Paramagnetic NMR study of Cu(2+)-IDA complex localization on a protein surface and its application to elucidate long distance information

The paramagnetic metal chelate complex Cu(2+)-iminodiacetic acid (Cu(2+)-IDA) was mixed with ubiquitin, a small globular protein. Quantitative analyses of (1)H and (15)N chemical shift changes and line broadenings induced by the paramagnetic effects indicated that Cu(2+)-IDA was localized to a histidine residue (His68) on the ubiquitin surface. The distances between the backbone amide proton and the Cu(2+) relaxation center were evaluated from the proton transverse relaxation rates enhanced by the paramagnetic effect. These correlated well with the distances calculated from the crystal structure up to 20 A. Here, we show that a Cu(2+)-IDA is the first paramagnetic reagent that specifically localizes to a histidine residue on the protein surface and gives the long-range distance information.     

Sorting of lipoproteins to the outer membrane in E. coli

Escherichia coli lipoproteins are anchored to the periplasmic surface of the inner or outer membrane depending on the sorting signal. An ATP-binding cassette (ABC) transporter, LolCDE, releases outer membrane-specific lipoproteins from the inner membrane, causing the formation of a complex between the released lipoproteins and the periplasmic molecular chaperone LolA. When this complex interacts with outer membrane receptor LolB, the lipoproteins are transferred from LolA to LolB and then localized to the outer membrane. The structures of LolA and LolB are remarkably similar to each other. Both have a hydrophobic cavity consisting of an unclosed beta-barrel and an alpha-helical lid. Structural differences between the two proteins reveal the molecular mechanisms underlying the energy-independent transfer of lipoproteins from LolA to LolB. Strong inner membrane retention of lipoproteins occurs with Asp at position 2 and a few limited residues at position 3. The inner membrane retention signal functions as a Lol avoidance signal and inhibits the recognition of lipoproteins by LolCDE, thereby causing their retention in the inner membrane. The positive charge of phosphatidylethanolamine and the negative charge of Asp at position 2 are essential for Lol avoidance. The Lol avoidance signal is speculated to cause the formation of a tight lipoprotein-phosphatidylethanolamine complex that has five acyl chains and therefore cannot be recognized by LolCDE.     

Hormonal control of C. elegans dauer formation and life span by a Rieske-like oxygenase

C. elegans diapause, gonadal outgrowth, and life span are regulated by a lipophilic hormone, which serves as a ligand to the nuclear hormone receptor DAF-12. A key step in hormone production is catalyzed by the CYP450 DAF-9, but the extent of the biosynthetic pathway is unknown. Here, we identify a conserved Rieske-like oxygenase, DAF-36, as a component in hormone metabolism. Mutants display larval developmental and adult aging phenotypes, as well as patterns of epistasis similar to that of daf-9. Larval phenotypes are potently reversed by crude lipid extracts, 7-dehydrocholesterol, and a recently identified DAF-12 sterol ligand, suggesting that DAF-36 works early in the hormone biosynthetic pathway. DAF-36 is expressed primarily within the intestine, a major organ of metabolic and endocrine control, distinct from DAF-9. These results imply that C. elegans hormone production has multiple steps and is distributed, and that it may provide one way that tissues register their current physiological state during organismal commitments.     

Efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2

The distribution of the severe acute respiratory syndrome coronavirus (SARS-CoV) receptor, an angiotensin-converting enzyme 2 (ACE2), does not strictly correlate with SARS-CoV cell tropism in lungs; therefore, other cellular factors have been predicted to be required for activation of virus infection. In the present study, we identified transmembrane protease serine 2 (TMPRSS2), whose expression does correlate with SARS-CoV infection in the upper lobe of the lung. In Vero cells expressing TMPRSS2, large syncytia were induced by SARS-CoV infection. Further, the lysosome-tropic reagents failed to inhibit, whereas the heptad repeat peptide efficiently inhibited viral entry into cells, suggesting that TMPRSS2 affects the S protein at the cell surface and induces virus-plasma membrane fusion. On the other hand, production of virus in TMPRSS2-expressing cells did not result in S-protein cleavage or increased infectivity of the resulting virus. Thus, TMPRSS2 affects the entry of virus but not other phases of virus replication. We hypothesized that the spatial orientation of TMPRSS2 vis-a-vis S protein is a key mechanism underling this phenomenon. To test this, the TMPRSS2 and S proteins were expressed in cells labeled with fluorescent probes of different colors, and the cell-cell fusion between these cells was tested. Results indicate that TMPRSS2 needs to be expressed in the opposing (target) cell membrane to activate S protein rather than in the producer cell, as found for influenza A virus and metapneumoviruses. This is the first report of TMPRSS2 being required in the target cell for activation of a viral fusion protein but not for the S protein synthesized in and transported to the surface of cells. Our findings suggest that the TMPRSS2 expressed in lung tissues may be a determinant of viral tropism and pathogenicity at the initial site of SARS-CoV infection.     

