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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. [...]

Abstract Background Histone H1 is an important constituent of chromatin, and is involved in regulation of its structure. During the cell cycle, chromatin becomes locally decondensed in S phase, highly condensed during metaphase, and again decondensed before re-entry into G1. This has been connected to increasing phosphorylation of H1 histones through the cell cycle. However, many of these experiments have been performed using cell-synchronization techniques and cell cycle-arresting drugs. In this study, we investigated the H1 subtype composition and phosphorylation pattern in the cell cycle of normal human activated T cells and Jurkat T-lymphoblastoid cells by capillary electrophoresis after sorting of exponentially growing cells into G1, S and G2/M populations. Results We found that the relative amount of H1.5 protein increased significantly after T-cell activation. Serine phosphorylation of H1 subtypes occurred to a large extent in late G1 or early S phase in both activated T cells and Jurkat cells. Furthermore, our data confirm that the H1 molecules newly synthesized during S phase achieve a similar phosphorylation pattern to the previous ones. Jurkat cells had more extended H1.5 phosphorylation in G1 compared with T cells, a difference that can be explained by faster cell growth and/or the presence of enhanced H1 kinase activity in G1 in Jurkat cells. Conclusion Our data are consistent with a model in which a major part of interphase H1 phosphorylation takes place in G1 or early S phase. This implies that H1 serine phosphorylation may be coupled to changes in chromatin structure necessary for DNA replication. In addition, the increased H1 phosphorylation of malignant cells in G1 may be affecting the G1/S transition control and enabling facilitated S-phase entry as a result of relaxed chromatin condensation. Furthermore, increased H1.5 expression may be coupled to the proliferative capacity of growth-stimulated T cells.

Abstract Background Covalent histone modifications are central to all DNA-dependent processes. Modifications of histones H3 and H4 are becoming well characterised, but knowledge of how H2A modifications regulate chromatin dynamics and gene expression is still very limited. Results To understand the function of H2A modifications, we performed a systematic analysis of the histone H2A methylation status. We identified and functionally characterised two new methylation sites in H2A: R11 (H2AR11) and R29 (H2AR29). Using an unbiased biochemical approach in combination with candidate assays we showed that protein arginine methyltransferase (PRMT) 1 and PRMT6 are unique in their ability to catalyse these modifications. Importantly we found that H2AR29me2 is specifically enriched at genes repressed by PRMT6, implicating H2AR29me2 in transcriptional repression. Conclusions Our data establishes R11 and R29 as new arginine methylation sites in H2A. We identified the specific modifying enzymes involved, and uncovered a novel functional role of H2AR29me2 in gene silencing in vivo. Thus this work reveals novel insights into the function of H2A methylation and in the mechanisms of PRMT6-mediated transcriptional repression.

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.)

Aspergillus fumigatus is the predominant mould pathogen for humans. Adaption to host-imposed iron limitation has previously been demonstrated to be essential for its virulence. [2Fe-2S] clusters are crucial as cofactors of several metabolic pathways and mediate cytosolic/nuclear iron sensing in fungi including A. fumigatus . [2Fe-2S] cluster trafficking has been shown to involve BolA family proteins in both mitochondria and the cytosol/nucleus. Interestingly, both A. fumigatus homologues, termed Bol1 and Bol3, possess mitochondrial targeting sequences, suggesting the lack of cytosolic/nuclear versions. Here, we show by the combination of mutational, proteomic and fluorescence microscopic analyses that expression of the Bol3 encoding gene leads to dual localization of gene products to mitochondria and the cytosol/nucleus via alternative translation initiation downstream of the mitochondrial targeting sequence, which appears to be highly conserved in various Aspergillus species. Lack of either mitochondrial Bol1 or Bol3 was phenotypically inconspicuous while lack of cytosolic/nuclear Bol3 impaired growth during iron limitation but not iron sensing which indicates a particular importance of [2Fe-2S] cluster trafficking during iron limitation. Remarkably, cytosolic/nuclear Bol3 differs from the mitochondrial version only by N-terminal acetylation, a finding that was only possible by mutational hypothesis testing.

