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Matrix-assisted laser desorption-ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool in histopathological characterization and represents a modern analytical technique, enabling two-dimensional detection of molecular components of biological samples. [...]

The Golgi apparatus is essential for protein sorting, yet its quality control mechanisms are poorly understood. Here we show that the Dsc ubiquitin ligase complex uses its rhomboid pseudo-protease subunit, Dsc2, to assess the hydrophobic length of α-helical transmembrane domains (TMDs) at the Golgi. Thereby the Dsc complex likely interacts with orphaned ER and Golgi proteins that have shorter TMDs and ubiquitinates them for targeted degradation. Some Dsc substrates will be extracted by Cdc48 for endosome and Golgi associated proteasomal degradation (EGAD), while others will undergo ESCRT dependent vacuolar degradation. Some substrates are degraded by both, EGAD- or ESCRT pathways. The accumulation of Dsc substrates entails a specific increase in glycerophospholipids with shorter and asymmetric fatty acyl chains. Hence, the Dsc complex mediates the selective degradation of orphaned proteins at the sorting center of cells, which prevents their spreading across other organelles and thereby preserves cellular membrane protein and lipid composition., (© 2024. The Author(s).)

The neurodevelopmental disorders Prader-Willi syndrome (PWS) and Schaaf-Yang syndrome (SYS) both arise from genomic alterations within human chromosome 15q11-q13. A deletion of the SNORD116 cluster, encoding small nucleolar RNAs, or frameshift mutations within MAGEL2 result in closely related phenotypes in individuals with PWS or SYS, respectively. By investigation of their subcellular localization, we observed that in contrast to a predominant cytoplasmic localization of wild-type (WT) MAGEL2, a truncated MAGEL2 mutant was evenly distributed between the cytoplasm and the nucleus. To elucidate regulatory pathways that may underlie both diseases, we identified protein interaction partners for WT or mutant MAGEL2, in particular the survival motor neuron protein (SMN), involved in spinal muscular atrophy, and the fragile-X-messenger ribonucleoprotein (FMRP), involved in autism spectrum disorders. The interactome of the non-coding RNA SNORD116 was also investigated by RNA-CoIP. We show that WT and truncated MAGEL2 were both involved in RNA metabolism, while regulation of transcription was mainly observed for WT MAGEL2. Hence, we investigated the influence of MAGEL2 mutations on the expression of genes from the PWS locus, including the SNORD116 cluster. Thereby, we provide evidence for MAGEL2 mutants decreasing the expression of SNORD116, SNORD115, and SNORD109A, as well as protein-coding genes MKRN3 and SNRPN, thus bridging the gap between PWS and SYS., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Spatiotemporal-controlled second messengers alter molecular interactions of central signaling nodes for ensuring physiological signal transmission. One prototypical second messenger molecule which modulates kinase signal transmission is the cyclic-adenosine monophosphate (cAMP). The main proteinogenic cellular effectors of cAMP are compartmentalized protein kinase A (PKA) complexes. Their cell-type specific compositions precisely coordinate substrate phosphorylation and proper signal propagation which is indispensable for numerous cell-type specific functions. Here we present evidence that TAF15, which is implicated in the etiology of amyotrophic lateral sclerosis, represents a novel nuclear PKA substrate. In cross-linking and immunoprecipitation experiments (iCLIP) we showed that TAF15 phosphorylation alters the binding to target transcripts related to mRNA maturation, splicing and protein-binding related functions. TAF15 appears to be one of multiple PKA substrates that undergo RNA-binding dynamics upon phosphorylation. We observed that the activation of the cAMP-PKA signaling axis caused a change in the composition of a collection of RNA species that interact with TAF15. This observation appears to be a broader principle in the regulation of molecular interactions, as we identified a significant enrichment of RNA-binding proteins within endogenous PKA complexes. We assume that phosphorylation of RNA-binding domains adds another layer of regulation to binary protein-RNAs interactions with consequences to RNA features including binding specificities, localization, abundance and composition., (© 2024. The Author(s).)

Introduction: The complement system is part of innate immunity and is comprised of an intricate network of proteins that are vital for host defense and host homeostasis. A distinct mechanism by which complement defends against invading pathogens is through the membrane attack complex (MAC), a lytic structure that forms on target surfaces. The MAC is made up of several complement components, and one indispensable component of the MAC is C7. The role of C7 in MAC assembly is well documented, however, inherent characteristics of C7 are yet to be investigated., Methods: To shed light on the molecular characteristics of C7, we examined the properties of serum-purified C7 acquired using polyclonal and novel monoclonal antibodies. The properties of serum‑purified C7 were investigated through a series of proteolytic analyses, encompassing Western blot and mass spectrometry. The nature of C7 protein-protein interactions were further examined by a novel enzyme-linked immunosorbent assay (ELISA), as well as size‑exclusion chromatography., Results: Protein analyses showcased an association between C7 and clusterin, an inhibitory complement regulator. The distinct association between C7 and clusterin was also demonstrated in serum-purified clusterin. Further assessment revealed that a complex between C7 and clusterin (C7-CLU) was detected. The C7-CLU complex was also identified in healthy serum and plasma donors, highlighting the presence of the complex in circulation., Discussion: Clusterin is known to dissociate the MAC structure by binding to polymerized C9, nevertheless, here we show clusterin binding to the native form of a terminal complement protein in vivo. The presented data reveal that C7 exhibits characteristics beyond that of MAC assembly, instigating further investigation of the effector role that the C7-CLU complex plays in the complement cascade., Competing Interests: ET is an employee of Hycult Biotech. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AZ declared a shared affiliation with the author M-OS to the handling editor at the time of review., (Copyright © 2024 Massri, Toonen, Sarg, Kremser, Grasse, Fleischer, Torres-Quesada, Hengst, Skjoedt, Bayarri-Olmos, Rosbjerg, Garred, Orth-Höller, Prohászka and Würzner.)

Shiga toxins (Stxs), especially the Stx2a subtype, are the major virulence factors involved in enterohemorrhagic Escherichia coli (EHEC)-associated hemolytic uremic syndrome (eHUS), a life-threatening disease causing acute kidney injury, especially in children. After oral transmission and colonization in the gut, EHEC release Stx. Intracellular cleavage of the Stx A subunit, when followed by reduction, boosts the enzymatic activity that causes damage to targeted cells. This cleavage was assumed to be mostly mediated by furin during Stx intracellular trafficking. To investigate whether this cleavage could occur in the intestine, even prior to entering target cells, Stx2a A subunit structure (intact or cleaved) was characterized after its exposure to specific host factors present in human stool. The molecular weight of Stx2a A subunit/fragments was determined by immunoblotting after electrophoretic separation under reducing conditions. In this study, it was demonstrated that Stx2a is cleaved by certain human stool components. Trypsin and chymotrypsin-like elastase 3B (CELA3B), two serine proteases, were identified as potential candidates that can trigger the extracellular cleavage of Stx2a A subunit directly after its secretion by EHEC in the gut. Whether the observed cleavage indeed translates to natural infections and plays a role in eHUS pathogenesis has yet to be determined. If so, it seems likely that a host's protease profile could affect disease development by changing the toxin's biological features.

