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Introduction: Dental pulp with special structure has become a good reference sample in paleomicrobiology‐related blood‐borne diseases, many pathogens were detected by different methods based on the diagnosis of nucleic acids and proteins. Objectives: This review aims to propose the preparation process from ancient teeth collection to organic molecule extraction of dental pulp and summary, analyze the methods that have been applied to detect septicemic pathogens through ancient dental pulps during the past 20 years following the first detection of an ancient microbe. Methods: The papers used in this review with two main objectives were obtained from PubMed and Google scholar with combining keywords: "ancient," "dental pulp," "teeth," "anatomy," "structure," "collection," "preservation," "selection," "photography," "radiography," "contamination," "decontamination," "DNA," "protein," "extraction," "bone," "paleomicrobiology," "bacteria," "virus," "pathogen," "molecular biology," "proteomics," "PCR," "MALDI‐TOF," "LC/MS," "ELISA," "immunology," "immunochromatography," "genome," "microbiome," "metagenomics." Results: The analysis of ancient dental pulp should have a careful preparation process with many different steps to give highly accurate results, each step complies with the rules in archaeology and paleomicrobiology. After the collection of organic molecules from dental pulp, they were investigated for pathogen identification based on the analysis of DNA and protein. Actually, DNA approach takes a principal role in diagnosis while the protein approach is more and more used. A total of seven techniques was used and ten bacteria (Yersinia pestis, Bartonella quintana, Salmonella enterica serovar Typhi, Salmonella enterica serovar Paratyphi C, Mycobacterium leprae, Mycobacterium tuberculosis, Rickettsia prowazeki, Staphylococcus aureus, Borrelia recurrentis, Bartonella henselae) and one virus (Anelloviridae) were identified. Y. pestis had the most published in quantity and all methods were investigated for this pathogen, S. aureus and B. recurrentis were identified by three different methods and others only by one. The combining methods interestingly increase the positive rate with ELISA, PCR and iPCR in Yersinia pestis diagnosis. Twenty‐seven ancient genomes of Y. pestis and one ancient genome of B. recurrentis were reconstructed. Comparing to the ancient bone, ancient teeth showed more advantage in septicemic diagnosis. Beside pathogen identification, ancient pulp help to distinguish species. Conclusions: Dental pulp with specific tissue is a suitable sample for detection of the blood infection in the past through DNA and protein identification with the correct preparation process, furthermore, it helps to more understand the pathogens of historic diseases and epidemics. [ABSTRACT FROM AUTHOR]

The current focus of many functional genomic studies is on the elucidation of gene regulatory networks. The functional analyses of transcription factors and their DNA-binding sites, in conjunction with genome-wide expression profiling, are crucial in understanding of gene regulatory networks. This paper describes an efficient and easy method for characterizing the DNA-binding sequence specificity of novel plant transcription factors. This new method is based on the fusion of a DNA-binding protein (DBP) to 6×His-tagged cellulase D (CELD), which serves both as a means for affinity purification of DBP-DNA complex in the selection of binding sites from a pool of biotinylated random-sequence oligonucleotides and as a reporter for measurement of DNA-binding activity. Thus, it eliminates the use of radioactivity and gel electrophoresis techniques currently used for purification of DBP-DNA complexes and assays of DNA-binding activity. The effectiveness of this method was demonstrated by the success of simultaneous selection of the binding sites of nine plant DBPs from four superfamilies (AP2, bHLH, NAC and MYB). The high-throughput capacity of CELD-based DNA-binding assays allows the quantitative analysis of the binding sequence specificity from a large number of DBP-selected oligonucleotides. The binding sequence specificity of three novel transcription factors (rice OsbHLH66, wheat TaNAC69 and TaMYB80), determined with this method, is presented. This new method provides the capacity of high-throughput analysis on the DNA-binding sequence specificity of a large number of putative transcription factors, predicted on the basis of conserved DNA-binding domains. [ABSTRACT FROM AUTHOR]

Efficient protein turnover is essential for cellular homeostasis and organ function. Loss of proteostasis is a hallmark of aging culminating in severe dysfunction of protein turnover. To investigate protein turnover dynamics as a function of age, we performed continuous in vivo metabolic stable isotope labeling in mice along the aging continuum. First, we discovered that the brain proteome uniquely undergoes dynamic turnover fluctuations during aging compared to heart and liver tissue. Second, trends in protein turnover in the brain proteome during aging showed sex-specific differences that were tightly tied to cellular compartments. Next, parallel analyses of the insoluble proteome revealed that several cellular compartments experience hampered turnover, in part due to misfolding. Finally, we found that age-associated fluctuations in proteasome activity were associated with the turnover of core proteolytic subunits, which was recapitulated by pharmacological suppression of proteasome activity. Taken together, our study provides a proteome-wide atlas of protein turnover across the aging continuum and reveals a link between the turnover of individual proteasome subunits and the age-associated decline in proteasome activity. Synopsis: Continuous pulse-step metabolic labeling with quantitative proteomics in mice reveals sex-specific differences in protein turnover linked to subcellular compartments. Age-related changes in protein turnover result in part from altered proteasome activity and subunit turnover. Brain protein turnover trends during aging display sex-specific differences tied to subcellular compartments. Misfolded proteins contribute to differential turnover trends in the brain. Reduced proteasome activity is associated with slowed turnover of proteasome subunits during aging. Continuous pulse-step metabolic labeling with quantitative proteomics in mice reveals sex-specific differences in protein turnover linked to subcellular compartments. Age-related changes in protein turnover result in part from altered proteasome activity and subunit turnover. [ABSTRACT FROM AUTHOR]

Transglutaminases form a large family of intracellular and extracellular enzymes that catalyse the Ca2+-dependent post-translational modification of proteins. Despite significant advances in our understanding of the biological role of most mammalian transglutaminase isoforms, recent findings suggest new scenarios, most notably for the ubiquitous tissue transglutaminase. It is becoming apparent that some transglutaminases, normally expressed at low levels in many tissue types, are activated and/or overexpressed in a variety of diseases, thereby resulting in enhanced concentrations of cross-linked proteins. As applies to all enzymes that exert their metabolic function by modifying the properties of target proteins, the identification and characterization of the modified proteins will cast light on the functions of transglutaminases and their involvement in human diseases. In this paper we review data on the properties of mammalian transglutaminases, particularly as regards their protein substrates and the relevance of transglutaminase-catalysed reactions in physiological and disease conditions. [ABSTRACT FROM AUTHOR]

SUMMARY: Identification of protein interactors is ideally suited for the functional characterization of small molecules. 3′,5′‐cAMP is an evolutionary ancient signaling metabolite largely uncharacterized in plants. To tap into the physiological roles of 3′,5′‐cAMP, we used a chemo‐proteomics approach, thermal proteome profiling (TPP), for the unbiased identification of 3′,5′‐cAMP protein targets. TPP measures shifts in the protein thermal stability upon ligand binding. Comprehensive proteomics analysis yielded a list of 51 proteins significantly altered in their thermal stability upon incubation with 3′,5′‐cAMP. The list contained metabolic enzymes, ribosomal subunits, translation initiation factors, and proteins associated with the regulation of plant growth such as CELL DIVISION CYCLE 48. To functionally validate obtained results, we focused on the role of 3′,5′‐cAMP in regulating the actin cytoskeleton suggested by the presence of actin among the 51 identified proteins. 3′,5′‐cAMP supplementation affected actin organization by inducing actin‐bundling. Consistent with these results, the increase in 3′,5′‐cAMP levels, obtained either by feeding or by chemical modulation of 3′,5′‐cAMP metabolism, was sufficient to partially rescue the short hypocotyl phenotype of the actin2 actin7 mutant, severely compromised in actin level. The observed rescue was specific to 3′,5′‐cAMP, as demonstrated using a positional isomer 2′,3′‐cAMP, and true for the nanomolar 3′,5′‐cAMP concentrations reported for plant cells. In vitro characterization of the 3′,5′‐cAMP–actin pairing argues against a direct interaction between actin and 3′,5′‐cAMP. Alternative mechanisms by which 3′,5′‐cAMP would affect actin dynamics, such as by interfering with calcium signaling, are discussed. In summary, our work provides a specific resource, 3′,5′‐cAMP interactome, as well as functional insight into 3′,5′‐cAMP‐mediated regulation in plants. Significance Statement: The present study provides the list of potential 3′,5′‐cAMP interactors pointing towards actin as one of them. We demonstrate that 3′,5′‐cAMP can regulate actin bundling in vivo. [ABSTRACT FROM AUTHOR]

The article features comments on EGFR signaling. By using protein microarray and quantitative proteomics technologies, a group has recently identified proteins associated with the cytosolic domains of the human EGFR family. In a related project, others have used protein microarrays to identify ErbB interactors. It is commented that the dramatic differences are due to the fact that the two groups looked at very different things. The paper indicates that systems biology is in its nascent stage.

