Search tips
Search criteria

Results 1-21 (21)

Clipboard (0)

Select a Filter Below

more »
Year of Publication
1.  Randomized Trial of Micafungin for the Prevention of Invasive Fungal Infection in High-Risk Liver Transplant Recipients 
In this randomized clinical trial comparing micafungin 100 mg with standard-care antifungal prophylaxis (fluconazole, liposomal amphotericin B, or caspofungin) in high-risk liver transplant patients, micafungin 100 mg was noninferior and had a better kidney safety profile.
Background. Invasive fungal infection (IFI) following liver transplant is associated with significant morbidity and mortality. Antifungal prophylaxis is rational for liver transplant patients at high IFI risk.
Methods. In this open-label, noninferiority study, patients were randomized 1:1 to receive intravenous micafungin 100 mg or center-specific standard care (fluconazole, liposomal amphotericin B, or caspofungin) posttransplant. The primary endpoint was clinical success (absence of a proven/probable IFI and no need for additional antifungals) at end of prophylaxis (EOP). Noninferiority (10% margin) of micafungin vs standard care was assessed in the per protocol and full analysis sets. Safety assessments included adverse events and liver and kidney function tests.
Results. The full analysis set comprised 344 patients (172 micafungin; 172 standard care). Mean age was 51.2 years; 48.0% had a Model for End-Stage Liver Disease score ≥20. At EOP (mean treatment duration, 17 days), clinical success was 98.6% for micafungin and 99.3% for standard care (Δ standard care – micafungin [95% confidence interval], 0.7% [−2.7% to 4.4%]) in the per protocol set and 96.5% and 93.6%, respectively (−2.9% [−8.0% to 1.9%]), in the full analysis set. Incidences of drug-related adverse events for micafungin and standard care were 11.6% and 16.3%, leading to discontinuation in 6.4% and 11.6% of cases, respectively. At EOP, liver function tests were similar but creatinine clearance was higher in micafungin- vs standard care–treated patients.
Conclusions. Micafungin was noninferior to standard care as antifungal prophylaxis in liver transplant patients at high risk for IFI. Adverse event profiles and liver function at EOP were similar, although kidney function was better with micafungin.
Clinical Trials Registration. NCT01058174.
PMCID: PMC4357288  PMID: 25520332
antifungal therapy; liver transplant; micafungin; prophylaxis; infection
2.  Quantitative cross-linking/mass spectrometry reveals subtle protein conformational changes 
Quantitative cross-linking/mass spectrometry (QCLMS) probes protein structural dynamics in solution by quantitatively comparing the yields of cross-links between different conformational statuses. We have used QCLMS to understand the final maturation step of the proteasome lid and also to elucidate the structure of complement C3(H2O). Here we benchmark our workflow using a structurally well-described reference system, the human complement protein C3 and its activated cleavage product C3b. We found that small local conformational changes affect the yields of cross-linking residues that are near in space while larger conformational changes affect the detectability of cross-links. Distinguishing between minor and major changes required robust analysis based on replica analysis and a label-swapping procedure. By providing workflow, code of practice and a framework for semi-automated data processing, we lay the foundation for QCLMS as a tool to monitor the domain choreography that drives binary switching in many protein-protein interaction networks.
PMCID: PMC5140025  PMID: 27976756
cross-linking/mass spectrometry; quantitative protein structure; conformational change; isotope labeled cross-linkers; automated data process
3.  Production Strategies and Applications of Microbial Single Cell Oils 
Polyunsaturated fatty acids (PUFAs) of the ω-3 and ω-6 class (e.g., α-linolenic acid, linoleic acid) are essential for maintaining biofunctions in mammalians like humans. Due to the fact that humans cannot synthesize these essential fatty acids, they must be taken up from different food sources. Classical sources for these fatty acids are porcine liver and fish oil. However, microbial lipids or single cell oils, produced by oleaginous microorganisms such as algae, fungi and bacteria, are a promising source as well. These single cell oils can be used for many valuable chemicals with applications not only for nutrition but also for fuels and are therefore an ideal basis for a bio-based economy. A crucial point for the establishment of microbial lipids utilization is the cost-effective production and purification of fuels or products of higher value. The fermentative production can be realized by submerged (SmF) or solid state fermentation (SSF). The yield and the composition of the obtained microbial lipids depend on the type of fermentation and the particular conditions (e.g., medium, pH-value, temperature, aeration, nitrogen source). From an economical point of view, waste or by-product streams can be used as cheap and renewable carbon and nitrogen sources. In general, downstream processing costs are one of the major obstacles to be solved for full economic efficiency of microbial lipids. For the extraction of lipids from microbial biomass cell disruption is most important, because efficiency of cell disruption directly influences subsequent downstream operations and overall extraction efficiencies. A multitude of cell disruption and lipid extraction methods are available, conventional as well as newly emerging methods, which will be described and discussed in terms of large scale applicability, their potential in a modern biorefinery and their influence on product quality. Furthermore, an overview is given about applications of microbial lipids or derived fatty acids with emphasis on food applications.
