Interaction of filipin with junctional membrane at different stages of the junction's life history

The relationship of filipin-sterol complexes to tight and gap junctions during their formation, maturation, internalization, and degradation was studied in separate cell lines. Filipin-sterol complexes tended to be excluded from mature junctions in tight junction forming COLO 316 cells and gap junction forming SW-13 cells. Once internalized, unlabeled junctional membrane appeared to fuse with heavily labeled vesicles, presumably lysosomes. Although the absence of filipin-sterol complexes from junctional membrane does not necessarily reflect the absolute sterol content of this membrane, the fact that filipin-sterol complexes are largely excluded from these areas indicates that this membrane is different from surrounding membrane. The absence of filipin-sterol complexes also permits the visualization of 'mixing' of this specialized unlabeled membrane domain with other filipin labeled membrane systems.     

Only the initial binding of cationic immune complexes to glomerular anionic sites is mediated by charge-charge interactions

The role of charge-charge interactions between cationic immune complexes and the anionic sites on the glomerular basement membrane was examined. For this purpose, soluble immune complexes at fivefold antigen excess were prepared with human serum albumin and cationized rabbit antibodies to this protein. When unrelated cationic proteins, protamine sulfate or cationized rabbit serum albumin, were given 1 min before the cationized immune complexes, glomerular immune deposits did not form. Cationic immune complexes allowed to deposit in glomeruli could readily be displaced by protamine sulfate or cationized rabbit serum albumin injected 1 min after the immune complexes. If the same cationic molecules were injected 1 hr after the immune complexes, the complexes could not be displaced from glomeruli. In contrast, cationic complexes that were deposited in glomeruli in the presence of a very high degree of antigen excess in circulation to prevent their condensation into larger complexes in glomeruli were readily displaced at 1 min and 1 hr with protamine sulfate or with cationized rabbit serum albumin. On the basis of these results, we concluded that the initial binding of cationic immune complexes to glomeruli occurs by charge-charge interactions. Once the immune complexes in glomeruli condense to larger deposits, forces other than charge-charge interactions are responsible for their retention in glomeruli.     

ATP-dependent chromatin remodeling enzymes: two heads are not better, just different

ATP-dependent chromatin remodeling complexes enable rapid rearrangements in chromatin structure in response to developmental cues. The ATPase subunits of remodeling complexes share homology with the helicase motifs of DExx box helicases. Recent single-molecule experiments indicate that, like helicases, many of these complexes use ATP to translocate on DNA. Despite sharing this fundamental property, two key classes of remodeling complexes, the ISWI class and the SWI/SNF class, generate distinct remodeled products. SWI/SNF complexes generate nucleosomes with altered positions, nucleosomes with DNA loops and nucleosomes that are capable of exchanging histone dimers or octamers. In contrast, ISWI complexes generate nucleosomes with altered positions but in standard structures. Here, we draw analogies to monomeric and dimeric helicases and propose that ISWI and SWI/SNF complexes catalyze different outcomes in part because some ISWI complexes function as dimers while SWI/SNF complexes function as monomers.     

溶酶体相关细胞器生物发生复合体相互作用组构成胞内体运输网络

随着高等生物中十几个新的参与囊泡运输的 Hermansky-Pudlak 综合征(HPS)蛋白质的发现, 认为可能存在一类新的囊泡运输通路。该通路主要由新近鉴定的 3 个被称为溶酶体相关细胞器生物发生复合体(BLOC)所组成, 被分别命名为BLOC-1、BLOC-2 和 BLOC-3。越来越多的证据表明这些复合体与以前认识较清楚的 AP3 和 HOPS 复合体共同在胞内体运输中起重要作用。这些复合体之间的相互作用构成了以胞内体和细胞骨架为连接纽带的参与蛋白质运输的复杂网络。该网络中的每个节点的相互作用可区分为复合体内和复合体外相互作用两大类。复合体之间的联系可以是来自不同复合体亚基间的直接相互作用, 也可以通过耦联的节点联结不同的复合体。解析这一复杂网络有助于进一步了解参与蛋白质和膜运输这一动态而精细网络的结构与功能。一旦该网络结构得到破坏, 则可能导致如 HPS 这类囊泡运输或细胞器发生障碍性疾病。     