Excessive fibrous capsule formation around silicone mammary implants (SMI) involves immune reactions to silicone. Capsular fibrosis, a common SMI complication linked to host responses, worsens with specific implant topographies. Our study with 10 patients investigated intra- and inter-individually, reduced surface roughness effects on disease progression, wound responses, chronic inflammation, and capsular composition. The results illuminate the significant impact of surface roughness on acute inflammatory responses, fibrinogen accumulation, and the subsequent fibrotic cascade. The reduction of surface roughness to an average roughness of 4 μm emerges as a promising approach for mitigating detrimental immune reactions, promoting healthy wound healing, and curbing excessive fibrosis. The identified proteins adhering to rougher surfaces shed light on potential mediators of pro-inflammatory and pro-fibrotic processes, further emphasizing the need for meticulous consideration of surface design. The composition of the implant capsule and the discovery of intracapsular HSP60 expression highlight the intricate web of stress responses and immune activation that can impact long-term tissue outcomes., Competing Interests: The 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., (Copyright © 2024 Schoberleitner, Faserl, Tripp, Pechriggl, Sigl, Brunner, Zelger, Hermann-Kleiter, Baier, Steinkellner, Sarg, Egle, Brunner and Wolfram.)

Breast cancer is the most common cancer in women globally, often necessitating mastectomy and subsequent breast reconstruction. Silicone mammary implants (SMIs) play a pivotal role in breast reconstruction, yet their interaction with the host immune system and microbiome remains poorly understood. This study investigates the impact of SMI surface topography on host antimicrobial responses, wound proteome dynamics, and microbial colonization. Biological samples were collected from ten human patients undergoing breast reconstruction with SMIs. Mass spectrometry profiles were analyzed for acute and chronic wound proteomes, revealing a nuanced interplay between topography and antimicrobial response proteins. 16S rRNA sequencing assessed microbiome dynamics, unveiling topography-specific variations in microbial composition. Surface topography alterations influenced wound proteome composition. Microbiome analysis revealed heightened diversity around rougher SMIs, emphasizing topography-dependent microbial invasion. In vitro experiments confirmed staphylococcal adhesion, growth, and biofilm formation on SMI surfaces, with increased texture correlating positively with bacterial colonization. This comprehensive investigation highlights the intricate interplay between SMI topography, wound proteome dynamics, and microbial transmission. The findings contribute to understanding host-microbe interactions on SMI surfaces, essential for optimizing clinical applications and minimizing complications in breast reconstruction.

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.)

Friedreich's ataxia (FRDA) is a severe multisystemic disorder caused by a deficiency of the mitochondrial protein frataxin. While some aspects of FRDA pathology are developmental, the causes underlying the steady progression are unclear. The inaccessibility of key affected tissues to sampling is a main hurdle. Skeletal muscle displays a disease phenotype and may be sampled in vivo to address open questions on FRDA pathophysiology. Thus, we performed a quantitative mass spectrometry-based proteomics analysis in gastrocnemius skeletal muscle biopsies from genetically confirmed FRDA patients ( n = 5) and controls. Obtained data files were processed using Proteome Discoverer and searched by Sequest HT engine against a UniProt human reference proteome database. Comparing skeletal muscle proteomics profiles between FRDA and controls, we identified 228 significant differentially expressed (DE) proteins, of which 227 were downregulated in FRDA. Principal component analysis showed a clear separation between FRDA and control samples. Interactome analysis revealed clustering of DE proteins in oxidative phosphorylation, ribosomal elements, mitochondrial architecture control, and fission/fusion pathways. DE findings in the muscle-specific proteomics suggested a shift toward fast-twitching glycolytic fibers. Notably, most DE proteins (169/228, 74%) are target of the transcription factor nuclear factor-erythroid 2. Our data corroborate a mitochondrial biosignature of FRDA, which extends beyond a mere oxidative phosphorylation failure. Skeletal muscle proteomics highlighted a derangement of mitochondrial architecture and maintenance pathways and a likely adaptive metabolic shift of contractile proteins. The present findings are relevant for the design of future therapeutic strategies and highlight the value of skeletal muscle-omics as disease state readout in FRDA., Competing Interests: The 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., (Copyright © 2023 Indelicato, Faserl, Amprosi, Nachbauer, Schneider, Wanschitz, Sarg and Boesch.)