PMCID: PMC5050229  PMID: 27761130
single cell oil; solid-state fermentation; submerged fermentation; downstream processing; food application
4.  Quantitative Cross-linking/Mass Spectrometry Using Isotope-labeled Cross-linkers and MaxQuant* 
The conceptually simple step from cross-linking/mass spectrometry (CLMS) to quantitative cross-linking/mass spectrometry (QCLMS) is compounded by technical challenges. Currently, quantitative proteomics software is tightly integrated with the protein identification workflow. This prevents automatically quantifying other m/z features in a targeted manner including those associated with cross-linked peptides. Here we present a new release of MaxQuant that permits starting the quantification process from an m/z feature list. Comparing the automated quantification to a carefully manually curated test set of cross-linked peptides obtained by cross-linking C3 and C3b with BS3 and isotope-labeled BS3-d4 revealed a number of observations: (1) Fully automated process using MaxQuant can quantify cross-links in our reference data set with 68% recall rate and 88% accuracy. (2) Hidden quantification errors can be converted into exposed failures by label-swap replica, which makes label-swap replica an essential part of QCLMS. (3) Cross-links that failed during automated quantification can be recovered by semi-automated re-quantification. The integrated workflow of MaxQuant and semi-automated assessment provides the maximum of quantified cross-links. In contrast, work on larger data sets or by less experienced users will benefit from full automation in MaxQuant.
PMCID: PMC4974350  PMID: 27302889
5.  Structure of Complement C3(H2O) Revealed By Quantitative Cross-Linking/Mass Spectrometry And Modeling* 
The slow but spontaneous and ubiquitous formation of C3(H2O), the hydrolytic and conformationally rearranged product of C3, initiates antibody-independent activation of the complement system that is a key first line of antimicrobial defense. The structure of C3(H2O) has not been determined. Here we subjected C3(H2O) to quantitative cross-linking/mass spectrometry (QCLMS). This revealed details of the structural differences and similarities between C3(H2O) and C3, as well as between C3(H2O) and its pivotal proteolytic cleavage product, C3b, which shares functionally similarity with C3(H2O). Considered in combination with the crystal structures of C3 and C3b, the QCMLS data suggest that C3(H2O) generation is accompanied by the migration of the thioester-containing domain of C3 from one end of the molecule to the other. This creates a stable C3b-like platform able to bind the zymogen, factor B, or the regulator, factor H. Integration of available crystallographic and QCLMS data allowed the determination of a 3D model of the C3(H2O) domain architecture. The unique arrangement of domains thus observed in C3(H2O), which retains the anaphylatoxin domain (that is excised when C3 is enzymatically activated to C3b), can be used to rationalize observed differences between C3(H2O) and C3b in terms of complement activation and regulation.
PMCID: PMC4974347  PMID: 27250206
6.  A Novel Glutamyl (Aspartyl)-Specific Aminopeptidase A from Lactobacillus delbrueckii with Promising Properties for Application 
PLoS ONE  2016;11(3):e0152139.
