Analysis of the Candida albicans Phosphoproteome.

Candida albicans is an important human fungal pathogen in both immunocompetent and immunocompromised individuals. C. albicans regulation has been studied in many contexts, including morphological transitions, mating competence, biofilm formation, stress resistance, and cell wall synthesis. Analysis of kinase- and phosphatase-deficient mutants has made it clear that protein phosphorylation plays an important role in the regulation of these pathways. In this study, to further our understanding of phosphorylation in C. albicans regulation, we performed a deep analysis of the phosphoproteome in C. albicans. We identified 19,590 unique peptides that corresponded to 15,906 unique phosphosites on 2,896 proteins. The ratios of serine, threonine, and tyrosine phosphosites were 80.01%, 18.11%, and 1.81%, respectively. The majority of proteins (2,111) contained at least two detected phosphorylation sites. Consistent with findings in other fungi, cytoskeletal proteins were among the most highly phosphorylated proteins, and there were differences in Gene Ontology (GO) terms for proteins with serine and threonine versus tyrosine phosphorylation sites. This large-scale analysis identified phosphosites in protein components of Mediator, an important transcriptional coregulatory protein complex. A targeted analysis of the phosphosites in Mediator complex proteins confirmed the large-scale studies, and further in vitro assays identified a subset of these phosphorylations that were catalyzed by Cdk8 (Ssn3), a kinase within the Mediator complex. These data represent the deepest single analysis of a fungal phosphoproteome and lay the groundwork for future analyses of the C. albicans phosphoproteome and specific phosphoproteins.

Bronchoalveolar neutrophils, interferon gamma-inducible protein 10 and interleukin-7 in AIDS-associated tuberculosis.

During advanced AIDS tuberculosis (TB) often presents atypically with smear-negative and non-cavitary disease, yet immune features associated with this change are poorly characterized. We examined the local immune response in a cohort of Tanzanian AIDS-associated TB patients who underwent bronchoalveolar lavage. TB infection was confirmed in bronchoalveolar lavage (BAL) fluid by culture, probe and polymerase chain reaction (PCR). Among TB patients CD4 count correlated positively with the extent of cavitary disease as well as BAL TB load (qPCR C(T)). TB patients had significantly higher granulocyte-macrophage colony-stimulating factor (GM-CSF) than non-TB patients, and those with non-cavitary TB had significantly higher BAL interferon gamma-inducible protein (IP-10) and interleukin (IL)-7 than those with cavities. BAL neutrophils were as prevalent as monocytes/macrophages or epithelial cells, and immunohistochemistry revealed that neutrophils, monocytes/macrophages, and epithelial cells were major sources of the IP-10 and IL-7. These data suggest a dysregulated cytokine profile may contribute to the TB of advanced AIDS.

Structural basis for the functional switch of the E. coli Ada protein.

The Escherichia coli protein Ada specifically repairs the S(p) diastereomer of DNA methyl phosphotriesters in DNA by direct and irreversible transfer of the methyl group to its own Cys 69 which is part of a zinc-thiolate center. The methyl transfer converts Ada into a transcriptional activator that binds sequence-specifically to promoter regions of its own gene and other methylation resistance genes. Ada thus acts as a chemosensor to activate repair mechanisms in situations of methylation damage. Here we present a highly refined solution structure of the 10 kDa N-terminal domain, N-Ada10, which reveals structural details of the nonspecific DNA interaction of N-Ada10 during the repair process and provides a basis for understanding the mechanism of the conformational switch triggered by methyl transfer. To further elucidate this, EXAFS (extended X-ray absorption fine structure) and XANES (X-ray absorption near-edge structure) data were acquired, which confirmed that the zinc-thiolate center is maintained when N-Ada is methylated. Thus, ligand exchange is not the mechanism that enhances sequence-specific DNA binding and transcriptional activation upon methylation of N-Ada. The mechanism of the switch was further elucidated by recording NOESY spectra of specifically labeled methylated-Ada/DNA complexes, which showed that the transferred methyl group makes many contacts within N-Ada but none with the DNA. This implies that methylation of N-Ada induces a structural change, which enhances the promoter affinity of a remodeled surface region that does not include the transferred methyl group.

Structural organization of yeast and mammalian mediator complexes.

Structures of yeast Mediator complex, of a related complex from mouse cells and of thyroid hormone receptor-associated protein complex from human cells have been determined by three-dimensional reconstruction from electron micrographs of single particles. All three complexes show a division in two parts, a "head" domain and a combined "middle-tail" domain. The head domains of the three complexes appear most similar and interact most closely with RNA polymerase II. The middle-tail domains show the greatest structural divergence and, in the case of the tail domain, may not interact with polymerase at all. Consistent with this structural divergence, analysis of a yeast Mediator mutant localizes subunits that are not conserved between yeast and mammalian cells to the tail domain. Biochemically defined Rgr1 and Srb4 modules of yeast Mediator are then assigned to the middle and head domains.