We recently identified protein kinase N1 (PKN1) as a negative gatekeeper of neuronal AKT protein kinase activity during postnatal cerebellar development. The developing cerebellum is specifically vulnerable to hypoxia-ischemia (HI), as it occurs during hypoxic-ischemic encephalopathy, a condition typically caused by oxygen deprivation during or shortly after birth. In that context, activation of the AKT cell survival pathway has emerged as a promising new target for neuroprotective interventions. Here, we investigated the role of PKN1 in an in vitro model of HI, using postnatal cerebellar granule cells (Cgc) derived from Pkn1 wildtype and Pkn1 -/- mice. Pkn1 -/- Cgc showed significantly higher AKT phosphorylation, resulting in reduced caspase-3 activation and improved survival after HI. Pkn1 -/- Cgc also showed enhanced axonal outgrowth on growth-inhibitory glial scar substrates, further pointing towards a protective phenotype of Pkn1 knockout after HI. The specific PKN1 phosphorylation site S374 was functionally relevant for the enhanced axonal outgrowth and AKT interaction. Additionally, PKN1pS374 shows a steep decrease during cerebellar development. In summary, we demonstrate the pathological relevance of the PKN1-AKT interaction in an in vitro HI model and establish the relevant PKN1 phosphorylation sites, contributing important information towards the development of specific PKN1 inhibitors.

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.

The etiology of exaggerated fibrous capsule formation around silicone mammary implants (SMI) is multifactorial but primarily induced by immune mechanisms towards the foreign material silicone. The aim of this work was to understand the disease progression from implant insertion and immediate tissue damage response reflected in (a) the acute wound proteome and (b) the adsorption of chronic inflammatory wound proteins at implant surfaces. An intraindividual relative quantitation TMT-liquid chromatography-tandem mass spectrometry approach was applied to the profile wound proteome formed around SMI in the first five days post-implantation. Compared to plasma, the acute wound profile resembled a more complex composition comprising plasma-derived and locally differentially expressed proteins (DEPs). DEPs were subjected to a functional enrichment analysis, which revealed the dysregulation of signaling pathways mainly involved in immediate inflammation response and ECM turnover. Moreover, we found time-course variations in protein enrichment immediately post-implantation, which were adsorbed to SMI surfaces after 6-8 months. Characterization of the expander-adhesive proteome by a label-free approach uncovered a long-term adsorbed acute wound and the fibrosis-associated proteome. Our findings propose a wound biomarker panel for the early detection and diagnosis of excessive fibrosis that could potentially broaden insights into the characteristics of fibrotic implant encapsulation.

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: Platelets (thrombocytes) are small anuclear cells that play an important role in blood clotting. They are activated and dysfunctional in brain disorders, such as Alzheimer's disease (AD) and depression. Platelets express the amyloid-precursor protein (APP) and release beta-amyloid40 into the blood. Recent evidence reports that platelets also express the microtubule-associated protein tau. In this study, we further characterized the molecular appearance of tau and examined its alterations in patients with neurocognitive impairment., Methods: Platelets were isolated from patients with AD, mild cognitive impairment (MCI) or depression and compared to healthy controls. Subsequently, FACS analysis was employed to characterize platelets for platelet surface P-selectin (CD62P). In order to enhance the detection levels, samples were pooled (15 samples per group) and analyzed by Lumipulse Assay, Western blots, and mass spectrometry., Results: Tau is expressed in human platelets and tau levels were decreased in platelets isolated from patients with AD and depression. Additionally, phospho-tau-181 was slightly increased in patients with depression. We show that tau is highly fragmented (20-40 kDa) in the platelet extracts using Western blot analysis. The mass spectrometry data did not show a clear identification of tau in the pooled platelet samples., Conclusions: Our data reveal that tau is found in platelets, possibly in a highly fragmented form. Tau levels may be used as a potential diagnostic approach to differentiate AD and depression from healthy controls., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s). Published by IMR Press.)

Background: In physoclist fishes filling of the swimbladder requires acid secretion of gas gland cells to switch on the Root effect and subsequent countercurrent concentration of the initial gas partial pressure increase by back-diffusion of gas molecules in the rete mirabile. It is generally assumed that the rete mirabile functions as a passive exchanger, but a detailed analysis of lactate and water movements in the rete mirabile of the eel revealed that lactate is diffusing back in the rete. In the present study we therefore test the hypothesis that expression of transport proteins in rete capillaries allows for back-diffusion of ions and metabolites, which would support the countercurrent concentrating capacity of the rete mirabile. It is also assumed that in silver eels, the migratory stage of the eel, the expression of transport proteins would be enhanced., Results: Analysis of the transcriptome and of the proteome of rete mirabile tissue of the European eel revealed the expression of a large number of membrane ion and metabolite transport proteins, including monocarboxylate and glucose transport proteins. In addition, ion channel proteins, Ca 2+ -ATPase, Na + /K + -ATPase and also F 1 F 0 -ATP synthase were detected. In contrast to our expectation in silver eels the expression of these transport proteins was not elevated as compared to yellow eels. A remarkable number of enzymes degrading reactive oxygen species (ROS) was detected in rete capillaries., Conclusions: Our results reveal the expression of a large number of transport proteins in rete capillaries, so that the back diffusion of ions and metabolites, in particular lactate, may significantly enhance the countercurrent concentrating ability of the rete. Metabolic pathways allowing for aerobic generation of ATP supporting secondary active transport mechanisms are established. Rete tissue appears to be equipped with a high ROS defense capacity, preventing damage of the tissue due to the high oxygen partial pressures generated in the countercurrent system., (© 2021. The Author(s).)

