A Proteomic Study on the Membrane Protein Fraction of T Cells Confirms High Substrate Selectivity for the ER Translocation Inhibitor Cyclotriazadisulfonamide.

Cyclotriazadisulfonamide (CADA) inhibits the cotranslational translocation of type I integral membrane protein human CD4 (huCD4) across the endoplasmic reticulum in a signal peptide (SP)-dependent way. Previously, sortilin was identified as a secondary substrate for CADA but showed reduced CADA sensitivity as compared with huCD4. Here, we performed a quantitative proteomic study on the crude membrane fraction of human T-cells to analyze how many proteins are sensitive to CADA. To screen for these proteins, we employed stable isotope labeling by amino acids in cell culture technique in combination with quantitative MS on CADA-treated human T-lymphoid SUP-T1 cells expressing high levels of huCD4. In line with our previous reports, our current proteomic analysis (data available via ProteomeXchange with identifier PXD027712) demonstrated that only a very small subset of proteins is depleted by CADA. Our data also confirmed that cellular expression of both huCD4 and sortilin are affected by CADA treatment of SUP-T1 cells. Furthermore, three additional targets for CADA are identified, namely, endoplasmic reticulum lectin 1 (ERLEC1), inactive tyrosine-protein kinase 7 (PTK7), and DnaJ homolog subfamily C member 3 (DNAJC3). Western blot and flow cytometry analysis of ERLEC1, PTK7, and DNAJC3 protein expression validated susceptibility of these substrates to CADA, although with varying degrees of sensitivity. Additional cell-free in vitro translation/translocation data demonstrated that the new substrates for CADA carry cleavable SPs that are targets for the cotranslational translocation inhibition exerted by CADA. Thus, our quantitative proteomic analysis demonstrates that ERLEC1, PTK7, and DNAJC3 are validated additional substrates of CADA; however, huCD4 remains the most sensitive integral membrane protein for the endoplasmic reticulum translocation inhibitor CADA. Furthermore, to our knowledge, CADA is the first compound that specifically interferes with only a very small subset of SPs and does not affect signal anchor sequences.

Flexible open conformation of the AP-3 complex explains its role in cargo recruitment at the Golgi.

Vesicle formation at endomembranes requires the selective concentration of cargo by coat proteins. Conserved adapter protein complexes at the Golgi (AP-3), the endosome (AP-1), or the plasma membrane (AP-2) with their conserved core domain and flexible ear domains mediate this function. These complexes also rely on the small GTPase Arf1 and/or specific phosphoinositides for membrane binding. The structural details that influence these processes, however, are still poorly understood. Here we present cryo-EM structures of the full-length stable 300 kDa yeast AP-3 complex. The structures reveal that AP-3 adopts an open conformation in solution, comparable to the membrane-bound conformations of AP-1 or AP-2. This open conformation appears to be far more flexible than AP-1 or AP-2, resulting in compact, intermediate, and stretched subconformations. Mass spectrometrical analysis of the cross-linked AP-3 complex further indicates that the ear domains are flexibly attached to the surface of the complex. Using biochemical reconstitution assays, we also show that efficient AP-3 recruitment to the membrane depends primarily on cargo binding. Once bound to cargo, AP-3 clustered and immobilized cargo molecules, as revealed by single-molecule imaging on polymer-supported membranes. We conclude that its flexible open state may enable AP-3 to bind and collect cargo at the Golgi and could thus allow coordinated vesicle formation at the trans-Golgi upon Arf1 activation.

The glucose-sensing transcription factor ChREBP is targeted by proline hydroxylation.

Cellular energy demands are met by uptake and metabolism of nutrients like glucose. The principal transcriptional regulator for adapting glycolytic flux and downstream pathways like de novo lipogenesis to glucose availability in many cell types is carbohydrate response element-binding protein (ChREBP). ChREBP is activated by glucose metabolites and post-translational modifications, inducing nuclear accumulation and regulation of target genes. Here we report that ChREBP is modified by proline hydroxylation at several residues. Proline hydroxylation targets both ectopically expressed ChREBP in cells and endogenous ChREBP in mouse liver. Functionally, we found that specific hydroxylated prolines were dispensable for protein stability but required for the adequate activation of ChREBP upon exposure to high glucose. Accordingly, ChREBP target gene expression was rescued by re-expressing WT but not ChREBP that lacks hydroxylated prolines in ChREBP-deleted hepatocytes. Thus, proline hydroxylation of ChREBP is a novel post-translational modification that may allow for therapeutic interference in metabolic diseases.

