Characterization of peptides resulting from digestion of human skin elastin with elastase

Several pathological disorders are associated with abnormalities in elastic fibers, which are mainly composed of elastin. Understanding the biochemical basis of such disorders requires information about the primary structure of elastin. Since the acquisition of structural information for elastin is hampered by its extreme insolubility in water or any organic solvent, in this study, human skin elastin was digested with elastase to produce water-soluble peptides. Tandem mass spectrometry (MS/MS) experiments were performed using conventional electrospray ionization (ESI) and nano-ESI techniques coupled with ion trap and quadrupole time-of-flight (qTOF) mass analyzers, respectively. The peptides were identified from the fragment spectra using database searching and/or de novo sequencing. The cleavage sites of the enzyme and, for the first time, the extent and location of proline hydroxylation in human skin elastin were determined. A total of 117 peptides were identified with sequence coverage of 58.8%. It has been observed that 25% of proline residues in the sequenced region are hydroxylated. Elastase cleaves predominantly at the C-terminals of the amino acids Gly, Val, Leu, Ala, and Ile, and to a lesser extent at Phe, Pro, Glu, and Arg. Our results confirm a previous report that human skin elastin lacks amino acid sequences expressed by exon 26A.     

Distribution of Dinophysis species and their association with lipophilic phycotoxins in plankton from the Argentine Sea

Dinophysis is a cosmopolitan genus of marine dinoflagellates, considered as the major proximal source of diarrheic shellfish toxins and the only producer of pectenotoxins (PTX). From three oceanographic expeditions carried out during autumn, spring and late summer along the Argentine Sea (∼38–56°S), lipophilic phycotoxins were determined by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) in size-fractionated plankton samples. Lipophilic toxin profiles were associated with species composition by microscopic analyses of toxigenic phytoplankton. Pectenotoxin-2 and PTX-11 were frequently found together with the presence of Dinophysis acuminata and Dinophysis tripos. By contrast, okadaic acid was rarely detected and only in trace concentrations, and dinophysistoxins were not found. The clear predominance of PTX over other lipophilic toxins in Dinophysis species from the Argentine Sea is in accordance with previous results obtained from north Patagonian Gulfs of the Argentine Sea, and from coastal waters of New Zealand, Chile, Denmark and United States. Dinophysis caudata was rarely found and it was confined to the north of the sampling area. Because of low cell densities, neither D. caudata nor Dinophysis norvegica could be biogeographically related to lipophilic toxins in this study. Nevertheless, the current identification of D. norvegica in the southern Argentine Sea is the first record for the southwestern Atlantic Ocean. Given the typical toxigenicity of this species on a global scale, this represents an important finding for future surveillance of plankton-toxin associations.     

Different methylation characteristics of protein arginine methyltransferase 1 and 3 toward the Ewing Sarcoma protein and a peptide

The multifunctional Ewing Sarcoma (EWS) protein, a member of a large family of RNA-binding proteins, is extensively asymmetrically dimethylated at arginine residues within RGG consensus sequences. Using recombinant proteins we examined whether type I protein arginine methyltransferase (PRMT)1 or 3 is responsible for asymmetric dimethylations of the EWS protein. After in vitro methylation of the EWS protein by GST-PRMT1, we identified 27 dimethylated arginine residues out of 30 potential methylation sites by mass spectrometry-based techniques (MALDI-TOF MS and MS/MS). Thus, PRMT1 recognizes most if not all methylation sites of the EWS protein. With GST-PRMT3, however, only nine dimethylated arginines, located mainly in the C-terminal region of EWS protein, could be assigned, indicating that structural determinants prevent complete methylation. In contrary to previous reports this study also revealed that trypsin is able to cleave after methylated arginines. Pull-down experiments showed that endogenous EWS protein binds efficiently to GST-PRMT1 but less to GST-PRMT3, which is in accordance to the in vitro methylation results. Furthermore, methylation of a peptide containing different methylation sites revealed differences in the site selectivity as well as in the kinetic properties of GST-PRMT1 and GST-PRMT3. Kinetic differences due to an inhibition effect of the methylation inhibitor S-adenosyl-L-homocysteine could be excluded by determining the corresponding K(i) values of the two enzymes and the K(d) values for the methyl donor S-adenosyl-L-methionine. The study demonstrates the strength of MS-based methods for a qualitative and quantitative analysis of enzymic arginine methylation, a posttranslational modification that becomes more and more the object of investigations.     

Mapping of Rpb3 and Rpb5 contact sites on two large subunits, Rpb1 and Rpb2, of the RNA polymerase II from fission yeast

[Rpb1 and Rpb2] Mapping of the contact sites␣on two large subunits of the fission yeast Schizosaccharomyces pombe RNA polymerase II with two small subunits, Rpb3 and Rpb5, was carried out using the two-hybrid screening system in the budding yeast Saccharomyces cerevisiae. Rpb5 was found to interact with any fragment of Rpb1 that contained the region H, which is conserved among the subunit 1 homologues of all RNA polymerases, including the β' subunit of prokaryotic RNA polymerases. In agreement with the fact that Rpb5 is shared among all three forms of eukaryotic RNA polymerases, the region H of RNA polymerase I subunit 1 (Rpa190) was also found to interact with Rpb5. On the other hand, two-hybrid screening of Rpb2 fragments from RNA polymerase II indicated the presence of an Rpb3 contact site in the region H which is conserved among the subunit 2 homologues of all RNA polymerases, including the β subunit of prokaryotic RNA polymerases. Possible functions of the regions H in the subunits 1 and 2 are discussed. Received: 10 December 1997 / Accepted: 14 April 1998     