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Dehydration of cells by acute hyperosmotic stress has profound effects upon cell structure and function. Interphase chromatin and mitotic chromosomes collapse ("congelation"). HL-60/S4 cells remain ~100% viable for, at least, 1 hour, exhibiting shrinkage to ~2/3 their original volume, when placed in 300mM sucrose in tissue culture medium. Fixed cells were imaged by immunostaining confocal and STED microscopy. At a "global" structural level (μm), mitotic chromosomes congeal into a residual gel with apparent (phase) separations of Ki67, CTCF, SMC2, RAD21, H1 histones and HMG proteins. At an "intermediate" level (sub-μm), radial distribution analysis of STED images revealed a most probable peak DNA density separation of ~0.16 μm, essentially unchanged by hyperosmotic stress. At a "local" structural level (~1-2 nm), in vivo crosslinking revealed essentially unchanged crosslinked products between H1, HMG and inner histones. Hyperosmotic cellular stress is discussed in terms of concepts of mitotic chromosome structure and liquid-liquid phase separation.

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., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Selectable markers are indispensable for genetic engineering, yet their number and variety are limited. Most selection procedures for prototrophic cells rely on the introduction of antibiotic resistance genes. New minimally invasive tools are needed to facilitate sophisticated genetic manipulations. Here, we characterized three endogenous genes in the human fungal pathogen Aspergillus fumigatus for their potential as markers for targeted genomic insertions of DNAs of interest (DOIs). Since these genes are involved in uptake and metabolization of pyrimidines, resistance to the toxic effects of prodrugs 5-fluorocytosine and 5-fluorouracil can be used to select successfully integrated DOIs. We show that DOI integration, resulting in the inactivation of these genes, caused no adverse effects with respect to nutrient requirements, stress resistance, or virulence. Beside the individual use of markers for site-directed integration of reporter cassettes, including the 17-kb penicillin biosynthetic cluster, we demonstrate their sequential use by inserting three genes encoding fluorescent proteins into a single strain for simultaneous multicolor localization microscopy. In addition to A. fumigatus , we validated the applicability of this novel toolbox in Penicillium chrysogenum and Fusarium oxysporum Enabling multiple targeted insertions of DOIs without the necessity for exogenous markers, this technology has the potential to significantly advance genetic engineering. IMPORTANCE This work reports the discovery of a novel genetic toolbox comprising multiple, endogenous selectable markers for targeted genomic insertions of DNAs of interest (DOIs). Marker genes encode proteins involved in 5-fluorocytosine uptake and pyrimidine salvage activities mediating 5-fluorocytosine deamination as well as 5-fluorouracil phosphoribosylation. The requirement for their genomic replacement by DOIs to confer 5-fluorocytosine or 5-fluorouracil resistance for transformation selection enforces site-specific integrations. Due to the fact that the described markers are endogenously encoded, there is no necessity for the exogenous introduction of commonly employed markers such as auxotrophy-complementing genes or antibiotic resistance cassettes. Importantly, inactivation of the described marker genes had no adverse effects on nutrient requirements, growth, or virulence of the human pathogen Aspergillus fumigatus Given the limited number and distinct types of selectable markers available for the genetic manipulation of prototrophic strains such as wild-type strains, we anticipate that the proposed methodology will significantly advance genetic as well as metabolic engineering of fungal species., (Copyright © 2020 Birštonas et al.)

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., (© 2018 The Authors. Electrophoresis published by Wiley-VCH Verlag GmbH & Co. KGaA.)