Lactic acid bacteria (LAB) are auxotrophic for a number of amino acids. Thus, LAB have one of the strongest proteolytic systems to acquit their amino acid requirements. One of the intracellular exopeptidases present in LAB is the glutamyl (aspartyl) specific aminopeptidase (PepA; EC Most of the PepA enzymes characterized yet, belonged to Lactococcus lactis sp., but no PepA from a Lactobacillus sp. has been characterized so far. In this study, we cloned a putative pepA gene from Lb. delbrueckii ssp. lactis DSM 20072 and characterized it after purification. For comparison, we also cloned, purified and characterized PepA from Lc. lactis ssp. lactis DSM 20481. Due to the low homology between both enzymes (30%), differences between the biochemical characteristics were very likely. This was confirmed, for example, by the more acidic optimum pH value of 6.0 for Lb-PepA compared to pH 8.0 for Lc-PepA. In addition, although the optimum temperature is quite similar for both enzymes (Lb-PepA: 60°C; Lc-PepA: 65°C), the temperature stability after three days, 20°C below the optimum temperature, was higher for Lb-PepA (60% residual activity) than for Lc-PepA (2% residual activity). EDTA inhibited both enzymes and the strongest activation was found for CoCl2, indicating that both enzymes are metallopeptidases. In contrast to Lc-PepA, disulfide bond-reducing agents such as dithiothreitol did not inhibit Lb-PepA. Finally, Lb-PepA was not product-inhibited by L-Glu, whereas Lc-PepA showed an inhibition.
PMCID: PMC4803204  PMID: 27003449
7.  A Study into the Collision-induced Dissociation (CID) Behavior of Cross-Linked Peptides* 
Cross-linking/mass spectrometry resolves protein–protein interactions or protein folds by help of distance constraints. Cross-linkers with specific properties such as isotope-labeled or collision-induced dissociation (CID)-cleavable cross-linkers are in frequent use to simplify the identification of cross-linked peptides. Here, we analyzed the mass spectrometric behavior of 910 unique cross-linked peptides in high-resolution MS1 and MS2 from published data and validate the observation by a ninefold larger set from currently unpublished data to explore if detailed understanding of their fragmentation behavior would allow computational delivery of information that otherwise would be obtained via isotope labels or CID cleavage of cross-linkers. Isotope-labeled cross-linkers reveal cross-linked and linear fragments in fragmentation spectra. We show that fragment mass and charge alone provide this information, alleviating the need for isotope-labeling for this purpose. Isotope-labeled cross-linkers also indicate cross-linker-containing, albeit not specifically cross-linked, peptides in MS1. We observed that acquisition can be guided to better than twofold enrich cross-linked peptides with minimal losses based on peptide mass and charge alone. By help of CID-cleavable cross-linkers, individual spectra with only linear fragments can be recorded for each peptide in a cross-link. We show that cross-linked fragments of ordinary cross-linked peptides can be linearized computationally and that a simplified subspectrum can be extracted that is enriched in information on one of the two linked peptides. This allows identifying candidates for this peptide in a simplified database search as we propose in a search strategy here. We conclude that the specific behavior of cross-linked peptides in mass spectrometers can be exploited to relax the requirements on cross-linkers.
PMCID: PMC4813691  PMID: 26719564
8.  Identification of a Novel Di-D-Fructofuranose 1,2’:2,3’ Dianhydride (DFA III) Hydrolysis Enzyme from Arthrobacter aurescens SK8.001 
PLoS ONE  2015;10(11):e0142640.
Previously, a di-D-fructofuranose 1,2’:2,3’ dianhydride (DFA III)-producing strain, Arthrobacter aurescens SK8.001, was isolated from soil, and the gene cloning and characterization of the DFA III-forming enzyme was studied. In this study, a DFA III hydrolysis enzyme (DFA IIIase)-encoding gene was obtained from the same strain, and the DFA IIIase gene was cloned and expressed in Escherichia coli. The SDS-PAGE and gel filtration results indicated that the purified enzyme was a homotrimer holoenzyme of 145 kDa composed of subunits of 49 kDa. The enzyme displayed the highest catalytic activity for DFA III at pH 5.5 and 55°C, with specific activity of 232 U mg-1. Km and Vmax for DFA III were 30.7 ± 4.3 mM and 1.2 ± 0.1 mM min-1, respectively. Interestingly, DFA III-forming enzymes and DFA IIIases are highly homologous in amino acid sequence. The molecular modeling and docking of DFA IIIase were first studied, using DFA III-forming enzyme from Bacillus sp. snu-7 as a template. It was suggested that A. aurescens DFA IIIase shared a similar three-dimensional structure with the reported DFA III-forming enzyme from Bacillus sp. snu-7. Furthermore, their catalytic sites may occupy the same position on the proteins. Based on molecular docking analysis and site-directed mutagenesis, it was shown that D207 and E218 were two potential critical residues for the catalysis of A. aurescens DFA IIIase.
