Structural organization and function of parasitic systems

The structural organization and functioning of parasitic systems are considered in the context of the general theory of systems. By using the parasitic system of an epizootic process of plague as an example, the essence of the concepts of the structural organization and functioning of the systems is determined, and a scheme of a relationship of elements in the trinomial parasitic systems is presented. The mechanism of parasite-host interaction at the molecular, cellular, body's, and populational levels is considered on the basis of the theory of immune-specific wave information in an living organism.     

Functional organization of the yeast proteome by a yeast interactome map

It is hoped that comprehensive mapping of protein physical interactions will facilitate insights regarding both fundamental cell biology processes and the pathology of diseases. To fulfill this hope, good solutions to 2 issues will be essential: (i) how to obtain reliable interaction data in a high-throughput setting and (ii) how to structure interaction data in a meaningful form, amenable to and valuable for further biological research. In this article, we structure an interactome in terms of predicted permanent protein complexes and predicted transient, nongeneric interactions between these complexes. The interactome is generated by means of an associated computational algorithm, from raw high-throughput affinity purification/mass spectrometric interaction data. We apply our technique to the construction of an interactome for Saccharomyces cerevisiae, showing that it yields reliability typical of low-throughput experiments from high-throughput data. We discuss biological insights raised by this interactome including, via homology, a few related to human disease.     

Supramolecular organization of the mammalian translation system.

Although evidence suggests that the protein synthetic machinery is organized within cells, this point has been difficult to prove because any organization that might exist is lost upon preparation of the cell-free systems usually used to study translation in vitro. To examine this process under conditions more representative of the intact cell, we have developed an active protein-synthesizing system using Chinese hamster ovary (CHO) cells permeabilized with the plant glycoside saponin. This procedure renders cells permeable to trypan blue and exogenous tRNA, but there is little release of endogenous macromolecules. Protein synthesis in this system proceeds at the same rate as that in intact cells and is about 40-fold faster than that in a cell-free system prepared from the same cells. Active protein synthesis in this system requires the addition of only Mg2+, K+, and creatine phosphate, with a small further stimulation by ATP and an amino acid mixture; no exogenous macromolecules are necessary. The proteins synthesized in this system are indistinguishable from those made by the intact cell, and the channeling of aminoacyl-tRNA observed in vivo is maintained. Our data suggest that the permeabilized cell system retains the protein-synthesizing capabilities of the intact cell and presumably its internal structure as well. Studies with this system demonstrate that the protein-synthesizing apparatus is highly organized and that its macromolecular components are not freely diffusible in mammalian cells.     

Tripartite organization of centromeric chromatin in budding yeast

The centromere is the genetic locus that organizes the proteinaceous kinetochore and is responsible for attachment of the chromosome to the spindle at mitosis and meiosis. In most eukaryotes, the centromere consists of highly repetitive DNA sequences that are occupied by nucleosomes containing the CenH3 histone variant, whereas in budding yeast, a ～120-bp centromere DNA element (CDE) that is sufficient for centromere function is occupied by a single right-handed histone variant CenH3 (Cse4) nucleosome. However, these in vivo observations are inconsistent with in vitro evidence for left-handed octameric CenH3 nucleosomes. To help resolve these inconsistencies, we characterized yeast centromeric chromatin at single base-pair resolution. Intact particles containing both Cse4 and H2A are precisely protected from micrococcal nuclease over the entire CDE of all 16 yeast centromeres in both solubilized chromatin and the insoluble kinetochore. Small DNA-binding proteins protect CDEI and CDEIII and delimit the centromeric nucleosome to the ～80-bp CDEII, only enough for a single DNA wrap. As expected for a tripartite organization of centromeric chromatin, loss of Cbf1 protein, which binds to CDEI, both reduces the size of the centromere-protected region and shifts its location toward CDEIII. Surprisingly, Cse4 overproduction caused genome-wide misincorporation of nonfunctional CenH3-containing nucleosomes that protect ～135 base pairs and are preferentially enriched at sites of high nucleosome turnover. Our detection of two forms of CenH3 nucleosomes in the yeast genome, a singly wrapped particle at the functional centromere and octamer-sized particles on chromosome arms, reconcile seemingly conflicting in vivo and in vitro observations.     