Mediator of transcriptional regulation.

Three lines of evidence have converged on a multiprotein Mediator complex as a conserved interface between gene-specific regulatory proteins and the general transcription apparatus of eukaryotes. Mediator was discovered as an activity required for transcriptional activation in a reconstituted system from yeast. Upon resolution to homogeneity, the activity proved to reside in a 20-protein complex, which could exist in a free state or in a complex with RNA polymerase II, termed holoenzyme. A second line of evidence came from screens in yeast for mutations affecting transcription. Two-thirds of Mediator subunits are encoded by genes revealed by these screens. Five of the genetically defined subunits, termed Srbs, were characterized as interacting with the C-terminal domain of RNA polymerase II in vivo, and were shown to bind polymerase in vitro. A third line of evidence has come recently from studies in mammalian transcription systems. Mammalian counterparts of yeast Mediator were shown to interact with transcriptional activator proteins and to play an essential role in transcriptional regulation. Mediator evidently integrates and transduces positive and negative regulatory information from enhancers and operators to promoters. It functions directly through RNA polymerase II, modulating its activity in promoter-dependent transcription. Details of the Mediator mechanism remain obscure. Additional outstanding questions include the patterns of promoter-specificity of the various Mediator subunits, the possible cell-type-specificity of Mediator subunit composition, and the full structures of both free Mediator and RNA polymerase II holoenzyme.

Mediator-nucleosome interaction.

Mediator, a multiprotein complex involved in the regulation of RNA polymerase II transcription, binds to nucleosomes and acetylates histones. Three lines of evidence identify the Nut1 subunit of Mediator as responsible for the histone acetyltransferase (HAT) activity. An "in-gel" HAT assay reveals a single band of the appropriate size. Sequence alignment shows significant similarity of Nut1 to the GCN5-related N-acetyltransferase superfamily. Finally, recombinant Nut1 exhibits HAT activity in an in-gel assay.

Conserved structures of mediator and RNA polymerase II holoenzyme.

Single particles of the mediator of transcriptional regulation (Mediator) and of RNA polymerase II holoenzyme were revealed by electron microscopy and image processing. Mediator alone appeared compact, but at high pH or in the presence of RNA polymerase II it displayed an extended conformation. Holoenzyme contained Mediator in a fully extended state, partially enveloping the globular polymerase, with points of apparent contact in the vicinity of the polymerase carboxyl-terminal domain and the DNA-binding channel. A similarity in appearance and conformational behavior of yeast and murine complexes indicates a conservation of Mediator structure among eukaryotes.

Mediator protein mutations that selectively abolish activated transcription.

Deletion of any one of three subunits of the yeast Mediator of transcriptional regulation, Med2, Pgd1 (Hrs1), and Sin4, abolished activation by Gal4-VP16 in vitro. By contrast, other Mediator functions, stimulation of basal transcription and of TFIIH kinase activity, were unaffected. A different but overlapping Mediator subunit dependence was found for activation by Gcn4. The genetic requirements for activation in vivo were closely coincident with those in vitro. A whole genome expression profile of a Deltamed2 strain showed diminished transcription of a subset of inducible genes but only minor effects on "basal" transcription. These findings make an important connection between transcriptional activation in vitro and in vivo, and identify Mediator as a "global" transcriptional coactivator.

Identification of new mediator subunits in the RNA polymerase II holoenzyme from Saccharomyces cerevisiae.

Mediator was isolated from yeast on the basis of its requirement for transcriptional activation in a fully defined system. We have now identified three new members of mediator in the low molecular mass range by peptide sequence determination. These are the products of the NUT2, CSE2, and MED11 genes. The product of the NUT1 gene is evidently a component of mediator as well. NUT1 and NUT2 were earlier identified as negative regulators of the HO promoter, whereas mutations in CSE2 affect chromosome segregation. MED11 is a previously uncharacterized gene. The existence of these proteins in the mediator complex was verified by copurification and co-immunoprecipitation with RNA polymerase II holoenzyme.

The Med proteins of yeast and their function through the RNA polymerase II carboxy-terminal domain.

Mediator was resolved from yeast as a multiprotein complex on the basis of its requirement for transcriptional activation in a fully defined system. Three groups of mediator polypeptides could be distinguished: the products of five SRB genes, identified as suppressors of carboxy-terminal domain (CTD)-truncation mutants; products of four genes identified as global repressors; and six members of a new protein family, termed Med, thought to be primarily responsible for transcriptional activation. Notably absent from the purified mediator were Srbs 8, 9, 10, and 11, as well as members of the SWI/SNF complex. The CTD was required for function of mediator in vitro, in keeping with previous indications of involvement of the CTD in transcriptional activation in vivo. Evidence for human homologs of several mediator proteins, including Med7, points to similar mechanisms in higher cells.