Structural changes of TasA in biofilm formation of Bacillus subtilis.

Microorganisms form surface-attached communities, termed biofilms, which can serve as protection against host immune reactions or antibiotics. Bacillus subtilis biofilms contain TasA as major proteinaceous component in addition to exopolysaccharides. In stark contrast to the initially unfolded biofilm proteins of other bacteria, TasA is a soluble, stably folded monomer, whose structure we have determined by X-ray crystallography. Subsequently, we characterized in vitro different oligomeric forms of TasA by NMR, EM, X-ray diffraction, and analytical ultracentrifugation (AUC) experiments. However, by magic-angle spinning (MAS) NMR on live biofilms, a swift structural change toward only one of these forms, consisting of homogeneous and protease-resistant, ß-sheet-rich fibrils, was observed in vivo. Thereby, we characterize a structural change from a globular state to a fibrillar form in a functional prokaryotic system on the molecular level.

Mussel-Inspired Polymerization of Peptides: The Chemical Activation Route as Key to Broaden the Sequential Space of Artificial Mussel-Glue Proteins.

A previously introduced tyrosinase-activated polymerization of Tyr- and Cys-bearing peptides yielding artificial mussel-glue proteins is realized without the need of the specific enzyme by a chemical activation route. This decouples the sequence of polymerizable peptides (unimers) from the constraints of tyrosinase substrates and enables the polymerization of minimal motifs such as Dopa-Lys-Cys (Umini *KC ) or Dopa-Gly-Cys (Umini *GC ). In the polymerization procedure, sodium periodate is used to oxidize Dopa residues of the unimers to Dopa-quinones to which the thiol of a Cys residue is added in a Michael-type reaction. The resulting polyUmini *KC and polyUmini *GC exhibit a thiol-catechol connectivity as a potent adhesive functionality at each repeat unit. QCM-D experiments show the excellent substrate adsorption properties of the products from the chemically activated polymerization. On aluminum oxide surfaces, polyUmini *KC rapidly forms a coating, even under seawater model conditions and the coating resists rinsing with hypersaline solution of 4.2 M salt mixtures. While the sodium periodate oxidation is less specific than the tyrosinase reaction and requires the implementation of Dopa instead of Tyr residues into the polymerizable unimers, the chemical route makes scale-up more easily accessible.

Oxidative inactivation of the endogenous antioxidant protein DJ-1 by the food contaminants 3-MCPD and 2-MCPD.

3-Chloro-1,2-propanediol (3-MCPD) and 2-chloro-1,3-propanediol (2-MCPD) are heat-induced food contaminants being present either as free substances or as fatty acid esters in numerous foods. 3-MCPD was classified to be possibly carcinogenic to humans (category 2B) with kidney and testis being the primary target organs according to animal studies. A previous 28-day oral feeding study with rats revealed that the endogenous antioxidant protein DJ-1 was strongly deregulated at the protein level in kidney, liver, and testis of the experimental animals that had been treated either with 3-MCPD, 2-MCPD or their dipalmitate esters. Here we show that this deregulation is due to the oxidation of a conserved, redox-active cysteine residue (Cys106) of DJ-1 to a cysteine sulfonic acid which is equivalent to loss of function of DJ-1. Irreversible oxidation of DJ-1 is associated with a number of oxidative stress-related diseases such as Parkinson, cancer, and type II diabetes. It is assumed that 3-MCPD or 2-MCPD do not directly oxidize DJ-1, but that these substances induce the formation of reactive oxygen species (ROS) which in turn trigger DJ-1 oxidation. The implications of 3-MCPD/2-MCPD-mediated ROS formation in vivo for the ongoing risk assessment of these compounds as well as the potential of oxidized DJ-1 to serve as a novel effect biomarker for 3-MCPD/2-MCPD toxicity are being discussed.