SUMMARY: Fusarium graminearum is a fungal pathogen that causes Fusarium head blight in cereal crops. The identification of proteins secreted from pathogens to overcome plant defenses and cause disease, collectively known as effectors, can reveal the etiology of a disease process. Proximity‐dependent biotin identification (BioID) was used to identify potential effector proteins secreted in planta by F. graminearum during the infection of Arabidopsis. Mass spectrometry analysis of streptavidin affinity‐purified proteins revealed over 300 proteins from F. graminearum, of which 62 were candidate effector proteins (CEPs). An independent analysis of secreted proteins from axenic cultures of F. graminearum showed a 42% overlap with CEPs, thereby assuring confidence in the BioID methodology. The analysis also revealed that 19 out of 62 CEPs (approx. 30%) had been previously characterized with virulence function in fungi. The functional characterization of additional CEPs was undertaken through deletion analysis by the CRISPR/Cas9 method, and by overexpression into Triticum aestivum (wheat) leaves by the Ustilago hordei delivery system. Deletion studies of 12 CEPs confirmed the effector function of three previously characterized CEPs and validated the function of another four CEPs on wheat inflorescence or vegetative tissues. Lastly, overexpression in wheat showed that all seven CEPs enhanced resistance against the bacterial pathogen Pseudomonas syringae DC3000. Significance Statement: Towards a better understanding of the Fusarium head blight disease, this study utilizes a proteomic tool called proximity‐dependent biotin identification, or BioID, to identify effector proteins from the pathogen Fusarium graminearum. Identification and characterization of effectors will enable pathologists to monitor the pathogen evolution, and use them as potential new targets for fungicide development as part of the integrated pest management programs. [ABSTRACT FROM AUTHOR]

Protein–protein interaction (PPI) plays a crucial role in most biological processes, including signal transduction and cell apoptosis. Importantly, the knowledge of PPIs can be useful for identification of multimeric protein complexes and elucidation of uncharacterized protein functions. Arabidopsis thaliana, the best‐characterized dicotyledonous plant, the steadily increasing amount of information on the levels of its proteome and signaling pathways is progressively enabling more researchers to construct models for cellular processes for the plant, which in turn encourages more experimental data to be generated. In this study, we performed an overview analysis of the 10 major organelles and their associated proteins of the dicotyledonous model plant Arabidopsis thaliana via PPI network, and found that PPI may play an important role in organelle communication. Further, multilocation proteins, especially phosphorylation‐related multilocation proteins, can function as a "needle and thread" via PPIs and play an important role in organelle communication. Similar results were obtained in a monocotyledonous model crop, rice. Furthermore, we provide a research strategy for multilocation proteins by LOPIT technique, proteomics, and bioinformatics analysis and also describe their potential role in the field of plant science. The results provide a new view that the phosphorylation‐related multilocation proteins play an important role in organelle communication and provide new insight into PPIs and novel directions for proteomic research. The research of phosphorylation‐related multilocation proteins may promote the development of organelle communication and provide an important theoretical basis for plant responses to external stress. [ABSTRACT FROM AUTHOR]

SUMMARY: Grain filling and grain development are essential biological processes in the plant's life cycle, eventually contributing to the final seed yield and quality in all cereal crops. Studies of how the different wheat (Triticum aestivum L.) grain components contribute to the overall development of the seed are very scarce. We performed a proteomics and metabolomics analysis in four different developing components of the wheat grain (seed coat, embryo, endosperm, and cavity fluid) to characterize molecular processes during early and late grain development. In‐gel shotgun proteomics analysis at 12, 15, 20, and 26 days after anthesis (DAA) revealed 15 484 identified and quantified proteins, out of which 410 differentially expressed proteins were identified in the seed coat, 815 in the embryo, 372 in the endosperm, and 492 in the cavity fluid. The abundance of selected protein candidates revealed spatially and temporally resolved protein functions associated with development and grain filling. Multiple wheat protein isoforms involved in starch synthesis such as sucrose synthases, starch phosphorylase, granule‐bound and soluble starch synthase, pyruvate phosphate dikinase, 14‐3‐3 proteins as well as sugar precursors undergo a major tissue‐dependent change in abundance during wheat grain development suggesting an intimate interplay of starch biosynthesis control. Different isoforms of the protein disulfide isomerase family as well as glutamine levels, both involved in the glutenin macropolymer pattern, showed distinct spatial and temporal abundance, revealing their specific role as indicators of wheat gluten quality. Proteins binned into the functional category of cell growth/division and protein synthesis/degradation were more abundant in the early stages (12 and 15 DAA). At the metabolome level all tissues and especially the cavity fluid showed highly distinct metabolite profiles. The tissue‐specific data are integrated with biochemical networks to generate a comprehensive map of molecular processes during grain filling and developmental processes. [ABSTRACT FROM AUTHOR]

Alternative roles for sweat production beyond thermoregulation, considered less frequently, include chemical signaling. We identified the presence of a well‐established rodent urinary pheromone, major urinary protein (MUP) in sweat ductules of the footpad dermal skin of mice. A hindpaw sweat proteomic analysis in hindpaw sweat samples collected in rats and generated by unmyelinated axon activation, identified seven lipocalin family members including MUP and 19 additional unique proteins. Behavioural responses to sniffing male mouse foot protein lysates suggested avoidance in a subset of male mice, but were not definitive. Rodent hindpaw sweat glands secrete a repertoire of proteins that include MUPs known to have roles in olfactory communication. [ABSTRACT FROM AUTHOR]

All neurodegenerative diseases feature aggregates, which usually contain disease‐specific diagnostic proteins; non‐protein constituents, however, have rarely been explored. Aggregates from SY5Y‐APPSw neuroblastoma, a cell model of familial Alzheimer's disease, were crosslinked and sequences of linked peptides identified. We constructed a normalized "contactome" comprising 11 subnetworks, centered on 24 high‐connectivity hubs. Remarkably, all 24 are nucleic acid‐binding proteins. This led us to isolate and sequence RNA and DNA from Alzheimer's and control aggregates. RNA fragments were mapped to the human genome by RNA‐seq and DNA by ChIP‐seq. Nearly all aggregate RNA sequences mapped to specific genes, whereas DNA fragments were predominantly intergenic. These nucleic acid mappings are all significantly nonrandom, making an artifactual origin extremely unlikely. RNA (mostly cytoplasmic) exceeded DNA (chiefly nuclear) by twofold to fivefold. RNA fragments recovered from AD tissue were ~1.5‐to 2.5‐fold more abundant than those recovered from control tissue, similar to the increase in protein. Aggregate abundances of specific RNA sequences were strikingly differential between cultured SY5Y‐APPSw glioblastoma cells expressing APOE3 vs. APOE4, consistent with APOE4 competition for E‐box/CLEAR motifs. We identified many G‐quadruplex and viral sequences within RNA and DNA of aggregates, suggesting that sequestration of viral genomes may have driven the evolution of disordered nucleic acid‐binding proteins. After RNA‐interference knockdown of the translational‐procession factor EEF2 to suppress translation in SY5Y‐APPSw cells, the RNA content of aggregates declined by >90%, while reducing protein content by only 30% and altering DNA content by ≤10%. This implies that cotranslational misfolding of nascent proteins may ensnare polysomes into aggregates, accounting for most of their RNA content. [ABSTRACT FROM AUTHOR]

This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach. 1. The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two-step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one. 2. α-Chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxytic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of α-chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll-400/Dextran-70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65-98 °C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date. [ABSTRACT FROM AUTHOR]