PMCID: PMC4640833  PMID: 26555784
9.  Serum Albumin Domain Structures in Human Blood Serum by Mass Spectrometry and Computational Biology* 
Chemical cross-linking combined with mass spectrometry has proven useful for studying protein-protein interactions and protein structure, however the low density of cross-link data has so far precluded its use in determining structures de novo. Cross-linking density has been typically limited by the chemical selectivity of the standard cross-linking reagents that are commonly used for protein cross-linking. We have implemented the use of a heterobifunctional cross-linking reagent, sulfosuccinimidyl 4,4′-azipentanoate (sulfo-SDA), combining a traditional sulfo-N-hydroxysuccinimide (sulfo-NHS) ester and a UV photoactivatable diazirine group. This diazirine yields a highly reactive and promiscuous carbene species, the net result being a greatly increased number of cross-links compared with homobifunctional, NHS-based cross-linkers. We present a novel methodology that combines the use of this high density photo-cross-linking data with conformational space search to investigate the structure of human serum albumin domains, from purified samples, and in its native environment, human blood serum. Our approach is able to determine human serum albumin domain structures with good accuracy: root-mean-square deviation to crystal structure are 2.8/5.6/2.9 Å (purified samples) and 4.5/5.9/4.8Å (serum samples) for domains A/B/C for the first selected structure; 2.5/4.9/2.9 Å (purified samples) and 3.5/5.2/3.8 Å (serum samples) for the best out of top five selected structures. Our proof-of-concept study on human serum albumin demonstrates initial potential of our approach for determining the structures of more proteins in the complex biological contexts in which they function and which they may require for correct folding. Data are available via ProteomeXchange with identifier PXD001692.
PMCID: PMC4813692  PMID: 26385339
10.  Investigation of the Germination of Barley and Wheat Grains with a Design of Experiments for the Production of Hydrolases 
Food Technology and Biotechnology  2015;53(2):127-135.
The production of hydrolases from cereals has been examined in order to investigate food-derived enzymes as an alternative source to microbial enzymes for the use in food processes. For that, the influence of temperature on the pretreatment, imbibition and germination of barley and wheat grains was determined by measuring the β-glucosidase, β-galactosidase and lipase activities using a design of experiments. The evaluation of the statistical model showed an increase of the β-glucosidase activity with low imbibition and low germination temperature for barley grains and low imbibition and high germination temperature for wheat grains. The maximum β-glucosidase activity in wheat extracts was (585±151) nkat per g of dry mass (dm), while in barley extracts it was (109±15) nkat per g of dm. The maximum β-galactosidase activities in barley and wheat extracts were (34±12) and (63±23) nkat per g of dm, respectively. The maximum lipase activities of (6.7±0.1) and (4.6±4.4) nkat per g of dm in barley and wheat extracts, respectively, were rather low compared to the glycosidase activities. The extracts were also tested for other hydrolase activities (e.g. peptidase and α-amylase activities). The insights obtained enable the basis for the potential use of cereal hydrolases in food processing, which might be attractive to consumers.
PMCID: PMC5068404  PMID: 27904341
cereal grain; glucosidase; galactosidase; design of experiments; germination; hydrolase production
11.  xiNET: Cross-link Network Maps With Residue Resolution* 
xiNET is a visualization tool for exploring cross-linking/mass spectrometry results. The interactive maps of the cross-link network that it generates are a type of node-link diagram. In these maps xiNET displays: (1) residue resolution positional information including linkage sites and linked peptides; (2) all types of cross-linking reaction product; (3) ambiguous results; and, (4) additional sequence information such as domains. xiNET runs in a browser and exports vector graphics which can be edited in common drawing packages to create publication quality figures. Availability: xiNET is open source, released under the Apache version 2 license. Results can be viewed by uploading data to or by downloading the software from and running it locally.
