The import receptor for the peroxisomal targeting signal 2 (PTS2) in Saccharomyces cerevisiae is encoded by the PAS7 gene

The import of peroxisomal matrix proteins is dependent on one of two targeting signals, PTS1 and PTS2. We demonstrate in vivo that not only the import of thiolase but also that of a chimeric protein consisting of the thiolase PTS2 (amino acids 1-18) fused to the bacterial protein β-lactamase is Pas7p dependent. In addition, using a combination of several independent approaches (two-hybrid system, co-immunoprecipitation, affinity chromatography and high copy suppression), we show that Pas7p specifically interacts with thiolase in vivo and in vitro. For this interaction, the N-terminal PTS2 of thiolase is both necessary and sufficient. The specific binding of Pas7p to thiolase does not require peroxisomes. Pas7p recognizes the PTS2 of thiolase even when this otherwise N-terminal targeting signal is fused to the C-terminus of other proteins, i.e. the activation domain of Gal4p or GST. These results demonstrate that Pas7p is the targeting signal-specific receptor of thiolase in Saccharomyces cerevisiae and, moreover, are consistent with the view that Pas7p is the general receptor of the PTS2. Our observation that Pas7p also interacts with the human peroxisomal thiolase suggests that in the human peroxisomal disorders characterized by an import defect for PTS2 proteins (classical rhizomelic chondrodysplasia punctata), a functional homologue of Pas7p may be impaired

Structure of the CaMKIIδ/Calmodulin Complex Reveals the Molecular Mechanism of CaMKII Kinase Activation

Structural and biophysical studies reveal how CaMKII kinases, which are important for cellular learning and memory, are switched on by binding of Ca2+/calmodulin

X-ray structure of engineered human Aortic Preferentially Expressed Protein-1 (APEG-1)

BACKGROUND: Human Aortic Preferentially Expressed Protein-1 (APEG-1) is a novel specific smooth muscle differentiation marker thought to play a role in the growth and differentiation of arterial smooth muscle cells (SMCs). RESULTS: Good quality crystals that were suitable for X-ray crystallographic studies were obtained following the truncation of the 14 N-terminal amino acids of APEG-1, a region predicted to be disordered. The truncated protein (termed ΔAPEG-1) consists of a single immunoglobulin (Ig) like domain which includes an Arg-Gly-Asp (RGD) adhesion recognition motif. The RGD motif is crucial for the interaction of extracellular proteins and plays a role in cell adhesion. The X-ray structure of ΔAPEG-1 was determined and was refined to sub-atomic resolution (0.96 Å). This is the best resolution for an immunoglobulin domain structure so far. The structure adopts a Greek-key β-sandwich fold and belongs to the I (intermediate) set of the immunoglobulin superfamily. The residues lying between the β-sheets form a hydrophobic core. The RGD motif folds into a 3(10 )helix that is involved in the formation of a homodimer in the crystal which is mainly stabilized by salt bridges. Analytical ultracentrifugation studies revealed a moderate dissociation constant of 20 μM at physiological ionic strength, suggesting that APEG-1 dimerisation is only transient in the cell. The binding constant is strongly dependent on ionic strength. CONCLUSION: Our data suggests that the RGD motif might play a role not only in the adhesion of extracellular proteins but also in intracellular protein-protein interactions. However, it remains to be established whether the rather weak dimerisation of APEG-1 involving this motif is physiogically relevant

The scientific impact of the Structural Genomics Consortium: a protein family and ligand-centered approach to medically-relevant human proteins

As many of the structural genomics centers have ended their first phase of operation, it is a good point to evaluate the scientific impact of this endeavour. The Structural Genomics Consortium (SGC), operating from three centers across the Atlantic, investigates human proteins involved in disease processes and proteins from Plasmodium falciparum and related organisms. We present here some of the scientific output of the Oxford node of the SGC, where the target areas include protein kinases, phosphatases, oxidoreductases and other metabolic enzymes, as well as signal transduction proteins. The SGC has aimed to achieve extensive coverage of human gene families with a focus on protein–ligand interactions. The methods employed for effective protein expression, crystallization and structure determination by X-ray crystallography are summarized. In addition to the cumulative impact of accelerated delivery of protein structures, we demonstrate how family coverage, generic screening methodology, and the availability of abundant purified protein samples, allow a level of discovery that is difficult to achieve otherwise. The contribution of NMR to structure determination and protein characterization is discussed. To make this information available to a wide scientific audience, a new tool for disseminating annotated structural information was created that also represents an interactive platform allowing for a continuous update of the annotation by the scientific community