A Complex of Htm1 and the Oxidoreductase Pdi1 Accelerates Degradation of Misfolded Glycoproteins.

A quality control system in the endoplasmic reticulum (ER) efficiently discriminates polypeptides that are in the process of productive folding from conformers that are trapped in an aberrant state. Only the latter are transported into the cytoplasm and degraded in a process termed ER-associated protein degradation (ERAD). In the ER, an enzymatic cascade generates a specific N-glycan structure of seven mannosyl and two N-acetylglucosamine residues (Man7GlcNAc2) on misfolded glycoproteins to facilitate their disposal. We show that a complex encompassing the yeast lectin-like protein Htm1 and the oxidoreductase Pdi1 converts Man8GlcNAc2 on glycoproteins into the Man7GlcNAc2 signal. In vitro the Htm1-Pdi1 complex processes both unfolded and native proteins albeit with a preference for the former. In vivo, elevated expression of HTM1 causes glycan trimming on misfolded and folded proteins, but only degradation of the non-native species is accelerated. Thus, modification with a Man7GlcNAc2 structure does not inevitably commit a protein for ER-associated protein degradation. The function of Htm1 in ERAD relies on its association with Pdi1, which appears to regulate the access to substrates. Our data support a model in which the balanced activities of Pdi1 and Htm1 are crucial determinants for the efficient removal of misfolded secretory glycoproteins.

Identification of Novel Nuclear Factor of Activated T Cell (NFAT)-associated Proteins in T Cells.

Transcription factors of the nuclear factor of activated T cell (NFAT) family are essential for antigen-specific T cell activation and differentiation. Their cooperative DNA binding with other transcription factors, such as AP1 proteins (FOS, JUN, and JUNB), FOXP3, IRFs, and EGR1, dictates the gene regulatory action of NFATs. To identify as yet unknown interaction partners of NFAT, we purified biotin-tagged NFATc1/αA, NFATc1/ßC, and NFATc2/C protein complexes and analyzed their components by stable isotope labeling by amino acids in cell culture-based mass spectrometry. We revealed more than 170 NFAT-associated proteins, half of which are involved in transcriptional regulation. Among them are many hitherto unknown interaction partners of NFATc1 and NFATc2 in T cells, such as Raptor, CHEK1, CREB1, RUNX1, SATB1, Ikaros, and Helios. The association of NFATc2 with several other transcription factors is DNA-dependent, indicating cooperative DNA binding. Moreover, our computational analysis discovered that binding motifs for RUNX and CREB1 are found preferentially in the direct vicinity of NFAT-binding motifs and in a distinct orientation to them. Furthermore, we provide evidence that mTOR and CHEK1 kinase activity influence NFAT's transcriptional potency. Finally, our dataset of NFAT-associated proteins provides a good basis to further study NFAT's diverse functions and how these are modulated due to the interplay of multiple interaction partners.

Glycoprotein B of equine herpesvirus type 1 has two recognition sites for subtilisin-like proteases that are cleaved by furin.

Glycoprotein B (gB) of equine herpesvirus type 1 (EHV-1) is predicted to be cleaved by furin in a fashion similar to that of related herpesviruses. To investigate the contribution of furin-mediated gB cleavage to EHV-1 growth, canonical furin cleavage sites were mutated. Western blot analysis of mutated EHV-1 gB showed that it was cleaved at two positions, 518RRRR521 and 544RLHK547, and that the 28 aa between the two sites were removed after cleavage. Treating infected cells with either convertase or furin inhibitors reduced gB cleavage efficiency. Further, removal of the first furin recognition motif did not affect in vitro growth of EHV-1, while mutation of the second motif greatly affected virus growth. In addition, a second possible signal peptide cleavage site was identified for EHV-1 gB between residues 98 and 99, which was 13 aa downstream of that previously identified.

SORLA regulates calpain-dependent degradation of synapsin.