PMCID: PMC4390258  PMID: 25648531
12.  Quantitative cross-linking/mass spectrometry using isotope-labelled cross-linkers☆ 
Journal of Proteomics  2013;88:120-128.
Dynamic proteins and multi-protein complexes govern most biological processes. Cross-linking/mass spectrometry (CLMS) is increasingly successful in providing residue-resolution data on static proteinaceous structures. Here we investigate the technical feasibility of recording dynamic processes using isotope-labelling for quantitation. We cross-linked human serum albumin (HSA) with the readily available cross-linker BS3-d0/4 in different heavy/light ratios. We found two limitations. First, isotope labelling reduced the number of identified cross-links. This is in line with similar findings when identifying proteins. Second, standard quantitative proteomics software was not suitable for work with cross-linking. To ameliorate this we wrote a basic open source application, XiQ. Using XiQ we could establish that quantitative CLMS was technically feasible.
Biological significance
Cross-linking/mass spectrometry (CLMS) has become a powerful tool for providing residue-resolution data on static proteinaceous structures. Adding quantitation to CLMS will extend its ability of recording dynamic processes. Here we introduce a cross-linking specific quantitation strategy by using isotope labelled cross-linkers. Using a model system, we demonstrate the principle and feasibility of quantifying cross-linking data and discuss challenges one may encounter while doing so. We then provide a basic open source application, XiQ, to carry out automated quantitation of CLMS data. Our work lays the foundations of studying the molecular details of biological processes at greater ease than this could be done so far.
This article is part of a Special Issue entitled: New Horizons and Applications for Proteomics [EuPA 2012].
Graphical abstract
•Quantitative cross-linking using isotope labelling is technically feasible•Commercial cross-linker BS3-d0/4 is suitable for quantitative cross-linking•XiQ, an open source script to extract quantitative data from raw files•Cross-link reference data set to test identification and quantitation software•Isotope labelling reduced the number of identified cross-links
PMCID: PMC3714596  PMID: 23541715
Quantitation; Cross-linking; Structural biology; Protein dynamics; Mass spectrometry; Proteomics
13.  Characterization of the Recombinant Exopeptidases PepX and PepN from Lactobacillus helveticus ATCC 12046 Important for Food Protein Hydrolysis 
PLoS ONE  2013;8(7):e70055.
The proline-specific X-prolyl dipeptidyl aminopeptidase (PepX; EC and the general aminopeptidase N (PepN; EC from Lactobacillus helveticus ATCC 12046 were produced recombinantly in E. coli BL21(DE3) via bioreactor cultivation. The maximum enzymatic activity obtained for PepX was 800 µkatH-Ala-Pro-pNA L−1, which is approx. 195-fold higher than values published previously. To the best of our knowledge, PepN was expressed in E. coli at high levels for the first time. The PepN activity reached 1,000 µkatH-Ala-pNA L−1. After an automated chromatographic purification, both peptidases were biochemically and kinetically characterized in detail. Substrate inhibition of PepN and product inhibition of both PepX and PepN were discovered for the first time. An apo-enzyme of the Zn2+-dependent PepN was generated, which could be reactivated by several metal ions in the order of Co2+>Zn2+>Mn2+>Ca2+>Mg2+. PepX and PepN exhibited a clear synergistic effect in casein hydrolysis studies. Here, the relative degree of hydrolysis (rDH) was increased by approx. 132%. Due to the remarkable temperature stability at 50°C and the complementary substrate specificities of both peptidases, a future application in food protein hydrolysis might be possible.
PMCID: PMC3716637  PMID: 23894590
14.  Biotransformation of caffeoyl quinic acids from green coffee extracts by Lactobacillus johnsonii NCC 533 
AMB Express  2013;3:28.
