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The molecular assembly of cells depends not only on the balance between anabolism and catabolism but to a large degree on the building blocks available in the environment. For cultured mammalian cells, this is largely determined by the composition of the applied growth medium. Here, we study the impact of lipids in the medium on mitochondrial membrane architecture and function by combining LC-MS/MS lipidomics and functional tests with lipid supplementation experiments in an otherwise serum-free and lipid-free cell culture model. We demonstrate that the composition of mitochondrial cardiolipins strongly depends on the lipid environment in cultured cells and favors the incorporation of essential linoleic acid over other fatty acids. Simultaneously, the mitochondrial respiratory complex I activity was altered, whereas the matrix-localized enzyme citrate synthase was unaffected. This raises the question on a link between membrane composition and respiratory control. In summary, we found a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium. This underlines the importance of considering these factors when using and establishing cell culture models in biomedical research. In summary, we found a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium., Graphical abstract

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the past decade. Interest in the bio-nano interface arises from the fact that the biomolecular corona confers a biological identity to NMs and thus causes the body to identify them as “self”. For example, previous studies have demonstrated that the proteins in the corona are capable of interacting with membrane receptors to influence cellular uptake and established that the corona is responsible for cellular trafficking of NMs and their eventual toxicity. To date, most research has focused upon the protein corona and overlooked the possible impacts of the metabolites included in the corona or synergistic effects between components in the complete biomolecular corona. As such, this work demonstrates methodologies to characterize both the protein and metabolite components of the biomolecular corona using bottom-up proteomics and metabolomics approaches in parallel. This includes an on-particle digest of the protein corona with a surfactant used to increase protein recovery, and a passive characterization of the metabolite corona by analyzing metabolite matrices before and after NM exposures. This work introduces capillary electrophoresis – mass spectrometry (CESI-MS) as a new technique for NM corona characterization. The protocols outlined here demonstrate how CESI-MS can be used for the reliable characterization of both the protein and metabolite corona acquired by NMs. The move to CESI-MS greatly decreases the volume of sample required (compared to traditional liquid chromatography – mass spectrometry (LC-MS) approaches) with multiple injections possible from as little as 5 µL of sample, making it ideal for volume limited samples. Furthermore, the environmental consequences of analysis are reduced with respect to LC-MS due to the low flow rates (

Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (mu(eff)) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The mu(eff) was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the mu(eff) for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics.

How cells adjust nutrient transport across their membranes is incompletely understood. Previously, we have shown thatS. cerevisiaebroadly re-configures the nutrient transporters at the plasma membrane in response to amino acid availability, through endocytosis of sugar- and amino acid transporters (AATs) (Müller et al., 2015). A genome-wide screen now revealed that the selective endocytosis of four AATs during starvation required the α-arrestin family protein Art2/Ecm21, an adaptor for the ubiquitin ligase Rsp5, and its induction through the general amino acid control pathway. Art2 uses a basic patch to recognize C-terminal acidic sorting motifs in AATs and thereby instructs Rsp5 to ubiquitinate proximal lysine residues. When amino acids are in excess, Rsp5 instead uses TORC1-activated Art1 to detect N-terminal acidic sorting motifs within the same AATs, which initiates exclusive substrate-induced endocytosis. Thus, amino acid excess or starvation activate complementary α-arrestin-Rsp5-complexes to control selective endocytosis and adapt nutrient acquisition.

How cells adjust transport across their membranes is incompletely understood. Previously, we have shown thatS.cerevisiaebroadly re-configures the nutrient transporters at the plasma membrane in response to amino acid availability, through selective endocytosis of sugar- and amino acid transporters (AATs) (Müller et al., 2015). A genome-wide screen now revealed that Art2/Ecm21, a member of the α-arrestin family of Rsp5 ubiquitin ligase adaptors, is required for the simultaneous endocytosis of four AATs and induced during starvation by the general amino acid control pathway. Art2 uses a basic patch to recognize C-terminal acidic sorting motifs in these AATs and instructs Rsp5 to ubiquitinate proximal lysine residues. In response to amino acid excess, Rsp5 instead uses TORC1-activated Art1 to detect N-terminal acidic sorting motifs within the same AATs, which initiates exclusive substrate-induced endocytosis of individual AATs. Thus, amino acid availability activates complementary α-arrestin-Rsp5-complexes to control selective endocytosis for nutrient acquisition.

Background The high molecular complexity of variably O-glycosylated and degraded pro B-type natriuretic peptide (proBNP) derived molecular forms challenges current immunoassays. Antibodies used show pronounced differences in cross-reactivities with these circulating fragments, which still need to be better characterized on a molecular level. To pave the way for advanced quantitative assays in the future, it is critical to fully understand these circulating forms. Methods Plasma samples were collected from 8 heart failure (HF) patients and 2 healthy controls. NT-proBNP and proBNP were purified by immunoprecipitation and analyzed by nano-flow liquid chromatography coupled to high-resolution mass spectrometry. Fragments formed during proteolysis in solution digestion were distinguished from naturally occurring peptides by using an 18O stable isotope labeling strategy. Results We detected 16 previously unknown circulating fragments of proBNP peptides (9 of which are located in the N-terminal and 7 in the C-terminal region), revealing a more advanced state of degradation than previously known. Two of these fragments are indicative of either unidentified processing modes or a far-reaching C-terminal degradation (or a combination thereof) of the precursor proBNP. Conclusions Our results further restrict ideal target epitopes for immunoassay antibodies and expand the current thinking of diversity, degradation, and processing of proBNP, as well as the distribution of circulating forms.

The precise interplay between the mRNA codon and the tRNA anticodon is crucial for ensuring efficient and accurate translation by the ribosome. The insertion of RNA nucleobase derivatives in the mRNA allowed us to modulate the stability of the codon-anticodon interaction in the decoding site of bacterial and eukaryotic ribosomes, allowing an in-depth analysis of codon recognition. We found the hydrogen bond between the N1 of purines and the N3 of pyrimidines to be sufficient for decoding of the first two codon nucleotides, whereas adequate stacking between the RNA bases is critical at the wobble position. Inosine, found in eukaryotic mRNAs, is an important example of destabilization of the codon-anticodon interaction. Whereas single inosines are efficiently translated, multiple inosines, e.g., in the serotonin receptor 5-HT2C mRNA, inhibit translation. Thus, our results indicate that despite the robustness of the decoding process, its tolerance toward the weakening of codon-anticodon interactions is limited., The recognition of the mRNA codon by the tRNA anticodon is crucial for protein synthesis. Here the authors introduce non-standard nucleotides in bacterial and eukaryotic mRNA to reveal the minimal hydrogen bond requirement of codon-anticodon interaction for efficient and accurate translation.