High-Affinity Inhibitors of Human NAD+-Dependent 15-Hydroxyprostaglandin Dehydrogenase: Mechanisms of Inhibition and Structure-Activity Relationships

BACKGROUND: 15-Hydroxyprostaglandin dehydrogenase (15-PGDH, EC 1.1.1.141) is the key enzyme for the inactivation of prostaglandins, regulating processes such as inflammation or proliferation. The anabolic pathways of prostaglandins, especially with respect to regulation of the cyclooxygenase (COX) enzymes have been studied in detail; however, little is known about downstream events including functional interaction of prostaglandin-processing and -metabolizing enzymes. High-affinity probes for 15-PGDH will, therefore, represent important tools for further studies. PRINCIPAL FINDINGS: To identify novel high-affinity inhibitors of 15-PGDH we performed a quantitative high-throughput screen (qHTS) by testing >160 thousand compounds in a concentration-response format and identified compounds that act as noncompetitive inhibitors as well as a competitive inhibitor, with nanomolar affinity. Both types of inhibitors caused strong thermal stabilization of the enzyme, with cofactor dependencies correlating with their mechanism of action. We solved the structure of human 15-PGDH and explored the binding modes of the inhibitors to the enzyme in silico. We found binding modes that are consistent with the observed mechanisms of action. CONCLUSIONS: Low cross-reactivity in screens of over 320 targets, including three other human dehydrogenases/reductases, suggest selectivity of the present inhibitors for 15-PGDH. The high potencies and different mechanisms of action of these chemotypes make them a useful set of complementary chemical probes for functional studies of prostaglandin-signaling pathways. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S2

Structural Basis for Substrate Specificity in Human Monomeric Carbonyl Reductases

Carbonyl reduction constitutes a phase I reaction for many xenobiotics and is carried out in mammals mainly by members of two protein families, namely aldo-keto reductases and short-chain dehydrogenases/reductases. In addition to their capacity to reduce xenobiotics, several of the enzymes act on endogenous compounds such as steroids or eicosanoids. One of the major carbonyl reducing enzymes found in humans is carbonyl reductase 1 (CBR1) with a very broad substrate spectrum. A paralog, carbonyl reductase 3 (CBR3) has about 70% sequence identity and has not been sufficiently characterized to date. Screening of a focused xenobiotic compound library revealed that CBR3 has narrower substrate specificity and acts on several orthoquinones, as well as isatin or the anticancer drug oracin. To further investigate structure-activity relationships between these enzymes we crystallized CBR3, performed substrate docking, site-directed mutagenesis and compared its kinetic features to CBR1. Despite high sequence similarities, the active sites differ in shape and surface properties. The data reveal that the differences in substrate specificity are largely due to a short segment of a substrate binding loop comprising critical residues Trp229/Pro230, Ala235/Asp236 as well as part of the active site formed by Met141/Gln142 in CBR1 and CBR3, respectively. The data suggest a minor role in xenobiotic metabolism for CBR3. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1

Search for New Color-Octet Vector Particle Decaying to ttbar in ppbar Collisions at s=1.96\sqrt{s}=1.96 TeV

Submitted to Phys. Lett. BWe present the result of a search for a massive color-octet vector particle, (e.g. a massive gluon) decaying to a pair of top quarks in proton-antiproton collisions with a center-of-mass energy of 1.96 TeV. This search is based on 1.9 fb$^{-1}$ of data collected using the CDF detector during Run II of the Tevatron at Fermilab. We study $t\bar{t}$ events in the lepton+jets channel with at least one $b$-tagged jet. A massive gluon is characterized by its mass, decay width, and the strength of its coupling to quarks. These parameters are determined according to the observed invariant mass distribution of top quark pairs. We set limits on the massive gluon coupling strength for masses between 400 and 800 GeV$/c^2$ and width-to-mass ratios between 0.05 and 0.50. The coupling strength of the hypothetical massive gluon to quarks is consistent with zero within the explored parameter space.We present the result of a search for a massive color-octet vector particle, (e.g. a massive gluon) decaying to a pair of top quarks in proton–antiproton collisions with a center-of-mass energy of 1.96 TeV. This search is based on 1.9 fb−1 of data collected using the CDF detector during Run II of the Tevatron at Fermilab. We study View the MathML source events in the lepton + jets channel with at least one b-tagged jet. A massive gluon is characterized by its mass, decay width, and the strength of its coupling to quarks. These parameters are determined according to the observed invariant mass distribution of top quark pairs. We set limits on the massive gluon coupling strength for masses between 400 and 800 GeV/c2 and width-to-mass ratios between 0.05 and 0.50. The coupling strength of the hypothetical massive gluon to quarks is consistent with zero within the explored parameter space.Peer reviewe