Institute for Biological Sciences

The ultimate goal of structural studies of proteins is to gain insight into protein three-dimensional structure at highresolution level. This can often be accomplished by the application of techniques such as X-ray crystallography or multidimensional nuclear magnetic resonance (NMR) spectroscopy. However, high-resolution studies of proteins are not always feasible. For example, crystallographic studies require high-quality single crystals which for many proteins (e.g., the vast majority of membrane proteins) are not available. Furthermore, the question arises as to whether the relatively “static” structure in single crystals adequately represents the protein conformation in a complex and dynamic environment of living cells. There is a growing realization [e.g., Martinek et al. (1989)l that in vivo most proteins act in an interfacial environmeht where they form dynamic complexes with biological membranes, nucleic acids, polysaccharides, or other proteins. Aqueous buffers, from which protein crystals are usually grown, do not necessarily mimic well the conditions of protein functioning in vivo. NMR offers a somewhat better flexibility in studying protein structure in “biologically relevant” environments. However, the interpretation of NMR spectra of larger proteins is very complex, and the assignment of interproton distances generated by the NMR experiment is not always feasible; at present the technique is restricted to small proteins of less than approximately 15-20 kDa.

HUPO initiated the Plasma Proteome Project (PPP) in 2002. Its pilot phase has (1) evaluated advantages and limitations of many depletion, fractionation, and MS technology platforms; (2) compared PPP reference specimens of human serum and EDTA, heparin, and citrate-anti-coagulated plasma; and (3) created a publicly-available knowledge base (www.bioinformatics.med.umich.edu/hupo/ppp; www.ebi.ac.uk/pride). Thirty-five participating laboratories in 13 countries submitted datasets. Working groups addressed (a) specimen stability and protein concentrations; (b) protein identifications from 18 MS/MS datasets; (c) independent analyses from raw MS-MS spectra; (d) search engine performance, subproteome analyses, and biological insights; (e) antibody arrays; and (f) direct MS/SELDI analyses. MS-MS datasets had 15 710 different International Protein Index (IPI) protein IDs; our integration algorithm applied to multiple matches of peptide sequences yielded 9504 IPI proteins identified with one or more peptides and 3020 proteins identified with two or more peptides (the Core Dataset). These proteins have been characterized with Gene Ontology, InterPro, Novartis Atlas, OMIM, and immunoassay-based concentration determinations. The database permits examination of many other subsets, such as 1274 proteins identified with three or more peptides. Reverse protein to DNA matching identified proteins for 118 previously unidentified ORFs. We recommend use of plasma instead of serum, with EDTA (or citrate) for anticoagulation. To improve resolution, sensitivity and reproducibility of peptide identifications and protein matches, we recommend combinations of depletion, fractionation, and MS/MS technologies, with explicit criteria for evaluation of spectra, use of search algorithms, and integration of homologous protein matches. This Special Issue of PROTEOMICS presents papers integral to the collaborative analysis plus many reports of supplementary work on various aspects of the PPP workplan. These PPP results on complexity, dynamic range, incomplete sampling, false-positive matches, and integration of diverse datasets for plasma and serum proteins lay a foundation for development and validation of circulating protein biomarkers in health and disease.

Infections caused by multidrug-resistant (MDR) Gram-negative bacteria represent a major global health problem. Polymyxin antibiotics such as colistin have resurfaced as effective last-resort antimicrobials for use against MDR Gram-negative pathogens, including Acinetobacter baumannii. Here we show that A. baumannii can rapidly develop resistance to polymyxin antibiotics by complete loss of the initial binding target, the lipid A component of lipopolysaccharide (LPS), which has long been considered to be essential for the viability of Gram-negative bacteria. We characterized 13 independent colistin-resistant derivatives of A. baumannii type strain ATCC 19606 and showed that all contained mutations within one of the first three genes of the lipid A biosynthesis pathway: lpxA, lpxC, and lpxD. All of these mutations resulted in the complete loss of LPS production. Furthermore, we showed that loss of LPS occurs in a colistin-resistant clinical isolate of A. baumannii. This is the first report of a spontaneously occurring, lipopolysaccharide-deficient, Gram-negative bacterium.