The metabotropic glutamate receptor 1 (mGlu 1 ) plays a pivotal role in synaptic transmission and neuronal plasticity. Despite the fact that several interacting proteins involved in the mGlu 1 subcellular trafficking and intracellular transduction mechanisms have been identified, the protein network associated with this receptor in specific brain areas remains largely unknown. To identify novel mGlu 1 -associated protein complexes in the mouse cerebellum, we used an unbiased tissue-specific proteomic approach, namely co-immunoprecipitation followed by liquid chromatography/tandem mass spectrometry analysis. Many well-known protein complexes as well as novel interactors were identified, including G-proteins, Homer, δ2 glutamate receptor, 14-3-3 proteins, and Na/K-ATPases. A novel putative interactor, KCTD12, was further investigated. Reverse co-immunoprecipitation with anti-KCTD12 antibodies revealed mGlu 1 in wild-type but not in KCTD12-knock-out homogenates. Freeze-fracture replica immunogold labeling co-localization experiments showed that KCTD12 and mGlu 1 are present in the same nanodomain in Purkinje cell spines, although at a distance that suggests that this interaction is mediated through interposed proteins. Consistently, mGlu 1 could not be co-immunoprecipitated with KCTD12 from a recombinant mammalian cell line co-expressing the two proteins. The possibility that this interaction was mediated via GABA B receptors was excluded by showing that mGlu 1 and KCTD12 still co-immunoprecipitated from GABA B receptor knock-out tissue. In conclusion, this study identifies tissue-specific mGlu 1 -associated protein clusters including KCTD12 at Purkinje cell synapses.

N -chlorotaurine (NCT) a long-lived oxidant generated by leukocytes, can be synthesized chemically and applied topically as an anti-infective to different body sites, including the lung via inhalation. Here, we demonstrate the activity of NCT against viruses causing acute respiratory tract infections, namely severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza viruses, and respiratory syncytial virus (RSV). Virucidal activity of NCT was tested in plaque assays, confirmed by RT-qPCR assays. Attack on virus proteins was investigated by mass spectrometry. NCT revealed broad virucidal activity against all viruses tested at 37°C and pH 7. A significant reduction in infectious particles of SARS-CoV-2 isolates from early 2020 by 1 log 10 was detected after 15 min of incubation in 1% NCT. Proteinaceous material simulating body fluids enhanced this activity by transchlorination mechanisms (1 -2 log 10 reduction within 1-10 min). Tested SARS-CoV-2 variants B.1.1.7 (Alpha) und B.1.351 (Beta) showed a similar susceptibility. Influenza virus infectious particles were reduced by 3 log 10 (H3N2) to 5 log 10 (H1N1pdm), RSV by 4 log 10 within a few min. Mass spectrometry of NCT-treated SARS-CoV-2 spike protein and 3C-like protease, influenza virus haemagglutinin and neuraminidase, and RSV fusion glycoprotein disclosed multiple sites of chlorination and oxidation as the molecular mechanism of action. Application of 1.0% NCT as a prophylactic and therapeutic strategy against acute viral respiratory tract infections deserves comprehensive clinical investigation.

Background: The presence of nuclear mitochondrial DNA (numtDNA) has been reported within several nuclear genomes. Next to mitochondrial protein-coding genes, numtDNA sequences also encode for mitochondrial tRNA genes. However, the biological roles of numtDNA remain elusive., Results: Employing in silico analysis, we identify 281 mitochondrial tRNA homologs in the human genome, which we term nimtRNAs (nuclear intronic mitochondrial-derived tRNAs), being contained within introns of 76 nuclear host genes. Despite base changes in nimtRNAs when compared to their mtRNA homologs, a canonical tRNA cloverleaf structure is maintained. To address potential functions of intronic nimtRNAs, we insert them into introns of constitutive and alternative splicing reporters and demonstrate that nimtRNAs promote pre-mRNA splicing, dependent on the number and positioning of nimtRNA genes and splice site recognition efficiency. A mutational analysis reveals that the nimtRNA cloverleaf structure is required for the observed splicing increase. Utilizing a CRISPR/Cas9 approach, we show that a partial deletion of a single endogenous nimtRNA Lys within intron 28 of the PPFIBP1 gene decreases inclusion of the downstream-located exon 29 of the PPFIBP1 mRNA. By employing a pull-down approach followed by mass spectrometry, a 3'-splice site-associated protein network is identified, including KHDRBS1, which we show directly interacts with nimtRNA Tyr by an electrophoretic mobility shift assay., Conclusions: We propose that nimtRNAs, along with associated protein factors, can act as a novel class of intronic splicing regulatory elements in the human genome by participating in the regulation of splicing.

How cells adjust nutrient transport across their membranes is incompletely understood. Previously, we have shown that S. cerevisiae broadly 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., Competing Interests: VI, JZ, SS, JK, SW, RG, TJ, LK, GB, HL, LH, SL, OS, DT No competing interests declared, (© 2020, Ivashov et al.)

The fungal class 1 lysine deacetylase (KDAC) RpdA is a promising target for prevention and treatment of invasive fungal infection. RpdA is essential for survival of the most common air-borne mold pathogen Aspergillus fumigatus and the model organism Aspergillus nidulans . In A. nidulans , RpdA depletion induced production of previously unknown small bioactive substances. As known from yeasts and mammals, class 1 KDACs act as components of multimeric protein complexes, which previously was indicated also for A. nidulans . Composition of these complexes, however, remained obscure. In this study, we used tandem affinity purification to characterize different RpdA complexes and their composition in A. nidulans . In addition to known class 1 KDAC interactors, we identified a novel RpdA complex, which was termed RcLS2F. It contains ScrC, previously described as suppressor of the transcription factor CrzA, as well as the uncharacterized protein FscA. We show that recruitment of FscA depends on ScrC and we provide clear evidence that Δ crzA suppression by ScrC depletion is due to a lack of transcriptional repression caused by loss of the novel RcLS2F complex. Moreover, RcLS2F is essential for sexual development and engaged in an autoregulatory feed-back loop., (Copyright © 2020 Bauer, Gross, Merschak, Kremser, Karahoda, Sarikaya Bayram, Abt, Binder, Gsaller, Lindner, Bayram, Brosch and Graessle.)