It was found that the DEAE-cellulose-treated UDP-Glc:protein transglucosylase I catalyzing the first step (reaction 1) in the formation of α-glucan bound to protein in potato tuber is not only specific for the glucosyl donor but also for the endogenous acceptor. A single radioactive 38-kDa macromolecular component appeared during denaturing polyacrytamide get electrophoresis of reaction 1 product. The labeled componem is probably the polypeptide subunit of the endogenous acceptor which is being glucosytated. The radioactivity incorporated in reaction 1 product was isolated from a protease digest as a low-molecular-mass glucopeptide fraction. A β-elimination reaction carried out in the presence of a reducing agent demonstrated that only one glucosyl moiety is transferred from UDP-Glc to the ammoacyl residue, thus forming an O-glucosidic linkage. ³H-labeled sodium borohydride showed that serine and threonine are involved in the peptide bond to glucose. Ion-exchange chromatography on DEAE-cellulose, affinity chromatography on concanavalin-A-Sepharose, gel filtration on Sephacryl S-300 and sucrose density gradient centrifugation failed to separate the enzyme catalyzing reaction 1 from the endogenous acceptor. [ABSTRACT FROM AUTHOR]

Partially purified enzymatic fractions from extracts of Escherichia coli B/r catalyse transfer of the isotope label from [adenine-2,8-3H]NAD+ to some bacterial proteins, as well as to hen egg-white lysozyme. The radio-active group in the modified lysozyme was identified as mono(ADP-ribose). Several bacterial proteins were labelled in vivo with 32P; the presence of the label in the form of an ADP-ribosyl group was shown in one of them. [ABSTRACT FROM AUTHOR]

The Heteroptera are a diverse suborder of phytophagous, hematophagous, and zoophagous insects. The shift to zoophagy can be traced back to the transformation of salivary glands into venom glands, but the venom is used not only to kill and digest invertebrate prey but also as a defense strategy, mainly against vertebrates. In this study, we used an integrated transcriptomics and proteomics approach to compare the composition of venoms from the anterior main gland (AMG) and posterior main gland (PMG) of the reduviid bugs Platymeris biguttatus L. and Psytalla horrida Stål. In both species, the AMG and PMG secreted distinct protein mixtures with few interspecific differences. PMG venom consisted mostly of S1 proteases, redulysins, Ptu1‐like peptides, and uncharacterized proteins, whereas AMG venom contained hemolysins and cystatins. There was a remarkable difference in biological activity between the AMG and PMG venoms, with only PMG venom conferring digestive, neurotoxic, hemolytic, antibacterial, and cytotoxic effects. Proteomic analysis of venom samples revealed the context‐dependent use of AMG and PMG venom. Although both species secreted PMG venom alone to overwhelm their prey and facilitate digestion, the deployment of defensive venom was species‐dependent. P. biguttatus almost exclusively used PMG venom for defense, whereas P. horrida secreted PMG venom in response to mild harassment but AMG venom in response to more intense harassment. This intriguing context‐dependent use of defensive venom indicates that future research should focus on species‐dependent differences in venom composition and defense strategies among predatory Heteroptera. [ABSTRACT FROM AUTHOR]

Background and Purpose: Δ9 -Tetrahydrocannabinolic acid (Δ9 -THCA-A), the precursor of Δ9 -THC, is a non-psychotropic phytocannabinoid that shows PPARγ agonist activity. Here, we investigated the ability of Δ9 -THCA-A to modulate the classic cannabinoid CB1 and CB2 receptors and evaluated its anti-arthritis activity in vitro and in vivo.Experimental Approach: Cannabinoid receptors binding and intrinsic activity, as well as their downstream signalling, were analysed in vitro and in silico. The anti-arthritis properties of Δ9 -THCA-A were studied in human chondrocytes and in the murine model of collagen-induced arthritis (CIA). Plasma disease biomarkers were identified by LC-MS/MS based on proteomic and elisa assays.Key Results: Functional and docking analyses showed that Δ9 -THCA-A can act as an orthosteric CB1 receptor agonist and also as a positive allosteric modulator in the presence of CP-55,940. Also, Δ9 -THCA-A seemed to be an inverse agonist for CB2 receptors. In vivo, Δ9 -THCA-A reduced arthritis in CIA mice, preventing the infiltration of inflammatory cells, synovium hyperplasia, and cartilage damage. Furthermore, Δ9 -THCA-A inhibited expression of inflammatory and catabolic genes on knee joints. The anti-arthritic effect of Δ9 -THCA-A was blocked by either SR141716 or T0070907. Analysis of plasma biomarkers, and determination of cytokines and anti-collagen antibodies confirmed that Δ9 -THCA-A mediated its activity mainly through PPARγ and CB1 receptor pathways.Conclusion and Implications: Δ9 -THCA-A modulates CB1 receptors through the orthosteric and allosteric binding sites. In addition, Δ9 -THCA-A exerts anti-arthritis activity through CB1 receptors and PPARγ pathways, highlighting its potential for the treatment of chronic inflammatory diseases such as rheumatoid arthritis. [ABSTRACT FROM AUTHOR]

Breast cancer is the second leading cause of death in the world. Breast cancer research is focused towards its early prediction, diagnosis, and prognosis. Breast cancer can be predicted on omics profiles, clinical tests, and pathological images. The omics profiles comprise of genomic, proteomic, and transcriptomic profiles that are available as high‐dimensional datasets. Survival prediction is carried out on omics data to predict early the onset of disease, relapse, reoccurrence of diseases, and biomarker identification. The early prediction of breast cancer is desired for the effective treatment of patients as delay can aggravate the staging of cancer. In this study, extreme learning machine (ELM) based model for breast cancer survival prediction named eBreCaP is proposed. It integrates the genomic (gene expression, copy number alteration, DNA methylation, protein expression) and pathological image datasets; and trains them using an ensemble of ELM with the six best‐chosen models suitable to be applied on integrated data. eBreCaP has been evaluated on nine performance parameters, namely sensitivity, specificity, precision, accuracy, Matthews correlation coefficient, area under curve, area under precision–recall, hazard ratio, and concordance Index. eBreCaP has achieved an accuracy of 85% for early breast cancer survival prediction using the ensemble of ELM with gradient boosting. [ABSTRACT FROM AUTHOR]

While it is generally accepted that α‐synuclein oligomers (αSOs) play an important role in neurodegeneration in Parkinson's disease, the basis for their cytotoxicity remains unclear. We have previously shown that docosahexaenoic acid (DHA) stabilizes αSOs against dissociation without compromising their ability to colocalize with glutamatergic synapses of primary hippocampal neurons, suggesting that they bind to synaptic proteins. Here, we develop a proteomic screen for putative αSO binding partners in rat primary neurons using DHA‐stabilized human αSOs as a bait protein. The protocol involved co‐immunoprecipitation in combination with a photoactivatable heterobifunctional sulfo‐LC‐SDA crosslinker which did not compromise neuronal binding and preserved the interaction between the αSOs‐binding partners. We identify in total 29 proteins associated with DHA‐αSO of which eleven are membrane proteins, including synaptobrevin‐2B (VAMP‐2B), the sodium–potassium pump (Na+/K+ ATPase), the V‐type ATPase, the voltage‐dependent anion channel and calcium‐/calmodulin‐dependent protein kinase type II subunit gamma; only these five hits were also found in previous studies which used unmodified αSOs as bait. We also identified Rab‐3A as a target with likely disease relevance. Three out of four selected hits were subsequently validated with dot‐blot binding assays. In addition, likely binding sites on these ligands were identified by computational analysis, highlighting a diversity of possible interactions between αSOs and target proteins. These results constitute an important step in the search for disease‐modifying treatments targeting toxic αSOs. [ABSTRACT FROM AUTHOR]