In this study we demonstrate the potential of sequential injection of samples in capillary electrophoresis-mass spectrometry 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 so that every 10 min 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 4800 proteins. These numbers could be increased to 62 000 peptides and more than 6100 proteins identified, when performing three experiments analyzing a total of 60 fractions, all within 12.5 h. We found that the electrophoretic mobility of peptides can be used to trace back peptides and assign them to the fraction they originate from., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.)

Serine protease inhibitors (serpin) have therapeutic potential in a variety of pathogenic processes, ranging from thrombosis and altered immune response to liver cirrhosis. To investigate the physiological effects of protein C inhibitor (PCI, serpinA5), its gene was inactivated in a mouse model, resulting in male infertility. In the present report, 2D differential gel electrophoresis was utilized to investigate the molecular mechanisms for PCI involvement in male reproduction. Comparing the testes proteomes of three PCI-knockout mice with three wild types demonstrated similar patterns with the exception of a massive upregulation of prostaglandin reductase 1 (tenfold; p < 0.002) and the complete shifts in the molecular weights of serpinA1C and serpinA3K. All these PCI-dependent proteome changes were immunologically verified. Unbiased proteome analysis indicated that inactivation of serpinA5 strongly influenced both the protein species pattern of other A-clade serpins as well as prostaglandin metabolism in the testes., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The polyene antifungal amphotericin B (AmB) is widely used to treat life-threatening fungal infections. Even though AmB resistance is exceptionally rare in fungi, most Aspergillus terreus isolates exhibit an intrinsic resistance against the drug in vivo and in vitro. Heat shock proteins perform a fundamental protective role against a multitude of stress responses, thereby maintaining protein homeostasis in the organism. In this study, we elucidated the role of heat shock protein 70 (Hsp70) family members and compared resistant and susceptible A. terreus clinical isolates. The upregulation of cytoplasmic Hsp70 members at the transcriptional as well as translational levels was significantly higher with AmB treatment than without AmB treatment, particularly in resistant A. terreus isolates, thereby indicating a role of Hsp70 proteins in the AmB response. We found that Hsp70 inhibitors considerably increased the susceptibility of resistant A. terreus isolates to AmB but exerted little impact on susceptible isolates. Also, in in vivo experiments, using the Galleria mellonella infection model, cotreatment of resistant A. terreus strains with AmB and the Hsp70 inhibitor pifithrin-μ resulted in significantly improved survival compared with that achieved with AmB alone. Our results point to an important mechanism of regulation of AmB resistance by Hsp70 family members in A. terreus and suggest novel drug targets for the treatment of infections caused by resistant fungal isolates., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of β-structure in the phosphorylated samples, concomitant to a decrease in α-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl2 and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Protein C inhibitor (PCI) is a serpin with broad protease reactivity. It binds glycosaminoglycans and certain phospholipids that can modulate its inhibitory activity. PCI can penetrate through cellular membranes via binding to phosphatidylethanolamine. The exact mechanism of PCI internalization and the intracellular role of the serpin are not well understood. Here we showed that testisin, a glycosylphosphatidylinositol-anchored serine protease, cleaved human PCI and mouse PCI (mPCI) at their reactive sites as well as at sites close to their N terminus. This cleavage was observed not only with testisin in solution but also with cell membrane-anchored testisin on U937 cells. The cleavage close to the N terminus released peptides rich in basic amino acids. Synthetic peptides corresponding to the released peptides of human PCI (His(1)-Arg(11)) and mPCI (Arg(1)-Ala(18)) functioned as cell-penetrating peptides. Because intact mPCI but not testisin-cleaved mPCI was internalized by Jurkat T cells, a truncated mPCI mimicking testisin-cleaved mPCI was created. The truncated mPCI lacking 18 amino acids at the N terminus was not taken up by Jurkat T cells. Therefore our model suggests that testisin or other proteases could regulate the internalization of PCI by removing its N terminus. This may represent one of the mechanisms regulating the intracellular functions of PCI., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Chicken erythrocyte nuclei were digested with micrococcal nuclease and fractionated by centrifugation in low-salt buffer into soluble and insoluble fractions. Post-translational modifications of the purified linker histones of both fractions were analyzed by LC-ESI-MS/MS. All six histone H1 subtypes (H1.01, H1.02, H1.03, H1.10, H1.1L and H1.1R) and histone H5 were identified. Mass spectrometry analysis enabled the identification of a wide range of PTMs, including N(α)-terminal acetylation, acetylation, formylation, phosphorylation and oxidation. A total of nine new modifications in chicken linker histones were mapped, most of them located in the N-terminal and globular domains. Relative quantification of the modified peptides showed that linker histone PTMs were differentially distributed among both chromatin fractions, suggesting their relevance in the regulation of chromatin structure. The analysis of our results combined with previously reported data for chicken and some mammalian species showed that most of the modified positions were conserved throughout evolution, highlighting their importance in specific linker histone functions and epigenetics., Biological Significance: Post-translational modifications of linker histones could have a role in the regulation of gene expression through the modulation of chromatin higher-order structure and chromatin remodeling. Finding new PTMs in linker histones is the first step to elucidate their role in the histone code. In this manuscript we report nine new post-translational modifications of the linker histones from chicken erythrocytes, one in H5 and eight in the H1 subtypes. Chromatin fractionated by centrifugation in low-salt buffer resulted in two fractions with different contents and compositions of linker histones and enriched in specific core histone PTMs. Of particular interest is the fact that linker histone PTMs were differentially distributed in both chromatin fractions, suggesting specific functions. Future studies are needed to establish the interplay between PTMs of linker and core histones in order to fully understand chromatin regulation. A protein sequence alignment summarizing the PTMs found to date in chicken, mouse, rat and humans showed that, while many of the modified positions were conserved between these species, the type of modification often varied depending on the species or the cellular type. This finding suggests an important role for the PTMs in the regulation of linker histone functions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The percent identity matrices of two sequence multiple alignments between linker histones from chicken and mammalian species are described. Linker histone protein sequences for chicken, mouse, rat and humans, available on public databases were used. This information is related to the research article entitled "Identification of novel post-translational modifications in linker histones from chicken erythrocytes"published in the Journal of Proteomics [1].