Isoelectric focusing of high-molecular-weight protein complex under native conditions using agarose gel

The isolation and characterization of protein complexes are essential steps toward understanding cellular functions. A method for separating and characterizing high-molecular-weight protein complexes using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with native agarose gel isoelectric focusing (IEF) is described. Using this method, fractions containing high-molecular-weight protein complexes were analyzed. The advantages of using native agarose gel IEF include the ability to concentrate the protein complexes and the ease of handling when performing 2D separations. Although limited with respect to the size of molecules and particles that may be separated, this method is useful for the isolation and characterization of high-molecular-weight protein complexes.     

Soluble Interleukin-15 Complexes Are Generated In Vivo by Type I Interferon Dependent and Independent Pathways

Interleukin (IL)-15 associates with IL-15Rα on the cell surface where it can be cleaved into soluble cytokine/receptor complexes that have the potential to stimulate CD8 T cells and NK cells. Unfortunately, little is known about the in vivo production of soluble IL-15Rα/IL-15 complexes (sIL-15 complexes), particularly regarding the circumstances that induce them and the mechanisms responsible. The main objective of this study was to elucidate the signals leading to the generation of sIL-15 complexes. In this study, we show that sIL-15 complexes are increased in the serum of mice in response to Interferon (IFN)-α. In bone marrow derived dendritic cells (BMDC), IFN-α increased the activity of ADAM17, a metalloproteinase implicated in cleaving IL-15 complexes from the cell surface. Moreover, knocking out ADAM17 in BMDCs prevented the ability of IFN-α to induce sIL-15 complexes demonstrating ADAM17 as a critical protease mediating cleavage of IL-15 complexes in response to type I IFNs. Type I IFN signaling was required for generating sIL-15 complexes as in vivo induction of sIL-15 complexes by Poly I:C stimulation or total body irradiation (TBI) was impaired in IFNAR-/- mice. Interestingly, serum sIL-15 complexes were also induced in mice infected with Vesicular stomatitis virus (VSV) or mice treated with agonistic CD40 antibodies; however, sIL-15 complexes were still induced in IFNAR-/- mice after VSV infection or CD40 stimulation indicating pathways other than type I IFNs induce sIL-15 complexes. Overall, this study has shown that type I IFNs, VSV infection, and CD40 stimulation induce sIL-15 complexes suggesting the generation of sIL-15 complexes is a common event associated with immune activation. These findings reveal an unrealized mechanism for enhanced immune responses occurring during infection, vaccination, inflammation, and autoimmunity.     

Single particle macromolecular structure determination via electron microscopy

Three-dimensional structure determination of macromolecules and macromolecular complexes is an integral part of understanding biological functions. For large protein and macromolecular complexes structure determination is often performed using electron cryomicroscopy where projection images of individual macromolecular complexes are combined to produce a three-dimensional reconstruction. Single particle methods have been devised to perform this structure determination for macromolecular complexes with little or no underlying symmetry. These computational methods generally involve an iterative process of aligning unique views of the macromolecular images followed by determination of the angular components that define those views. In this review, this structure determination process is described with the aim of clarifying a seemingly complex structural method.     

Unusual Cu(III)—Schiff's base complexes

A new series of CU(III) complexes of the type [Cu(SB)]ClO₄, where SB is a dibasic tetradentate Schiff's base, were synthesized and characterized by elemental analysis, IR and electronic spectra. The prepared complexes are compared with Cu(II) complexes of the same ligands. The room temperature magnetic susceptibility and the x-band EPR spectra of the two types of complexes showed different behaviour. Cu(II) complexes were non conducting whereas Cu(III) complexes gave a conductance corresponding to 1:1 electrolyte. In fact this is the first report on the Cu(III) complexes with the tetradentate Schiff base ligands.     