Hybrid cluster proteins (HCPs) are metalloproteins characterized by the presence of an iron‐sulfur‐oxygen cluster. These proteins occur in all three domains of life. In eukaryotes, HCPs have so far been found only in a few anaerobic parasites and photosynthetic microalgae. With respect to all species harboring an HCP, the green microalga Chlamydomonas reinhardtii stands out by the presence of four HCP genes. The study of the gene and protein structures as well as the phylogenetic analyses strongly support a model in which the HCP family in the alga has emerged from a single gene of alpha proteobacterial origin and then expanded by several rounds of duplications. The spectra and redox properties of HCP1 and HCP3, produced heterologously in Escherichia coli, were analyzed by electron paramagnetic resonance spectroscopy on redox‐titrated samples. Both proteins contain a [4Fe‐4S]‐cluster as well as a [4Fe‐2O‐2S]‐hybrid cluster with paramagnetic properties related to those of HCPs from Desulfovibrio species. Immunoblotting experiments combined with mass spectrometry‐based proteomics showed that both nitrate and darkness contribute to the strong upregulation of the HCP levels in C. reinhardtii growing under oxic conditions. The link to the nitrate metabolism is discussed in the light of recent data on the potential role of HCP in S‐nitrosylation in bacteria. [ABSTRACT FROM AUTHOR]

Depression is a commonly occurring neuropsychiatric disease with an increasing incidence rate. Saikosaponin A (SA), a major bioactive component extracted from Radix Bupleuri, possesses anti‐malignant cell proliferation, anti‐inflammation, anti‐oxidation and liver protective effects. However, few studies have investigated SA's antidepressant effects and pharmacological mechanisms of action. Our study aimed to explore the anti‐depression effect of SA and screen the target proteins regulated by SA in a rat model of chronic unpredictable mild stress (CUMS)‐induced depression. Results showed that 8‐week CUMS combined with separation could successfully produce depressive‐like behaviours and cause a decrease of dopamine (DA) in rat hippocampus, and 4‐week administration of SA could relieve CUMS rats' depressive symptoms and up‐regulated DA content. There were 15 kinds of significant differentially expressed proteins that were detected not only between the control and CUMS groups, but also between the CUMS and SA treatment groups. Proline‐rich transmembrane protein 2 (PRRT2) was down‐regulated by CUMS while up‐regulated by SA. These findings reveal that SA may exert antidepressant effects by up‐regulating the expression level of PRRT2 and increasing DA content in hippocampus. The identification of these 15 differentially expressed proteins, including PRRT2, provides further insight into the treatment mechanism of SA for depression. [ABSTRACT FROM AUTHOR]

Summary: Stress responses in plants imply spatio‐temporal changes in enzymes and metabolites, including subcellular compartment‐specific re‐allocation processes triggered by sudden changes in environmental parameters. To investigate interactions of primary metabolism with abiotic stress, the gin2‐1 mutant, defective in the sugar sensor hexokinase 1 (HXK1) was compared with its wildtype Landsberg erecta (Ler) based on time resolved, compartment‐specific metabolome and proteome data obtained over a full diurnal cycle. The high light sensitive gin2‐1 mutant was substantially delayed in subcellular re‐distribution of metabolites upon stress, and this correlated with a massive reduction in proteins belonging to the ATP producing electron transport chain under high light, while fewer changes occurred in the cold. In the wildtype, compounds specifically protecting individual compartments could be identified, e.g., maltose and raffinose in plastids, myo‐inositol in mitochondria, but gin2‐1 failed to recruit these substances to the respective compartments, or responded only slowly to high irradiance. No such delay was obtained in the cold. At the whole cell level, concentrations of the amino acids, glycine and serine, provided strong evidence for an important role of the photorespiratory pathway during stress exposure, and different subcellular allocation of serine may contribute to the slow growth of the gin2‐1 mutant under high irradiance. Significance Statement: Combining proteome and metabolome analysis at subcellular resolution revealed that the hexokinase‐1 mutant of Arabidopsis, gin2‐1, cannot respond adequately to sudden changes of light intensity, thus explaining its high light sensitivity. Failure of recruiting compartment‐specific protective compounds resulted in massive loss of electron transport chain components under high light stress, while the response to cold was not impaired in the mutant. [ABSTRACT FROM AUTHOR]

Summary: The Russian dandelion Taraxacum koksaghyz synthesizes considerable amounts of high‐molecular‐weight rubber in its roots. The characterization of factors that participate in natural rubber biosynthesis is fundamental for the establishment of T. koksaghyz as a rubber crop. The cis‐1,4‐isoprene polymers are stored in rubber particles. Located at the particle surface, the rubber transferase complex, member of the cis‐prenyltransferase (cisPT) enzyme family, catalyzes the elongation of the rubber chains. An active rubber transferase heteromer requires a cisPT subunit (CPT) as well as a CPT‐like subunit (CPTL), of which T. koksaghyz has two homologous forms: TkCPTL1 and TkCPTL2, which potentially associate with the rubber transferase complex. Knockdown of TkCPTL1, which is predominantly expressed in latex, led to abolished poly(cis‐1,4‐isoprene) synthesis but unaffected dolichol content, whereas levels of triterpenes and inulin were elevated in roots. Analyses of latex from these TkCPTL1‐RNAi plants revealed particles that were similar to native rubber particles regarding their particle size, phospholipid composition, and presence of small rubber particle proteins (SRPPs). We found that the particles encapsulated triterpenes in a phospholipid shell stabilized by SRPPs. Conversely, downregulating the low‐expressed TkCPTL2 showed no altered phenotype, suggesting its protein function is redundant in T. koksaghyz. MS‐based comparison of latex proteomes from TkCPTL1‐RNAi plants and T. koksaghyz wild‐types discovered putative factors that convert metabolites in biosynthetic pathways connected to isoprenoids or that synthesize components of the rubber particle shell. Significance Statement: Factors and mechanisms related to the biosynthesis of natural rubber are still not fully elucidated. We show that only TkCPTL1 is necessary for rubber synthesis in the Russian dandelion Taraxacum koksaghyz, while its homolog TkCPTL2 is dispensable for generating poly(cis‐1,4‐isoprene). In‐depth characterization of TkCPTL1‐depleted plants revealed further insights into the distribution of carbon between rubber, triterpenes, and inulin as well as provided further understanding of rubber particle function in storing latex metabolites. [ABSTRACT FROM AUTHOR]

Background/Purpose: Uveitis often is the only initial clinical manifestation of JIA for years. There are no reliable markers of JIA-associated uveitis and impossibility to overcome ocular barriers impede diagnosis and treatment leading to blindness. During the last years the number of proteins identified in tears increased from 200 (Herber et al., 2001) of only 17 (Zhou et al., 2003) different molecular weights (MW) to 491 (de Souza et al., 2006) with 80 chemokines, cytokines, and growth factors (Sack et al., 2005). Objective of the study is to reveal protein markers of JIA-associated uveitis in tears using high-resolution mass-spectrometry and hierarchical clustering methodology. Methods: Standard clinical protocol was used to diagnose uveitis and JIA. Tear samples drawn from 17 JIA-patients with uveitis, 4 JIA-patients without uveitis, 3 patients with systemic vasculitis, 4 patients with idiopathic uveitis and 3 healthy subjects were analyzed. Tears were collected on a filter paper, digested with trypsin and the peptides were purified on Zip tips. The samples were loaded to nano C18 column attached to Shimadzu nano LC coupled in-line to LTQ Orbitrap XL tandem mass spectrometer (Thermo Fischer Scientific, GA, USA). The mass spectra of the peptides were detected with a data-dependent 4-event scan method (a survey FT-MS parent scan followed by sequential datadependent FT-MS/MS scans on the three most abundant peptide ions from the parent scan). Proteins were identified from the mass spectra results with Proteome Discoverer software for protein database search using the International Protein Index (IPI) Human Protein Database (version 1.79). Quantification was conducted using SIEVE. Results: About 3000 proteins were detected in tears. Among those, 236 proteins (MW 7.4-466 kDa) were chosen as candidates for early diagnosis of the JIA-associated uveitis. Besides the rheumatoid factors RFET12, RF-IP14, RF-IP24, cytokines and T-cell receptor alpha-chain, we identified anti-CD40 single-chain antibody fragment A49, inhibiting protein CD40 which is a member of the TNF-receptor superfamily, lipocalin-1 precursor, associated with immune response and prostaglandin synthesis, clusterin inhibiting the cytolytic reaction of complement components, anti-TNF-alfa antibody light chain Fab fragment, protein phosphatase, regulatory subunit 15B, which plays a role in cell progression, apoptosis and regulation of membrane receptors and channels, DMBT/8kb.2 protein, which takes part in the interaction of tumor cells and the immune system. Conclusion: The result of our study demonstrates the benefits of high resolution mass- spectrometry for analysis and development of molecular signatures which can be used in diagnostics. Hierarchical clustering methodology allowed grouping according to expression profiles and the dendrogram construction procedure revealed clusters of proteins associated with iron metabolism as the most promising markers of JIA-associated uveitis. Earlier it was shown that induced anterior uveitis in the eyes of rats can be described in terms of proteins drawn from the anterior chamber (Sobn et al., 2000), which result is close to our terms of description. [ABSTRACT FROM AUTHOR]