Significance Cardiolipins are a unique class of phospholipids in mitochondrial membranes that are crucial for cellular bioenergetics as they stabilize respiratory chain complexes. In contrast to most other phospholipids, cardiolipins are substituted with four, rather than only two fatty acyl side chains. Consequently, this opens up a vast number of different theoretically possible molecular lipid species. Experimentally assessing the molecular diversity of cardiolipin species is analytically challenging. In this study we successfully combine tandem mass spectrometry with a mathematical structural modeling approach, to achieve the comprehensive characterization of complex biological cardiolipin compositions., Current strategies used to quantitatively describe the biological diversity of lipids by mass spectrometry are often limited in assessing the exact structural variability of individual molecular species in detail. A major challenge is represented by the extensive isobaric overlap present among lipids, hampering their accurate identification. This is especially true for cardiolipins, a mitochondria-specific class of phospholipids, which are functionally involved in many cellular functions, including energy metabolism, cristae structure, and apoptosis. Substituted with four fatty acyl side chains, cardiolipins offer a particularly high potential to achieve complex mixtures of molecular species. Here, we demonstrate how systematically generated high-performance liquid chromatography-mass spectral data can be utilized in a mathematical structural modeling approach, to comprehensively analyze and characterize the molecular diversity of mitochondrial cardiolipin compositions in cell culture and disease models, cardiolipin modulation experiments, and a broad variety of frequently studied model organisms.

Capillary electrophoresis coupled to mass spectrometry is a very efficient analytical method for the analysis of post-translational modifications because of its high separation efficiency and high detection sensitivity. Here we applied CE-MS using three differently coated separation capillaries for in-depth analysis of a set of 70 synthetic post-translationally modified peptides (including phosphorylation, acetylation, methylation, and nitration). We evaluated the results in terms of peptide detection and separation characteristics and found that the use of a neutrally coated capillary resulted in highest overall signal intensity of singly modified peptides. In contrast, the use of a bare-fused silica capillary was superior in the identification of multi-phosphorylated peptides (12 out of 15 were identified). Fast separations of approximately 12 min could be achieved using a positively coated capillary, however, at the cost of separation efficiency. A comparison to nanoLC-MS revealed that multi-phosphorylated peptides interact with the RP material very poorly so that these peptides were either washed out or elute as very broad peaks from the nano column which results in a reduced peptide identification rate (7 out of 15). Moreover, the methods applied were found to be very well suited for the analysis of the acetylated, nitrated and methylated peptides. All 36 synthetic peptides, which exhibit one of those modifications, could be identified regardless of the method applied. As a final step in this study and as a proof of principle, the phosphoproteome enriched from PC-12 pheochromocytoma cells was analyzed by CE-MS resulting in 5686 identified and 4088 quantified phosphopeptides. We compared the characterized analytes to those identified by a nanoLC-MS proteomics study and found that less than one third of the phosphopeptides were identical, which demonstrates the benefit by combining different approaches quite impressively.

Reversed-phase high-performance liquid chromatography (RP-HPLC) in combination with mass spectrometry (MS) is typically employed for mapping modifications in proteins and peptides. Here we applied a low-flow capillary electrophoresis (CE) -electrospray ionization interface coupled to Orbitrap mass spectrometers to analyze challenging modifications such as asparagine deamidation, aspartate isomerization, arginine citrullination, and phosphopeptide isomers. We achieved excellent resolution of asparagine (Asn), aspartic acid (Asp) and isoaspartic acid (iso-Asp) containing peptides using a synthetic peptide mixture. The migration order in CE enabled a clear assignment of in vitro deamidation/isomerization sites in a protein standard mixture of intermediate complexity (48 proteins) as well as the determination of the in vivo deamidation rate of histone H1.0 directly in a crude nuclear protein fraction. Besides these well-known modifications citrullination, a post-translational modification which changes the positively charged guanidinium group of arginine to the uncharged ureido group of citrulline, was investigated. Applying CE-MS for fast and sensitive analyses of various post-translational modifications of intact and enzymatically digested histone H4, we were able to detect a variety of citrullinated proteoforms. MS/MS analysis with electron transfer dissociation (ETD) fragmentation identified the presence of deiminated Arg at position 3 and 17 of histone H4. Moreover, based on CE-MS, isobaric mono-phosphorylated peptides obtained in the course of a kinase activity study were separated and individual positional isomers quantified.

The analysis, identification and quantification of histones and their post-translational modifications plays a central role in chromatin research and in studying epigenetic regulations during physiological processes. In the last decade analytical strategies based on mass spectrometry have been greatly improved for providing a global view of single modification abundances or to determine combinatorial patterns of modifications. Presented here is a newly developed strategy for histone protein analysis and a number of applications are illustrated with an emphasis on PTM characterization. Capillary electrophoresis is coupled to mass spectrometry (CE-MS) and has proven to be a very promising concept as it enables to study intact histones (top-down proteomics) as well as the analysis of enzymatically digested proteins (bottom-up proteomics). This technology combines highly efficient low-flow CE separations with ionization in a single device and offers an orthogonal separation principle to conventional LC-MS analysis, thus expanding the existing analytical repertoire in a perfect manner.

latworms can very rapidly attach to and detach from many substrates. In the presented work, we analysed the adhesive system of the marine proseriate flatworm Minona ileanae. We used light-, scanning- and transmission electron microscopy to analyse the morphology of the adhesive organs, which are located at the ventral side of the tail-plate. We performed transcriptome sequencing and differential RNA-seq for the identification of tail-specific transcripts. Using in situ hybridization expression screening, we identified nine transcripts that were expressed in the cells of the adhesive organs. Knock-down of five of these transcripts by RNA interference led to a reduction of the animal's attachment capacity. Adhesive proteins in footprints were confirmed using mass spectrometry and antibody staining. Additionally, lectin labelling of footprints revealed the presence of several sugar moieties. Furthermore, we determined a genome size of about 560 Mb for M. ileanae. We demonstrated the potential of Oxford Nanopore sequencing of genomic DNA as a cost-effective tool for identifying the number of repeats within an adhesive protein and for combining transcripts that were fragments of larger genes. A better understanding of the molecules involved in flatworm bioadhesion can pave the way towards developing innovative glues with reversible adhesive properties. This article is part of the theme issue ‘Transdisciplinary approaches to the study of adhesion and adhesives in biological systems’., Philosophical Transactions of the Royal Society B: Biological Sciences, 374 (1784), ISSN:0962-8436, ISSN:1471-2970, ISSN:0080-4622

Activation of G-protein coupled receptors elevates cAMP levels promoting dissociation of protein kinase A (PKA) holoenzymes and release of catalytic subunits (PKAc). This results in PKAc-mediated phosphorylation of compartmentalized substrates that control central aspects of cell physiology. The mechanism of PKAc activation and signaling have been largely characterized. However, the modes of PKAc inactivation by regulated proteolysis were unknown. Here, we identify a regulatory mechanism that precisely tunes PKAc stability and downstream signaling. Following agonist stimulation, the recruitment of the chaperone-bound E3 ligase CHIP promotes ubiquitylation and proteolysis of PKAc, thus attenuating cAMP signaling. Genetic inactivation of CHIP or pharmacological inhibition of HSP70 enhances PKAc signaling and sustains hippocampal long-term potentiation. Interestingly, primary fibroblasts from autosomal recessive spinocerebellar ataxia 16 (SCAR16) patients carrying germline inactivating mutations of CHIP show a dramatic dysregulation of PKA signaling. This suggests the existence of a negative feedback mechanism for restricting hormonally controlled PKA activities., How intracellular cAMP activate PKA is well-characterized, but PKA inactivation remains poorly understood. Here, Rinaldi et al. show that CHIP/HSP70 ubiquitinates the catalytic subunit of PKA, with implications for the human disease spinocerebellar ataxia 16, as patients often have CHIP mutations.

