data is a likelihood function (9, 10) that accounts for the effect of measurement errors in the observed diffraction intensities (11). Potential solutions are scored by the log-likelihood-gain on intensities (LLGI), the sum of the log-likelihoods for individual reflections minus the log-likelihoods for an uninformative model (Methods).Success in MR depends on the signal-to-noise of the search, which varies according to two parameters in the likelihood function: D obs characterizes the precision of each measurement, taking values near 1 for moderately well-measured data and only taking values near 0 for extremely weak data; σ A measures the quality of the model in terms of the fraction of a crystallographic structure factor that it explains. The resolution-dependent value of σ A for each reflection can be estimated from the fraction (f P ) of the X-ray scattering power accounted for by the model (where the total scattering power is the sum of the squares of the scattering factors for the atoms in the crystal), its estimated accuracy (rms error Δ), and the resolution (d) of the reflection (9), with (optionally) a correction for...

Background/Aims: Solute carrier family 12 member 3 (SLC12A3) encodes a sodium/chloride transporter in kidneys. Previous reports suggest that Arg913Gln polymorphism in this gene is associated with diabetic nephropathy (DN), but the data appear to be inconsistent. Up to now, there is no biological evidence concerning the effects of SLC12A3 in DN. In this study, we aim to evaluate the genetic effects of the SLC12A3 gene and its Arg913Gln polymorphism with genetic and functional analyses. Methods: We genotyped SLC12A3 genetic polymorphisms including Arg913Gln in 784 non-diabetes controls and 633 type 2 diabetes (T2D) subjects with or without DN in a Malaysian population and performed a meta-analysis of the present and previous studies. We further analyzed the role of slc12a3 in kidney development and progress of DN in zebrafish and db/db mice. Results: We found that SLC12A3 Arg913Gln polymorphism was associated with T2D (p = 0.028, OR = 0.772, 95% CI = 0.612-0.973) and DN (p = 0.038, OR = 0.547, 95% CI = 0.308-0.973) in the Malaysian cohort. The meta-analysis confirmed the protective effects of SLC12A3 913Gln allele in DN (Z-value = -1.992, p = 0.046, OR = 0.792). Furthermore, with knockdown of zebrafish ortholog, slc12a3 led to structural abnormality of kidney pronephric distal duct at 1-cell stage. Slc12a3 mRNA and protein expression levels were upregulated in kidneys of db/db mice from 6, 12, and 26 weeks at the age. Conclusion: The present study provided the first biological and further genetic evidence that SLC12A3 has genetic susceptibility in the development of DN, while the minor 913Gln allele in this gene confers a protective effect in the disease. i 2014 S. Karger AG, Basel

Matrix metalloproteinases (MMPs) contribute to the breakdown of tissue structures such as the basement membrane, promoting tissue fibrosis. Here we developed an electrospun membrane biofunctionalized with a fragment of the laminin β1-chain to modulate the expression of MMP2 in this context. We demonstrate that interfacing of the β1-fragment with the mesothelium of the peritoneal membrane via a biomaterial abrogates the release of active MMP2 in response to transforming growth factor β1 and rescues tissue integrity ex vivo and in vivo in a mouse model of peritoneal fibrosis. Importantly, our data demonstrate that the membrane inhibits MMP2 expression. Changes in the expression of epithelial-to-mesenchymal transition (EMT)-related molecules further point towards a contribution of the modulation of EMT. Biomaterial-based presentation of regulatory basement membrane signals directly addresses limitations of current therapeutic approaches by enabling a localized and specific method to counteract MMP2 release applicable to a broad range of therapeutic targets.