Glucocorticoid receptor binding to calf thymus DNA. 2. Role of a DNA-binding activity factor in receptor heterogeneity and a multistep mechanism of receptor activation.

In the preceding paper [Cavanaugh, A. H., & Simons, S. S., Jr. (1990) Biochemistry (preceding paper in this issue)], we characterized an apparently identical factor in the cytosol and the nuclear extract of HTC cells that is required for the DNA binding of approximately 43% of the activated receptor-glucocorticoid complexes. In the present study, both those activated complexes that are influenced by this factor and the role of this factor in the process of activation are examined. We find that sodium arsenite inhibits only the DNA binding of those complexes that require factor. Conversely, methyl methane-thiolsulfonate inhibits the DNA binding of only those complexes that are independent of factor. These results provide direct chemical evidence for two populations of activated complexes. Double-reciprocal plots revealed that the increase in DNA binding with endogenous factor occurred by recruiting new complexes for DNA binding as opposed to increasing the binding affinity of existing complexes. These results further suggest that factor associates only with the receptor-steroid complex and does not additionally interact with DNA. A saturable association of factor with complexes was indicated since the amount of available factor in cytosolic solutions decreased after activation of the complexes. Sodium molybdate is known to inhibit the activation of HTC cell receptor-steroid complexes. When factor was added to complexes that had been subjected to activating conditions in the presence of the inhibitor sodium molybdate, no increased DNA binding was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein composition of DNA-matrix complexes with "strong" and "weak" types of bonds, isolated from Ehrlich ascites carcinoma cells

A preparative procedure for obtaining tightly and loosely bound DNA-matrix complexes was developed. To this end, the DNA-matrix complexes were separated from DNA excess by restrictase digestion and solubilized in 0.1% sodium dodecyl sulfate + 8 M urea. To obtain tightly bound complexes, the lysate was transferred to a solution containing 2 M LiCl and 4 mM urea and gel filtered in the same medium. Tightly bound complexes were eluted in the same peak with DNA. Loosely bound complexes were obtained by gel filtration in 0.1% sodium dodecyl sulfate + 8 M urea after preliminary digestion of the original complexes with DNAase I which selectively destroys the tight bonds. Within the composition of loosely bound DNA-matrix complexes, the polypeptides with Mr of 180, 65-75, 58 and 47-50 kDa were identified, whereas tightly bound complexes were shown to contain polypeptides with molecular masses of 180, 65-75, 63, 61, 58 and 52-53 kDa.     

Comparing the spectroscopic and electrochemical properties of ruthenium and osmium complexes of the tridentate polyazine ligands 2,2′:6′,2″-terpyridine and 2,3,5,6-tetrakis(2-pyridyl)pyrazine

A number of osmium and ruthenium complexes of the tridentate ligands 2,2′:6′,2″-terpyridine (tpy) and 2,3,5,6-tetrakis(2-pyridyl)pyrazine (tpp) have been prepared and characterized by our laboratory. All these complexes possess metal based oxidations and ligand based reductions localized on each polyazine ligand. Polymetallic complexes bridged by the tpp ligand exhibit two sequential tpp based reductions prior to the reduction of other polyazine ligands in these complexes. The spectroscopy of these complexes is dominated by ligand based π-π^* transitions in the ultraviolet and MLCT (metal-to-ligand charge transfer) bands terminating on each polyzine ligand in the visible. For the complexes reported herein the lowest lying optical transitionis a M → BL CT band. For most of the complexes reported, occupation of this excited state gives rise to an observable emission at room temperature. For ruthenium complexes of these tridentate ligands, the presence of a low-lying LF state shortens the excited state lifetimes of these chromophores. This gives rise to ruthenium complexes that display shorter excited state lifetimes than the analogous osmium based systems. Incorporation of tpp based chromophores into polymetallic frameworks leads to the production of bimetallic species with long-lived excited states, 100 ns at room temperature. This makes these chromophores good candidates for the development of stereochemically defined supramolecular complexes. It is possible to measure an electrochemical HOMO-LUMO energy gap and a correlation between this electrochemically measured energy gap and the spectroscopic energy associated with this HOMO→LUMO transition are reported herein (HOMO== highest occupied molecular orbital, LUMO = lowest unoccupied molecular orbital).     