Increased understanding of the role of the nanomaterial protein corona in driving nanomaterial uptake into, and impacts on, cells and organisms, and the consequent need for characterization of the corona, has led to a flourishing of methods for isolation and analysis of the constituent proteins over the past decade. However, despite over 700 corona studies to date, very little is understood in terms of which methods provide the most precise and comprehensive characterization of the corona. With the increasing importance of the modeling of corona formation and its correlation with biological impacts, it is timely to properly characterize and validate the isolation approaches used to determine the protein corona. The current work introduces Capillary Electrophoresis with Electro Spray Ionization Mass Spectrometry (CESI-MS) as a novel method for protein corona characterizations and develops an on-particle tryptic digestion method, comparing peptide solubilization solutions and characterizing the recovery of proteins from the nanomaterial surface. The CESI-MS was compared to the gold standard nano-LC-MS for corona analysis and maintained a high degree of reproducibility, while increasing throughput by &gt, 3-fold. The on-particle digestion is compared to an in-solution digestion and an in-gel digestion of the protein corona. Interestingly, a range of different protein classes were found to be recovered to greater or lesser extents among the different methods. Apolipoproteins were detected at lower concentrations when a surfactant was used to solubilize peptides, whereas immunoglobulins in general have a high affinity for nanomaterials, and thus show lower recovery using on-particle digestion. The optimized on-particle digestion was validated using 6 nanomaterials and proved capable of recovering in excess of 97% of the protein corona. These are important factors to consider when designing corona studies and modeling corona formation and impacts, highlighting the significance of a comprehensive validation of nanomaterial corona analysis methods.

Significance Synthetic adhesives are widely used in our daily lives, in medicine and industry. These man-made glues contain toxic or carcinogenic components. In contrast, biological adhesives produced by animals and plants are nontoxic and tissue-compatible, and are able to function under wet conditions. However, little is known about the mechanisms underlying biological adhesives. We characterized adhesion and release in our model system Macrostomum lignano. We used a state-of-the-art toolbox to identify the involved adhesive and release molecules. We aim for understanding the fundamental mechanisms that mediate adhesion and release in flatworms, with the future goal of generating a flatworm-derived biomimetic glue that can be applied in biomedicine and industry., The flatworm Macrostomum lignano features a duo-gland adhesive system that allows it to repeatedly attach to and release from substrates in seawater within a minute. However, little is known about the molecules involved in this temporary adhesion. In this study, we show that the attachment of M. lignano relies on the secretion of two large adhesive proteins, M. lignano adhesion protein 1 (Mlig-ap1) and Mlig-ap2. We revealed that both proteins are expressed in the adhesive gland cells and that their distribution within the adhesive footprints was spatially restricted. RNA interference knockdown experiments demonstrated the essential function of these two proteins in flatworm adhesion. Negatively charged modified sugars in the surrounding water inhibited flatworm attachment, while positively charged molecules impeded detachment. In addition, we found that M. lignano could not adhere to strongly hydrated surfaces. We propose an attachment–release model where Mlig-ap2 attaches to the substrate and Mlig-ap1 exhibits a cohesive function. A small negatively charged molecule is secreted that interferes with Mlig-ap1, inducing detachment. These findings are of relevance for fundamental adhesion science and efforts to mitigate biofouling. Further, this model of flatworm temporary adhesion may serve as the starting point for the development of synthetic reversible adhesion systems for medicinal and industrial applications.

Significance Translation termination is a crucial process during protein synthesis. Class I release factors (RFs) are in charge of recognizing stop codons and consequently hydrolyzing the peptidyl-tRNA at the ribosomal P site. High-resolution crystal- and cryo-EM structures of RFs bound to the ribosome revealed a network of potential interactions that is formed between the mRNA and RFs; however, it remained enigmatic which interactions are critical for accurate stop codon recognition and peptide release. By using chemically modified stop codon nucleotides, the importance and the contribution of single hydrogen bonds to stop codon recognition was investigated. This approach revealed a detailed picture of chemical groups defining a stop codon and contributing to the discrimination against sense codons during prokaryotic and eukaryotic translation termination., Termination of protein synthesis is triggered by the recognition of a stop codon at the ribosomal A site and is mediated by class I release factors (RFs). Whereas in bacteria, RF1 and RF2 promote termination at UAA/UAG and UAA/UGA stop codons, respectively, eukaryotes only depend on one RF (eRF1) to initiate peptide release at all three stop codons. Based on several structural as well as biochemical studies, interactions between mRNA, tRNA, and rRNA have been proposed to be required for stop codon recognition. In this study, the influence of these interactions was investigated by using chemically modified stop codons. Single functional groups within stop codon nucleotides were substituted to weaken or completely eliminate specific interactions between the respective mRNA and RFs. Our findings provide detailed insight into the recognition mode of bacterial and eukaryotic RFs, thereby revealing the chemical groups of nucleotides that define the identity of stop codons and provide the means to discriminate against noncognate stop codons or UGG sense codons.

Rapid inactivation of antimicrobial test agents after exact incubation times with microorganisms is required in time-kill assays. Sodium thiosulphate and a combination of methionine and histidine were compared for neutralisation of active halogen compounds. Test oxidants were mixed with surplus sodium thiosulphate (3%-6%) or histidine/methionine (1% each) in phosphate-buffered saline and incubated for different times, followed by addition of Staphylococcus aureus, Escherichia coli, or Pseudomonas aeruginosa at 1000CFU/ml. After further incubation, quantitative cultures were performed. Thiosulphate did not sufficiently inactivate chlorine and bromine compounds, indicated by a 10-fold (S. aureus) up to >100-fold (E. coli, P. aeruginosa) reduction of CFU. This was particularly true for high concentrations of the oxidants of about 50mM, for highly reactive agents (HOCl and bromamine T) more than for chloramine T and N-chlorotaurine, and for short pre-incubation times before addition of the bacteria. By contrast, histidine/methionine proved to be suitable for chloramines and bromamine T and for low concentrations of HOCl (0.07%). HOCl at 0.7% could neither be inactivated completely by thiosulphate nor by histidine/methionine. In contrast to chlorine and bromine compounds, iodine was neutralized by thiosulphate, but not by histidine/methionine. Histidine/methionine is superior to inactivate chlorine and bromine and should replace sodium thiosulphate at least in killing tests with high concentrations of these disinfectants. Inclusion of a short reaction time (maximum one minute) of test oxidant and neutralising substance before addition of bacteria is decisive in inactivation tests to obtain reliable results.