MARCO is a class A scavenger receptor capable of binding both Gram-negative and -positive bacteria. Using the surface plasmon resonance technique, we show here that a recombinant, soluble form of MARCO, sMARCO, binds the major Gram-negative and -positive bacterial surface components, lipopolysaccharide and lipoteichoic acid. Yet, the interaction of these two polyanions with sMARCO is of much lower affinity than that of polyinosinic acid, a polyanionic inhibitor of bacterial binding to MARCO. To further elucidate the ligand-binding functions of MARCO, we performed a phage display screen with sMARCO. The screening resulted in the enrichment of only a handful of phage clones. Contrary to expectations, no polyanionic peptides, but only those with a predominantly hydrophobic nature, were enriched. One peptide, VRWGSFAAWL, was displayed on two-thirds of the phages recovered after four rounds of screening. The VRWGSFAAWL phage-sMARCO interaction had significantly slower dissociation kinetics than that between sMARCO and lipopolysaccharide or lipoteichoic acid. Further work with this phage, and the second most enriched phage, displaying the peptide RLNWAWWLSY, demonstrated that both peptides bind to the SRCR domain of MARCO, and that they probably bind to the same site. Data base searches suggested that the VRWGSFAAWL peptide represents complement component C4, but we could not convincingly confirm this suggestion. A study with chimeric scavenger receptors indicated that even minor sequence changes in the MARCO scavenger receptor cysteine-rich (SRCR) domain can have profound effects on the binding of the prototypic scavenger receptor ligand, acetylated low density lipoprotein. As shown by differential binding of glutathione S-transferase-VR-WGSFAAWL, these differences were very likely due to conformational changes. These findings led to experiments that demonstrated a crucial role of the SRCR domain for acetylated low density lipoprotein binding in MARCO. Thus, our results strengthen the notion that the SRCR domain is the major ligand-binding domain in MARCO. Furthermore, they suggest that the domain may contain multiple ligand-binding sites.MARCO, a close relative of scavenger receptor A (see Ref. 1), is a trimeric type II transmembrane protein with an N-terminal intracellular domain, a transmembrane domain, and an extracellular portion composed of a short spacer domain, a long triple-helical collagenous domain, and a C-terminal scavenger receptor cysteine-rich (SRCR) 3 domain (2). MARCO has a very restricted expression pattern in adult mice living under pathogen-free conditions. It is expressed at significant levels only in the marginal zone macrophages of the spleen, in macrophages of the medullary cord of lymph nodes, and in the peritoneal macrophages (2). 4 In bacterial infections MARCO expression is up-regulated in macrophages of most tissues (3-6). Cells transfected with a plasmid encoding MARCO avidly bind both Gram-negative and -positive bacteria, but not yeast (2, 3). MARCO also has other than microbial ligand...

Podocin is a key protein of the kidney podocyte slit diaphragm protein complex, an important part of the glomerular filtration barrier. Mutations in the human podocin gene NPHS2 cause familial or sporadic forms of renal disease owing to the disruption of filtration barrier integrity. The exclusive expression of NPHS2 in podocytes reflects its unique function and raises interesting questions about its transcriptional regulation. Here, we further define a 2.5-kb zebrafish nphs2 promoter fragment previously described and identify a 49-bp podocyte-specific transcriptional enhancer using Tol2-mediated G 0 transgenesis in zebrafish. Within this enhancer, we identified a cis-acting element composed of two adjacent DNA-binding sites (FLAT-E and forkhead) bound by transcription factors Lmx1b and FoxC. In zebrafish, double knockdown of Lmx1b and FoxC orthologs using morpholino doses that caused no or minimal phenotypic changes upon individual knockdown completely disrupted podocyte development in 40% of injected embryos. Cooverexpression of the two genes potently induced endogenous nphs2 expression in zebrafish podocytes. We found that the NPHS2 promoter also contains a cis-acting Lmx1b-FoxC motif that binds LMX1B and FoxC2. Furthermore, a genome-wide search identified several genes that carry the Lmx1b-FoxC motif in their promoter regions. Among these candidates, motif-driven podocyte enhancer activity of CCNC and MEIS2 was functionally analyzed in vivo. Our results show that podocyte expression of some genes is combinatorially regulated by two transcription factors interacting synergistically with a common enhancer. This finding provides insights into transcriptional mechanisms required for normal and pathologic podocyte functions. Normal glomerular filtration function depends on structural integrity of the filtration barrier. Glomerular podocytes play a key role in establishing and maintaining this unique filtration barrier structure. Mature podocytes are characterized by cell cycle arrest, foot process formation, and the presence of the slit diaphragm, 1 which bridges the gaps between the interdigitating foot processes of neighboring podocytes and functions as a size-selective filtration barrier. 2,3 For their differentiation, as well as for the maintenance of their complex architecture, podocytes require the expression of several specific genes in a correct spatial and temporal fashion. This notion is supported by the identification of many mutations in podocyte-expressed genes as the underlying cause of inherited renal diseases. 4 Moreover, recent studies from genetically modified mice

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