Yeast RNA polymerase II transcription reconstituted with purified proteins.

Protocols are presented for the preparation of a fully defined yeast RNA polymerase II transcription system, consisting of essentially pure TFIIB, -E, -F, and -H, TATA-binding protein, RNA polymerase II, and mediator of transcriptional regulation. This system, comprising 44 polypeptides, is able to initiate transcription at any of a dozen yeast and mammalian promoters thus far tested and responds to a variety of transcriptional activator proteins.

Identification of Rox3 as a component of mediator and RNA polymerase II holoenzyme.

Yeast Rox3 protein, implicated by genetic evidence in both negative and positive transcriptional regulation, is identified as a mediator subunit by peptide sequence determination and is shown to copurify and co-immunoprecipitate with RNA polymerase II holoenzyme.

Backbone dynamics, amide hydrogen exchange, and resonance assignments of the DNA methylphosphotriester repair domain of Escherichia coli Ada using NMR.

The 10kDa amino-terminal fragment of Escherichia coli Ada protein (N-Ada10) repairs methyl phosphotriesters in DNA and possesses a tightly bound zinc ion. The complete resonance assignments of this protein domain have been obtained using multidimensional homonuclear and heteronuclear NMR experiments. The assignments served to study the internal mobility of this protein domain via 15N relaxation experiments. This involved the measurement of longitudinal and transverse 15N relaxation rates, as well as the amide proton solvent exchange rates. Relaxation rates in the rotating frame, R1 rho, of 15N nuclei were measured at different spin-lock field strengths, leading to the detection of two slow conformational exchange processes at Gly-25 and Gln-73. For the latter, which is next to the active site of this protein domain, the characteristic time of this process was found to be around 60 microseconds. The other relaxation experiments unveiled some regions of fast internal motions, faster than the overall correlation time. These motions were found in the N- and C- terminal tails, in segment 33-35 which forms the turn between beta-strands S1 and S2, and residues 47-52 located in a long loop preceding strand S3. The latter loop belongs to the potential DNA binding surface of N-Ada10. While the structure from residue 18 to residue 26 appears not well defined in the calculated structure, the relaxation experiments do not indicate higher mobility for this region. Residues at the N-terminal portion, including the first helix, the sequentially adjacent loop, and part of the second helix, exhibit internal motions close to the time scale of the overall rotational correlation time. This appears to be related to the fact that the first helix has no hydrogen bonds or salt bridges to the rest of the protein and is stabilized only by the involvement of some of its side chains in a hydrophobic core consisting of the side chains of two phenylalanines, a tryptophan, a leucine, and a valine. The four cysteines which bind the zinc show motions on different time scales ranging from microseconds to picoseconds. Thus the motions in the immediate region around the bound zinc of the DNA methyl phosphotriester repair domain are of relatively small amplitude but take place over a wide time range. On the other hand, high mobility is found in the turn connecting S1 and S2 and in the loop preceding S3, regions of the potential DNA binding surface.

Metal dependence of transcriptional switching in Escherichia coli Ada.

The Escherichia coli Ada protein repairs methylphosphotriesters in DNA by direct, irreversible methyl transfer to one of its own cysteine residues. The methyl transfer process is autocatalyzed by coordination of the acceptor residue, Cys69, to a tightly bound zinc ion. Kinetic data reveal a 4-fold reduction in the methylphosphotriester repair activity for the Cd(II) form of Ada versus the native Zn(II)-bound form, thus confirming a direct role for the metal in autocatalysis. Quantitative electrophoretic mobility shift assays reveal that the specific DNA affinity of the protein is increased 10(3)-fold by transfer of a methyl group to Cys69; the Cd(II) and the Zn(II) forms of the protein behave similarly in this respect. This methylation-sensitive stimulation of binding underlies the ability of Ada to activate inducibly the transcription of a methylation-dependent regulon. We conclude that the chemical properties of the bound metal influence the transition state for autocatalytic methyl transfer, but not the structure that ultimately results from this process.

Metal-coordination sphere in the methylated Ada protein-DNA co-complex.