Metal detoxification is crucial for animals to cope with environmental exposure. In snails, a pivotal role in protection against cadmium (Cd) is attributed to metallothioneins (MTs). Some gastropod species express, in a lineage-specific manner, Cd-selective MTs devoted exclusively to the binding and detoxification of this single metal, whereas other species of snails possess non-selective MTs, but still show a high tolerance against Cd. An explanation for this may be that invertebrates and in particular snails may also synthetize phytochelatins (PCs), originally known to be produced by plants, to provide protection against metal or metalloid toxicity. Here we demonstrate that despite the fact that similar mechanisms for Cd inactivation exist in snail species through binding of the metal to MTs, the actual detoxification pathways for this metal may follow different traits in a species-specific manner. In particular, this depends on the detoxification capacity of MTs due to their Cd-selective or non-specific binding features. In the terrestrial slug Arion vulgaris, for example, Cd is solely detoxified by a Cd-selective MT isoform (AvMT1). In contrast, the freshwater snail Biomphalaria glabrata activates an additional pathway for metal inactivation by synthesizing phytochelatins, which compensate for the insufficient capacity of its non-selective MT system to detoxify Cd. We hypothesize that in other snails and invertebrate species, too, an alternative inactivation of the metal by PCs may occur, if their MT system is not Cd-selective enough, or its Cd loading capacity is exhausted.

The transcription factor FOXO3 is associated with poor outcome in high-stage neuroblastoma (NB), as it facilitates chemoprotection and tumor angiogenesis. In other tumor entities, FOXO3 stimulates metastasis formation, one of the biggest challenges in the treatment of aggressive NB. However, the impact of FOXO3 on the metastatic potential of neuronal tumor cells remains largely unknown. In the present study, we uncover the small leucine-rich proteoglycan family member lumican (LUM) as a FOXO3-regulated gene that stimulates cellular migration in NB. By a drug-library screen we identified the small molecular weight compound repaglinide (RPG) as a putative FOXO3 inhibitor. Here, we verify that RPG binds to the FOXO3-DNA-binding-domain (DBD) and thereby silences the transcriptional activity of FOXO3. Consistent with the concept that the FOXO3/LUM axis enhances the migratory capacity of aggressive NB cells, we demonstrate that stable knockdown of LUM abrogates the FOXO3-mediated increase in cellular migration. Importantly, FOXO3 inhibition by RPG represses the binding of FOXO3 to the LUM promoter, inhibits FOXO3-mediated LUM RNA and protein expression, and efficiently abrogates FOXO3-triggered cellular &ldquo, wound healing&rdquo, as well as spheroid-based 3D-migration. Thus, silencing the FOXO3/LUM axis by the FDA-approved compound RPG represents a promising strategy for novel therapeutic interventions in NB and other FOXO3-dependent tumors.

We present the first comprehensive capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS) analysis of post-translational modifications derived from H1 and core histones. Using a capillary electrophoresis system equipped with a sheathless high-sensitivity porous sprayer and nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-ESI-MS) as two complementary techniques, we characterized H1 histones isolated from rat testis. Without any pre-separation of the perchloric acid extraction, a total of 70 different modified peptides, including 50 phosphopeptides, were identified in the rat linker histones H1.0, H1a-H1e, and H1t. Out of the 70 modified H1 histone peptides, 27 peptides could be identified with CESI-MS only, and 11 solely with LC-ESI-MS. Immobilized metal-affinity chromatography enrichment prior to MS analysis yielded a total of 55 phosphopeptides; 22 of these peptides could be identified only by CESI-MS, and 19 only by LC-ESI-MS, showing the complementarity of the two techniques. We mapped 42 H1 modification sites, including 31 phosphorylation sites, of which 8 were novel sites. For the analysis of core histones, we chose a different strategy. In a first step, the sulfuric-acid-extracted core histones were pre-separated using reverse-phase high-performance liquid chromatography. Individual rat testis core histone fractions obtained in this way were digested and analyzed via bottom-up CESI-MS. This approach yielded the identification of 42 different modification sites including acetylation (lysine and N(α)-terminal); mono-, di-, and trimethylation; and phosphorylation. When we applied CESI-MS for the analysis of intact core histone subtypes from butyrate-treated mouse tumor cells, we were able to rapidly detect their degree of modification, and we found this method very useful for the separation of isobaric trimethyl and acetyl modifications. Taken together, our results highlight the need for additional techniques for the comprehensive analysis of post-translational modifications. CESI-MS is a promising new proteomics tool as demonstrated by this, the first comprehensive analysis of histone modifications, using rat testis as an example.

BACKGROUND Currently, N-terminal pro–B-type natriuretic peptide (NT-proBNP) and its physiologically active counterpart, BNP, are most frequently used as biomarkers for diagnosis, prognosis, and disease monitoring of heart failure (HF). Commercial NT-proBNP and BNP immunoassays cross-react to varying degrees with unprocessed proBNP, which is also found in the circulation. ProBNP processing and immunoassay response are related to O-linked glycosylation of NT-proBNP and proBNP. There is a clear and urgent need to identify the glycosylation sites in the endogenously circulating peptides requested by the community to gain further insights into the different naturally occurring forms. METHODS The glycosylation sites of (NT-) proBNP (NT-proBNP and/or proBNP) were characterized in leftovers of heparinized plasma samples of severe HF patients (NT-proBNP: >10000 ng/L) by using tandem immunoaffinity purification, sequential exoglycosidase treatment for glycan trimming, β-elimination and Michael addition chemistry, as well as high-resolution nano-flow liquid chromatography electrospray multistage mass spectrometry. RESULTS We describe 9 distinct glycosylation sites on circulating (NT-) proBNP in HF patients. Differentially glycosylated variants were detected based on highly accurate mass determination and multistage mass spectrometry. Remarkably, for each of the identified proteolytic glycopeptides, a nonglycosylated form also was detectable. CONCLUSIONS Our results directly demonstrate for the first time a rather complex distribution of the endogenously circulating glycoforms by mass spectrometric analysis in HF patients, and show 9 glycosites in human (NT-) proBNP. This information may also have an impact on commercial immunoassays applying antibodies specific for the central region of (NT-) proBNP, which detect mostly nonglycosylated forms.