Toll-like receptor 5 (TLR5) recognizes an evolutionarily conserved site on bacterial flagellin that is required for flagellar filament assembly and motility. The alpha and epsilon Proteobacteria, including the important human pathogens Campylobacter jejuni, Helicobacter pylori, and Bartonella bacilliformis, require flagellar motility to efficiently infect mammalian hosts. In this study, we demonstrate that these bacteria make flagellin molecules that are not recognized by TLR5. We map the site responsible for TLR5 evasion to amino acids 89-96 of the N-terminal D1 domain, which is centrally positioned within the previously defined TLR5 recognition site. Salmonella flagellin is strongly recognized by TLR5, but mutating residues 89-96 to the corresponding H. pylori flaA sequence abolishes TLR5 recognition and also destroys bacterial motility. To preserve bacterial motility, alpha and epsilon Proteobacteria possess compensatory amino acid changes in other regions of the flagellin molecule, and we engineer a mutant form of Salmonella flagellin that evades TLR5 but retains motility. These results suggest that TLR5 evasion is critical for the survival of this subset of bacteria at mucosal sites in animals and raise the intriguing possibility that flagellin receptors provided the selective force to drive the evolution of these unique subclasses of bacterial flagellins.

The ability to respond to differential levels of oxygen is important to all respiring cells. The response to oxygen deficiency, or hypoxia, takes many forms and ranges from systemic adaptations to those that are cell autonomous. Perhaps the most ancient of the cell-autonomous adaptations to hypoxia is a metabolic one: the Pasteur effect, which includes decreased oxidative phosphorylation and an increase in anaerobic fermentation. Because anaerobic fermentation produces far less ATP than oxidative phosphorylation per molecule of glucose, increased activity of the glycolytic pathway is necessary to maintain free ATP levels in the hypoxic cell. Here, we present genetic and biochemical evidence that, in mammalian cells, this metabolic switch is regulated by the transcription factor HIF-1. As a result, cells lacking HIF-1alpha exhibit decreased growth rates during hypoxia, as well as decreased levels of lactic acid production and decreased acidosis. We show that this decrease in glycolytic capacity results in dramatically lowered free ATP levels in HIF-1alpha-deficient hypoxic cells. Thus, HIF-1 activation is an essential control element of the metabolic state during hypoxia; this requirement has important implications for the regulation of cell growth during development, angiogenesis, and vascular injury.

A wide variety of compounds can be biodegraded via reductive removal of halogen substituents. This process can degrade toxic pollutants, some of which are not known to be biodegraded by any other means. Reductive dehalogenation of aromatic compounds has been found primarily in undefined, syntrophic anaerobic communities. We discuss ecological and physiological principles which appear to be important in these communities and evaluate how widely applicable these principles are. Anaerobic communities that catalyze reductive dehalogenation appear to differ in many respects. A large number of pure cultures which catalyze reductive dehalogenation of aliphatic compounds are known, in contrast to only a few organisms which catalyze reductive dehalogenation of aromatic compounds. Desulfomonile tiedjei DCB-1 is an anaerobe which dehalogenates aromatic compounds and is physiologically and morphologically unusual in a number of respects, including the ability to exploit reductive dehalogenation for energy metabolism. When possible, we use D. tiedjei as a model to understand dehalogenating organisms in the above-mentioned undefined systems. Aerobes use reductive dehalogenation for substrates which are resistant to known mechanisms of oxidative attack. Reductive dehalogenation, especially of aliphatic compounds, has recently been found in cell-free systems. These systems give us an insight into how and why microorganisms catalyze this activity. In some cases transition metal complexes serve as catalysts, whereas in other cases, particularly with aromatic substrates, the catalysts appear to be enzymes.

The cellular alterations associated with skeletal muscle differentiation share a high degree of similarity with key phenotypic changes usually ascribed to apoptosis. For example, actin fiber disassembly/reorganization is a conserved feature of both apoptosis and differentiating myoblasts and the conserved muscle contractile protein, myosin light chain kinase, is required for the apoptotic feature of membrane blebbing. As such, these observations suggest that the induction of differentiation and apoptosis in the myogenic lineage may use overlapping cellular mechanisms. Here, we report that skeletal muscle differentiation depends on the activity of the key apoptotic protease, caspase 3. Peptide inhibition of caspase 3 activity or homologous deletion of caspase 3 leads to dramatic reduction in both myotube/myofiber formation and expression of muscle-specific proteins. Subsequently, we have identified Mammalian Sterile Twenty-like kinase as a crucial caspase 3 effector in this cellular process. Mammalian Sterile Twenty-like kinase is cleavage-activated by caspase 3, and restoration of this truncated kinase in caspase 3 null myoblasts restores the differentiation phenotype. Taken together, these results confirm a unique and unanticipated role for a caspase 3-mediated signal cascade in the promotion of myogenesis.