INTRODUCTION: Sorting-related receptor with A-type repeats (SORLA) is an intracellular sorting receptor in neurons and a major risk factor for Alzheimer disease. METHODS: Here, we performed global proteome analyses in the brain of SORLA-deficient mice followed by biochemical and histopathologic studies to identify novel neuronal pathways affected by receptor dysfunction. RESULTS: We demonstrate that the lack of SORLA results in accumulation of phosphorylated synapsins in cortex and hippocampus. We propose an underlying molecular mechanism by demonstrating that SORLA interacts with phosphorylated synapsins through 14-3-3 adaptor proteins to deliver synapsins to calpain-mediated proteolytic degradation. DISCUSSION: Our results suggest a novel function for SORLA which is in control of synapsin degradation, potentially impacting on synaptic vesicle endocytosis and/or exocytosis.

Improved two-dimensional reversed phase-reversed phase LC-MS/MS approach for identification of peptide-protein interactions.

Quantitative mass spectrometry (MS) in combination with affinity purification approaches allows for an unbiased study of protein-protein and peptide-protein interactions. In shotgun approaches that are based on proteolytic digestion of complex protein mixtures followed by two-dimensional liquid-phase chromatography, the separation effort prior to MS analysis is focused on tryptic peptides. Here we developed an improved offline 2-D liquid chromatography-MS/MS approach for the identification and quantification of binding proteins utilizing reversed-phase capillary columns with acidic acetonitrile-containing eluents in both chromatographic dimensions. A specific fractionation scheme was applied in order to obtain samples with evenly distributed peptides and to fully utilize the separation space in the second dimension nanoLC-MS/MS. We report peptide-protein interaction studies to identify phosphorylation-dependent binding partners of the T cell adapter protein ADAP. The results of the SILAC-based pull-down experiments show this approach is well suited for distinguishing phosphorylation-specific interactions from unspecific binding events. The data provide further evidence that phosphorylated Tyr 595 of ADAP may serve as a direct binding site for the SH2 domains of the T cell proteins SLP76 and NCK. From a technical point of view we provide a detailed protocol for an offline 2-D RP-RP LC-MS/MS method that offers a robust and time-saving alternative for quantitative interactome analysis.

Structural basis of phosphatidylinositol 3-kinase C2α function.

Phosphatidylinositol 3-kinase type 2α (PI3KC2α) is an essential member of the structurally unresolved class II PI3K family with crucial functions in lipid signaling, endocytosis, angiogenesis, viral replication, platelet formation and a role in mitosis. The molecular basis of these activities of PI3KC2α is poorly understood. Here, we report high-resolution crystal structures as well as a 4.4-Å cryogenic-electron microscopic (cryo-EM) structure of PI3KC2α in active and inactive conformations. We unravel a coincident mechanism of lipid-induced activation of PI3KC2α at membranes that involves large-scale repositioning of its Ras-binding and lipid-binding distal Phox-homology and C-C2 domains, and can serve as a model for the entire class II PI3K family. Moreover, we describe a PI3KC2α-specific helical bundle domain that underlies its scaffolding function at the mitotic spindle. Our results advance our understanding of PI3K biology and pave the way for the development of specific inhibitors of class II PI3K function with wide applications in biomedicine.

Identification of biomarkers of brown adipose tissue aging highlights the role of dysfunctional energy and nucleotide metabolism pathways.

Brown adipose tissue function declines during aging and may contribute to the onset of metabolic disorders such as diabetes and obesity. Only limited understanding of the mechanisms leading to the metabolic impairment of brown adipocytes during aging exists. To this end, interscapular brown adipose tissue samples were collected from young and aged mice for quantification of differential gene expression and metabolite levels. To identify potential processes involved in brown adipocyte dysfunction, metabolite concentrations were correlated to aging and significantly changed candidates were subsequently integrated with a non-targeted proteomic dataset and gene expression analyses. Our results include novel age-dependent correlations of polar intermediates in brown adipose tissue. Identified metabolites clustered around three biochemical processes, specifically energy metabolism, nucleotide metabolism and vitamin metabolism. One mechanism of brown adipose tissue dysfunction may be linked to mast cell activity, and we identify increased histamine levels in aged brown fat as a potential biomarker. In addition, alterations of genes involved in synthesis and degradation of many metabolites were mainly observed in the mature brown adipocyte fraction as opposed to the stromal vascular fraction. These findings may provide novel insights on the molecular mechanisms contributing to the impaired thermogenesis of brown adipocytes during aging.