Ergothioneine (EGT; 2-mercaptohistidine trimethylbetaine) is a trimethylated and sulphurised histidine derivative which exhibits antioxidant properties. Here we report that deletion of Aspergillus fumigatus egtA (AFUA_2G15650), which encodes a trimodular enzyme, abrogated EGT biosynthesis in this opportunistic pathogen. EGT biosynthetic deficiency in A. fumigatus significantly reduced resistance to elevated H2O2 and menadione, respectively, impaired gliotoxin production and resulted in attenuated conidiation. Quantitative proteomic analysis revealed substantial proteomic remodelling in ΔegtA compared to wild-type under both basal and ROS conditions, whereby the abundance of 290 proteins was altered. Specifically, the reciprocal differential abundance of cystathionine γ-synthase and β-lyase, respectively, influenced cystathionine availability to effect EGT biosynthesis. A combined deficiency in EGT biosynthesis and the oxidative stress response regulator Yap1, which led to extreme oxidative stress susceptibility, decreased resistance to heavy metals and production of the extracellular siderophore triacetylfusarinine C and increased accumulation of the intracellular siderophore ferricrocin. EGT dissipated H2O2in vitro, and elevated intracellular GSH levels accompanied abrogation of EGT biosynthesis. EGT deficiency only decreased resistance to high H2O2 levels which suggests functionality as an auxiliary antioxidant, required for growth at elevated oxidative stress conditions. Combined, these data reveal new interactions between cellular redox homeostasis, secondary metabolism and metal ion homeostasis.

We used linker histone-depleted normal human fibroblast nuclei as templates to study how phosphorylation affects histone H5 binding to chromatin in situ. Permeabilized cells were treated with 0.7 M NaCl to extract the native linker histones. Histone H5 was purified from chicken erythrocytes and phosphorylated in vitro by recombinant cdk5/p35 kinase. High performance capillary electrophoresis (HPCE) showed that the phosphorylated protein contained a mixture of multiply phosphorylated forms. Control experiments, using mass spectrometry, revealed that up to five SPXK motifs in the C terminus were phosphorylated, but also that about 10% of the protein contained one phosphoserine in the N-terminus. Reconstitution of H1-depleted fibroblast nuclei with nonphosphorylated or phosphorylated H5 was performed at physiological ionic strength. The bound H5 was then extracted using NaCl concentrations in the range of 0.15 to 0.7 M. The release of the H5 molecules was monitored by DAPI staining and image cytofluorometry. Our results show that H5 phosphorylation substantially reduced its affinity for chromatin in situ, which support previous observations indicating that C-terminal phosphorylation may be essential for the biological functions of linker histones.

Investigation of site-specific protein O-glycosylation remains a formidable task in post-translational modification-centred proteomics. In particular, the determination of O-glycosylated amino acids in mucin-like glycopeptides lags far behind the techniques for phosphorylation site and N-glycosylation site identification, for which well-established enrichment techniques are available. The present work investigated β-elimination of mucin-like O-glycopeptides with a mild alkylamine base and concomitant Michael-type addition using 2-mercaptoethanol as nucleophile applied to synthetic GalNAcylated O-glycopeptides as well as exoglycosidase-treated endogenous peptides isolated from human blood plasma. This strategy permits O-glycosylated sites to be unambiguously localized, even in multiple-glycosylated peptides. Peptides covalently modified with the glycan surrogate exhibit excellent backbone fragmentation in MS/MS due to their stability during CID.

In this study we have evaluated the suitability of a sheathless capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS) interface with a porous tip as the nanospray emitter for use in peptide analysis. A positively charged capillary coating and 0.1% formic acid as background electrolyte were used for separation upstream from mass spectrometry characterization. The influence of the distance between emitter tip and MS inlet, ESI voltage applied, and of the electroosmotic flow (EOF) on electrospray performance and efficiency of the system was investigated in detail. Under optimized conditions, less than 30 amol of a model peptide (angiotensin I) was required for a detection in the base peak electropherogram and positive identification via tandem MS. Three different cationic capillary coatings were investigated for stability, resolution, and EOF and were found to enable reproducible separations by CE-ESI-MS. After optimizing MS settings, the effectiveness of the CE-ESI-MS method developed was compared with a state-of-the-art nano-liquid chromatography (LC)-ESI-MS method by analyzing Arg-C-digested rat testis linker histones with both systems. With comparable amounts of sample applied, the number of identified peptides increased by more than 60% when using CE-ESI-MS. We found that low molecular mass peptides (below 1400 Da) were preferentially identified by CE-ESI-MS, since this group of peptides poorly interacted with the reversed-phase material in the nano-LC system. Finally, total analysis time in LC-ESI-MS for three runs including equilibration was nearly 4 times longer than that of CE-ESI-MS: 246 versus 66 min.

The opportunistic fungal pathogen Aspergillus fumigatus produces four types of siderophores, low-molecular-mass iron chelators: it excretes fusarinine C (FsC) and triacetylfusarinine C (TAFC) for iron uptake and accumulates ferricrocin (FC) for hyphal and hydroxyferricrocin (HFC) for conidial iron distribution and storage. Siderophore biosynthesis has recently been shown to be crucial for fungal virulence. Here we identified a new component of the fungal siderophore biosynthetic machinery: AFUA_1G04450, termed SidL. SidL is conserved only in siderophore-producing ascomycetes and shows similarity to transacylases involved in bacterial siderophore biosynthesis and the N 5 -hydroxyornithine:anhydromevalonyl coenzyme A- N 5 -transacylase SidF, which is essential for TAFC biosynthesis. Inactivation of SidL in A. fumigatus decreased FC biosynthesis during iron starvation and completely blocked FC biosynthesis during iron-replete growth. In agreement with these findings, SidL deficiency blocked conidial accumulation of FC-derived HFC under iron-replete conditions, which delayed germination and decreased the size of conidia and their resistance to oxidative stress. Remarkably, the sidL gene is not clustered with other siderophore-biosynthetic genes, and its expression is not affected by iron availability. Tagging of SidL with enhanced green fluorescent protein suggested a cytosolic localization of the FC-biosynthetic machinery. Taken together, these data suggest that SidL is a constitutively active N 5 -hydroxyornithine-acetylase required for FC biosynthesis, in particular under iron-replete conditions. Moreover, this study revealed the unexpected complexity of siderophore biosynthesis, indicating the existence of an additional, iron-repressed N 5 -hydroxyornithine-acetylase.

Alkylglycerol monooxygenase (glyceryl-ether monooxygenase, EC 1.14.16.5) is the only enzyme known to cleave the O-alkyl bond of ether lipids which are essential components of brain membranes, protect the eye from cataract, interfere or mediate signalling processes, and are required for spermatogenesis. Along with phenylalanine hydroxylase, tyrosine hydroxylase, tryptophan hydroxylase, and nitric oxide synthase, alkylglycerol monooxygenase is one of five known enzymatic reactions which depend on tetrahydrobiopterin. Although first described in 1964, no sequence had been assigned to this enzyme so far since it lost activity upon protein purification attempts. A functional library screen using pools of plasmids of a rat liver expression library transfected to CHO cells was also unsuccessful. We therefore selected human candidate genes by bioinformatic approaches and by proteomic analysis of partially purified enzyme and tested alkylglycerol monooxygenase activity in CHO cells transfected with expression plasmids. Transmembrane protein 195, a predicted membrane protein with unassigned function which occurs in bilateral animals, was found to encode for tetrahydrobiopterin-dependent alkylglycerol monooxygenase. This sequence assignment was confirmed by injection of transmembrane protein 195 cRNA into Xenopus laevis oocytes. Transmembrane protein 195 shows no sequence homology to aromatic amino acid hydroxylases or nitric oxide synthases, but contains the fatty acid hydroxylase motif. This motif is found in enzymes which contain a diiron center and which carry out hydroxylations of lipids at aliphatic carbon atoms like alkylglycerol monooxygenase. This sequence assignment suggests that alkylglycerol monooxygenase forms a distinct third group among tetrahydrobiopterin-dependent enzymes.