Dimeric organization of the yeast oligosaccharyl transferase complex

The enzyme complex oligosaccharyl transferase (OT) catalyzes N-glycosylation in the lumen of the endoplasmic reticulum. The yeast OT complex is composed of nine subunits, all of which are transmembrane proteins. Several lines of evidence, including our previous split-ubiquitin studies, have suggested an oligomeric organization of the OT complex, but the exact oligomeric nature has been unclear. By FLAG epitope tagging the Ost4p subunit of the OT complex, we purified the OT enzyme complex by using the nondenaturing detergent digitonin and a one-step immunoaffinity technique. The digitonin-solubilized OT complex was catalytically active, and all nine subunits were present in the enzyme complex. The purified OT complex had an apparent mass of approximately 500 kDa, suggesting a dimeric configuration, which was confirmed by biochemical studies. EM showed homogenous individual particles and revealed a dimeric structure of the OT complexes that was consistent with our biochemical studies. A 3D structure of the dimeric OT complex at 25-A resolution was reconstructed from EM images. We suggest that the dimeric structure of OT might be required for effective association with the translocon dimer and for its allosteric regulation during cotranslational glycosylation.     

Alternative branch points are selected during splicing of a yeast pre-mRNA in mammalian and yeast extracts.

Pre-mRNA splicing in yeast and higher eukaryotes proceeds by similar pathways, in which a probable splicing intermediate and the excised intron are in a lariat configuration. To compare the pre-mRNA splicing mechanisms in yeast and higher eukaryotes, we have analyzed the RNA products resulting from in vitro processing of a yeast intron-containing pre-mRNA in HeLa cell and yeast extracts. In yeast, the RNA branch (2'-5' phosphodiester bond) of the RNA lariat forms at the third adenosine of the TACTAAC box in vivo and in vitro. In contrast, in the HeLa cell extract, the yeast pre-mRNA is accurately spliced, but the RNA lariats contain RNA branches located significantly closer to the 3' splice site than the TACTAAC box. In yeast, mutant pre-mRNAs that lack the TACTAAC box are not spliced in vivo or in vitro. However, these same mutant pre-mRNAs are accurately spliced in the HeLa cell extract. Therefore, although pre-mRNA splicing in yeast and higher eukaryotes proceeds by the same basic pathway, there are substantial differences in the specificity of the biochemical components that mediate the formation of the RNA processing products.     

Morphological, chemical, and antigenic organization of mammalian C-type viruses.

New features in the architecture of mammalian type C viruses, in particular knoblike surface projections and hexagonally arranged subunits on the core shell could be demonstrated by electron microscopy, taking advantage of newly developed preparation techniques. As examples, murine leukemia viruses (MuLVs) and newly isolated porcine and bovine C viruses are presented. The major proteins of a MuLV were isolated and partially characterized in chemical terms and with respect to their serological and other biological activities, such as interfering and hemagglutinating (HA) capacity. Most of the characterized proteins could be localized in particular substructures of the virion either by selective removal or isolation of electron microscopically identifiable constituents. The information obtained allowed the design of a more detailed model of mammalian C viruses. Special attention was devoted to the further characterization of interspecies antigens of mammalian C viruses. Different antigenic determinants were revealed. Their distribution allows further subgrouping of mammalian C viruses.     

Structural organization of a filamentous influenza A virus

Influenza is a lipid-enveloped, pleomorphic virus. We combine electron cryotomography and analysis of images of frozen-hydrated virions to determine the structural organization of filamentous influenza A virus. Influenza A/Udorn/72 virions are capsule-shaped or filamentous particles of highly uniform diameter. We show that the matrix layer adjacent to the membrane is an ordered helix of the M1 protein and its close interaction with the surrounding envelope determines virion morphology. The ribonucleoprotein particles (RNPs) that package the genome segments form a tapered assembly at one end of the virus interior. The neuraminidase, which is present in smaller numbers than the hemagglutinin, clusters in patches and are typically present at the end of the virion opposite to RNP attachment. Incubation of virus at low pH causes a loss of filamentous morphology, during which we observe a structural transition of the matrix layer from its helical, membrane-associated form to a multilayered coil structure inside the virus particle. The polar organization of the virus provides a model for assembly of the virion during budding at the host membrane. Images and tomograms of A/Aichi/68 X-31 virions show the generality of these conclusions to non-filamentous virions.     