Preeclampsia (PE) is one of the most significant pregnancy‐related hypertensive disorders. Currently, there are no useful markers to predict the onset of the condition in pregnant women. To provide further insights into the pathogenesis of PE and identify biomarkers of the condition, we used isobaric tags for relative and absolute quantitation (iTRAQ) proteomics coupled with 2‐D LC‐MS/MS, to analyze urinary protein profiles from 7 PE patients and 7 normotensive pregnant women. A total of 294 proteins were abnormally expressed in PE patients. Of these, 233 were significantly down‐regulated and 61 proteins were significantly up‐regulated. Bioinformatics analysis using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database, found that the most differentially expressed proteins (DEPs) were involved in coagulation and complement pathways, the renin‐angiotensin system and cell adhesion molecules (CAMs) pathways. We further validated three of the DEPs, including serotransferrin (TF) and complement factor B (CFB) by immunoblottingand serum paraoxonase/arylesterase 1 (PON1) by ELISA using 14 pairs of urine samples from PE patients and normal pregnant women. Taken together, our results provide the basis for further understanding the pathogenesis of PE and identifying predictive biomarkers. [ABSTRACT FROM AUTHOR]

Purpose: To obtain insights on the protein composition of posterior capsular plaques (PCP) in congenital unilateral cataract with anterior vitreolenticular interface dysgenesis (AVLID). Methods: Posterior capsular plaque's were collected during surgery in children presenting with congenital unilateral cataract. Surgeries were analysed focusing on the type of cataract, the integrity of the posterior capsule after peeling the PCP and the presence of vitreolenticular adherences when performing primary posterior capsulorhexis. Proteome analysis was performed on the collected PCPs. Results: Posterior capsular plaques collection and proteome analysis were feasible from four children presenting with unilateral idiopathic congenital cataract and AVLID. A large portion of the proteins found in the PCPs was similar to the proteins known to be present in lens epithelial cells and fibres. Proteins like vimentin, fibronectin, collagen type I, collagen type VI and lumican were also found, which typically are present in mesenchymal tissue but not in lens tissue or capsule. Conclusion: Posterior capsular plaques in cases of unilateral idiopathic congenital cataract of the AVLID type present a protein composition of mainly proteins found in lens epithelial cells and fibres. Some proteins however are a specific for lens tissue and are typically seen in mesenchymal tissue. [ABSTRACT FROM AUTHOR]

Abstract: Quantitative mass spectrometry enables to monitor the abundance of thousands of proteins across biological conditions. Currently, most data analysis approaches rely on the assumption that the majority of the observed proteins remain unchanged across compared samples. Thus, gross morphological differences between cell states, deriving from, e.g., differences in size or number of organelles, are often not taken into account. Here, we analyzed multiple published datasets and frequently observed that proteins associated with a particular cellular compartment collectively increase or decrease in their abundance between conditions tested. We show that such effects, arising from underlying morphological differences, can skew the outcome of differential expression analysis. We propose a method to detect and normalize morphological effects underlying proteomics data. We demonstrate the applicability of our method to different datasets and biological questions including the analysis of sub‐cellular proteomes in the context of Caenorhabditis elegans aging. Our method provides a complementary perspective to classical differential expression analysis and enables to uncouple overall abundance changes from stoichiometric variations within defined group of proteins. [ABSTRACT FROM AUTHOR]

Proteomic analyses of the excretory/secretory proteins from the RH strain of Toxoplasma gondii have been performed to understand their functions in the host-parasite interaction. A total of 34 proteins were identified from LC/MS/MS analysis and their abundance was estimated by spectral counting methods. Among them, 8 species of micronemal proteins (MICs), 2 species of rhoptry proteins (ROPs), and 6 species of dense granular proteins (GRAs) were confirmed. Besides these, 18 species of protein were newly identified, and their cellular functions were estimated from sequence analysis. The three most abundant of the 34 identified extractor/secretory proteins GRA1, GRA7 and GRA2 were confirmed to be highly expressed in T. gondii using the spectral count method. This phenomenon is another demonstration of the importance of GRA proteins for the penetration and survival of T. gondii. [ABSTRACT FROM AUTHOR]

Abscisic acid (ABA) mediates resistance to abiotic stress and controls developmental processes in plants. The group-A PP2Cs, of which ABI1 is the prototypical member, are protein phosphatases that play critical roles as negative regulators very early in ABA signal transduction. Because redundancy is thought to limit the genetic dissection of early ABA signalling, to identify redundant and early ABA signalling proteins, we pursued a proteomics approach. We generated YFP-tagged ABI1 Arabidopsis expression lines and identified in vivo ABI1-interacting proteins by mass-spectrometric analyses of ABI1 complexes. Known ABA signalling components were isolated including SnRK2 protein kinases. We confirm previous studies in yeast and now show that ABI1 interacts with the ABA-signalling kinases OST1, SnRK2.2 and SnRK2.3 in plants. Interestingly, the most robust in planta ABI1-interacting proteins in all LC-MS/MS experiments were nine of the 14 PYR/PYL/RCAR proteins, which were recently reported as ABA-binding signal transduction proteins, providing evidence for in vivo PYR/PYL/RCAR interactions with ABI1 in Arabidopsis. ABI1–PYR1 interaction was stimulated within 5 min of ABA treatment in Arabidopsis. Interestingly, in contrast, PYR1 and SnRK2.3 co-immunoprecipitated equally well in the presence and absence of ABA. To investigate the biological relevance of the PYR/PYLs, we analysed pyr1/pyl1/pyl2/pyl4 quadruple mutant plants and found strong insensitivities in ABA-induced stomatal closure and ABA-inhibition of stomatal opening. These findings demonstrate that ABI1 can interact with several PYR/PYL/RCAR family members in Arabidopsis, that PYR1–ABI1 interaction is rapidly stimulated by ABA in Arabidopsis and indicate new SnRK2 kinase-PYR/PYL/RCAR interactions in an emerging model for PYR/PYL/RCAR-mediated ABA signalling. [ABSTRACT FROM AUTHOR]

The maize endosperm transcriptome was investigated through cDNA libraries developed at three characteristic stages: (i) lag phase [10 days after pollination (DAP)]; (ii) beginning of storage (14 DAP); and (iii) maximum starch accumulation rate (21 DAP). Expressed sequence tags for 711, 757 and 384 relevant clones, respectively, were obtained and checked manually. The proportion of sequences with no clear function decreased from 35% to 20%, and a large increase in storage protein sequences (i.e. 5% to 38%) was observed from stages (i) to (iii). The remaining major categories included metabolism (11%–13%), transcription–RNA processing–protein synthesis (13%–20%), protein destination (5%–9%), cellular communication (3%–9%) and cell rescue–defence (4%). Good agreement was generally found between category rank in the 10-DAP transcriptome and the recently reported 14-DAP proteome, except that kinases and proteins for RNA processing were not detected in the latter. In the metabolism category, the respiratory pathway transcripts represented the largest proportion (25%–37%), and showed a shift in favour of glycolysis at 21 DAP. At this stage, amino acid metabolism increased to 17%, whereas starch metabolism surprisingly decreased to 7%. A second experiment focused on carbohydrate metabolism by comparing gene expression at three levels (transcripts, proteins and enzyme activities) in relation to substrate or product from 10 to 40 DAP. Here, two distinct patterns were observed: invertases and hexoses were predominant at the beginning, whereas enzyme patterns in the starch pathway, at the three levels, anticipated and paralleled starch accumulation, suggesting that, in most cases, transcriptional control is responsible for the regulation of starch biosynthesis. [ABSTRACT FROM AUTHOR]