Penicillium antifungal protein (PAF) is a promising antimycotic without toxic effects on mammalian cells and therefore may represent a drug candidate against the often lethal Aspergillus infections that occur in humans. The pathogenesis of PAF on sensitive fungi involves G-protein coupled signalling followed by apoptosis. In the present study, the solution structure of this small, cationic, antifungal protein from Penicillium chrysogenum is determined by NMR. We demonstrate that PAF belongs to the structural classification of proteins fold class of its closest homologue antifungal protein from Aspergillus giganteus. PAF comprises five beta-strands forming two orthogonally packed beta-sheets that share a common interface. The ambiguity in the assignment of two disulfide bonds out of three was investigated by NMR dynamics, together with restrained molecular dynamics calculations. The clue could not be resolved: the two ensembles with different disulfide patterns and the one with no S-S bond exhibit essentially the same fold. (15)N relaxation dispersion and interference experiments did not reveal disulfide bond rearrangements via slow exchange. The measured order parameters and the 3.0 ns correlation time are appropriate for a compact monomeric protein of this size. Using site-directed mutagenesis, we demonstrate that the highly-conserved and positively-charged lysine-rich surface region enhances the toxicity of PAF. However, the binding capability of the oligosaccharide/oligonucleotide binding fold is reduced in PAF compared to antifungal protein as a result of less solvent-exposed aromatic regions, thus explaining the absence of chitobiose binding. The present study lends further support to the understanding of the documented substantial differences between the mode of action of two highly homologous antifungal proteins.

Cellular senescence is known as a potent mechanism of tumor suppression, and cellular senescence in vitro also reflects at least some features of aging in vivo. The Free Radical Theory of aging suggests that reactive oxygen species are important causative agents of aging and cellular senescence. Besides damage of nucleic acids and lipids, also oxidative modifications of proteins have been described as potential causative events in the senescence response. However, the identity of protein targets for post-translational modifications in senescent cells has remained unclear. In the present communication, we analyzed the occurrence of oxidative posttranslational modifications in senescent human endothelial cells and dermal fibroblasts. We found a significant increase in the level of protein carbonyls and AGE modification with senescence in both cell types. Using 2D-Gel electrophoresis and Western Blot we found that heat shock cognate protein 70 is a bona fide target for AGE modification in human fibroblasts.

Hyaluronidase from honey bee was recombinantly expressed as a secreted glycoprotein in Pichia pastoris. The active enzyme was produced in milligram quantities per liter of primary culture. When changing the codons of the original transcript to triplet sequences preferred by P. pastoris, no further increase of protein product could be achieved. After expression of a fusion protein by linking hyaluronidase and human serum albumin together with the recognition sequence for the protease, factorXa, fragmented protein products were obtained in the culture supernatant. Only after replacement of the hinge region with a serine-glycine-rich linker, stable full-length fusion protein could be generated. The protein products were purified by cation exchange chromatography at pH 5.0 and pure enzyme fractions were further characterized in detail. The biochemical properties of the product matched those of crude hyaluronidase within bee venom: the native and the recombinant enzyme exhibited activity over a pH range from 3 to 8 (maximum: 3.8), at temperatures as low as 4 degrees C and up to 90 degrees C (maximum 62 degrees C), and at ionic strength as high as 2 M salt. Recombinant bee hyaluronidase efficiently degrades 6-S-chondroitin sulfate (chondroitin sulfate C) as well as 4-S-chondroitin sulfate (chondroitin sulfate A), the latter to a lesser extent. Only very little hydrolase activity towards chondroitin sulfate B (dermatan sulfate) was detectable.

[99mTc-EDDA–HYNIC-D-Phe1,Tyr3]octreotide (99mTc-EDDA/HYNIC–TOC) is a promising new agent with the potential to replace [111In-DTPA-D-Phe1]-octreotide in somatostatin receptor scintigraphy. This hydrazinonicotinic acid derivatized somatostatin complex contains ethylenediamine N,N′ diacetic acid (EDDA) as a coligand resulting in a high in vitro and in vivo stability. Since direct 99mTc-labelling of HYNIC–TOC with EDDA results in low labelling yields, in this study we describe the preparation of 99mTc-EDDA/HYNIC-TOC via coligand exchange from Tricine for EDDA. Exchange of coligands is achieved at elevated temperature and under optimized conditions of pH, EDDA concentration and stannous ion. High labelling yields (mean 95.9%) were achieved at high specific activities (>37GBq/µmol). Characterization via HPLC, receptor binding and LC–MS of the resulting complex is described. The formulation developed enables rapid and simple labelling of 99mTc-EDDA/HYNIC–TOC in a manner suitable for a clinical setting. Copyright © 2003 John Wiley & Sons, Ltd.

In this study we demonstrate the potential of sequential injection of samples in capillary electrophoresis-mass spectrometry (CE-MS) for rapid and sensitive proteome characterization of human lymphoblastic T-cells (line CCRF-CEM). Proteins were extracted, enzymatically digested, and the resulting peptides fractionated by RP-HPLC. Twenty fractions were thereafter analyzed by CE-MS within a single MS analysis. The CE-MS method was designed that every 10 minutes a new fraction was injected into the CE system. Without any rinsing or equilibration steps we were able to generate a continuous stream of peptides feeding the mass analyzer. In 250 min, the total analysis time of a single sequential injection experiment, we were able to identify roughly 28.000 peptide sequences counting for 4.800 proteins. These numbers could be increased to 62.000 peptides and more than 6.100 proteins identified, when performing three experiments analyzing a total of 60 fractions, all within 12.5 hours. We found that the electrophoretic mobility of peptides can be used to trace back peptides and assign them to the fraction they originate from. This article is protected by copyright. All rights reserved

Myelin basic protein (MBP) is a multifunctional protein involved in maintaining the stability and integrity of the myelin sheath by a variety of interactions with membranes and other proteins. MBP is subjected to extensive posttranslational modifications (PTMs) that are known to be crucial for the regulation of these interactions. Here, we report capillary electrophoresis-mass spectrometric (CE-MS) analysis for the separation and identification of MBP peptides that incorporate the same PTM at different sites, creating multiple localization variants, and the ability to analyze challenging modifications such as asparagine and glutamine deamidation, isomerization, and arginine citrullination. Moreover, we observed site-specific alterations in the modification level of MBP purified from brain of mice of different age. In total, we identified 40 modifications at 33 different sites, which include both previously reported and seven novel modifications. The identified modifications include Nα-terminal acetylation, mono- and dimethylation, phosphorylation, oxidation, deamidation, and citrullination. Notably, some new sites of arginine methylation overlap with the sites of citrullination. Our results highlight the need for sensitive and efficient techniques for a comprehensive analysis of PTMs.