Double PHD Fingers Protein DPF2 Recognizes Acetylated Histones and Suppresses the Function of Estrogen-related Receptor α through Histone Deacetylase 1

Estrogen-related receptor α (ERRα) is a member of the nuclear receptor superfamily and regulates many physiological functions, including mitochondrial biogenesis and lipid metabolism. ERRα enhances the transactivation function without endogenous ligand by associating with coactivators such as peroxisome proliferator-activated receptor γ coactivator 1 α and β (PGC-1α and -β) and members of the steroid receptor coactivator family. However, the molecular mechanism by which the transactivation function of ERRα is converted from a repressive state to an active state is poorly understood. Here we used biochemical purification techniques to identify ERRα-associated proteins in HeLa cells stably expressing ERRα. Interestingly, we found that double PHD fingers protein DPF2/BAF45d suppressed PGC-1α-dependent transactivation of ERRα by recognizing acetylated histone H3 and associating with HDAC1. DPF2 directly bound to ERRα and suppressed the transactivation function of nuclear receptors such as androgen receptor. DPF2 was recruited to ERR target gene promoters in myoblast cells, and knockdown of DPF2 derepressed the level of mRNA expressed by target genes of ERRα. These results show that DPF2 acts as a nuclear receptor-selective co-repressor for ERRα by associating with both acetylated histone H3 and HDAC1.     

Covalent Bond between Ligand and Receptor Required for Efficient Activation in Rhodopsin

Rhodopsin is an extensively studied member of the G protein-coupled receptors (GPCRs). Although rhodopsin shares many features with the other GPCRs, it exhibits unique features as a photoreceptor molecule. A hallmark in the molecular structure of rhodopsin is the covalently bound chromophore that regulates the activity of the receptor acting as an agonist or inverse agonist. Here we show the pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of bovine rhodopsin, by use of a rhodopsin mutant K296G reconstituted with retinylidene Schiff bases. Our results show that photoreceptive functions of rhodopsin, such as regiospecific photoisomerization of the ligand, and its quantum yield were not affected by the absence of the covalent bond, whereas the activation mechanism triggered by photoisomerization of the retinal was severely affected. Furthermore, our results show that an active state similar to the Meta-II intermediate of wild-type rhodopsin did not form in the bleaching process of this mutant, although it exhibited relatively weak G protein activity after light irradiation because of an increased basal activity of the receptor. We propose that the covalent bond is required for transmitting structural changes from the photoisomerized agonist to the receptor and that the covalent bond forcibly keeps the low affinity agonist in the receptor, resulting in a more efficient G protein activation.     

Cell surface-expressed moesin-like HDL/apoA-I binding protein promotes cholesterol efflux from human macrophages