Echinoderms, such as the rock-boring sea urchin Paracentrotus lividus , attach temporarily to surfaces during locomotion using their tube feet. They can attach firmly to any substrate and release from it within seconds through the secretion of unknown molecules. The composition of the adhesive, as well as the releasing secretion, remains largely unknown. This study re-analyzed a differential proteome dataset from Lebesgue et al. by mapping mass spectrometry-derived peptides to a P. lividus de novo transcriptome generated in this study. This resulted in a drastic increase in mapped proteins in comparison to the previous publication. The data were subsequently combined with a differential RNAseq approach to identify potential adhesion candidate genes. A gene expression analysis of 59 transcripts using whole mount in situ hybridization led to the identification of 16 transcripts potentially involved in bioadhesion. In the future these data could be useful for the production of synthetic reversible adhesives for industrial and medical purposes.

Late endosomal/lysosomal adaptor and MAPK and mTOR activator (LAMTOR/Ragulator) is a scaffold protein complex that anchors and regulates multiprotein signaling units on late endosomes/lysosomes. To identify LAMTOR-modulated endolysosomal proteins, primary macrophages were derived from bone marrow of conditional knockout mice carrying a specific deletion of LAMTOR2 in the monocyte/macrophage cell lineage. Affymetrix-based transcriptomic analysis and quantitative iTRAQ-based organelle proteomic analysis of endosomes derived from macrophages were performed. Further analyses showed that LAMTOR could be a novel regulator of foam cell differentiation. The lipid droplet formation phenotype observed in macrophages was additionally confirmed in MEFs, where lipidomic analysis identified cholesterol esters as specifically downregulated in LAMTOR2 knockout cells. The data obtained indicate a function of LAMTOR2 in lipid metabolism., (© 2019 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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.

Centromeres are specialized chromosomal regions epigenetically defined by the presence of the histone H3 variant CENP-A. CENP-A is required for kinetochore formation which is essential for chromosome segregation during mitosis. Spatial restriction of CENP-A to the centromere is tightly controlled. Its overexpression results in ectopic incorporation and the formation of potentially deleterious neocentromeres in yeast, flies and in various human cancers. While the contribution of posttranslational modifications of CENP-A to these processes has been studied in yeast and mammals to some extent, very little is known about Drosophila melanogaster. Here, we show that CENP-A is phosphorylated at serine 20 (S20) by casein kinase II and that in mitotic cells, the phosphorylated form is enriched on chromatin. Importantly, our results reveal that S20 phosphorylation regulates the turn-over of prenucleosomal CENP-A by the SCFPpa-proteasome pathway and that phosphorylation promotes removal of CENP-A from ectopic but not from centromeric sites in chromatin. We provide multiple lines of evidence for a crucial role of S20 phosphorylation in controlling restricted incorporation of CENP-A into centromeric chromatin in flies. Modulation of the phosphorylation state of S20 may provide the cells with a means to fine-tune CENP-A levels in order to prevent deleterious loading to extra-centromeric sites., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Flatworms 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'.

In filamentous fungi, arginine methylation has been implicated in morphogenesis, mycotoxin biosynthesis, pathogenicity, and stress response although the exact role of this posttranslational modification in these processes remains obscure. Here, we present the first genome-wide transcriptome analysis in filamentous fungi that compared expression levels of genes regulated by type I and type II protein arginine methyltransferases (PRMTs). In Aspergillus nidulans, three conserved type I and II PRMTs are present that catalyze asymmetric or symmetric dimethylation of arginines. We generated a double type I mutant (ΔrmtA/rmtB) and a combined type I and type II mutant (ΔrmtB/rmtC) to perform genome-wide comparison of their effects on gene expression, but also to monitor putative overlapping activities and reciprocal regulations of type I and type II PRMTs in Aspergillus. Our study demonstrates, that rmtA and rmtC as type I and type II representatives act together as repressors of proteins that are secreted into the extracellular region as the majority of up-regulated genes are mainly involved in catabolic pathways that constitute the secretome of Aspergillus. In addition to a strong up-regulation of secretory genes we found a significant enrichment of down-regulated genes involved in processes related to oxidation-reduction, transmembrane transport and secondary metabolite biosynthesis. Strikingly, nearly 50% of down-regulated genes in both double mutants correspond to redox reaction/oxidoreductase processes, a remarkable finding in light of our recently observed oxidative stress phenotypes of ΔrmtA and ΔrmtC. Finally, analysis of nuclear and cytoplasmic extracts for mono-methylated proteins revealed the presence of both, common and specific substrates of RmtA and RmtC. Thus, our data indicate that type I and II PRMTs in Aspergillus seem to co-regulate the same biological processes but also specifically affect other pathways in a non-redundant fashion., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Cellular homeostasis requires the ubiquitin-dependent degradation of membrane proteins. This was assumed to be mediated exclusively either by endoplasmic reticulum-associated degradation (ERAD) or by endosomal sorting complexes required for transport (ESCRT)-dependent lysosomal degradation. We identified in Saccharomyces cerevisiae an additional pathway that selectively extracts membrane proteins at Golgi and endosomes for degradation by cytosolic proteasomes. One endogenous substrate of this endosome and Golgi-associated degradation pathway (EGAD) is the ER-resident membrane protein Orm2, a negative regulator of sphingolipid biosynthesis. Orm2 degradation is initiated by phosphorylation, which triggers its ER export. Once on Golgi and endosomes, Orm2 is poly-ubiquitinated by the membrane-embedded "Defective in SREBP cleavage" (Dsc) ubiquitin ligase complex. Cdc48/VCP then extracts ubiquitinated Orm2 from membranes, which is tightly coupled to the proteasomal degradation of Orm2. Thereby, EGAD prevents the accumulation of Orm2 at the ER and in post-ER compartments and promotes the controlled de-repression of sphingolipid biosynthesis. Thus, the selective degradation of membrane proteins by EGAD contributes to proteostasis and lipid homeostasis in eukaryotic cells., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Fusarinine C (FSC) has recently been shown to be a promising and novel chelator for 89 Zr. Here, FSC has been further derivatized to optimize the complexation properties of FSC-based chelators for 89 Zr-labeling by introducing additional carboxylic groups. These were expected to improve the stability of 89 Zr-complexes by saturating the 8-coordination sphere of [ 89 Zr] Zr 4+ , and also to introduce functionalities suitable for conjugation to targeting vectors such as monoclonal antibodies. For proof of concept, succinic acid derivatization at the amine groups of FSC was carried out, resulting in FSC(succ)₂ and FSC(succ)₃. FSC(succ)₂ was further derivatized to FSC(succ)₂ AA by reacting with acetic anhydride (AA). The Zr 4+ complexation properties of these chelators were studied by reacting with ZrCl₄. Partition coefficient, protein binding, serum stability, acid dissociation, and transchelation studies of 89 Zr-complexes were carried out in vitro and the results were compared with those for 89 Zr-desferrioxamine B ([ 89 Zr]Zr-DFO) and 89 Zr-triacetylfusarinine C ([ 89 Zr]Zr-TAFC). The in vivo properties of [ 89 Zr]Zr-FSC(succ)₃ were further compared with [ 89 Zr]Zr-TAFC in BALB/c mice using micro-positron emission tomography/computer tomography (microPET/CT) imaging. Fusarinine C (succ)₂AA and FSC(succ)₃ were synthesized with satisfactory yields. Complexation with ZrCl₄ was achieved using a simple strategy resulting in high-purity Zr-FSC(succ)₂AA and Zr-FSC(succ)₃ with 1:1 stoichiometry. Distribution coefficients of 89 Zr-complexes revealed increased hydrophilic character compared to [ 89 Zr]Zr-TAFC. All radioligands showed high stability in phosphate buffered saline (PBS) and human serum and low protein-bound activity over a period of seven days. Acid dissociation and transchelation studies exhibited a range of in vitro stabilities following the order: [ 89 Zr]Zr-FSC(succ)₃ > [ 89 Zr]Zr-TAFC > [ 89 Zr]Zr-FSC(succ)₂AA >> [ 89 Zr]Zr-DFO. Biodistribution studies of [ 89 Zr]Zr-FSC(succ)₃ revealed a slower excretion pattern compared to [ 89 Zr]Zr-TAFC. In conclusion, [ 89 Zr]Zr-FSC(succ)₃ showed the best stability and inertness. The promising results obtained with [ 89 Zr]Zr-FSC(succ)₂AA highlight the potential of FSC(succ)₂ as a monovalent chelator for conjugation to targeted biomolecules, in particular, monoclonal antibodies.