Nonenzymatic deamidation of peptides and proteins represents an important degradation reaction occurring in vitro in the course of isolation or storage and in vivo during development and/or aging of cells. This review first presents a synopsis of the influence of structure on deamidation reaction proceeding via a five-membered succinimide intermediate, followed by an outline of procedures for separation and detection of deamidated forms. Selected examples for in vitro and in vivo deamidation are reviewed including the possible biological consequences of this protein degradation. Finally, the reaction of protein methyltransferase with L-isoaspartyl- and D-aspartyl residues and its possible role in protein repair is elucidated.

Histone H1(0) is known to consist of two subfractions named H1(0)a and H1(0)b. The present work was performed with the aim of elucidating the nature of these two subfractions. By using reversed-phase high performance liquid chromatography in combination with hydrophilic interaction liquid chromatography, we fractionated human histone H1(0) into even four subfractions. Hydrophilic interaction liquid chromatographic analysis of the peptide fragments obtained after cleavage with cyanogen bromide and digestion with chymotrypsin suggested that the four H1(0) subfractions differ only in their small N-terminal end of the H1(0) molecule (30 residues). Edman degradation of the N-terminal H1(0) peptide fragments and mass spectra analysis have indicated that human histone H1(0) consists of intact histones H1(0) (named H1(0) Asn-3) and deamidated H1(0) forms (H1(0) Asp-3) having an aspartic acid residue at position 3 instead of asparagine. Moreover, both H1(0) Asn-3 and H1(0) Asp-3 are blocked (H1(0)a Asn-3, H1(0)a Asp-3) and unblocked (H1(0)b Asn-3, H1(0)b Asp-3) on their N terminus. Acid-urea gel electrophoretic analysis has shown that the histone subfraction, in the literature originally named H1(0)a, actually consists of a mixture of H1(0)a Asn-3 and H1(0)a Asp-3, whereas H1(0)b consists of H1(0)b Asn-3 and H1(0)b Asp-3. Furthermore, we found that hydrophilic interaction liquid chromatography separates rat and mouse histone H1(0) just like human H1(0) into four subfractions. Hydrophilic interaction liquid chromatographic analysis of brain and liver histone H1(0) from rats of different ages revealed an age-dependent increase of both the N-terminally acetylated and the deamidated forms of H1(0). In addition, we found that the relative proportions of the four forms of H1(0) histones differ from tissue to tissue.

The Transient Receptor Potential Vanilloid 1 (TRPV1, vanilloid receptor 1) ion channel plays a key role in the perception of thermal and inflammatory pain, however, its molecular environment in dorsal root ganglia (DRG) is largely unexplored. Utilizing a panel of sequence-directed antibodies against TRPV1 protein and mouse DRG membranes, the channel complex from mouse DRG was detergent-solubilized, isolated by immunoprecipitation and subsequently analyzed by mass spectrometry. A number of potential TRPV1 interaction partners were identified, among them cytoskeletal proteins, signal transduction molecules, and established ion channel subunits. Based on stringent specificity criteria, the voltage-gated K+ channel beta 2 subunit (Kvβ2), an accessory subunit of voltage-gated K+ channels, was identified of being associated with native TRPV1 channels. Reverse co-immunoprecipitation and antibody co-staining experiments confirmed TRPV1/Kvβ2 association. Biotinylation assays in the presence of Kvβ2 demonstrated increased cell surface expression levels of TRPV1, while patch-clamp experiments resulted in a significant increase of TRPV1 sensitivity to capsaicin. Our work shows, for the first time, the association of a Kvβ subunit with TRPV1 channels, and suggests that such interaction may play a role in TRPV1 channel trafficking to the plasma membrane.

The opportunistic fungal pathogen Aspergillus fumigatus produces siderophores for uptake and storage of iron, which is essential for its virulence. The main precursor of siderophore biosynthesis (SB), ornithine, can be produced from glutamate in the mitochondria or by cytosolic hydrolysis of ornithine-derived arginine. Here, we studied the impact of mitochondrial versus cytosolic ornithine biosynthesis on SB by comparison of the arginine auxotrophic mutants ΔargEF and ΔargB, which lack and possess mitochondrial ornithine production, respectively. Deficiency in argEF (encoding acetylglutamate kinase and acetylglutamyl-phosphate-reductase), but not argB (encoding ornithine transcarbamoyl transferase) decreased (i) the cellular ornithine content, (ii) extra- and intracellular SB, (iii) growth under harsh iron starvation, (iv) resistance to the ornithine decarboxylase inhibitor eflornithine, and (v) virulence in the Galleria mellonella larvae model. These lines of evidence indicate that SB is mainly fueled by mitochondrial rather than cytosolic ornithine production and underline the role of SB in virulence. Ornithine content and SB of ΔargB increased with declining arginine supplementation indicating feedback-inhibition of mitochondrial ornithine biosynthesis by arginine. In contrast to SB, the arginine and polyamine contents were only mildly affected in ΔargEF, indicating prioritization of the latter two ornithine-consuming pathways over SB. These data highlight the metabolic differences between the two arginine auxotrophic mutants ΔargEF and ΔargB and demonstrate that supplementation of an auxotrophic mutant does not restore the wild type metabolism at the molecular level, a fact to be considered when working with auxotrophic mutants. Moreover, cross pathway control-mediating CpcA was found to influence the ornithine pool as well as biosynthesis of siderophores and polyamines.

Hydrophilic-interaction liquid chromatography (HILIC) has recently been introduced as a highly efficient chromatographic technique for the separation of a wide range of solutes. The present work was performed with the aim of evaluating the potential utility of HILIC for the separation of posttranslationally acetylated histones. The protein fractionations were generally achieved by using a weak cation-exchange column and an increasing sodium perchlorate gradient system in the presence of acetonitrile (70%, v/v) at pH 3.0. In combination with reversed-phase high-performance liquid chromatography (RP-HPLC) we have successfully separated various H2A variants and posttranslationally acetylated forms of H2A variants and H4 proteins in very pure form. An unambiguous assignment of the histone fractions obtained was performed using high-performance capillary and acid-urea—Triton gel electrophoresis. Our results demonstrate that for the analysis and isolation of modified core histone variants HILIC provides a new and important alternative to traditional separation techniques and will be useful in studying the biological function of histone acetylation.