Patients with chronic kidney disease (CKD) show a panel of partially de-regulated serum markers indicative for bone metabolism disorders and cardiovascular diseases (CVDs). This review provides an overview of currently reported biomarker candidates at the interface of kidney disease, bone metabolism disorders and CVDs, and gives details on their functional interplay on the level of protein-protein interaction data. We retrieved 13 publications from 1999 to 2006 reporting 31 genes associated with CVDs, and 46 genes associated with bone metabolism disorders in patients with CKD. We identified these genes to be functionally involved in signal transduction processes, cell communication, immunity and defence, as well as skeletal development. On the basis of the given set of 77 genes further 276 interacting proteins were identified using reference data on known protein interactions. Their functional interplay was estimated by linking properties reflected by gene expression data characterizing CKD, gene ontology terms as provided by the gene ontology consortium and transcription factor binding site profiles. Highly connected sub-networks of proteins associated with CKD, CVDs or bone metabolism disorders were detected involving proteins like collagens (COL1A1, COL1A2), fibronectin, transforming growth factor-β1, or components of fibrinogen (FG-α, FG-γ, FG-γ). A systems biology approach provides a methodological framework for linking singular biomarker candidates towards deriving functional dependencies among clinically interlinked diseases. [ABSTRACT FROM AUTHOR]

For the characterization of protein sequences and post-translational modifications by MS, the ‘top-down’ proteomics approach utilizes molecular and fragment ion mass data obtained by ionizing and dissociating a protein in the mass spectrometer. This requires more complex instrumentation and methodology than the far more widely used ‘bottom-up’ approach, which instead uses such data of peptides from the protein's digestion, but the top-down data are far more specific. The ESI MS spectrum of a 14 protein mixture provides full separation of its molecular ions for MS/MS dissociation of the individual components. False-positive rates for the identification of proteins are far lower with the top-down approach, and quantitation of multiply modified isomers is more efficient. Bottom-up proteolysis destroys the information on the size of the protein and the connectivities of the peptide fragments, but it has no size limit for protein digestion. In contrast, the top-down approach has a ∼ 500 residue, ∼ 50 kDa limitation for the extensive molecular ion dissociation required. Basic studies indicate that this molecular ion intractability arises from greatly strengthened electrostatic interactions, such as hydrogen bonding, in the gas-phase molecular ions. This limit is now greatly extended by variable thermal and collisional activation just after electrospray (‘prefolding dissociation’). This process can cleave 287 inter-residue bonds in the termini of a 1314 residue (144 kDa) protein, specify previously unidentified disulfide bonds between eight of 27 cysteines in a 1714 residue (200 kDa) protein, and correct sequence predictions in two proteins, one of 2153 residues (229 kDa). [ABSTRACT FROM AUTHOR]

Advances in proteomic techniques have allowed the large-scale identification of phosphorylation sites in complex protein samples, but new biological insight requires an understanding of their in vivo dynamics. Here, we demonstrate the use of a stable isotope-based quantitative approach for pathway discovery and structure–function studies in Arabidopsis cells treated with the bacterial elicitor flagellin. The quantitative comparison identifies individual sites on plasma membrane (PM) proteins that undergo rapid phosphorylation or dephosphorylation. The data reveal both divergent dynamics of different sites within one protein and coordinated regulation of homologous sites in related proteins, as found for the PM H+-ATPases AHA1, 2 and 3. Strongly elicitor-responsive phosphorylation sites may reflect direct regulation of protein activity. We confirm this prediction for RbohD, an NADPH oxidase that mediates the rapid production of reactive oxygen species (ROS) in response to elicitors and pathogens. Plant NADPH oxidases are structurally distinct from their mammalian homologues, and regulation of the plant enzymes is poorly understood. On RbohD, we found both unchanging and strongly induced phosphorylation sites. By complementing an RbohD mutant plant with non-phosphorylatable forms of RbohD, we show that only those sites that undergo differential regulation are required for activation of the protein. These experiments demonstrate the potential for use of quantitative phosphoproteomics to determine regulatory mechanisms at the molecular level and provide new insights into innate immune responses. [ABSTRACT FROM AUTHOR]

Phytoremediation is a relatively new approach to remove polycyclic aromatic hydrocarbons (PAHs) from the environment. When plants are grown under pyrene treatment, they respond by synthesizing a set of protective proteins. To learn more about protein changes in response to pyrene treatment, we extracted total proteins from the leaves of maize ( Zea mays L.) 1 week after pyrene treatment. The proteins extracted were separated with two-dimensional gel electrophoresis. In total, approximately 54 protein spots were found by comparing gels from treated and control groups. According to the isoelectric point, molecular weight, and abundance of these protein spots, 20 pyrene-induced proteins were found to have changed abundance. Of these, 15 protein spots were increased and five protein spots were newly appeared in pyrene-treated plant leaves. Six model upregulated protein spots of different molecular weights were excised from the gels and subjected to trypsin digestion followed by peptide separation using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Peptide masses were used to search the matrix-science database for protein identification. Two of the proteins were identified on the basis of the homology of their peptide profiles with existing protein sequences as pyruvate orthophosphate dikinase and the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit. These proteins are involved in the regulation of carbohydrate and energy metabolism. The present study gives new insights into the pyrene stress response in maize leaves and demonstrates the power of the proteomic approach in phytoremediation of PAHs. [ABSTRACT FROM AUTHOR]

The two-hybrid method detects the interaction of two proteins by their ability to reconstitute the activity of a split transcription factor, thus allowing the use of a simple growth selection in yeast to identify new interactions. Since its introduction about 15 years ago, the assay largely has been applied to single proteins, successfully uncovering thousands of novel protein partners. In the last few years, however, two-hybrid experiments have been scaled up to focus on the entire complement of proteins found in an organism. Although a single such effort can itself result in thousands of interactions, the validity of these high-throughput approaches has been questioned as a result of the prevalence of numerous false positives in these large data sets. Such artifacts may not be an obstacle to continued scale-up of the method, because the classification of true and false positives has proven to be a computational challenge that can be met by a growing number of creative strategies. Two examples are provided of this combination of high-throughput experimentation and computational analysis, focused on the interaction of Plasmodium falciparum proteins and of Saccharomyces cerevisiae membrane proteins. [ABSTRACT FROM AUTHOR]

New technologies enable acquisition of large data-sets containing genomic, proteomic and metabolic information that describe the state of a cell. These data-sets call for systematic methods enabling relevant information about the inner workings of the cell to be extracted. One important issue at hand is the understanding of the functional interactions between genes, proteins and metabolites. We here present a method for identifying the dynamic interactions between biochemical components within the cell, in the vicinity of a steady-state. Key features of the proposed method are that it can deal with data obtained under perturbations of any system parameter, not only concentrations of specific components, and that the direct effect of the perturbations does not need to be known. This is important as concentration perturbations are often difficult to perform in biochemical systems and the specific effects of general type perturbations are usually highly uncertain, or unknown. The basis of the method is a linear least-squares estimation, using time-series measurements of concentrations and expression profiles, in which system states and parameter perturbations are estimated simultaneously. An important side-effect of also employing estimation of the parameter perturbations is that knowledge of the system's steady-state concentrations, or activities, is not required and that deviations from steady-state prior to the perturbation can be dealt with. Time derivatives are computed using a zero-order hold discretization, shown to yield significant improvements over the widely used Euler approximation. We also show how network interactions with dynamics that are too fast to be captured within the available sampling time can be determined and excluded from the network identification. Known and unknown moiety conservation relationships can be processed in the same manner. The method requires that the number of samples equals at least the number of network components and, hence, is at present restricted to relatively small-scale networks. We demonstrate herein the performance of the method on two small-scalein silicogenetic networks. [ABSTRACT FROM AUTHOR]

The key concept of proteomics (looking at many proteins at once) opens new avenues in the search for clinically useful biomarkers of disease, treatment response and ageing. As the number of proteins that can be detected in plasma or serum (the primary clinical diagnostic samples) increases towards 1000, a paradoxical decline has occurred in the number of new protein markers approved for diagnostic use in clinical laboratories. This review explores the limitations of current proteomics protein discovery platforms, and proposes an alternative approach, applicable to a range of biological/physiological problems, in which quantitative mass spectrometric methods developed for analytical chemistry are employed to measure limited sets of candidate markers in large sets of clinical samples. A set of 177 candidate biomarker proteins with reported associations to cardiovascular disease and stroke are presented as a starting point for such a‘directed proteomics’ approach. [ABSTRACT FROM AUTHOR]