Effects of thawing, refreezing and storage conditions of tissue samples and protein extracts on 2-DE spot intensity.

We report that reliable quantitative proteome analyses can be performed with tissue samples stored at -80 degrees C for up to 10 years. However, storing protein extracts at 4 degrees C for 24 h and freezing protein extracts at -80 degrees C and thawing them significantly altered 41.6 and 17.5% of all spot intensities on 2-DE gels, respectively. Fortunately, these storing effects did not impair the reliability of quantifying 2-DE experiments. Nonetheless, the results show that freezing and storage conditions should be carefully controlled in proteomic experiments.

Equine Herpesvirus Type 1 Modulates Cytokine and Chemokine Profiles of Mononuclear Cells for Efficient Dissemination to Target Organs.

Equine herpesvirus type 1 (EHV-1) causes encephalomyelopathy and abortion, for which cell-associated viremia and subsequent virus transfer to and replication in endothelial cells (EC) are responsible and prerequisites. Viral and cellular molecules responsible for efficient cell-to-cell spread of EHV-1 between peripheral blood mononuclear cells (PBMC) and EC remain unclear. We have generated EHV-1 mutants lacking ORF1, ORF2, and ORF17 genes, either individually or in combination. Mutant viruses were analyzed for their replication properties in cultured equine dermal cells, PBMC infection efficiency, virus-induced changes in the PBMC proteome, and cytokine and chemokine expression profiles. ORF1, ORF2, and ORF17 are not essential for virus replication, but ORF17 deletion resulted in a significant reduction in plaque size. Deletion of ORF2 and ORF17 gene significantly reduced cell-to-cell virus transfer from virus-infected PBMC to EC. EHV-1 infection of PBMC resulted in upregulation of several pathways such as Ras signaling, oxidative phosphorylation, platelet activation and leukocyte transendothelial migration. In contrast, chemokine signaling, RNA degradation and apoptotic pathways were downregulated. Deletion of ORF1, ORF2 and ORF17 modulated chemokine signaling and MAPK pathways in infected PBMC, which may explain the impairment of virus spread between PBMC and EC. The proteomic results were further confirmed by chemokine assays, which showed that virus infection dramatically reduced the cytokine/chemokine release in infected PBMC. This study uncovers cellular proteins and pathways influenced by EHV-1 after PBMC infection and provide an important resource for EHV-1 pathogenesis. EHV-1-immunomodulatory genes could be potential targets for the development of live attenuated vaccines or therapeutics against virus infection.

Fish and Clips: A Convenient Strategy to Identify Tyrosinase Substrates with Rapid Activation Behavior for Materials Science Applications.

Peptides with suitable substrate properties for a specific tyrosinase are selected by combinatorial means from a one-bead-one-compound (OBOC) peptide library. The identified sequences exhibit tyrosine residues that are rapidly oxidized to 3,4-dihydroxyphenylalanine (Dopa), making the peptides interesting for enzyme-activated adhesives. The selection process of peptides involves tyrosinase oxidation of tyrosine-bearing sequences on a solid support, yielding dopaquinone residues (fish from the sequence pool), to which thiol-functional fluorescent probes attach by Michael-reaction (clip to mark). Labeled supports are isolated and sequence readout is feasible by MALDI-TOF-MS/MS to reveal peptides, while activation kinetics as well as enzyme-activated coating behavior are verifying the proper selection.

Identification of functional lipid metabolism biomarkers of brown adipose tissue aging.