The potential of Lactobacillus johnsonii NCC 533 to metabolize chlorogenic acids from green coffee extract was investigated. Two enzymes, an esterase and a hydroxycinnamate decarboxylase (HCD), were involved in this biotransformation. The complete hydrolysis of 5-caffeoylquinic acid (5-CQA) into caffeic acid (CA) by L. johnsonii esterase occurred during the first 16 h of reaction time. No dihydrocaffeic acid was identified in the reaction mixture. The decarboxylation of CA into 4-vinylcatechol (4-VC) started only when the maximum concentration of CA was reached (10 μmol/ml). CA was completely transformed into 4-VC after 48 h of incubation. No 4-vinylphenol or other derivatives could be identified in the reaction media. In this study we demonstrate the capability of L. johnsonii to transform chlorogenic acids from green coffee extract into 4-VC in two steps one pot reaction. Thus, the enzymatic potential of certain lactobacilli might be explored to generate flavor compounds from plant polyphenols.
PMCID: PMC3679781  PMID: 23692950
Chlorogenic acid; 4-vinylcatechol; Esterase; Decarboxylase; L. johnsonii
15.  Production, active staining and gas chromatography assay analysis of recombinant aminopeptidase P from Lactococcus lactis ssp. lactis DSM 20481 
AMB Express  2012;2:39.
The aminopeptidase P (PepP, EC gene from Lactococcus lactis ssp. lactis DSM 20481 was cloned, sequenced and expressed recombinantly in E. coli BL21 (DE3) for the first time. PepP is involved in the hydrolysis of proline-rich proteins and, thus, is important for the debittering of protein hydrolysates. For accurate determination of PepP activity, a novel gas chromatographic assay was established. The release of L-leucine during the hydrolysis of L-leucine-L-proline-L-proline (LPP) was examined for determination of PepP activity. Sufficient recombinant PepP production was achieved via bioreactor cultivation at 16 °C, resulting in PepP activity of 90 μkatLPP Lculture-1. After automated chromatographic purification by His-tag affinity chromatography followed by desalting, PepP activity of 73.8 μkatLPP Lculture-1 was achieved. This was approximately 700-fold higher compared to the purified native PepP produced by Lactococcus lactis ssp. lactis NCDO 763 as described in literature. The molecular weight of PepP was estimated to be ~ 40 kDa via native-PAGE together with a newly developed activity staining method and by SDS-PAGE. Furthermore, the kinetic parameters Km and Vmax were determined for PepP using three different tripeptide substrates. The purified enzyme showed a pH optimum between 7.0 and 7.5, was most active between 50°C and 60°C and exhibited reasonable stability at 0°C, 20°C and 37°C over 15 days. PepP activity could be increased 6-fold using 8.92 mM MnCl2 and was inhibited by 1,10-phenanthroline and EDTA.
PMCID: PMC3418211  PMID: 22853547
Lactococcus lactis; Aminopeptidase P; PepP; Gas chromatographic assay; Activity staining; LPP
16.  The use of concentrated heat after insect bites/stings as an alternative to reduce swelling, pain, and pruritus: an open cohort-study at German beaches and bathing-lakes 
Swelling, pain, and pruritus are the most relevant symptoms after insect bites/stings. Glucocorticoids and antihistamines are well established in insect sting treatment. Bite Away® is a CE-certified medical device of class 2A (noninvasive device intended for administration to the body, which exchanges energy with the patient in a therapeutic manner) to reduce swelling, pruritus, and pain after insect bites/stings via non-invasive administration of concentrated heat to the skin. We therefore performed a prospective, non-interventional, single-arm cohort study with 146 volunteers using the visual analog scale (VAS) for insect bites/stings to study the reduction of swelling, pruritus, and pain. Demographic data, time from insect sting to treatment, number and duration of administrations of concentrated heat, relevant symptoms, and the development of a VAS score of swelling, pruritus, and pain on baseline, after 2, 5, and 10 minutes after administration, were registered.
In total 146 subjects with a mean age of 29.4 ± 20.7 years (range 2–81) were enrolled in the study. Ninety-three (63.7%) of the subjects were stung by wasps, 33 (22.6%) of the subjects were bitten by mosquitoes, and eight suffered bee stings (5.3%). VAS score swelling decreased with statistical significance after the use of Bite Away® from 4 before treatment to 2 and 1 after 2–5 and 10 minutes, respectively. VAS pain score was 6 before treatment, 2 after 2 minutes, 1 after 5 minutes, and 0 after 10 minutes (median). VAS pruritus score was only available for 52 (35.2%) of the patients. The score decreased from 5 before treatment, to 2 after 2 minutes, and 0 after 5 and 10 minutes.