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., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Arion vulgaris is a land-living European slug belonging to the gastropod clade of Stylommatophora. The species is known as an efficient pest organism in vegetable gardening and horticulture, which may in part be the consequence of its genetically based innate immunity, along with its high ability to withstand toxic metal stress by intracellular detoxification. Like many species of terrestrial snails, slugs possess a distinct capacity for Cd accumulation in their midgut gland, where the metal is stored and inactivated, conferring to these animals an increased metal tolerance. Although midgut gland Cd fractions in slugs have been shown to be variably allocated between different metal-binding protein pools, depending on the level of environmental metal contamination, a true metallothionein (MT) was so far never characterized from slugs. Instead, the Cd binding proteins identified so far were described as Metallothionein-like proteins (MTLPs). In the present study, the slug A. vulgaris was used as a model organism, in order to verify the presence of true MTs in experimentally metal-exposed slugs. We wanted to find out if these suggested slug MTs have similar metal binding properties and metal-selective features like those previously reported from helicid snails. To this aim, two MT isoform genes (AvMT1 and AvMT2) were characterized from midgut gland extracts and localized in the cells of this tissue. The AvMT1 and AvMT2 proteins were purified and partially sequenced, and their metal-binding features analysed after recombinant expression. Eventually, we wanted to understand if and by how much the metal binding features of the two MT isoforms of A. vulgaris may be related, owing to their reciprocal amino acid sequence similarities, to the binding properties of metal-specific MTs from terrestrial snails.

Positron emission tomography (PET) with radiolabelled peptide-based tracers has attracted great interest in oncology over the past decades. The success of imaging is closely related to sufficient uptake of the radiotracer in malignant tissue and for this sufficient biological half-life, particularly in the bloodstream, is mandatory. Fast enzymatic degradation during circulation leading to insufficient imaging abilities of peptide-based radioligands remains a major issue. The design of multimeric constructs, bearing multiple targeting moieties, has been widely applied to improve target interaction. This concept may also be applied to prolong the biological half-life of peptide-based radiopharmaceuticals as enzymatic degradation can result in formation of metabolites still capable to interact with the target binding site. In this study we aimed to identify such metabolites and therefore we utilized the siderophore-based bifunctional chelator fusarinine C (FSC) for the design of novel mono- and multimeric constructs, bearing minigastrin (MG) analogues as targeting moieties to address cholecystokinin-2 receptors (CCK2R) which are overexpressed in a variety of cancerous diseases and are well known for fast enzymatic degradation, particularly for truncated des-(Glu)5-MG members, such as MG11. FSC-based imaging probes were radiolabelled with gallium-68 and characterized in vitro (logD, protein binding, affinity and cell-uptake studies, stability and metabolite studies, as well as generation of corresponding metabolites by artificial enzymatic degradation) and in vivo (biodistribution in A431-CCK2R/A431-mock tumour xenografted BALB/c nude mice and stability in blood of living BALB/c mice and analysis of corresponding organ homogenates and urine to identify degradation products). In summary, multimerization was accompanied by partial improvement towards targeting abilities. Identified metabolites formed by artificial enzymatic cleavage of trimeric FSC-MG conjugates in vitro contained intact binding sequences for the receptor. Furthermore, the 68Ga-labelled trimers exhibiting increasing uptake of radioligand in tumour tissue over time and improved in vivo stability in blood samples of living animals of the trimers compared to corresponding mono- and dimers, strongly supporting our hypothesis., Competing Interests: The authors have declared that no competing interests exist.

Nucleosomes consisting of a short piece of deoxyribonucleic acid (DNA) wrapped around an octamer of histone proteins form the fundamental unit of chromatin in eukaryotes. Their role in DNA compaction comes with regulatory functions that impact essential genomic processes such as replication, transcription, and repair. The assembly of nucleosomes obeys a precise pathway in which tetramers of histones H3 and H4 bind to the DNA first to form tetrasomes, and two dimers of histones H2A and H2B are subsequently incorporated to complete the complex. As viable intermediates, we previously showed that tetrasomes can spontaneously flip between a left-handed and right-handed conformation of DNA-wrapping. To pinpoint the underlying mechanism, here we investigated the role of the H3-H3 interface for tetramer flexibility in the flipping process at the single-molecule level. Using freely orbiting magnetic tweezers, we studied the assembly and structural dynamics of individual tetrasomes modified at the cysteines close to this interaction interface by iodoacetamide (IA) in real time. While such modification did not affect the structural properties of the tetrasomes, it caused a 3-fold change in their flipping kinetics. The results indicate that the IA-modification enhances the conformational plasticity of tetrasomes. Our findings suggest that subnucleosomal dynamics may be employed by chromatin as an intrinsic and adjustable mechanism to regulate DNA supercoiling.