Functional involvement of polypyrimidine tract-binding protein in translation initiation complexes with the internal ribosome entry site of foot-and-mouth disease virus.

The synthesis of picornavirus polyproteins is initiated cap independently far downstream from the 5' end of the viral RNA at the internal ribosome entry site (IRES). The cellular polypyrimidine tract-binding protein (PTB) binds to the IRES of foot-and-mouth disease virus (FMDV). In this study, we demonstrate that PTB is a component of 48S and 80S ribosomal initiation complexes formed with FMDV IRES RNA. The incorporation of PTB into these initiation complexes is dependent on the entry of the IRES RNA, since PTB and IRES RNA can be enriched in parallel either in 48S or 80S ribosomal complexes by stage-specific inhibitors of translation initiation. The formation of the ribosomal initiation complexes with the IRES occurs slowly, is temperature dependent, and correlates with the incorporation of PTB into these complexes. In a first step, PTB binds to the IRES, and then the small ribosomal subunit encounters this PTB-IRES complex. Mutations in the major PTB-binding site interfere simultaneously with the formation of initiation complexes, translation efficiency, and PTB cross-linking. PTB stimulates translation directed by the FMDV IRES in a rabbit reticulocyte lysate depleted of internal PTB, and the efficiency of translation can be restored to the original level by the addition of PTB. These results indicate that PTB plays an important role in the formation of initiation complexes with FMDV IRES RNA and in stimulation of internal translation initiation with this picornavirus.     

Iron sources forming nitrosyl complexes in animal tissues

Treatment of perfused mouse liver with nitric oxide does not change the intensities of ESR signals of iron-sulfur proteins characteristic of this tissue. Proceeding from this evidence and also from the ratio between the iron content in these proteins and dinitrosyl iron complexes (complexes 2.03) formed in the liver when it contacts with NO, it is concluded that iron-sulfur proteins are not involved in the formation of complexes 2.03. It seems that only the loosely bound form of non-heme iron-free iron is involved in this process.     

Oxygen radical-mediated DNA damage by redox-active Cr(III) complexes.

The mechanism of DNA damage induced by Cr(III) complexes is currently unknown even though it is considered to be the ultimate biologically active oxidation state of chromium. In this study, we have employed the Salmonella reversion assay to identify mutagenic Cr(III) complexes. Cyclic voltammetry was used to differentiate the redox kinetics between mutagenic and selected nonmutagenic Cr(III) species. Plasmid relaxation of supercoiled DNA was employed to show in vitro interactions with plasmid DNA and correlate the interactions with the electrochemical behavior and biological activity. The results of this study demonstrate that the mutagenic Cr(III) complexes identified in the Salmonella reversion assay display characteristics of reversibility and positive shifts of the Cr(III)/Cr(II) redox couple consistent with the ability of these Cr(III) complexes to serve as cyclical electron donors in a Fenton-like reaction. These same mutagenic complexes display an ability to relax supercoiled DNA in vitro, presumably by the induction of single-strand breaks. Nonmutagenic complexes were selected to test different ligands to determine how the ligand directs the activity of Cr(III) complexes. All nonmutagenic complexes tested thus far have shown classical irreversibility, more negative reduction potentials, and an inability to relax supercoiled plasmid DNA. These results suggest that the mechanism by which chromium complexes potentiate mutagenesis involves an oxygen radical as an active intermediate. These data also demonstrate the effect of associated ligands with regard to the ability of a metal to generate an active redox center.     