Locally administrated concentrated heat leads to fast amelioration of symptoms. Usually an absence of symptoms is noticeable 10 minutes after administration. Pain reduction is the dominant effect. Compared with alternatives of pruritus and pain treatment after insect bites/stings, Bite Away® seems to be the fastest treatment option available.
PMCID: PMC3257884  PMID: 22253544
insect bites/stings; swelling; pain; pruritus; concentrated heat; VAS
17.  The Protein Composition of Mitotic Chromosomes Determined Using Multiclassifier Combinatorial Proteomics 
Cell  2010;142(5):810-821.
Despite many decades of study, mitotic chromosome structure and composition remain poorly characterized. Here, we have integrated quantitative proteomics with bioinformatic analysis to generate a series of independent classifiers that describe the ∼4,000 proteins identified in isolated mitotic chromosomes. Integrating these classifiers by machine learning uncovers functional relationships between protein complexes in the context of intact chromosomes and reveals which of the ∼560 uncharacterized proteins identified here merits further study. Indeed, of 34 GFP-tagged predicted chromosomal proteins, 30 were chromosomal, including 13 with centromere-association. Of 16 GFP-tagged predicted nonchromosomal proteins, 14 were confirmed to be nonchromosomal. An unbiased analysis of the whole chromosome proteome from genetic knockouts of kinetochore protein Ska3/Rama1 revealed that the APC/C and RanBP2/RanGAP1 complexes depend on the Ska complex for stable association with chromosomes. Our integrated analysis predicts that up to 97 new centromere-associated proteins remain to be discovered in our data set.
Graphical Summary
► Method to define the protein composition of a complex nonpurifiable organelle ► Combines genetics and proteomics to study complexes in whole chromosomes ► Use of machine learning to uncover functional relationships between proteins ► Comprehensive list of >4000 mitotic chromosome-associated proteins
PMCID: PMC2982257  PMID: 20813266
18.  Architecture of the RNA polymerase II–TFIIF complex revealed by cross-linking and mass spectrometry 
The EMBO Journal  2010;29(4):717-726.
Higher-order multi-protein complexes such as RNA polymerase II (Pol II) complexes with transcription initiation factors are often not amenable to X-ray structure determination. Here, we show that protein cross-linking coupled to mass spectrometry (MS) has now sufficiently advanced as a tool to extend the Pol II structure to a 15-subunit, 670 kDa complex of Pol II with the initiation factor TFIIF at peptide resolution. The N-terminal regions of TFIIF subunits Tfg1 and Tfg2 form a dimerization domain that binds the Pol II lobe on the Rpb2 side of the active centre cleft near downstream DNA. The C-terminal winged helix (WH) domains of Tfg1 and Tfg2 are mobile, but the Tfg2 WH domain can reside at the Pol II protrusion near the predicted path of upstream DNA in the initiation complex. The linkers between the dimerization domain and the WH domains in Tfg1 and Tfg2 are located to the jaws and protrusion, respectively. The results suggest how TFIIF suppresses non-specific DNA binding and how it helps to recruit promoter DNA and to set the transcription start site. This work establishes cross-linking/MS as an integrated structure analysis tool for large multi-protein complexes.
PMCID: PMC2810376  PMID: 20094031
higher-order protein complex; integrated structure analysis; mass spectrometry; multi-dimensional structure and dynamics of biological macromolecules; transcription and its regulation
19.  Molecular pharmacological profile of the nonredox-type 5-lipoxygenase inhibitor CJ-13,610 
British Journal of Pharmacology  2004;142(5):861-868.