Signaling from lysosomes controls cellular clearance and energy metabolism. Lysosomal malfunction has been implicated in several pathologies, including neurodegeneration, cancer, infection, immunodeficiency, and obesity. Interestingly, many functions are dependent on the organelle position. Lysosomal motility requires the integration of extracellular and intracellular signals that converge on a competition between motor proteins that ultimately control lysosomal movement on microtubules. Here, we identify a novel upstream control mechanism of Arl8b-dependent lysosomal movement toward the periphery of the cell. We show that the C-terminal domain of lyspersin, a subunit of BLOC-1-related complex (BORC), is essential and sufficient for BORC-dependent recruitment of Arl8b to lysosomes. In addition, we establish lyspersin as the linker between BORC and late endosomal/lysosomal adaptor and mitogen activated protein kinase and mechanistic target of rapamycin activator (LAMTOR) complexes and show that epidermal growth factor stimulation decreases LAMTOR/BORC association, thereby promoting BORC- and Arl8b-dependent lysosomal centrifugal transport., (© 2017 Filipek et al.)

The LAMTOR [late endosomal and lysosomal adaptor and MAPK (mitogen-activated protein kinase) and mTOR (mechanistic target of rapamycin) activator] complex, also known as "Ragulator," controls the activity of mTOR complex 1 (mTORC1) on the lysosome. The crystal structure of LAMTOR consists of two roadblock/LC7 domain-folded heterodimers wrapped and apparently held together by LAMTOR1, which assembles the complex on lysosomes. In addition, the Rag guanosine triphosphatases (GTPases) associated with the pentamer through their carboxyl-terminal domains, predefining the orientation for interaction with mTORC1. In vitro reconstitution and experiments with site-directed mutagenesis defined the physiological importance of LAMTOR1 in assembling the remaining components to ensure fidelity of mTORC1 signaling. Functional data validated the effect of two short LAMTOR1 amino acid regions in recruitment and stabilization of the Rag GTPases., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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., (© 2016 American Association for Clinical Chemistry.)

Dysfunctional mitochondria contribute to the development of many diseases and pathological conditions through the excessive production of reactive oxygen species (ROS), and, where studied, ablation of p66Shc (p66) was beneficial. p66 translocates to the mitochondria and oxidizes cytochrome c to yield H 2 O 2 , which in turn initiates cell death. PKCβ-mediated phosphorylation of serine 36 in p66 has been implicated as a key regulatory step preceding mitochondrial translocation, ROS production, and cell death, and PKCβ thus may provide a target for therapeutic intervention. We performed a reassessment of PKCβ regulation of the oxidoreductase activity of p66. Although our experiments did not substantiate Ser 36 phosphorylation by PKCβ, they instead provided evidence for Ser 139 and Ser 213 as PKCβ phosphorylation sites regulating the pro-oxidant and pro-apoptotic function of p66. Mutation of another predicted PKCβ phosphorylation site also located in the phosphotyrosine binding domain, threonine 206, had no phenotype. Intriguingly, p66 with Thr 206 and Ser 213 mutated to glutamic acid showed a gain-of-function phenotype with significantly increased ROS production and cell death induction. Taken together, these data argue for a complex mechanism of PKCβ-dependent regulation of p66 activation involving Ser 139 and a motif surrounding Ser 213 ., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Nuclear factor kappa B (NF-κB) is an inducible transcription factor that plays critical roles in immune and stress responses and is often implicated in pathologies, including chronic inflammation and cancer. Although much has been learned about NF-κB-activating pathways, the specific repression of NF-κB is far less well understood. Here we identified the type I protein arginine methyltransferase 1 (PRMT1) as a restrictive factor controlling TNFα-induced activation of NF-κB. PRMT1 forms a cellular complex with NF-κB through direct interaction with the Rel homology domain of RelA. We demonstrate that PRMT1 methylates RelA at evolutionary conserved R30, located in the DNA-binding L1 loop, which is a critical residue required for DNA binding. Asymmetric R30 dimethylation inhibits the binding of RelA to DNA and represses NF-κB target genes in response to TNFα. Molecular dynamics simulations of the DNA-bound RelA:p50 predicted structural changes in RelA caused by R30 methylation or a mutation that interferes with the stability of the DNA-NF-κB complex. Our findings provide evidence for the asymmetric arginine dimethylation of RelA and unveil a unique mechanism controlling TNFα/NF-κB signaling.

Introduction: A variety of radiolabelled minigastrin analogues targeting the cholecystokinin 2 (CCK2) receptor were developed and compared in a concerted preclinical testing to select the most promising radiotracer for diagnosis and treatment of medullary thyroid carcinoma (MTC). DOTA-DGlu-DGlu-DGlu-DGlu-DGlu-DGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2 (CP04) after labelling with (111)In displayed excellent characteristics, such as high stability, receptor affinity, specific and persistent tumour uptake and low kidney retention in animal models. Therefore, it was selected for further clinical evaluation within the ERA-NET project GRAN-T-MTC. Here we report on the development of a pharmaceutical freeze-dried formulation of the precursor CP04 for a first multi-centre clinical trial with (111)In-CP04 in MTC patients., Materials and Methods: The kit formulation was optimised by adjustment of buffer, additives and radiolabelling conditions. Three clinical grade batches of a final kit formulation with two different amounts of peptide (10 or 50 μg) were prepared and radiolabelled with (111)In. Quality control and stability assays of both the kits and the resulting radiolabelled compound were performed by HPLC analysis., Results: Use of ascorbic acid buffer (pH4.5) allowed freeze-drying of the kit formulation with satisfactory pellet-formation. Addition of methionine and gentisic acid as well as careful selection of radiolabelling temperature was required to avoid extensive oxidation of the Met(11)-residue. Trace metal contamination, in particular Zn, was found to be a major challenge during the pharmaceutical filling process in particular for the 10 μg formulation. The final formulations contained 10 or 50 μg CP04, 25mg ascorbic acid, 0.5mg gentisic acid and 5mg L-methionine. The radiolabelling performed by incubation of 200-250 MBq (111)InCl3 at 90 °C for 15 min resulted in reproducible radiochemical purity (RCP) >94%. Kit-stability was proven for >6 months at +5 °C and at +25 °C. The radiolabelled product was stable for >4h at +25 °C., Conclusion: A kit formulation to prepare (111)In-CP04 for clinical application was developed, showing high stability of the kit as well as high RCP of the final product., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