Active cell death in hormone-dependent cells was studied using cultured human mammary carcinoma cells (MCF-7) treated with the anti-estrogens (AEs) tamoxifen (TAM), 4-hydroxy-tamoxifen (OH-TAM) or ICI 164 384 (10(-8)-10(-5) M) as a model. The following results were obtained. (i) In untreated MCF-7 cells a wave of replication occurred in the first 5 days of culture. All three AEs caused a dose-dependent inhibition of cell replication. (ii) TAM and OH-TAM at 10(-5) M, but not ICI 164 384, caused lytic cell death (necrosis) within 24 h, which was not inhibited by estradiol (10(-9)-10(-6)M). (iii) Lower concentrations of TAM or OH-TAM (up to 10(-6) M) or ICI 164 384 induced a more gradual appearance of cell death beginning at day 3. This type of cell death was inhibited by estradiol (10(-9) M), indicating its active nature. (iv) Nuclei showed two distinct patterns of alteration: (a) apoptosis-like condensation and fragmentation of chromatin to crescent masses abutting the nuclear envelope; (b) condensation of the chromatin to a single, pyknotic mass in the center of the nucleus, detached from the nuclear envelope. Quantitative histological evaluation revealed the predominance of pyknosis. (v) Biochemical DNA analysis revealed that only a relatively small amount of the total DNA was finally degraded into low molecular weight fragments (20 kb and less). (vi) Active cell death, with both apoptotic and pyknotic nuclear morphology, was associated with extensive formation of autophagic vacuoles (AV).3-Methyladenine, a known inhibitor of AV formation, partially prevented cell death as detected by nuclear changes. (vii) ICI 164 384 was about 10 times more effective than TAM or OH-TAM at inhibiting DNA synthesis, but had equal potency in inducing active cell death. It is concluded that AEs have anti-proliferative and anti-survival effects on MCF-7 human mammary cancer cells in culture. These two effects are under separate control because they differ by kinetics, dose dependence and sensitivity to the various AEs. Active cell death in MCF-7 cells seems to be initiated by autophagy, in contrast to concepts of apoptosis, and thus corresponds to autophagic/ lysosomal or type II death as previously defined. This may be important because of biochemical and molecular differences between these various subtypes of active cell death.

The present study describes the antimicrobial activity and free radical scavenging capacity (RSC) of essential oils from Mentha aquatica L., Mentha longifolia L., and Mentha piperita L. The chemical profile of each essential oil was determined by GC-MS and TLC. All essential oils exhibited very strong antibacterial activity, in particularly against Esherichia coli strains. The most powerful was M. piperita essential oil, especially towards multiresistant strain of Shigella sonei and Micrococcus flavus ATTC 10,240. All tested oils showed significant fungistatic and fungicidal activity [expressed as minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) values, respectively], that were considerably higher than those of the commercial fungicide bifonazole. The essential oils of M. piperita and M. longifolia were found to be more active than the essential oil of M. aquatica. Especially low MIC (4 microL/mL) and MFC (4 microL/mL) were found with M. piperita oil against Trichophyton tonsurans and Candida albicans (both 8 microL/mL). The RSC was evaluated by measuring the scavenging activity of the essential oils on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and OH radicals. All examined essential oils were able to reduce DPPH radicals into the neutral DPPH-H form, and this activity was dose-dependent. However, only the M. piperita oil reduced DPPH to 50 % (IC50 = 2.53 microg/mL). The M. piperita essential oil also exhibited the highest OH radical scavenging activity, reducing OH radical generation in the Fenton reaction by 24 % (pure oil). According to GC-MS and TLC (dot-blot techniques), the most powerful scavenging compounds were monoterpene ketones (menthone and isomenthone) in the essential oils of M. longifolia and M. piperita and 1,8-cineole in the oil of M. aquatica.