5-Lipoxygenase (5-LO) is a crucial enzyme in the synthesis of the bioactive leukotrienes (LTs) from arachidonic acid (AA), and inhibitors of 5-LO are thought to prevent the untowarded pathophysiological effects of LTs.In this study, we present the molecular pharmacological profile of the novel nonredox-type 5-LO inhibitor CJ-13,610 that was evaluated in various in vitro assays.In intact human polymorphonuclear leukocytes (PMNL), challenged with the Ca2+-ionophore A23187, CJ-13,610 potently suppressed 5-LO product formation with an IC50=0.07 μM. Supplementation of exogenous AA impaired the efficacy of CJ-13,610, implying a competitive mode of action.In analogy to ZM230487 and L-739.010, two closely related nonredox-type 5-LO inhibitors, CJ-13,610 up to 30 μM failed to inhibit 5-LO in cell-free assay systems under nonreducing conditions, but inclusion of peroxidase activity restored the efficacy of CJ-13,610 (IC50=0.3 μM).In contrast to ZM230487 and L-739.010, the potency of CJ-13,610 does not depend on the cell stimulus or the activation pathway of 5-LO. Thus, 5-LO product formation in PMNL induced by phosphorylation events was equally suppressed by CJ-13,610 as compared to Ca2+-mediated 5-LO activation. In transfected HeLa cells, CJ-13,610 only slightly discriminated between phosphorylatable wild-type 5-LO and a 5-LO mutant that lacks phosphorylation sites.In summary, CJ-13,610 may possess considerable potential as a potent orally active nonredox-type 5-LO inhibitor that lacks certain disadvantages of former representatives of this class of 5-LO inhibitors.
PMCID: PMC1575070  PMID: 15197110
5-Lipoxygenase; leukotriene; CJ-13,610; polymorphonuclear leukocyte; inflammation
20.  Coupling of boswellic acid-induced Ca2+ mobilisation and MAPK activation to lipid metabolism and peroxide formation in human leucocytes 
British Journal of Pharmacology  2003;141(2):223-232.
We have previously shown that 11-keto boswellic acids (11-keto-BAs), the active principles of Boswellia serrata gum resins, activate p38 MAPK and p42/44MAPK and stimulate Ca2+ mobilisation in human polymorphonuclear leucocytes (PMNL).In this study, we attempted to connect the activation of MAPK and mobilisation of Ca2+ to functional responses of PMNL, including the formation of reactive oxygen species (ROS), release of arachidonic acid (AA), and leukotriene (LT) biosynthesis.We found that, in PMNL, 11-keto-BAs stimulate the formation of ROS and cause release of AA as well as its transformation to LTs via 5-lipoxygenase.Based on inhibitor studies, 11-keto-BA-induced ROS formation is Ca2+-dependent and is mediated by NADPH oxidase involving PI 3-K and p42/44MAPK signalling pathways. Also, the release of AA depends on Ca2+ and p42/44MAPK, whereas the pathways stimulating 5-LO are not readily apparent.Pertussis toxin, which inactivates Gi/0 protein subunits, prevents MAPK activation and Ca2+ mobilisation induced by 11-keto-BAs, implying the involvement of a Gi/0 protein in BA signalling.Expanding studies on differentiated haematopoietic cell lines (HL60, Mono Mac 6, BL41-E-95-A) demonstrate that the ability of BAs to activate MAPK and to mobilise Ca2+ may depend on the cell type or the differentiation status.In summary, we conclude that BAs act via Gi/0 protein(s) stimulating signalling pathways that control functional leucocyte responses, in a similar way as chemoattractants, that is, N-formyl-methionyl-leucyl-phenylalanine or platelet-activating factor.
PMCID: PMC1574191  PMID: 14691050
Boswellic acids; leucocytes; MAPK; Ca2+; reactive oxygen species; lipoxygenase; arachidonic acid
21.  Production of a New d-Amino Acid Oxidase from the Fungus Fusarium oxysporum 
The fungus Fusarium oxysporum produced a d-amino acid oxidase (EC in a medium containing glucose as the carbon and energy source and ammonium sulfate as the nitrogen source. The specific d-amino acid oxidase activity was increased up to 12.5-fold with various d-amino acids or their corresponding derivatives as inducers. The best inducers were d-alanine (2.7 μkat/g of dry biomass) and d-3-aminobutyric acid (2.6 μkat/g of dry biomass). The addition of zinc ions was necessary to permit the induction of peroxisomal d-amino acid oxidase. Bioreactor cultivations were performed on a 50-liter scale, yielding a volumetric d-amino acid oxidase activity of 17 μkat liter−1 with d-alanine as an inducer. Under oxygen limitation, the volumetric activity was increased threefold to 54 μkat liter−1 (3,240 U liter−1).
PMCID: PMC91565  PMID: 10427080

Results 1-21 (21)