The incorporation of CENP-A into centromeric chromatin is an essential prerequisite for kinetochore formation. Yet, the molecular mechanisms governing this process are surprisingly divergent in different organisms. While CENP-A loading mechanisms have been studied in some detail in mammals, there are still large gaps to our understanding of CENP-A/Cid loading pathways in Drosophila. Here, we report on the characterization and delineation of at least three different CENP-A preloading complexes in Drosophila. Two complexes contain the CENP-A chaperones CAL1, FACT and/or Caf1/Rbap48. Notably, we identified a novel complex consisting of the histone acetyltransferase Hat1, Caf1 and CENP-A/H4. We show that Hat1 is required for proper CENP-A loading into chromatin, since knock-down in S2 cells leads to reduced incorporation of newly synthesized CENP-A. In addition, we demonstrate that CENP-A/Cid interacts with the HAT1 complex via an N-terminal region, which is acetylated in cytoplasmic but not in nuclear CENP-A. Since Hat1 is not responsible for acetylation of CENP-A/Cid, these results suggest a histone acetyltransferase activity-independent escort function for Hat1. Thus, our results point toward intriguing analogies between the complex processing pathways of newly synthesized CENP-A and canonical histones., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

p66Shc-dependent ROS production contributes to many pathologies including ischemia/reperfusion injury (IRI) during solid organ transplantation. Inhibiting p66Shc activation may provide a novel therapeutic approach to prevent damage, which is poorly managed by antioxidants in vivo. Previous work suggested that pro-oxidant and a pro-apoptotic function of p66Shc required mitochondrial import, which depended on serine 36 phosphorylation. PKCß has been proposed as S36 kinase but cJun N-terminal kinases (JNKs) may also phosphorylate this residue. To simulate the early stages of ischemia/reperfusion (IR) we either used H2O2 treatment or hypoxia/reoxygenation (HR). As during reperfusion in vivo, we observed increased JNK and p38 activity in mouse embryonic fibroblasts (MEFs) and HL-1 cardiomyocytes along with significantly increased p66ShcS36 phosphorylation, ROS production and cell damage. Application of specific inhibitors caused a pronounced decrease in p66ShcS36 phosphorylation only in the case of JNK1/2. Moreover, S36 phosphorylation of recombinant p66Shc by JNK1 but not PKCß was demonstrated. We further confirmed JNK1/2-dependent regulation of p66ShcS36 phosphorylation, ROS production and cell death using JNK1/2 deficient MEFs. Finally, the low ROS phenotype of JNK1/2 knockout MEFs was reversed by the phosphomimetic p66ShcS36E mutant. Inhibiting JNK1/2-regulated p66Shc activation may thus provide a therapeutic approach for the prevention of oxidative damage.

The sodium-activated potassium channels Slick (Slo2.1, KCNT2) and Slack (Slo2.2, KCNT1) are paralogous channels of the Slo family of high-conductance potassium channels. Slick and Slack channels are widely distributed in the mammalian CNS and they play a role in slow afterhyperpolarization, generation of depolarizing afterpotentials and in setting and stabilizing the resting potential. In the present study we used a combined approach of (co)-immunoprecipitation studies, Western blot analysis, double immunofluorescence and mass spectrometric sequencing in order to investigate protein-protein interactions of the Slick and Slack channels. The data strongly suggest that Slick and Slack channels co-assemble into identical cellular complexes. Double immunofluorescence experiments revealed that Slick and Slack channels co-localize in distinct mouse brain regions. Moreover, we identified the small cytoplasmic protein beta-synuclein and the transmembrane protein 263 (TMEM 263) as novel interaction partners of both, native Slick and Slack channels. In addition, the inactive dipeptidyl-peptidase (DPP 10) and the synapse associated protein 102 (SAP 102) were identified as constituents of the native Slick and Slack channel complexes in the mouse brain. This study presents new insights into protein-protein interactions of native Slick and Slack channels in the mouse brain.

The degradation and recycling of cellular components is essential for cell growth and survival. Here we show how selective and non-selective lysosomal protein degradation pathways cooperate to ensure cell survival upon nutrient limitation. A quantitative analysis of starvation-induced proteome remodeling in yeast reveals comprehensive changes already in the first three hours. In this period, many different integral plasma membrane proteins undergo endocytosis and degradation in vacuoles via the multivesicular body (MVB) pathway. Their degradation becomes essential to maintain critical amino acids levels that uphold protein synthesis early during starvation. This promotes cellular adaptation, including the de novo synthesis of vacuolar hydrolases to boost the vacuolar catabolic activity. This order of events primes vacuoles for the efficient degradation of bulk cytoplasm via autophagy. Hence, a catabolic cascade including the coordinated action of the MVB pathway and autophagy is essential to enter quiescence to survive extended periods of nutrient limitation.

In this work, we evaluate the incorporation of an ultralow flow interface for coupling capillary electrophoresis (CE) and mass spectrometry (MS), in combination with reversed-phase high-pressure liquid chromatography (HPLC) fractionation as an alternate workflow for quantitative proteomics. Proteins, extracted from a SILAC (stable isotope labeling by amino acids in cell culture) labeled and an unlabeled yeast strain were mixed and digested enzymatically in solution. The resulting peptides were fractionated using RP-HPLC and analyzed by CE-MS yielding a total of 28 538 quantified peptides that correspond to 3 272 quantified proteins. CE-MS analysis was performed using a neutral capillary coating, providing the highest separation efficiency at ultralow flow conditions (<10 nL/min). Moreover, we were able to demonstrate that CE-MS is a powerful method for the identification of low-abundance modified peptides within the same sample. Without any further enrichment strategies, we succeeded in quantifying 1 371 phosphopeptides present in the CE-MS data set and found 49 phosphopeptides to be differentially regulated in the two yeast strains. Including acetylation, phosphorylation, deamidation, and oxidized forms, a total of 8 106 modified peptides could be identified in addition to 33 854 unique peptide sequences found. The work presented here shows the first quantitative proteomics approach that combines SILAC labeling with CE-MS analysis.