Structural organization of the human androgen receptor gene

The complete coding region of the human androgen receptor gene has been isolated from a genomic library. The information for the androgen receptor was found to be divided over eight exons and the total length of the gene exceeded 90 kb. The sequence encoding the N-terminal region is present in one large exon. The two putative DNA-binding fingers are encoded separately by two small exons. The information for the hormone-binding domain is split over five exons. Positions of introns are identical to those reported for the chicken progesterone receptor and the human oestrogen receptor genes. Southern blot analysis of genomic DNA with various specific probes reveal that the human androgen receptor is encoded by a single-copy gene.     

Structural basis of flocculin-mediated social behavior in yeast

In the budding yeast Saccharomyces cerevisiae, self-recognition and the thereby promoted aggregation of thousands of cells into protective flocs is mediated by a family of cell-surface adhesins, the flocculins (Flo). Based on this social behavior FLO genes fulfill the definition of “greenbeard” genes, which direct cooperation toward other carriers of the same gene. The process of flocculation plays an eminent role in the food industry for the production of beer and wine. However, the precise mode of flocculin-mediated surface recognition and the exact structure of cognate ligands have remained elusive. Here, we present structures of the adhesion domain of a flocculin complexed to its cognate ligands derived from yeast high-mannose oligosaccharides at resolutions up to 0.95 Å. Besides a PA14-like architecture, the Flo5A domain reveals a previously undescribed lectin fold that utilizes a unique DcisD calcium-binding motif for carbohydrate binding and that is widely spread among pro- and eukaryotes. Given the high abundance of high-mannose oligosaccharides in yeast cell walls, the Flo5A structure suggests a model for recognition, where social non-self- instead of unsocial self-interactions are favored.     

Structural basis for induced formation of the inflammatory mediator prostaglandin E2

Prostaglandins (PG) are bioactive lipids produced from arachidonic acid via the action of cyclooxygenases and terminal PG synthases. Microsomal prostaglandin E synthase 1 (MPGES1) constitutes an inducible glutathione-dependent integral membrane protein that catalyzes the oxidoreduction of cyclooxygenase derived PGH(2) into PGE(2). MPGES1 has been implicated in a number of human diseases or pathological conditions, such as rheumatoid arthritis, fever, and pain, and is therefore regarded as a primary target for development of novel antiinflammatory drugs. To provide a structural basis for insight in the catalytic mechanism, we determined the structure of MPGES1 in complex with glutathione by electron crystallography from 2D crystals induced in the presence of phospholipids. Together with results from site-directed mutagenesis and activity measurements, we can thereby demonstrate the role of specific amino acid residues. Glutathione is found to bind in a U-shaped conformation at the interface between subunits in the protein trimer. It is exposed to a site facing the lipid bilayer, which forms the specific environment for the oxidoreduction of PGH(2) to PGE(2) after displacement of the cytoplasmic half of the N-terminal transmembrane helix. Hence, insight into the dynamic behavior of MPGES1 and homologous membrane proteins in inflammation and detoxification is provided.     

Point mutations in TOR confer Rheb-independent growth in fission yeast and nutrient-independent mammalian TOR signaling in mammalian cells

Rheb is a unique member of the Ras superfamily GTP-binding proteins. We as well as others previously have shown that Rheb is a critical component of the TSC/TOR signaling pathway. In fission yeast, Rheb is encoded by the rhb1 gene. Rhb1p is essential for growth and directly interacts with Tor2p. In this article, we report identification of 22 single amino acid changes in the Tor2 protein that enable growth in the absence of Rhb1p. These mutants also exhibit decreased mating efficiency. Interestingly, the mutations are located in the C-terminal half of the Tor2 protein, clustering mainly within the FAT and kinase domains. We noted some differences in the effect of a mutation in the FAT domain (L1310P) and in the kinase domain (E2221K) on growth and mating. Although the Tor2p mutations bypass Rhb1p's requirement for growth, they are incapable of suppressing Rhb1p's requirement for resistance to stress and toxic amino acids, pointing to multiple functions of Rhb1p. In mammalian systems, we find that mammalian target of rapamycin (mTOR) carrying analogous mutations (L1460P or E2419K), although sensitive to rapamycin, exhibits constitutive activation even when the cells are starved for nutrients. These mutations do not show significant difference in their ability to form complexes with Raptor, Rictor, or mLST8. Furthermore, we present evidence that mutant mTOR can complex with wild-type mTOR and that this heterodimer is active in nutrient-starved cells.     