HDL and its major component, apolipoprotein A-I (apoA-I), play a central role in reverse cholesterol transport. We recently reported the involvement of a glycosylphosphatidylinositol anchor (GPI anchor) in the binding of HDL and apoA-I on human macrophages, and purified an 80 kDa HDL/apoA-I binding protein. In the present study, we characterized the GPI-anchored HDL/apoA-I binding protein from macrophages. The HDL/apoA-I binding protein was purified from macrophages and digested with endopeptidase, and the resultant fragments were sequenced. Cholesterol efflux, flow cytometry, immunoblotting, and immunohistochemical analyses were performed to characterize the HDL/apoA-I binding protein. Two parts of seven amino acid sequences completely matched those of moesin. Flow cytometry, immunoblotting, and immunohistochemistry using anti-moesin antibody showed that the HDL/apoA-I binding protein was N-glycosylated and expressed on the cell surface. It was termed moesin-like protein. Treatment of macrophages with anti-moesin antibody blocked the binding of HDL/apoA-I and suppressed cholesterol efflux. The moesin-like protein was exclusively expressed on macrophages and was upregulated by cholesterol loading and cell differentiation. Our results indicate that the moesin-like HDL/apoA-I binding protein is specifically expressed on the surface of human macrophages and promotes cholesterol efflux from macrophages.-Matsuyama, A, N. Sakai, H. Hiraoka, K-i. Hirano, and S. Yamashita. Cell surface-expressed moesin-like HDL/apoA-I binding protein promotes cholesterol efflux from human macrophages.     

Genetic analysis of the mode of interplay between an ATPase subunit and membrane subunits of the lipoprotein-releasing ATP-binding cassette transporter LolCDE

The LolCDE complex, an ATP-binding cassette (ABC) transporter, releases lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane of Escherichia coli. The LolCDE complex is composed of two copies of an ATPase subunit, LolD, and one copy each of integral membrane subunits LolC and LolE. LolD hydrolyzes ATP on the cytoplasmic side of the inner membrane, while LolC and/or LolE recognize and release lipoproteins anchored to the periplasmic leaflet of the inner membrane. Thus, functional interaction between LolD and LolC/E is critically important for coupling of ATP hydrolysis to the lipoprotein release reaction. LolD contains a characteristic sequence called the LolD motif, which is highly conserved among LolD homologs but not other ABC transporters of E. coli. The LolD motif is suggested to be a region in contact with LolC/E, judging from the crystal structures of other ABC transporters. To determine the functions of the LolD motif, we mutagenized each of the 32 residues of the LolD motif and isolated 26 dominant-negative mutants, whose overexpression arrested growth despite the chromosomal lolD(+) background. We then selected suppressor mutations of the lolC and lolE genes that correct the growth defect caused by the LolD mutations. Mutations of the lolC suppressors were mainly located in the periplasmic loop, whereas ones of lolE suppressors were mainly located in the cytoplasmic loop, suggesting that the mode of interaction with LolD differs between LolC and LolE. Moreover, the LolD motif was found to be critical for functional interplay with LolC/E, since some LolD mutations lowered the ATPase activity of LolCDE without affecting that of LolD.     

Roles of the hydrophobic cavity and lid of LolA in the lipoprotein transfer reaction in Escherichia coli

LolA, a periplasmic chaperone, binds to outer membrane-specific lipoproteins released from the inner membrane through the action of an ATP-binding cassette transporter, LolCDE and then transfers them to the outer membrane receptor LolB, thereby mediating the inner to outer membrane transport of lipoproteins. The crystal structure of free LolA revealed that it has an internal hydrophobic cavity, which is surrounded by hydrophobic residues and closed by a lid comprising alpha-helices. The hydrophobic cavity most likely represents the binding site for the lipid moiety of a lipoprotein. It is speculated that the lid undergoes opening and closing upon the binding and transfer of lipoproteins, respectively. To determine the functions of the hydrophobic cavity and lid in detail, 14 residues involved in the formation of these structures were subjected to random mutagenesis. Among the obtained 21 LolA derivatives that did not support growth, 14 were active as to the binding of lipoproteins but defective in the transfer of lipoproteins to LolB, causing the periplasmic accumulation of a lipoprotein as a complex with a LolA derivative. A LolA derivative, I93G, bound lipoproteins faster than wild-type LolA did, whereas it did not transfer associated lipoproteins to LolB. When I93G and wild type LolA co-existed, lipoproteins were bound only to I93G; which therefore exhibited a dominant negative property. Another derivative, L59R, was also defective in the transfer of lipoproteins to LolB but did not exhibit a dominant negative property. Taken together, these results indicate that both the hydrophobic cavity and the lid are critically important for not only the binding of lipoproteins but also their transfer.