New biomarkers are needed to improve the specificity of prostate cancer detection and characterisation of individual tumors. In a proteomics profiling approach using MALDI-MS tissue imaging on frozen tissue sections, we identified discriminating masses. Imaging analysis of cancer, non-malignant benign epithelium and stromal areas of 15 prostatectomy specimens in a test and 10 in a validation set identified characteristic m/z peaks for each tissue type, e.g. m/z 10775 for benign epithelial, m/z 6284 and m/z 6657.5 for cancer and m/z 4965 for stromal tissue. A 10-fold cross-validation analysis showed highest discriminatory ability to separate tissue types for m/z 6284 and m/z 6657.5, both overexpressed in cancer, and a multicomponent mass peak cluster at m/z 10775-10797.4 overexpressed in benign epithelial tissue. ROC AUC values for these three masses ranged from 0.85 to 0.95 in the discrimination of malignant and non-malignant tissue. To identify the underlying proteins, prostate whole tissue extract was separated by nano-HPLC and subjected to MALDI TOF/TOF analysis. Proteins in fractions containing discriminatory m/z masses were identified by MS/MS analysis and candidate marker proteins subsequently validated by immunohistochemistry (IHC). Biliverdin reductase B (BLVRB) turned out to be overexpressed in PCa tissue., Biological Significance: In this study on cryosections of radical prostatectomies of prostate cancer patients, we performed a MALDI-MS tissue imaging analysis and a consecutive protein identification of significant m/z masses by nano-HPLC, MALDI TOF/TOF and MS/MS analysis. We identified BLVRB as a potential biomarker in the discrimination of PCa and benign tissue, also suggesting BVR as a feasible therapeutic target., (© 2013. Published by Elsevier B.V. All rights reserved.)

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.

LAMTOR3 (MP1) and LAMTOR2 (p14) form a heterodimer as part of the larger Ragulator complex that is required for MAPK and mTOR1 signaling from late endosomes/lysosomes. Here, we show that loss of LAMTOR2 (p14) results in an unstable cytosolic monomeric pool of LAMTOR3 (MP1). Monomeric cytoplasmic LAMTOR3 is rapidly degraded in a proteasome-dependent but lysosome-independent manner. Mutational analyses indicated that the turnover of the protein is dependent on ubiquitination of several lysine residues. Similarly, other Ragulator subunits, LAMTOR1 (p18), LAMTOR4 (c7orf59), and LAMTOR5 (HBXIP), are degraded as well upon the loss of LAMTOR2. Thus the assembly of the Ragulator complex is monitored by cellular quality control systems, most likely to prevent aberrant signaling at the convergence of mTOR and MAPK caused by a defective Ragulator complex.

N-chlorotaurine (NCT), the main representative of long-lived oxidants produced by granulocytes and monocytes, is known to exert broad-spectrum microbicidal activity. Here we show that NCT directly inactivates Shiga toxin 2 (Stx2), used as a model toxin secreted by enterohemorrhagic Escherichia coli (EHEC). Bacterial growth and Stx2 production were both inhibited by 2 mM NCT. The cytotoxic effect of Stx2 on Vero cells was removed by ≥5.5 mM NCT. Confocal microscopy and FACS analyses showed that the binding of Stx2 to human kidney glomerular endothelial cells was inhibited, and no NCT-treated Stx2 entered the cytosol. Mass spectrometry displayed oxidation of thio groups and aromatic amino acids of Stx2 by NCT. Therefore, long-lived oxidants may act as powerful tools of innate immunity against soluble virulence factors of pathogens. Moreover, inactivation of virulence factors may contribute to therapeutic success of NCT and novel analogs, which are in development as topical antiinfectives.

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.

Introduction: Lack of correlation between in vitro and in vivo stability is a general problem for the development of radiopeptides especially in the case of minigastrin derivatives for therapeutic applications. In this study, we compared the influence of experimental conditions on radiopeptide stability results in vitro using a model Minigastrin (MG) analogue labelled with Lu-177. Additionally, we attempted to characterize the main serum enzymatic cleavage sites by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) analysis., Methods: In vitro stability of a DOTA-minigastrin derivative ((177)Lu-DOTA-His-His-Glu-Ala-Tyr-Gly-Trp-NIe-Asp-Phe-NH(2)) was tested in serum, rat tissue homogenates and two different standardised enzymatic mixtures. Quantification of the metabolised radiopeptides at different time intervals was performed using reversed-phase high-performance liquid chromatography (RP-HPLC). Metabolites were characterised by MALDI-TOF-MS. Urine was collected after 15 min p.i. into the mice and compared with in vitro metabolites by RP-HPLC., Results: Faster degradation of the radiopeptide was found in blood in comparison with plasma and serum incubation and in components from rats faster than from human origin. Fast degradation was observed in kidney and liver homogenates as well as in standardised enzymatic mixtures, also revealing variations in the metabolic profile. In urine, no intact peptide was detected already 5 min post injection. MALDI-TOF-MS revealed major cleavage sites at the carboxy terminus of the peptide., Conclusion: Very variable results may be found when different kind of incubation media for testing radiopeptide stabilities is used. Serum incubation studies may overestimate stability; therefore, results should be interpreted with care and combined with alternative in vitro and in vivo investigations., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Context: Previous studies have implicated a deficiency in the inflammatory response in women who develop endometriosis. The specific immunological deficits have not been completely elucidated., Objective: Our objective was to identify differences in protein expression in serum that might shed light on the pathophysiology of endometriosis., Design and Setting: This cross-sectional study of women undergoing laparoscopy between 2003 and 2005 took place at a university medical center., Patients: Patients included consenting women age 18-49 yr undergoing surgery for pain and/or infertility or elective tubal ligation. Women with acute or chronic medical conditions were excluded., Intervention: Blood was collected preoperatively., Main Outcome Measure: Proteomic analysis of serum was done using two-dimensional difference gel electrophoresis., Results: We found 25 protein spots with a significant difference in abundance between women with endometriosis and controls, including acute-phase proteins and complement components. The abundance of vitamin D-binding protein was higher in all endometriosis pools by a factor of approximately 3 compared with the control pool (P < 0.02). Analysis of specific allele products using nano-scale liquid chromatography-electrospray ionization-mass spectrometry indicated that it was the GC*2 allele product that was in greater concentration in serum pools, as well as in single validation samples, in women with endometriosis (P = 0.006). In contrast to the GC*1 allele product, which is readily converted to a potent macrophage factor (Gc protein-derived macrophage-activating factor), the GC*2 allele product undergoes practically no such conversion., Conclusions: We speculate that the inability to sufficiently activate macrophages' phagocytotic function in those carrying the GC*2 polymorphism (more prevalent in endometriosis) may allow endometriotic tissues to implant in the peritoneal cavity. Future studies evaluating specific vitamin D-binding protein polymorphisms as a risk factor for endometriosis in larger populations of women are warranted.