Despite rapidly decreasing costs and innovative technologies, sequencing of angiosperm genomes is not yet undertaken lightly. Generating larger amounts of sequence data more quickly does not address the difficulties of sequencing and assembling complex genomes de novo. The cotton ( Gossypium spp.)

The plant hormone abscisic acid (ABA) induces gene expression via the ABA-response element (ABRE) present in the promoters of ABA-regulated genes. A group of bZIP proteins have been identified as ABRE-binding factors (ABFs) that activate transcription through this cis element. A rice ABF, TRAB1, has been shown to be activated via ABA-dependent phosphorylation. While a large number of signalling factors have been identified that are involved in stomatal regulation by ABA, relatively less is known about the ABA-signalling pathway that leads to gene expression. We have shown recently that three members of the rice SnRK2 protein kinase family, SAPK8, SAPK9 and SAPK10, are activated by ABA signal as well as by hyperosmotic stress. Here we show that transient overexpression in cultured cell protoplasts of these ABA-activated SnRK2 protein kinases leads to the activation of an ABRE-regulated promoter, suggesting that these kinases are involved in the gene-regulation pathway of ABA signalling. We further show several lines of evidence that these ABA-activated SnRK2 protein kinases directly phosphorylate TRAB1 in response to ABA. Kinetic analysis of SAPK10 activation and TRAB1 phosphorylation indicated that the latter immediately followed the former. TRAB1 was found to be phosphorylated not only in response to ABA, but also in response to hyperosmotic stress, which was interpreted as the consequence of phosphorylation of TRAB1 by hyperosmotically activated SAPKs. Physical interaction between TRAB1 and SAPK10 in vivo was demonstrated by a co-immunoprecipitation experiment. Finally, TRAB1 was phosphorylated in vitro by the ABA-activated SnRK2 protein kinases at Ser102, which is phosphorylated in vivo in response to ABA and is critical for the activation function.

Colletotrichum higginsianum is a fungal pathogen that infects a wide variety of cruciferous plants, causing important crop losses. We have used map-based cloning and natural variation analysis of 19 Arabidopsis ecotypes to identify a dominant resistance locus against C. higginsianum. This locus named RCH2 (for recognition of C. higginsianum) maps in an extensive cluster of disease-resistance loci known as MRC-J in the Arabidopsis ecotype Ws-0. By analyzing natural variations within the MRC-J region, we found that alleles of RRS1 (resistance to Ralstonia solanacearum 1) from susceptible ecotypes contain single nucleotide polymorphisms that may affect the encoded protein. Consistent with this finding, two susceptible mutants, rrs1-1 and rrs1-2, were identified by screening a T-DNA-tagged mutant library for the loss of resistance to C. higginsianum. The screening identified an additional susceptible mutant (rps4-21) that has a 5-bp deletion in the neighboring gene, RPS4-Ws, which is a well-characterized R gene that provides resistance to Pseudomonas syringae pv. tomato strain DC3000 expressing avrRps4 (Pst-avrRps4). The rps4-21/rrs1-1 double mutant exhibited similar levels of susceptibility to C. higginsianum as the single mutants. We also found that both RRS1 and RPS4 are required for resistance to R. solanacearum and Pst-avrRps4. Thus, RPS4-Ws and RRS1-Ws function as a dual resistance gene system that prevents infection by three distinct pathogens.

Mass spectrometry investigations of partially purified Campylobacter jejuni protein PEB3 showed it to be partially modified with an Asn-linked glycan with a mass of 1406 Da and composed of one hexose, five N-acetylhexosamines and a species of mass 228 Da, consistent with a trideoxydiacetamidohexose. By means of soybean lectin affinity chromatography, a mixture of glycoproteins was obtained from a glycine extract, and two-dimensional gel proteomics analysis led to the identification of at least 22 glycoproteins, predominantly annotated as periplasmic proteins. Glycopeptides were prepared from the glycoprotein mixture by Pronase digestion and gel filtration. The structure of the glycan was determined by using nano-NMR techniques to be GalNAc-alpha1,4-GalNAc-alpha1,4-[Glcbeta1,3-]GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac-beta1,N-Asn-Xaa, where Bac is bacillosamine, 2,4-diacetamido-2,4,6-trideoxyglucopyranose. Protein glycosylation was abolished when the pglB gene was mutated, providing further evidence that the enzyme encoded by this gene is responsible for formation of the glycopeptide N-linkage. Comparison of the pgl locus with that of Neisseria meningitidis suggested that most of the homologous genes are probably involved in the biosynthesis of bacillosamine.