OBJECTIVE: Aging is accompanied by loss of brown adipocytes and a decline in their thermogenic potential, which may exacerbate the development of adiposity and other metabolic disorders. Presently, only limited evidence exists describing the molecular alterations leading to impaired brown adipogenesis with aging and the contribution of these processes to changes of systemic energy metabolism. METHODS: Samples of young and aged murine brown and white adipose tissue were used to compare age-related changes of brown adipogenic gene expression and thermogenesis-related lipid mobilization. To identify potential markers of brown adipose tissue aging, non-targeted proteomic and metabolomic as well as targeted lipid analyses were conducted on young and aged tissue samples. Subsequently, the effects of several candidate lipid classes on brown adipocyte function were examined. RESULTS: Corroborating previous reports of reduced expression of uncoupling protein-1, we observe impaired signaling required for lipid mobilization in aged brown fat after adrenergic stimulation. Omics analyses additionally confirm the age-related impairment of lipid homeostasis and reveal the accumulation of specific lipid classes, including certain sphingolipids, ceramides, and dolichols in aged brown fat. While ceramides as well as enzymes of dolichol metabolism inhibit brown adipogenesis, inhibition of sphingosine 1-phosphate receptor 2 induces brown adipocyte differentiation. CONCLUSIONS: Our functional analyses show that changes in specific lipid species, as observed during aging, may contribute to reduced thermogenic potential. They thus uncover potential biomarkers of aging as well as molecular mechanisms that could contribute to the degradation of brown adipocytes, thereby providing potential treatment strategies of age-related metabolic conditions.

The whereabouts of transmembrane proteins from rat brain synaptosomes during two-dimensional gel electrophoresis.

Little is known about what happens to transmembrane proteins (TMP) in 2-DE. In order to obtain more insight into the whereabouts of these proteins we prepared membrane-enriched synaptosomes from rat frontal cortex and washed them with 7 M urea or Na(2)CO(3). From each preparation, 200 microg protein was loaded on 2-DE gels covering the 4-7 and 6-11 pH ranges, respectively. MALDI-MS/MS analysis detected only 3 TMP among 421 identified spots. However, when the samples had been washed with Na(2)CO(3), only few well-focused spots remained detectable on the gel covering the pH 6-11 range. Instead, a heavily ruthenium-stained smear became visible at the upper edge of the gel at the location where the samples had been applied by cup loading. LC-MS/MS analysis revealed that this smear contained 38 unfocused TMP with up to 12 transmembrane helices. After transfer to the second dimension, no major areas of protein staining were left on the IPG strips. This indicates that after extraction and denaturation the TMP may form high-molecular aggregates, due to their "hydrophobic interactions". These aggregates enter the IPG strips, but do not focus regularly. They are then transferred onto the 2-DE-gels, where they remain caught at the upper edge.

In-cell NMR in mammalian cells: part 3.

Irrespective of how isotope-labeled proteins are delivered into mammalian cells, laboratory routines are needed to assess the quality of the resulting in-cell NMR samples. These include methods to evaluate overall cell viability, protein transduction efficiency, intracellular protein concentration, localization, and stability. In addition, quality control experiments to assess protein leakage from manipulated cells are of particular importance for in-cell NMR experiments. The purpose of this chapter is to outline qualitative and quantitative methods to determine general biological properties of in-cell NMR samples in order to ensure the highest possible standards for in-cell NMR studies.

The relative influence of phosphorylation and methylation on responsiveness of peptides to MALDI and ESI mass spectrometry.

Qualitative and quantitative analysis of post-translational protein modifications by mass spectrometry is often hampered by changes in the ionization/detection efficiencies caused by amino acid modifications. This paper reports a comprehensive study of the influence of phosphorylation and methylation on the responsiveness of peptides to matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. Using well-characterized synthetic peptide mixtures consisting of modified peptides and their unmodified analogs, relative ionization/detection efficiencies of phosphorylated, monomethylated, and dimethylated peptides were determined. Our results clearly confirm that the ion yields are generally lower and the signal intensities are reduced with phosphopeptides than with their nonphosphorylated analogs and that this has to be taken into account in MALDI and ESI mass spectrometry. However, the average reduction of ion yield caused by phosphorylation is more pronounced with MALDI than with ESI. The unpredictable impact of phosphorylation does not depend on the hydrophobicity and net charge of the peptide, indicating that reliable quantification of phosphorylation by mass spectrometry requires the use of internal standards. In contrast to phosphorylation, mono- and dimethylated peptides frequently exhibit increased signal intensities in MALDI mass spectrometry (MALDI-MS). Despite minor matrix-dependent variability, MALDI methods are well suited for the sensitive detection of dimethylated arginine and lysine peptides. Mono- and dimethylation of the arginine guanidino group did not significantly influence the ionization efficiency of peptides in ESI-MS.