BACKGROUND: The Ada protein of Escherichia coli repairs methyl phosphotriesters in DNA by direct, irreversible methyl transfer to one of its own cysteine residues. This residue, Cys69, is ligated to a tightly bound zinc ion in the protein. After methyl transfer, Ada can bind DNA sequence-specifically, inducing the transcription of genes that confer resistance to the toxic effects of methylating agents. Coordination of zinc via a thioether-S is exceedingly rare. We therefore investigated whether methylation causes ligand exchange of Cys69, replacing the thioether with a new zinc ligand with higher affinity for the metal. RESULTS: We added a 13C-labeled methyl group to Cys69 of Ada and used isotope-edited NMR to observe the behavior of its protons. Comparison of the spectra for the Zn- and 112Cd-bound forms of the methylated protein with that of the 113Cd-bound form provided clear evidence that S-Me-Cys69 is coordinated to the metal in Ada when Ada is bound specifically to DNA. CONCLUSIONS: The transcriptionally competent form of Ada, in which Cys69 is methylated and the protein is bound to DNA, maintains the coordination of S-Me-Cys69 to the metal ion. Thus, ligand exchange is not responsible for switching Ada from a DNA-repair protein to a transcriptional activator. We propose that the lability of the thioether-zinc coordinate bond may provide a mechanism for down-regulation of the adaptive response by inactivation of the Ada DNA-binding domain.

Solution structure of the DNA methyl phosphotriester repair domain of Escherichia coli Ada.

The Escherichia coli Ada protein repairs methyl phosphotriesters in DNA by direct, irreversible methyl transfer to one of its own cysteine residues. The methyl-transfer process appears to be autocatalyzed by coordination of the acceptor residue, Cys-69, to a tightly bound zinc ion. Upon methyl transfer, Ada acquires the ability to bind DNA sequence-specifically and thereby to induce genes that confer resistance to methylating agents. The solution structure of an N-terminal 10-kDa fragment of Ada, which retains zinc binding and DNA methyl phosphotriester repair activities, was determined using multidimensional heteronuclear nuclear magnetic resonance techniques. The structure reveals a zinc-binding motif unlike any observed thus far in transcription factors or zinc-containing enzymes and provides insight into the mechanism of metalloactivated DNA repair.

Repair of DNA methylphosphotriesters through a metalloactivated cysteine nucleophile.

The Escherichia coli Ada protein repairs methylphosphotriesters in DNA by direct, irreversible methyl transfer to one of its own cysteines. Upon methyl transfer, Ada acquires the ability to bind specific DNA sequences and thereby to induce genes that confer resistance to methylating agents. The amino-terminal domain of Ada, which comprises the methylphosphotriester repair and sequence-specific DNA binding elements, contains a tightly bound zinc ion. Analysis of the zinc binding site by cadmium-113 nuclear magnetic resonance and site-directed mutagenesis revealed that zinc participates in the autocatalytic activation of the active site cysteine and may also function as a conformational switch.

Raman and infrared studies of wet-spun films of Na-hyaluronate.

Raman and infrared (IR) studies of Na-hyaluronate films have been performed as a function of relative humidity (RH) and temperature. These data show a number of vibrational modes. Certain of these modes are evident in both the Raman and IR data. Changes in the Raman spectra are observed at the order-disorder phase transition (between 84 and 90% RH) and suggest that the molecular conformation changes at the phase transition.

Head trauma. Perioperative nursing implications.

1. The highest mortality rate in the adult trauma patient population is among those presenting with acute head trauma and associated multiple trauma. 2. Motor vehicle crashes are the leading cause of head trauma, with alcohol being a primary associated factor. The other major mechanisms of head trauma are falls and assaults. 3. Secondary brain injury is a result of brain swelling and intracranial hemorrhage causing hypoxia and ischemia of the brain tissue. 4. The goals of surgical intervention are to evacuate intracranial hematoma and prevent increases in intracranial pressure.

Zinc binding by the methylation signaling domain of the Escherichia coli Ada protein.

The Escherichia coli Ada protein repairs O6-methylguanine residues and methyl phosphotriesters in DNA by direct transfer of the methyl group to a cysteine residue located in its C- or N-terminal domain, respectively. Methyl transfer to the N-terminal domain causes it to acquire a sequence-specific DNA binding activity, which directs binding to the regulatory region of several methylation-resistance genes. In this paper we show that the N-terminal domain of Ada contains a high-affinity binding site for a single zinc atom, whereas the C-terminal domain is free of zinc. The metal-binding domain is apparently located within the first 92 amino acids of Ada, which contains four conserved cysteine residues. We propose that these four cysteines serve as the zinc ligand residues, coordinating the metal in a tetrahedral arrangement. One of the putative ligand residues, namely, Cys69, also serves as the acceptor site for a phosphotriester-derived methyl group. This raises the possibility that methylation-dependent ligand reorganization about the metal plays a role in the conformational switching mechanism that converts Ada from a non-sequence-specific to a sequence-specific DNA-binding protein.