High-salinity stress affects plant growth and development. We have previously reported that overexpression of the salinity-responsive DWARF AND DELAYED FLOWERING 1 (DDF1) gene, encoding an AP2 transcription factor of the DREB1/CBF subfamily, causes dwarfism mainly by levels of reducing bioactive gibberellin (GA) in transgenic Arabidopsis. Here, we found that the GA 2-oxidase 7 gene (GA2ox7), which encodes a C20-GA deactivation enzyme, is strongly upregulated in DDF1-overexpressing transgenic plants. A loss-of-function mutation of GA2ox7 (ga2ox7-2) suppressed the dwarf phenotype of DDF1-overexpressing plants, indicating that their GA deficiency is due to overexpression of GA2ox7. Transient overexpression of DDF1 activated the promoter of GA2ox7 in Arabidopsis leaves. A gel shift assay showed that DDF1 binds DRE-like motifs (GCCGAC and ATCGAC) in the GA2ox7 promoter. In Arabidopsis under high-salinity stress, six GA2ox genes, including GA2ox7, were upregulated. Furthermore, the ga2ox7-2 mutant was less growth retarded than wild-type Col under high-salinity stress. These results demonstrate that, under salinity stress, Arabidopsis plants actively reduce endogenous GA levels via the induction of GA 2-oxidase, with the result that growth is repressed for stress adaptation.

The effects of topoisomerase II-reactive epipodophyllotoxins etoposide and teniposide as well as amsacrine on the viability of thymocytes in primary culture has been examined. All three drugs were shown to produce DNA cleavage detectable by resolving isolated DNA by pulsed field agarose gel electrophoresis. The DNA cleavage was found to have two components. The first was due to the interaction of the drugs with topoisomerase II, whereas the second component was due to endonuclease cleavage caused by the drug-induced entry of the thymocytes into programmed cell death or apoptosis. This second component of the DNA cleavage was also detected in thymocytes undergoing apoptosis following exposure to the glucocorticoid analogue, dexamethasone. The effect of the drugs on programmed cell death is dependent upon new protein and RNA synthesis, indicating that topoisomerase II has a role in the very first stages of the process. These results are discussed in terms of the use of this class of topoisomerase II-reactive drugs in chemotherapy.

Flagellins from three strains of Campylobacter jejuni and one strain of Campylobacter coli were shown to be extensively modified by glycosyl residues, imparting an approximate 6000-Da shift from the molecular mass of the protein predicted from the DNA sequence. Tryptic peptides from C. jejuni 81-176 flagellin were subjected to capillary liquid chromatography-electrospray mass spectrometry with a high/low orifice stepping to identify peptide segments of aberrant masses together with their corresponding glycosyl appendages. These modified peptides were further characterized by tandem mass spectrometry and preparative high performance liquid chromatography followed by nano-NMR spectroscopy to identify the nature and precise site of glycosylation. These analyses have shown that there are 19 modified Ser/Thr residues in C. jejuni 81-176 flagellin. The predominant modification found on C. jejuni flagellin was O-linked 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid (pseudaminic acid, Pse5Ac7Ac) with additional heterogeneity conferred by substitution of the acetamido groups with acetamidino and hydroxyproprionyl groups. In C. jejuni 81-176, the gene Cj1316c, encoding a protein of unknown function, was shown to be involved in the biosynthesis and/or the addition of the acetamidino group on Pse5Ac7Ac. Glycosylation is not random, since 19 of the total 107 Ser/Thr residues are modified, and all but one of these are restricted to the central, surface-exposed domain of flagellin when folded in the filament. The mechanism of attachment appears unrelated to a consensus peptide sequence but is rather based on surface accessibility of Ser/Thr residues in the folded protein.

