Adaptor protein complexes and intracellular transport

The AP (adaptor protein) complexes are heterotetrameric protein complexes that mediate intracellular membrane trafficking along endocytic and secretory transport pathways. There are five different AP complexes: AP-1, AP-2 and AP-3 are clathrin-associated complexes; whereas AP-4 and AP-5 are not. These five AP complexes localize to different intracellular compartments and mediate membrane trafficking in distinct pathways. They recognize and concentrate cargo proteins into vesicular carriers that mediate transport from a donor membrane to a target organellar membrane. AP complexes play important roles in maintaining the normal physiological function of eukaryotic cells. Dysfunction of AP complexes has been implicated in a variety of inherited disorders, including: MEDNIK (mental retardation, enteropathy, deafness, peripheral neuropathy, ichthyosis and keratodermia) syndrome, Fried syndrome, HPS (Hermansky–Pudlak syndrome) and HSP (hereditary spastic paraplegia).     

MULTIPLE CORE COMPLEXES IN GRASSHOPPER SPERMATOCYTES AND SPERMATIDS

At meiotic prophase, the grasshopper Chorthippus longicornis has normal synaptinemal complexes inside paired homologous chromosomes. Evidence is presented that short single cores and small multiple core complexes occur inside metaphase I chromosomes. At first anaphase, interphase, and early spermatid stage, large multiple core complexes are located in the cytoplasm. It is speculated that the multiple core complexes have some structural elements in common with the synaptinemal complexes, but that different forms of pairing behavior are exhibited by the different complexes.     

Inhibition of yeast growth by molybdenum-hydroxylamido complexes correlates with their presence in media at differing pH values

The effects of Mo-hydroxylamido complexes on cell growth were determined in Saccharomyces cerevisiae to investigate the biological effects of four different Mo complexes as a function of pH. Studies with yeast, an eukaryotic cell, are particularly suited to examine growth at different pH values because this organism grows well from pH 3 to 6.5. Studies can therefore be performed both in the presence of intact complexes and when the complexes have hydrolyzed to ligand and free metal ion. One of the complexes we examined was structurally characterized by X-ray crystallography. Yeast growth was inhibited in media solutions containing added Mo-dialkylhydroxylamido complexes at pH 3-7. When combining the yeast growth studies with a systematic study of the Mo-hydroxylamido complexes' stability as a function of pH and an examination of their speciation in yeast media, the effects of intact complexes can be distinguished from that of ligand and metal. This is possible because different effects are observed with complex present than when ligand or metal alone is present. At pH 3, the growth inhibition is attributed to the forms of molybdate ion that exist in solution because most of the complexes have hydrolyzed to oxomolybdate and ligand. The monoalkylhydroxylamine ligand inhibited yeast growth at pH 5, 6 and 7, while the dialkylhydroxylamine ligands had little effect on yeast growth. Growth inhibition of the Mo-dialkylhydroxylamido complexes is observed when a complex exists in the media. A complex that is inert to ligand exchange is not effective even at pH 3 where other Mo-hydroxylamido complexes show growth inhibition as molybdate. These results show that the formation of some Mo complexes can protect yeast from the growth inhibition observed when either the ligand or Mo salt alone are present.     

Respiratory Chains from Aerobic Thermophilic Prokaryotes

Thermophiles are organisms that grow optimally above 50 degrees C and up to approximately 120 degrees C. These extreme conditions must have led to specific characteristics of the cellular components. In this paper we extensively analyze the types of respiratory complexes from thermophilic aerobic prokaryotes. The different membrane-bound complexes so far characterized are described, and the genomic data available for thermophilic archaea and bacteria are analyzed. It is observed that no specific characteristics can be associated to thermophilicity as the different types of complexes I-IV are present randomly in thermophilic aerobic organisms, as well as in mesophiles. Rather, the extensive genomic analyses indicate that the differences concerning the several complexes are related to the organism phylogeny, i.e., to evolution and lateral gene transfer events.     