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., (© 2013.)

Nogo-A is the largest isoform of the Nogo/RTN4 (reticulon 4) proteins and has been characterized as a major myelin-associated inhibitor of regenerative nerve growth in the adult CNS (central nervous system). Apart from the myelin sheath, Nogo-A is expressed at high levels in principal neurons of the CNS. The specificity of Nogo-A resides in its central domain, NiG. We identified Apg-1, a member of the stress-induced Hsp110 (heat-shock protein of 110 kDa) family, as a novel interactor of NiG/Nogo-A. The interaction is selective because Apg-1 interacts with Nogo-A/RTN4-A, but not with RTN1-A, the closest paralogue of Nogo-A. Conversely, Nogo-A binds to Apg-1, but not to Apg-2 or Hsp105, two other members of the Hsp110 family. We characterized the Nogo-A-Apg-1 interaction by affinity precipitation, co-immunoprecipitation and proximity ligation assay, using primary hippocampal neurons derived from Nogo-deficient mice. Under conditions of hypoxic and oxidative stress we found that Nogo-A and Apg-1 were tightly co-regulated in hippocampal neurons. Although both proteins were up-regulated under hypoxic conditions, their expression levels were reduced upon the addition of hydrogen peroxide. Taken together, we suggest that Nogo-A is closely involved in the neuronal response to hypoxic and oxidative stress, an observation that may be of relevance not only in stroke-induced ischaemia, but also in neuroblastoma formation.

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.

RTN1A is a reticulon protein with predominant localization in the endoplasmic reticulum (ER). It was previously shown that RTN1A is expressed in neurons of the mammalian central nervous system but functional information remains sparse. To elucidate the neuronal function of RTN1A, we chose to focus our investigation on identifying possible novel binding partners specifically interacting with the unique N-terminus of RTN1A. Using a nonbiased approach involving GST pull-downs and MS analysis, we identified the intracellular calcium release channel ryanodine receptor 2 (RyR2) as a direct binding partner of RTN1A. The RyR2 binding site was localized to a highly conserved 150-amino acid residue region. RTN1A displays high preference for RyR2 binding in vitro and in vivo and both proteins colocalize in hippocampal neurons and Purkinje cells. Moreover, we demonstrate the precise subcellular localization of RTN1A in Purkinje cells and show that RTN1A inhibits RyR channels in [(3)H]ryanodine binding studies on brain synaptosomes. In a functional assay, RTN1A significantly reduced RyR2-mediated Ca(2+) oscillations. Thus, RTN1A and RyR2 might act as functional partners in the regulation of cytosolic Ca(2+) dynamics the in neurons., (Copyright © 2013 Elsevier B.V. All rights reserved.)