The effects of different buffer concentrations and compositions on the elution order and separation of H1 histone subtypes and their phosphorylated modifications isolated from several species was studied using high-performance capillary electrophoresis (CE). Various cations and anions were tested in an untreated silica capillary and low pH buffers, in the presence of the dynamic coating agent hydroxypropylmethyl cellulose. It was found that the cations and anions of buffers have a remarkable influence on both the efficiency and the selectivity of protein separations. A triethylammonium methanephosphonate system proved efficacious for the separation of rat histone subtype H1c from H1e and a perchlorate/triethylammonium phosphate system for the analysis of chicken and mouse linker histones. CE provides an attractive alternative to high-performance liquid chromatography and conventional gel electrophoresis.

Trypanosoma brucei brucei, a protozoan parasite of wild and domestic animals in Africa, is related to the pathogenic agent of human sleeping sickness. Four H1 histone proteins were isolated from nuclei of procyclic culture forms and cleaved with proteases. Amino acid sequence analysis of purified fragments indicated the presence of variants which displayed sequence identities as compared to the C-terminal domain of human H1. Substitutions of amino acids and posttranslational modifications of the histones in T b brucei H1 may influence protein conformation and histone-histone as well as histone-DNA interactions in the chromatin of the parasite. Digestion of soluble chromatin with immobilized trypsin at low and high ionic strengths indicated an internal localization of H1 in the condensed chromatin. The influence of histone H1 of T b brucei on the compaction pattern of the chromatin was investigated by dissociation and reconstitution experiments. Electron microscopy revealed that trypanosome H1 was able to induce condensation of the chromatin of the parasite and of rat liver into dense tangles. After dephosphorylation of H1, 30 nm fibers were induced in rat liver chromatin, while the resulting fibers were distinctly thinner in T b brucei. It can be concluded that the absence of 30 nm fibers in T b brucei chromatin cannot be explained by the divergent variants and posttranslational phosphorylations of H1 only but rather by the influence of both, the divergent core histones, previously described, and H1 properties.

Major contractile proteins were purified from relaxed actomyosin extracted from molluscan catch muscle myofibrils using ammonium sulfate fractionation and divalent cation precipitation. A fraction of this actomyosin was precipitated and purified as a supramolecular complex composed of twitchin (TW), myosin (MY), and myorod (MR). Another TW-MR complex was obtained via the removal of myosin. These supramolecular complexes and filaments assembled from purified myosin contained an endogenous protein kinase that phosphorylated myosin and myorod. Significantly, the activity of this novel myosin-associated (MA) kinase was inhibited at calcium concentrations of >0.1 microM. After partial purification of the kinase, we established that the inhibition resulted from binding of calcium to the substrate (myosin) and not from the binding to the enzyme (kinase). No inhibition was observed when myorod was used as a substrate, although the latter is identical to the rod portion of myosin lacking the head domains. Phosphorylation sites of myorod were identified, three at the C-terminal tip and three at the N-terminal domain. In the presence of calcium, addition of myosin to the TW-MR complex resulted in inhibition of this phosphorylation, while in the absence of myosin, this inhibition was negligible. Added myosin also inhibited phosphorylation of twitchin by PKA-like kinase, the latter also present in the complex. The opposite was true with the TW-MY-MR complex; that is, phosphorylation of myosin was inhibited by twitchin and/or myorod. Thus, in parallel to the well-established direct activation by calcium, molluscan catch muscle myosin also regulated its own phosphorylation. Therefore, in addition to the established phosphorylation of twitchin by PKA-like kinase, phosphorylation of myosin and myorod by myosin-associated kinase appears to play an important role in the development of the catch state.

High-performance capillary electrophoresis (HPCE) was used to separate successfully distinct phosphorylated derivatives of individual histone HI variants. With an untreated capillary (50 cm x 75 μm I.D.) the electrophoresis was performed in about 15 min. Inconvenient interactions of these highly basic proteins with the capillary wall were eliminated by using 0.1 M sodium phosphate buffer (pH 2.0) containing 0.03% hydroxypropylmethylcellulose. Under these experimental conditions the histone H1 variants Hlb and Hlc obtained from mitotic enriched NIH 3T3 fibroblasts and isolated by reversed-phase high-performance liquid chromatography were clearly separated in their non-phosphorylated and different phosphorylated forms. This result was confirmed by acid-urea gel electrophoresis, comparison with non-phosphorylated histones H1b and H1c, isolated from quiescent NIH 3T3 cells, and incubation of multi-phosphorylated histone H1b with alkaline phosphatase and subsequent acid-urea and capillary electrophoresis. The results illustrate that the application of HPCE to the analysis of histone modifications provides a new alternative to traditional gel electrophoresis.

Fatty aldehyde dehydrogenase (EC 1.2.1.48) converts long-chain fatty aldehydes to the corresponding acids. Deficiency in this enzyme causes the Sjogren Larsson Syndrome, a rare inherited disorder characterized by ichthyosis, spasticity, and mental retardation. Using a fluorescent aldehyde, pyrenedecanal, and HPLC with fluorescence detection, we developed a novel method to monitor fatty aldehyde dehydrogenase activity by quantification of the product pyrenedecanoic acid together with the substrate pyrenedecanal and possible side products, such as aldehyde adducts. As shown with recombinant enzymes, pyrenedecanal showed a high preference for fatty aldehyde dehydrogenase compared with other aldehyde dehydrogenases. The method allowed detection of fatty aldehyde dehydrogenase activity in nanogram amounts of microsomal or tissue protein and microgram amounts of Sjogren Larsson syndrome patients' skin fibroblast protein. It could successfully be adapted for the analysis of fatty aldehyde dehydrogenase activity in gel slices derived from low-temperature SDS-PAGE, showing that fatty aldehyde dehydrogenase activity from solubilized rat liver microsomes migrates as a dimer. Thus, monitoring of pyrenedecanoic acid formation from pyrenedecanal by HPLC with fluorescence detection provides a robust and sensitive method for determination of fatty aldehyde dehydrogenase activity.-Keller, M. A., K. Watschinger, G. Golderer, M. Maglione, B. Sarg, H. H. Lindner, G. Werner-Felmayer, A. Terrinoni, R. J. A. Wanders, and E. R. Werner. Monitoring of fatty aldehyde dehydrogenase by formation of pyrenedecanoic acid from pyrenedecanal. J. Lipid Res. 2010. 51: 1554-1559

Iron is an essential metal for virtually all organisms. Iron acquisition is well characterized for various organisms, whereas intracellular iron distribution is poorly understood. In contrast to bacteria, plants, and animals, most fungi lack ferritin-mediated iron storage but possess an intracellular siderophore shown to be involved in iron storage. Here we demonstrate that deficiency in the intracellular siderophore ferricrocin causes iron starvation in conidia of Aspergillus fumigatus , demonstrating that ferricrocin is also involved in intra- and transcellular iron distribution. Thus, ferricrocin represents the first intracellular iron transporter identified in any organism.

The influence of the eluent composition, ion-pair concentration, and temperature on the fractionation of chromosomal proteins by reversed-phase high performance liquid chromatography (HPLC) has been investigated. Based on this examination methods for the troublesome separation of histones H1o/H1b, H2A/H4, and H5.1/H5.2 have been developed.