Design of copper-based anti-inflammatory drugs

It has been shown that the inflammation associated with rheumatoid arthritis can be reduced using copper complexes. In order to improve the bioavailability of copper and hence efficacy of these complexes we have synthesized three different series of ligands, each having different characteristics. Thermodynamic results for copper(II) complexes for these polyamino, diaminodiamido and triaminodiamido ligands are presented. The polyamino ligands form the most stable complexes in vivo but tissue distribution studies in mice show that [Cu(3,6,9,12-tetraazatetradecanedioate)] is excreted rapidly, unchanged in the urine. The diamino ligand complexes are much less stable than their polyamino analogues and animal studies using [Cu(N,N'-bis[2-(dimethylamino)ethyl]-ethanediamide)H2] indicate that the complex dissociates in vivo and is excreted slowly via the liver. The triaminodiamido copper(II) complexes are approximately 2 log units more stable than their diamino analogues. Computer simulation calculations indicate that these complexes are also likely to dissociate in plasma. Measured partition coefficients, however, suggest the possibility of dermal absorption.     

Enhanced nuclear import and transfection efficiency of TAT peptide-based gene delivery systems modified by additional nuclear localization signals

Cellular uptake and nuclear localization are two major barriers in gene delivery. In order to evaluate whether additional nuclear localization signals (NLSs) can improve gene transfection efficiency, we introduced different kinds of NLSs to TAT-based gene delivery systems to form three kinds of complexes, including TAT-PV/DNA, TAT/DNA/PV, and TAT/DNA/HMGB1. The DNA binding ability of different vectors was evaluated by agarose gel electrophoresis. The in vitro transfections mediated by different complexes under different conditions were carried out. The cells treated by different complexes were observed by confocal microscopy. The MTT assay showed that all complexes did not exhibit apparent cytotoxicity in both HeLa and Cos7 cell lines even at high N/P ratios. The luciferase reporter gene expression mediated by TAT-PV/DNA complexes exhibited about 200-fold enhancement as compared with TAT/DNA complexes. Confocal study showed that, except TAT/DNA/PV, all other complexes exhibited enhanced nuclear accumulation and cellular uptake in both HeLa and Cos7 cell lines. These results indicated that the introduction of nuclear localization signals could enhance the transfection efficacy of TAT-based peptides, implying that the TAT peptide-based vectors demonstrated here have promising potential in gene delivery.     

Defining interactions and distributions of cadherin and catenin complexes in polarized epithelial cells

The cadherin/catenin complex plays important roles in cell adhesion, signal transduction, as well as the initiation and maintenance of structural and functional organization of cells and tissues. In the preceding study, we showed that the assembly of the cadherin/catenin complex is temporally regulated, and that novel combinations of catenin and cadherin complexes are formed in both Triton X-100-soluble and - insoluble fractions; we proposed a model in which pools of catenins are important in regulating assembly of E-cadherin/catenin and catenin complexes. Here, we sought to determine the spatial distributions of E- cadherin, alpha-catenin, beta-catenin, and plakoglobin, and whether different complexes of these proteins accumulate at steady state in polarized Madin-Darby canine kidney cells. Protein distributions were visualized by wide field, optical sectioning, and double immunofluorescence microscopy, followed by reconstruction of three- dimensional images. In cells that were extracted with Triton X-100 and then fixed (Triton X-100-insoluble fraction), more E-cadherin was concentrated at the apical junction relative to other areas of the lateral membrane. alpha-Catenin and beta-catenin colocalize with E- cadherin at the apical junctional complex. There is some overlap in the distribution of these proteins in the lateral membrane, but there are also areas where the distributions are distinct. Plakoglobin is excluded from the apical junctional complex, and its distribution in the lateral membrane is different from that of E-cadherin. Cells were also fixed and then permeabilized to reveal the total cellular pool of each protein (Triton X-100-soluble and -insoluble fractions). This analysis showed lateral membrane localization of alpha-catenin, beta- catenin, and plakoglobin, and it also revealed that they are distributed throughout the cell. Chemical cross-linking of proteins and analysis with specific antibodies confirmed the presence at steady state of E-cadherin/catenin complexes containing either beta-catenin or plakoglobin, and catenin complexes devoid of E-cadherin. Complexes containing E-cadherin/beta-catenin and E-cadherin/alpha-catenin are present in both the Triton X-100-soluble and -insoluble fractions, but E-cadherin/plakoglobin complexes are not detected in the Triton X-100- insoluble fraction. Taken together, these results show that different complexes of cadherin and catenins accumulate in fully polarized epithelial cells, and that they distribute to different sites. We suggest that cadherin/catenin and catenin complexes at different sites have specialized roles in establishing and maintaining the structural and functional organization of polarized epithelial cells.     