Structural basis for multifunctional roles of mammalian aminopeptidase N

Mammalian aminopeptidase N (APN) plays multifunctional roles in many physiological processes, including peptide metabolism, cell motility and adhesion, and coronavirus entry. Here we determined crystal structures of porcine APN at 1.85 Å resolution and its complexes with a peptide substrate and a variety of inhibitors. APN is a cell surface-anchored and seahorse-shaped zinc-aminopeptidase that forms head-to-head dimers. Captured in a catalytically active state, these structures of APN illustrate a detailed catalytic mechanism for its aminopeptidase activity. The active site and peptide-binding channel of APN reside in cavities with wide openings, allowing easy access to peptides. The cavities can potentially open up further to bind the exposed N terminus of proteins. The active site anchors the N-terminal neutral residue of peptides/proteins, and the peptide-binding channel binds the remainder of the peptides/proteins in a sequence-independent fashion. APN also provides an exposed outer surface for coronavirus binding, without its physiological functions being affected. These structural features enable APN to function ubiquitously in peptide metabolism, interact with other proteins to mediate cell motility and adhesion, and serve as a coronavirus receptor. This study elucidates multifunctional roles of APN and can guide therapeutic efforts to treat APN-related diseases.Mammalian aminopeptidase N (APN) plays pivotal roles in many physiological processes, such as pain sensation, blood pressure regulation, tumor angiogenesis and metastasis, immune cell chemotaxis, sperm motility, cell-cell adhesion, and coronavirus entry (1). Accordingly, APN is a major target for treatment of diseases that are related to the above physiological processes. It is puzzling how APN is able to possess such a wide range of physiological functions, some of which are seemingly unrelated to its aminopeptidase activity. This study determines the atomic structures of mammalian APN and its complexes with a variety of APN-targeting ligands, providing structural basis for the multifunctional roles of APN and for the development of novel therapy strategies to treat APN-related diseases.The M1-family of metalloenzymes consists of a large number of zinc-dependent aminopeptidases containing a zinc-binding HEXXH motif. As the most extensively studied member in this family, mammalian APN (also known as CD13 or alanine aminopeptidase) is widely expressed on cell surfaces of tissues, such as intestinal epithelia and the nervous system (1). APN preferentially cleaves neutral amino acids, most notably alanine, off the N terminus of peptides. The general catalytic mechanism of M1-family metalloenzymes is believed to be similar to that of prototypic zinc-peptidase thermolysin, which involves catalytic water attacking scissile peptide bonds (2), but detailed catalytic mechanisms of these enzymes remain elusive. To date, crystal structures are available for several members of the M1-family metalloenzymes (3–8). However, these enzymes are monomeric intracellular enzymes with specific physiological roles, whereas mammalian APN is a dimeric cell-surface ectoenzyme with multifunctional roles (1). This study investigates the structural differences between mammalian APN and other M1-family metalloenzymes that account for their functional differences.Mammalian APN functions ubiquitously in different peptide metabolism pathways. First, APN plays important roles in pain sensation and mood regulation by catalyzing the metabolism of neuropeptides that process sensory information. One of these neuropeptides is enkephalin, which binds to opioid receptors and has pain-relief and mood-regulating effects (9). APN degrades and shortens the in vivo life of enkephalin, and hence enhances pain sensation and regulates mood. Second, APN is involved in blood pressure regulation. APN degrades vasoconstrictive peptide angiotensin-III, causing vasodilation and lowered blood pressure (10). An endogenous APN inhibitor, substance P, blocks both the enkephalin-dependent and angiotensin-dependent pathways (11, 12). Third, APN is overexpressed on the cell surfaces of almost all major cancer forms and is essential for tumor angiogenesis by degrading angiogenic peptides (13). A natural APN inhibitor, bestatin, is a substrate analog for APN and demonstrates antitumor activities (14). Additionally, APN is involved in many other peptide metabolism pathways by catalyzing the degradation of peptides related to these pathways. These peptides include tuftsin, kinins, glutathione, somatostatin, thymopentin, neurokinin A, splenopentin, nociceptin FQ, and more (1). It is not clear how APN is able to access so many peptides from various peptide metabolism pathways.Curiously, APN also functions in cell motility and adhesion, and many of these functions are seemingly independent of its aminopeptidase activity. For example, APN participates in tumor cell motility as revealed in several studies: stable expression of APN on tumor cell surfaces greatly increases their migratory capacity, and knocking out APN expression or using anti-APN antibodies can block tumor migration (13, 15–17). The mechanisms whereby APN mediates tumor migration have been partially linked to its aminopeptidase activity, as it was shown that APN degrades extracellular matrix proteins (18, 19). However, an unknown activity of APN independent from its aminopeptidase activity also contributes to tumor migration, as suggested by several other studies: APN-mediated tumor migration can be blocked by antibodies that do not target the aminopeptiase activity of APN, and tumor cells expressing an enzymatically inactive APN also show enhanced migration (16, 20). Besides mediating tumor migration, APN also mediates other cell motility processes, such as immune cell chemotaxis and sperm motility (21–25). Additionally, APN mediates cell-cell adhesion by interacting with other cell-surface proteins (16, 26–28). How APN mediates cell motility and adhesion is largely a mystery.Interestingly, mammalian APN serves as a receptor for coronaviruses, including human respiratory coronavirus 229E (HCoV-229E), porcine transmissible gastroenteritis virus (TGEV), feline enteric coronavirus (FCoV), and canine enteric coronavirus (CCoV) (29–31). Coronaviruses are a family of positive-stranded RNA viruses that infect many mammalian and avian species. They exploit diverse cellular receptors for host cell entry through a receptor-binding domain (RBD) on their envelope-anchored spike glycoproteins. Crystal structures are available for several coronavirus receptors, either by themselves or complexed with coronavirus RBDs, illustrating detailed mechanisms whereby these receptors are recognized by coronaviruses (32–37). Mutagenesis has been used to characterize APN/coronavirus interactions, revealing multiple coronavirus-binding sites on APN (38, 39). It is not known how coronavirus binding affects physiological functions of APN.Here we report the crystal structures of porcine APN and its complexes with peptide substrate poly-alanine, amino acid alanine, substrate analog bestatin, and peptide inhibitor substance P. These structures provide a basis for understanding the multifunctional roles of mammalian APN and for the development of novel therapy strategies to treat APN-related diseases.     