Heterologous Expression of Mycobacterial Esx Complexes in Escherichia coli for Structural Studies Is Facilitated by the Use of Maltose Binding Protein Fusions

The expression of heteroligomeric protein complexes for structural studies often requires a special coexpression strategy. The reason is that the solubility and proper folding of each subunit of the complex requires physical association with other subunits of the complex. The genomes of pathogenic mycobacteria encode many small protein complexes, implicated in bacterial fitness and pathogenicity, whose characterization may be further complicated by insolubility upon expression in Escherichia coli, the most common heterologous protein expression host. As protein fusions have been shown to dramatically affect the solubility of the proteins to which they are fused, we evaluated the ability of maltose binding protein fusions to produce mycobacterial Esx protein complexes. A single plasmid expression strategy using an N-terminal maltose binding protein fusion to the CFP-10 homolog proved effective in producing soluble Esx protein complexes, as determined by a small-scale expression and affinity purification screen, and coupled with intracellular proteolytic cleavage of the maltose binding protein moiety produced protein complexes of sufficient purity for structural studies. In comparison, the expression of complexes with hexahistidine affinity tags alone on the CFP-10 subunits failed to express in amounts sufficient for biochemical characterization. Using this strategy, six mycobacterial Esx complexes were expressed, purified to homogeneity, and subjected to crystallization screening and the crystal structures of the Mycobacterium abscessus EsxEF, M. smegmatis EsxGH, and M. tuberculosis EsxOP complexes were determined. Maltose binding protein fusions are thus an effective method for production of Esx complexes and this strategy may be applicable for production of other protein complexes.     

An evolutionary conservation-based method for refining and reranking protein complex structures

Detection of protein complexes and their structures is crucial for understanding their role in the basic biology of organisms. Computational docking methods can provide researchers with a good starting point for the analysis of protein complexes. However, these methods are often not accurate and their results need to be further refined to improve interface packing. In this paper, we introduce a refinement method that incorporates evolutionary information into a novel scoring function by employing Evolutionary Trace (ET)-based scores. Our method also takes Van der Waals interactions into account to avoid atomic clashes in refined structures. We tested our method on docked candidates of eight protein complexes and the results suggest that the proposed scoring function helps bias the search toward complexes with native interactions. We show a strong correlation between evolutionary-conserved residues and correct interface packing. Our refinement method is able to produce structures with better lRMSD (least RMSD) with respect to the known complexes and lower energies than initial docked structures. It also helps to filter out false-positive complexes generated by docking methods, by detecting little or no conserved residues on false interfaces. We believe this method is a step toward better ranking and prediction of protein complexes.     

Respiratory supercomplexes: structure,function and assembly

The mitochondrial respiratory chain consists of 5 enzyme complexes that are responsible for ATP generation. The paradigm of the electron transport chain as discrete enzymes diffused in the inner mitochondrial membrane has been replaced by the solid state supercomplex model wherein the respiratory complexes associate with each other to form supramolecular complexes. Defects in these supercomplexes, which have been shown to be functionally active and required for forming stable respiratory complexes, have been associated with many genetic and neurodegenerative disorders demonstrating their biomedical significance. In this review, we will summarize the functional and structural significance of supercomplexes and provide a comprehensive review of their assembly and the assembly factors currently known to play a role in this process.     

铽(Ⅲ)离子及其复合物:从发光特性到传感应用

稀土元素也称镧系元素,因其独特的发光性质和配位性质,其发光复合物被广泛研究于生物技术领域。其中稀土铽(Ⅲ)离子复合物因具有优异的光谱特性,关于其研究呈现出快速的发展趋势。主要从其发光特性的角度出发,探讨了其发光机理,并对铽(Ⅲ)离子与不同有机化合物结合形成的发光铽配合物以及铽(Ⅲ)离子及其配合物与不同纳米材料形成的复合物进行了分类综述。此外,还详细地阐述了铽离子及其复合物在荧光探针、生物传感器、药物递送、细胞成像、癌症治疗等相关领域的应用。最后,对其今后发展趋势和潜在的研究价值进行了展望。