A novel gibberellin (GA)-deficient mutant designated dwarf and delayed-flowering 1 (ddf1) was isolated from a library of activation-tagged Arabidopsis. This mutant showed dwarfism and late-flowering, but the phenotype was rescued by exogenous GA3 like known mutants defective in GA biosynthesis. The contents of bioactive GA4 and GA1 were in fact decreased in ddf1 at least partially through the repression of biosynthetic steps catalyzed by GA 20-oxidase (GA20ox). Genetic and molecular analyses revealed that the ddf1 phenotypes are caused by increased or ectopic expression of a putative AP2 transcription factor. Overexpression of DDF2, encoding another putative AP2 transcription factor closely related to DDF1, also conferred the ddf1-like phenotype. Among genes encoding (putative) AP2 transcription factors in Arabidopsis, DDFs are phylogenetically close to dehydration-responsive element binding protein (DREB1)/C-repeat binding factor (CBF) genes, which are known to be involved in stress responses. The ddf1 mutation upregulates a stress-related gene RD29A. DDF1 mRNA is strongly induced by high-salinity stress within 1 h. Moreover, transgenic plants overexpressing DDF1 showed increased tolerance to high-salinity stress. These results suggest that DDF1 is involved in the regulation of GA biosynthesis and stress tolerance. The possible relation between the contents of endogenous GAs and acquisition of stress protection is discussed.

BNIP3 (formerly NIP3) is a pro-apoptotic, mitochondrial protein classified in the Bcl-2 family based on limited sequence homology to the Bcl-2 homology 3 (BH3) domain and COOH-terminal transmembrane (TM) domain. BNIP3 expressed in yeast and mammalian cells interacts with survival promoting proteins Bcl-2, Bcl-X(L), and CED-9. Typically, the BH3 domain of pro-apoptotic Bcl-2 homologues mediates Bcl-2/Bcl-X(L) heterodimerization and confers pro-apoptotic activity. Deletion mapping of BNIP3 excluded its BH3-like domain and identified the NH(2) terminus (residues 1-49) and TM domain as critical for Bcl-2 heterodimerization, and either region was sufficient for Bcl-X(L) interaction. Additionally, the removal of the BH3-like domain in BNIP3 did not diminish its killing activity. The TM domain of BNIP3 is critical for homodimerization, pro-apoptotic function, and mitochondrial targeting. Several TM domain mutants were found to disrupt SDS-resistant BNIP3 homodimerization but did not interfere with its killing activity or mitochondrial localization. Substitution of the BNIP3 TM domain with that of cytochrome b(5) directed protein expression to nonmitochondrial sites and still promoted apoptosis and heterodimerization with Bcl-2 and Bcl-X(L). We propose that BNIP3 represents a subfamily of Bcl-2-related proteins that functions without a typical BH3 domain to regulate apoptosis from both mitochondrial and nonmitochondrial sites by selective Bcl-2/Bcl-X(L) interactions.

Pathophysiology of the neurovascular unit (NVU) is commonly seen in neurological diseases. The typical features of NVU pathophysiology include tissue hypoxia, inflammatory and angiogenic activation, as well as initiation of complex molecular interactions between cellular (brain endothelial cells, astroctyes, pericytes, inflammatory cells, and neurons) and acellular (basal lamina) components of the NVU, jointly resulting in increased blood-brain barrier permeability, brain edema, neurovascular uncoupling, and neuronal dysfunction and damage. The evidence of important role of the brain vascular compartment in disease pathogenesis has elicited the debate whether the primary vascular events may be a cause of the neurological disease, as opposed to a mere participant recruited by a primary neuronal origin of pathology? Whereas some hereditary and acquired cerebral angiopathies could be considered a primary cause of neurological symptoms of the disease, the epidemiological studies showing a high degree of comorbidity among vascular disease and dementias, including Alzheimer's disease, as well as migraine and epilepsy, suggested that primary vascular pathology may be etiological factor causing neuronal dysfunction or degeneration in these diseases. This review focuses on recent hypotheses and evidence, suggesting that pathophysiology of the NVU may be initiating trigger for neuronal pathology and subsequent neurological manifestations of the disease.

This review highlights the challenges of analyzing lignin–carbohydrate bonds, which are hypothesized to contribute to the recalcitrance in biomass fractionation.