Interface of apoptotic protein complexes has distinct properties

Apoptosis is a programmed mechanism of cell death that is a normal component of development and health of multi-cellular organisms. In this study, we ask if interface properties of apoptotic protein complexes are different from protein complexes in general. We find that although in apoptotic protein complexes the overall distribution of interface size, surface complementarity, hydrogen bonding, hydrophobicity are similar to general interface properties, apoptotic complexes tend to have more fragmented interfaces and different secondary structural preferences. The statistics on the number of interfaces where specific amino acid(s) occur with significantly enhanced frequency suggest that Arg, Met and Asp are most important functional residues. The role of Met is believed to be unique, as evidenced from the existing data on hot spot potential of residues. These findings together provide insight into the possible role of various physico-chemical attributes at the protein interface in regulation of the apoptosis process.     

Synthesis, crystal structure, and DNA-cleaving behavior of 5-substituted benzene-1,3-bis(methylene)-spaced dinuclear copper(II) complexes

Three meta-dicopper complexes, 1-3, based on 5-substituted 1,3-xylylene spacer have been synthesized. These complexes are capable of inducing the transformation of supercoiled DNA (pUC19) to its nicked and linear DNA form in the presence of ascorbate, and their DNA nicking efficiency can be correlated to their DNA-binding ability. The cleavage mechanism is similar to that of the non-substituted meta-dicopper complex A. Amongst the three complexes, 5-(aminomethyl)-substituted complex 3 displayed a higher DNA-binding ability and nicking efficiency than unsubstituted complex A. The CD-spectroscopic study and structural analysis imply that the different CuCu distances and DNA binding modes induced by different 5-substituents on benzene-1,3-bis(methylene) spacer may be responsible for the different DNA cleaving behavior of meta-dicopper complexes.     

COMBINE analysis of the specificity of binding of Ras proteins to their effectors

The small guanosine triphosphate (GTP)-binding proteins of the Ras family are involved in many cellular pathways leading to cell growth, differentiation, and apoptosis. Understanding the interaction of Ras with other proteins is of importance not only for studying signalling mechanisms but also, because of their medical relevance as targets, for anticancer therapy. To study their selectivity and specificity, which are essential to their signal transfer function, we performed COMparative BINding Energy (COMBINE) analysis for 122 different wild-type and mutant complexes between the Ras proteins, Ras and Rap, and their effectors, Raf and RalGDS. The COMBINE models highlighted the amino acid residues responsible for subtle differences in binding of the same effector to the two different Ras proteins, as well as more significant differences in the binding of the two different effectors (RalGDS and Raf) to Ras. The study revealed that E37, D38, and D57 in Ras are nonspecific hot spots at its effector interface, important for stabilization of both the RalGDS-Ras and Raf-Ras complexes. The electrostatic interaction between a GTP analogue and the effector, either Raf or RalGDS, also stabilizes these complexes. The Raf-Ras complexes are specifically stabilized by S39, Y40, and D54, and RalGDS-Ras complexes by E31 and D33. Binding of a small molecule in the vicinity of one of these groups of amino acid residues could increase discrimination between the Raf-Ras and RalGDS-Ras complexes. Despite the different size of the RalGDS-Ras and Raf-Ras complexes, we succeeded in building COMBINE models for one type of complex that were also predictive for the other type of protein complex. Further, using system-specific models trained with only five complexes selected according to the results of principal component analysis, we were able to predict binding affinities for the other mutants of the particular Ras-effector complex. As the COMBINE analysis method is able to explicitly reveal the amino acid residues that have most influence on binding affinity, it is a valuable aid for protein design.     