Summary The newly sequenced genome of Streptomyces coelicolor is estimated to encode 7825 theoretical proteins. We have mapped approximately 10% of the theoretical proteome experimentally using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Products from 770 different genes were identified, and the types of proteins represented are discussed in terms of their anno-tated functional classes. An average of 1.2 proteins per gene was observed, indicating extensive post-translational regulation. Examples of modification by N-acetylation, adenylylation and proteolytic processing were characterized using mass spectrometry. Proteins from both primary and certain secondary metabolic pathways are strongly represented on the map, and a number of these enzymes were identified at more than one two-dimensional gel location. Post-translational modification mechanisms may therefore play a significant role in the regulation of these pathways. Unexpectedly, one of the enzymes for synthesis of the actinorhodin polyketide antibiotic appears to be located outside the cytoplasmic compartment, within the cell wall matrix. Of 20 gene clusters encoding enzymes characteristic of secondary metabolism, eight are represented on the proteome map, including three that specify the production of novel metabolites. This information will be valuable in the characterization of the new metabolites. [ABSTRACT FROM AUTHOR]

T7 endonuclease I is a nuclease that is selective for the structure of the four-way DNA junction. The active site is similar to those of a number of restriction enzymes. We have solved the crystal structure of endonuclease I with a wild-type active s [ABSTRACT FROM AUTHOR]

Accurate tRNA 3′ end maturation is essential for aminoacylation and thus for protein synthesis in all organisms. Here we report the first identification of protein and DNA sequences for tRNA 3′‐processing endonucleases (RNase Z). Purification of RNase Z from wheat identified a 43 kDa protein correlated with the activity. Peptide sequences obtained from the purified protein were used to identify the corresponding gene. In vitro expression of the homologous proteins from Arabidopsis thaliana and Methano coccus janaschii confirmed their tRNA 3′‐processing activities. These RNase Z proteins belong to the ELAC1/2 family of proteins and to the cluster of orthologous proteins COG 1234. The RNase Z enzymes from A.thaliana and M.janaschii are the first members of these families to which a function can now be assigned. Proteins with high sequence similarity to the RNase Z enzymes from A.thaliana and M.janaschii are present in all three kingdoms. [ABSTRACT FROM AUTHOR]

We have utilized a T7 polymerase/promoter system for the high-level incorporation of methionine analogs with suitable labels for structural research (X-ray and NMR studies) on recombinant annexin V produced in Escherichia coli. Here, we describe, to our knowledge, the first biosynthetic high-level substitution of methionine by 2-aminohexanoic acid (norleucine), ethionine and telluromethionine in a protein. The replacement has been confirmed by electrospray mass spectroscopy, amino acid analysis and X-ray structural analysis. Conditions for expression were optimized concerning the frequency of appearance of revertants, high-level replacement and maximal protein yield. For the incorporation of norleucine and ethionine, E. coli B834 (DE3)(hsd metB), which is auxotrophic for methionine, was grown under methionine-limited conditions with an excess of the analog in the culture medium, and the expression of protein under the control of the T7 promoter was induced after the methionine supply had been exhausted. The factor limiting the high-level incorporation of telluromethionine into protein is its sensitivity towards oxidation. To overcome this problem, bacteria were grown with a limited amount of methionine, harvested after its exhaustion and resuspended in fresh media without methionine; telluromethionine was added and protein synthesis induced. Under these conditions, significant amounts of protein can be expressed before telluromethionine has been completely degraded (within hours). Biosynthetic incorporation of heavy atoms such as tellurium into recombinant proteins can accelerate the process of obtaining heavy-atom derivatives suitable for X-ray structural analysis, supplementing the traditional trial-and-error preparation of heavy-atom derivatives for the method of multiple isomorphous replacement. Furthermore, the successful high-level incorporation of amino acid analogs can provide single-atom mutations for the detailed study of the structure and function of proteins. [ABSTRACT FROM AUTHOR]

The in vivo effects of ultraviolet-B radiation (280–320 nm) on photosystem-II activity and degradation of the D1 protein are investigated and compared with the in vitro results on isolated thylakoids and other detergent-extracted photosystem-II preparations. A cleavage site in the second transmembrane segment of the D1 protein, giving rise to a 20-kDa C-terminal and a 13-kDa N-terminal fragment pair, is detected after irradiation of entire leaves as well as in all photosystem-II preparations, irrespective of their actual ability to evolve oxygen but depending on the presence of Mn ions associated with the water-splitting system. Damage to the plastoquinone moiety, observed by other authors, is confirmed and is proposed to be responsible for the impairment of electron-transport activity, but not for the observed cleavage of the D1 protein. [ABSTRACT FROM AUTHOR]

Procolipase is the precursor of colipase, which acts as protein cofactor for the activity of pancreatic lipase. The solution structure of procolipase has been determined by ¹H NMR using two- and three-dimensional measurements. The secondary structure determination identified two separate three-stranded β-sheet regions with concomitant hydrogen bond patterns. The tertiary structure of the protein was determined using 863 non-trivial proton-proton distance constraints, 14 hydrogen bond distance constraints and 55 Φ and 25 X1 dihedral constraints. The structure that was obtained from distance geometry and energy refinement contains three highly disordered loops as well as a disordered N- and C-terminal region. The remaining part of the structure is well defined with a root-mean-square deviation (rmsd) relative to the average of 0.09±0.02 nm for backbone atoms (residues 11–30, 37–50, 57–69, 83–89). The protein comprises two identical domains, each containing a three-strand β-sheet and two disulfide bonds: a 15-residue region in each domain superimposes with 0.07 nm rmsd, measured on backbone atoms. The solution structure is nearly identical to the crystal structure. It is in agreement with previous NMR data and, in combination with these data, supports the current model of procolipase micelle interaction and the lipase activation by colipase. [ABSTRACT FROM AUTHOR]

A full-length cDNA clone for a cysteine proteinase inhibitor (cystatin) was isolated from a λgt10 cDNA library of immature corn kernels by screening with a mixture of cDNA inserts for oryzacystatins I and II. The cDNA clone spans 960 base pairs, encoding a 135-amino-acid protein containing a signal peptide fragment. The protein, named corn cystatin I, is considered to be a member of the cystatin superfamily, since it contains the commonly conserved Gtn-Val-VaI-Ala-Gly region that exists in most known cystatins as a probable binding site and is significantly similar to other cystatins in its overall amino acid sequence. Corn cystatin I expressed in Escherichia coli showed a strong papain-inhibitory activity. Northern blot analysis showed that the amount of mRNA for corn cystatin I reaches a maximum 2 weeks after flowering and then decreases gradually. [ABSTRACT FROM AUTHOR]

The thioredoxin/thioredoxin reductase system has been studied as regenerative machinery for proteins inactivated by oxidative stress in vitro and in cultured endothelial cells. Mammalian glyceraldehyde-3-phosphate dehydrogenase was used as the main model enzyme for monitoring the oxidative damage and the regeneration. Thioredoxin and its reductase purified from bovine liver were used as the regenerating system. The physiological concentrations (2–14 μM) of reduced thioredoxin, with 0.125 μM thioredoxin reductase and 0.25 mM NADPH, regenerated H2O2-inactivated glyceraldehyde-3-phosphate dehydrogenase and other mammalian enzymes almost completely within 20 min at 37°C. Although the treatment of endothelial cells with 0.2–12 mM H2O2 for 5 min resulted in a marked decrease in the activity of glyceraldehyde-3-phosphate dehydrogenase, it had no effect on the activities of thioredoxin and thioredoxin reductase. Essentially all of the thioredoxin in endothelial ceils at control state was in the reduced form and 70–85% remained in the reduced form even after the H2O2 treatment. The inactivated glyceraldehyde-3-phosphate dehydrogenase in a cell lysate prepared from the H2O2-treated endothelial cells was regenerated by incubating the lysate with 3 mM NADPH at 37 °C and the antiserum raised against bovine liver thioredoxin inhibited the regeneration. The inhibition of thioredoxin reductase activity by 13-cis-retinoic acid resulted in a decrease in the regeneration of glyceraldehyde-3-phosphate dehydrogenase in the H2O2-treated endothelial cells. The present findings provide evidence that thioredoxin is involved in the regeneration of proteins inactivated by oxidative stress in endothelial cells. [ABSTRACT FROM AUTHOR]