We discuss the progress during the last 4 years in the analysis of viruses by electrophoresis in capillaries and microfluidic devices. The paper is the continuation of a review published in this journal in 2005 [Kremser, L., Blaas, D., Kenndler, E., Electrophoresis 2004, 25, 2282-2291]. Eighteen papers on the topic have appeared since; the majority deals with zone electrophoresis and three reports are on IEF. These methods have been applied to human rhinoviruses, poliovirus Semliki Forest virus, norovirus-like particles, and the two bacteriophages MS2 and T5. A main finding was that addition of detergents and salts to the BGEs are essential for the robustness of the CE analysis. Analyte detection was usually via UV absorbance but there are some examples where the viruses were rendered fluorescent via modification of the capsid proteins with reactive dyes and/or by non-covalent attachment of intercalating fluorescent compounds to the nucleic acids making up the viral genome. Interestingly, some viruses are permeable to small molecular mass components; this allows fluorescent dyes to diffuse into the intact virus where they attach to the nucleic acid. Release of a viral genome upon heating was also monitored by using similar methodologies. Interactions of viruses and subviral particles with antibodies, receptors, and receptor-decorated liposomes were investigated with CE methods, all by using a non-equilibrium approach (i.e. co-incubation of the components prior to CE separation). Viruses are multivalent (i.e. possess many identical surface-exposed patches) and most of them are composed of defined numbers of identical subunits. The high resolution of CE has been most remarkably demonstrated by the separation of stoichiometric complexes between virus and a distinct number of soluble recombinant receptors and revealed their concentration-dependent distribution.

The electrophoretic properties of boron cluster compounds were determined in water, methanol and ACN as solvents of the BGE and discussed based on the principles of ion migration. Two types of boron cluster compounds were investigated. One type consisted of derivatives of the nido-7,8-dicarbaundecaborate cluster, the other types are derivatized cobalt bis(dicarbollide) ions (COSANs) whose central cobalt atom is sandwiched by two 7,8-dicarbaundecaborate clusters. The BGE in all solvents was acetate/acetic acid buffer with pH 4.75 in water, 9.7 in methanol and 22.3 in ACN, respectively, at different ionic strength between 5 and 30 mM. The dependence of the mobility on ionic strength could not be explained by the theory of Debye, Hückel and Onsager, but good agreement was found upon considering an ion size parameter. Limiting mobilities were derived by curve fitting, and by the aid of the solvent viscosities the hydrodynamic radii of the analyte anions were calculated. They are between 0.25 and 0.48 nm, and were nearly independent of the solvent. Electrophoresis of the analytes in a BGE consisting of 6 mM perchloric acid in ACN allows the conclusion that the present boron cluster compounds behave as stronger acids than perchloric acid.

The atypical antipsychotic drug Ziprasidone was determined by capillary zone electrophoresis in pharmaceutical formulations. Extraction of the drug from the formulation consisted in a simple dissolution step with methanol as solvent, and enables determination of the drug without any interference from the excipients. It was found that at pH of the background electrolyte above 5 the peak of the drug exhibited a tailing, at pH 6 or higher even a disappearance of the peak in the electropherogram was observed. This behaviour was related to the concomitant reduction of the solubility of the drug in the background electrolyte upon deprotonation at higher pH. As a consequence, analyses were carried out with formate buffer, pH 3.0, and enabled run times of about 3 min. The method was validated in terms of stability, specificity, precision, accuracy, linearity, quantitation limits, and robustness, and was applied to the analysis of different commercial capsules.

Exploiting the advantages of miniaturization of analytical devices we worked out conditions for the analysis of viruses, subviral particles, and virus-receptor complexes on microfluidic chips. To allow for detection via laser-induced fluorescence, the viral capsids were labelled with the fluorescent dye Cy5. We analyzed human rhinovirus serotype 2 and subviral particles, followed the complexation of the virus with a synthetic fragment of the VLDL-receptor, and tracked the heat-induced conversion of intact virions into empty capsids. In contrast to fused silica capillaries, the glass micro-channels allowed for electrophoresis of the analytes without detergent, and analyses were accomplished within few tens of seconds. This opens the avenue towards the analytics of membrane-enveloped viruses and other biological assemblies that are not stable in the presence of detergent. The chip format has the additional advantage of containment and easy disposal, making it particularly attractive for the analysis of infectious material.

We report the analysis of human rhinovirus serotype 2 (HRV2) on a commercially available lab-on-a-chip instrument. Due to lack of sufficient native fluorescence, the proteinaceous capsid of HRV2 was labeled with Cy5 for detection by the red laser (lambda ex 630 nm) implemented in the instrument. On the microdevice, electrophoresis of the labeled virus was possible in a BGE without stabilizing detergents, which is in contrast to conventional CE; moreover, analysis times were drastically shortened to the few 10 s range. Resolution of the sample constituents (virions, a contaminant present in all virus preparations, and excess dye) was improved upon adaptation of the separation conditions, mainly by adjusting the SDS concentration of the BGE. Purity of fractions from size-exclusion chromatography after labeling of virus was assessed, and affinity complex formation of the labeled virus with various recombinant very-low-density lipoprotein receptor derivatives differing in the number of concatenated V3 ligand binding repeats was monitored. Virus analysis on microchip devices is of particular interest for experiments with infectious material because of easy containment and disposal of samples. Thus, the employment of microchip devices in routine analysis of viruses appears to be exceptionally attractive.

The different electrophoretic behavior of the members of two groups of proteins with respect to the absence or presence of detergent additives in the BGE was explored. Recombinant soluble concatemers of repeat 3 of the very low density lipoprotein (VLDL)-receptor fused at their N-terminus to maltose-binding protein (MBP) exhibited different electrophoretic mobilities in borate buffer (pH 8.3) in the absence and in the presence of dodecyl-PEG ether (D-PEG). This enabled the separation of the receptor fragments from MBP after enzymatic cleavage. In the presence of SDS, the mobilities of all proteins approached the same values with increase in detergent concentrations. In contrast, viral capsid proteins of a human rhinovirus (HRV) exhibited different migration in the presence of the additive. For the receptor proteins, extreme apparent high plate numbers were observed when the SDS concentration in the sample and the separation buffer differed. This effect might be erroneously interpreted as a high efficiency. However, it is due to the conductivity boundaries caused by the sample and leads to a total loss of separation.