Gold(III) Compounds as New Family of Anticancer Drugs

Gold(III) complexes are emerging as a new class of metal complexes with outstanding cytotoxic properties and are presently being evaluated as potential antitumor agents. This renewed interest is the result of recent studies in which various gold(III) complexes have been shown to be stable under physiological conditions and to manifest relevant antiproliferative properties against selected human tumor cell lines. The pharmacological investigation of some representative gold(III) complexes has been extended to consider their effects on the cell cycle and to reveal induction of apoptosis. Remarkably, preliminary studies suggest that the interactions in vitro of gold(Ill) complexes with calf thymus DNA are weak whereas significant binding to model proteins takes place. Our findings imply that the mechanism of action of cytotoxic gold(Ill) complexes might be substantially different from that of clinically established platinum compounds.     

Protein complexes of the Escherichia coli cell envelope

Protein complexes are an intrinsic aspect of life in the membrane. Knowing which proteins are assembled in these complexes is therefore essential to understanding protein function(s). Unfortunately, recent high throughput protein interaction studies have failed to deliver any significant information on proteins embedded in the membrane, and many membrane protein complexes remain ill defined. In this study, we have optimized the blue native-PAGE technique for the study of membrane protein complexes in the inner and outer membranes of Escherichia coli. In combination with second dimension SDS-PAGE and mass spectrometry, we have been able to identify 43 distinct protein complexes. In addition to a number of well characterized complexes, we have identified known and orphan proteins in novel oligomeric states. For two orphan proteins, YhcB and YjdB, our findings enable a tentative functional assignment. We propose that YhcB is a hitherto unidentified additional subunit of the cytochrome bd oxidase and that YjdB, which co-localizes with the ZipA protein, is involved in cell division. Our reference two-dimensional blue native-SDS-polyacrylamide gels will facilitate future studies of the assembly and composition of E. coli membrane protein complexes during different growth conditions and in different mutant backgrounds.     

Identification and characterization of protein subcomplexes in yeast

Protein complexes are major components of cellular organization. Based on large-scale protein complex data, we present the first statistical procedure to find insightful substructures in protein complexes: we identify protein subcomplexes (SCs), i.e., multiprotein assemblies residing in different protein complexes. Four protein complex datasets with different origins and variable reliability are separately analyzed. Our method identifies well-characterized protein assemblies with known functions, thereby confirming the utility of the procedure. In addition, we also identify hitherto unknown functional entities consisting of either functionally unknown proteins or proteins with different functional annotation. We show that SCs represent more reliable protein assemblies than the original complexes. Finally, we demonstrate unique properties of subcomplex proteins that underline the distinct roles of SCs: (i) SCs are functionally and spatially more homogeneous than complete protein complexes (this fact is utilized to predict functional roles and subcellular localizations for so far unannotated proteins); (ii) the abundance of subcomplex proteins is less variable than the abundance of other proteins; (iii) SCs are enriched with essential and synthetic lethal proteins; and (iv) mutations in SC-proteins have higher fitness effects than mutations in other proteins.     

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.     

Decomposition of overlapping protein complexes: A graph theoretical method for analyzing static and dynamic protein associations

Background  

Most cellular processes are carried out by multi-protein complexes, groups of proteins that bind together to perform a specific task. Some proteins form stable complexes, while other proteins form transient associations and are part of several complexes at different stages of a cellular process. A better understanding of this higher-order organization of proteins into overlapping complexes is an important step towards unveiling functional and evolutionary mechanisms behind biological networks.