The yeast analog of mammalian cyclin/proliferating-cell nuclear antigen interacts with mammalian DNA polymerase delta.

DNA polymerase III from Saccharomyces cerevisiae is analogous to the mammalian DNA polymerase delta by several criteria, including an increased synthetic activity on poly(dA).oligo(dT) (40:1 nucleotide ratio) in the presence of calf thymus proliferating-cell nuclear antigen (PCNA), or cyclin. This stimulation assay has been used to purify the yeast analog of PCNA/cyclin (yPCNA) to homogeneity. yPCNA is a trimer or tetramer (Mr approximately 82,000) of identical subunits with a denatured Mr of 26,000. On a molar basis yPCNA and calf thymus PCNA/cyclin are equally active in stimulating DNA synthesis by DNA polymerase III. About 10 times more yPCNA than calf thymus PCNA/cyclin is needed, however, to stimulate calf thymus DNA polymerase delta, and the degree of stimulation obtained at saturating levels of yPCNA is a factor of 2-3 less than with calf thymus PCNA/cyclin. Both stimulatory proteins exert their effect in an identical fashion, i.e., by increasing the processivity of the DNA polymerase. Yeast DNA polymerases I and II and calf thymus DNA polymerase alpha are not stimulated by yPCNA. Treatment of logarithmic-phase cells with hydroxyurea blocks them in the S phase and produces a 4- to 5-fold increase in yPCNA.