An improved procedure for reincorporation of isolated uncoupling protein (UCP) from brown adipose tissue into phospholipid vesicles is reported and H + uptake in K +-driven exchange diffusion quantitatively analyzed. UCP is isolated and reconstituted with medium-length linear-chain alkyl polyoxyethylcne. In the critical step of vesicle formation, the stepwise removal of the detergent by polystyrene beads is applied. Vesicles are generated in the presence of solutes and buffers to be internalized which are then removed by gel filtration. The internal volume is about 4 µl/mg phospholipid with a vesicle diameter of 100 nm. One vesicle contains, on average, six molecules UCP. The best results are obtained with purified egg yolk phosphatidylcholine. Addition of PtdEtn, PtdSer decreases the vesicle size and, still more, H +-transport activity by UCP. Asolectin completely inactivates UCP. K +-gradient-driven H+ uptake is 80% inhibited by external GTP and 95% by internal plus external GTP. When H + transport is recorded externally by a pH electrode and internally by pyranine, the kinetics show no delay resulting from intervening membrane-bound H+ pools. Total H+ uptake after addition of carbonylcyanine m-chlorophenylhydrazone (CCCP) and valinomycin corresponds to the diffusion between H+ and K+ and is unchanged by GTP. The linear correlation of H+-transport inhibition to GTP binding demonstrates that all UCP molecules incorporated are equally active. The exchange diffusion between H+ uptake and K+ efflux is demonstrated using a K + electrode and 86Rb measurements. Recording Δψ using 3,3'-diispropylthiadicarbocyanine shows a rapid generation of Δψ on valinomycin addition, which decreases only slightly with H+ uptake, even after addition of CCCP or gramicidin.The 31 Δψ collapses only after addition of external K+. By demonstrating that valinomycin-induced K+ and H+ fluxes reflect relaxation into the diffusion equilibrium state, the transport rate of UCP can be evaluated as a first-order rate, VH+/CH+, in which the rate, VH+, is related to H +-uptake capacity, CH+. This allows quantitative comparison of transport rates independently of the variable CH+. The dependence on Δψ of H+ transport is measured by varying external K + concentration. A virtually linear relation of the rate to the K +-diffusion potential is observed, although the capacity is only slightly changed. The linear VaH+/Δψ relationship resembles an open-channel type of transport, but is discussed in terms of a low-activation-barrier type of carrier mechanism, in contrast to the log (VH+/Δψ) relation found for the ADP/ATP carrier with high activation barriers. [ABSTRACT FROM AUTHOR]

The nearly complete amino acid sequence obtained for murine calcyclin from Ehrlich ascites tumor cells reveals a very strong similarity with the rat and human sequences previously deduced from corresponding cDNA clones. While mouse and rat calcyclins are identical, the human protein shows at three positions a conservative amino acid replacement. Using a mouse calcyclin affinity matrix, two proteins with molecular masses of about 36 kDa have been purified from Ehrlich ascites tumor cells. The interaction between these two proteins and the immobilized calcyclin is strictly Ca2+-dependent. Immunological criteria and partial sequence data identify the two calcyclin-binding proteins as the phospholipid-binding protein annexin II (p36) and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. These observations suggest that calcyclin may exert its physiological function by a Ca2+-dependent interaction with cellular targets, e.g. annexin II or glyceraldehyde-3-phosphate dehydrogenase. [ABSTRACT FROM AUTHOR]

Structural and functional characteristics of subunits of bovine heart cytochrome-c reductase have been investigated by controlled digestion of soluble and membrane-reconstituted purified hc1 complex and direct amino acid sequencing of native and digested protein subunits. The results obtained show that the N-terminal segments of core protein Ii and the 14-kDa protein extend at the periphery of the complex. protruding into the inner matrix space. The Fe-S protein, located at the outer C-periphery of the complex, is shown to be anchored to other subunits of the complex by the amphipathic N- terminal region. Proteolytic cleavage of 7–11 residues from the N-terminal segment of the 14-kDa protein is apparently associated with decoupling of redox-linked proton pumping. Partial digestion of core protein II, the 6.4-kDa protein, and the C-terminal region of the 9.2-kDa protein, is without effect on the redox and proton- motive activity of the complex. [ABSTRACT FROM AUTHOR]

Protein phosphatases-1, 2A and 2B have been identified in membrane and soluble fractions of Drosophila melanogaster heads. Similarities between Drosophila and mammalian protein phosphatase-1 included specificity for the β subunit of phosphorylase kinase, sensitivity to inhibitor-1 and inhibitor-2, inhibition by protamine, retention by heparin-Sepharose and selective interaction with membranes. In addition, an inactive form of protein phosphatase-l, termed protein phosphatase-lI, was detected in the soluble fraction that could be activated by preincubation with MgATP and mammalian glycogen synthase kinase-3. Inhibitor-2 partially purified from Drosophila had an identical molecular mass to its mammalian counterpart, and recombined with mammalian protein phosphatase-1 to form a hybrid protein phosphatase-lI. Similarities between Drosophila and mammalian protein phosphatase-2A included preferential dephosphorylation of the α subunit of phosphorylase kinase, insensitivity to inhibitors-1 and -2, activation by protamine, exclusion from heparin-Sepharose and apparent molecular mass. A Ca2+-dependent calmodulin-stimulated protein phosphatase (protein phosphatase-2B) that was inhibited by trifluoperazine was identified in the soluble fraction. The remarkable similarities between Drosophila protein phosphatases and their mammalian counterparts are indicative of strict phylogenetic conservation and demonstrate that the procedures used to classify mammalian protein phosphatases have a wider application. Characterisation of the Drosophila phosphatases will facilitate genetic analysis of dephosphorylation systems and their possible roles in neuronal and behavioural plasticity in Drosophila. [ABSTRACT FROM AUTHOR]

Two derivatives of Hansenula anomala Flavocytochrome b2 have been prepared, one deprived of the flavin prosthetic group (deflavocytochrome B2), and the other consisting of the heme-h-carrying globule (b2 core). The redox potential of the heme in the two derivatives is -5 (±5) mV and -10 (±5) mV respectively, fairly similar to the value of -20 (±5) mV reported for the holoenzyme, indicating a minor effect of the flavin and of the flavodehydrogenase domain on heme potential. The kinetic of azurin and stellacyanin reduction by both derivatives have been investigated. At pH 7.0, I= 0.2 M and 20°C the second-order rate constants are k = 8 x 105M-1s-1 for azurin reduction by deflavocytochrome b2; k = 1.6 x 106 M-1s-1 for azurin reduction by b2 core; k = 1 x 107 M-1s-1 for stellacyanin reduction by deflavocytochrome b2; k = 3 x 107 M-1s-1 for stellacyanin reduction by b2 core. The change in pH markedly affects the kinetics in the case of azurin, but has no effect on stellacyanin reduction. The change in ionic strength has a significant effect when deflavocytochrome b2 is the reductant, indicating that the flavodehydrogenase domain plays a role in the stabilization of the transient kinetic complex by means of electrostatic interactions. The kinetic results are discussed in the framework of the Marcus theory. [ABSTRACT FROM AUTHOR]

The changes in the degree of phosphorylation of ribosomal protein S6 during the life cycle of the aquatic fungus Blastocladiella emersonii were analyzed by two-dimensional gel electrophoresis. Three phosphorylated derivatives of S6 are present throughout the entire life cycle. However, under certain germination condition, more highly phosphorylated derivatives of S6 appear. Nonetheless, the resumption of protein synthesis that occurs during germination is not dependent on those highly phosphorylated derivatives of S6. The pattern and sites of phosphorylation of S6 labelled in vivo with [32P]orthophosphate have been compared with those of 40S ribosomal subunit labelled in vitro by partially purified protein kinases. Three major phosphopeptides were found in S6 isolated from the zoospore, while six phosphopeptides were found after zoospore germination (in germling cells). The phosphopeptide patterns of S6 phosphorylated by the cAMP-dependent protein kinase and by casein kinases I and II were completely distinct. Only the cAMP-dependent protein kinase gives rise to a phosphopeptide found in 32P-labelled cells, indicating that one of sites phosphorylated in vivo is also phoshorylated in vitro by the cAMP-dependent protein kinase. [ABSTRACT FROM AUTHOR]