Binding and structural studies of the complexes of type 1 ribosome inactivating protein from Momordica balsamina with cytosine,cytidine, and cytidine diphosphate

The type 1 ribosome inactivating protein from Momordica balsamina (MbRIP1) has been shown to interact with purine bases, adenine and guanine of RNA/DNA. We report here the binding and structural studies of MbRIP1 with a pyrimidine base, cytosine; cytosine containing nucleoside, cytidine; and cytosine containing nucleotide, cytidine diphosphate. All three compounds bound to MbRIP1 at the active site with dissociation constants of 10^?4 M–10^?7 M. As reported earlier, in the structure of native MbRIP1, there are 10 water molecules in the substrate binding site. Upon binding of cytosine to MbRIP1, four water molecules were dislodged from the substrate binding site while five water molecules were dislodged when cytidine bound to MbRIP1. Seven water molecules were dislocated when cytidine diphosphate bound to MbRIP1. This showed that cytidine diphosphate occupied a larger space in the substrate binding site enhancing the buried surface area thus making it a relatively better inhibitor of MbRIP1 as compared to cytosine and cytidine. The key residues involved in the recognition of cytosine, cytidine and cytidine diphosphate were Ile71, Glu85, Tyr111 and Arg163. The orientation of cytosine in the cleft is different from that of adenine or guanine indicating a notable difference in the modes of binding of purine and pyrimidine bases. Since adenine containing nucleosides/nucleotides are suitable substrates, the cytosine containing nucleosides/nucleotides may act as inhibitors.     

Substrate/inhibition studies of bacteriophage T7 RNA polymerase with the 5′-triphosphate derivative of a ring-expanded (‘fat’) nucleoside possessing potent antiviral and anticancer activities

As part of an effort to explore the mechanism of potent, broad spectrum antiviral and anticancer activities of a number of ring-expanded (‘fat’) nucleosides that we recently reported, a representative ‘fat’ nucleoside 4,6-diamino-8-imino-8H-1-β- -ribofuranosylimidazo[4,5-e][1,3]diazepine (1) was converted to its 5′-triphosphate derivative (2), and biochemically screened for possible inhibition of nucleic acid polymerase activity, employing synthetic DNA templates and the bacteriophage T7 RNA polymerase as a representative polymerase. Our results suggest that 2 is a moderate inhibitor of T7 RNA polymerase, and that the 5′-triphosphate moiety of 2 appears to be essential for inhibition as nucleoside Scheme 1 and Scheme 2 alone failed to inhibit the polymerase reaction.

Scheme 2.     

Similarities between a predicted secondary structure for the M1 RNA ribozyme and the tRNA binding center of 16 S rRNA from E. coli

We propose a new model for the secondary structure of the M1 RNA component of E. coli RNase P which is based on significant sequence homologies with parts of the E. coli 16 S rRNA. A large domain of the new model resembles closely the secondary structure of the tRNA binding center of 16 S rRNA. We suggest that this domain of M1 RNA when functioning as a ribozyme binds the mature part of the precursor tRNA.     

Characterization of Protein Methyltransferases Rkm1, Rkm4, Efm4, Efm7, Set5 and Hmt1 Reveals Extensive Post-Translational Modification

Protein methylation is one of the major post-translational modifications (PTMs) in the cell. In Saccharomyces cerevisiae, over 20 protein methyltransferases (MTases) and their respective substrates have been identified. However, the way in which these MTases are modified and potentially subject to regulation remains poorly understood. Here, we investigated six overexpressed S. cerevisiae protein MTases (Rkm1, Rkm4, Efm4, Efm7, Set5 and Hmt1) to identify PTMs of potential functional relevance. We identified 48 PTM sites across the six MTases, including phosphorylation, acetylation and methylation. Forty-two sites are novel. We contextualized the PTM sites in structural models of the MTases and revealed that many fell in catalytic pockets or enzyme–substrate interfaces. These may regulate MTase activity. Finally, we compared PTMs on Hmt1 with those on its human homologs PRMT1, PRMT3, CARM1, PRMT6 and PRMT8. This revealed that several PTMs are conserved from yeast to human, whereas others are only found in Hmt1. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD006767.     

Mechanism of U Insertion RNA Editing in Trypanosome Mitochondria: The Bimodal TUTase Activity of the Core Complex

Expression of the trypanosomal mitochondrial genome requires the insertion and deletion of uridylyl residues at specific sites in pre-mRNAs. RET2 terminal uridylyl transferase is an integral component of the RNA editing core complex (RECC) and is responsible for the guide-RNA-dependent U insertion reaction. By analyzing RNA-interference-based knock-in Trypanosoma brucei cell lines, purified editing complex, and individual protein, we have investigated RET2's association with the RECC. In addition, the U insertion activity exhibited by RET2 as an RECC subunit was compared with characteristics of the monomeric protein. We show that interaction of RET2 with RECC is accomplished via a protein-protein contact between its middle domain and a structural subunit, MP81. The recombinant RET2 catalyzes a faithful editing on gapped (precleaved) double-stranded RNA substrates, and this reaction requires an internal monophosphate group at the 5′ end of the mRNA 3′ cleavage fragment. However, RET2 processivity is limited to insertion of three Us. Incorporation into the RECC voids the internal phosphate requirement and allows filling of longer gaps similar to those observed in vivo. Remarkably, monomeric and RECC-embedded enzymes display a similar bimodal activity: the distributive insertion of a single uracil is followed by a processive extension limited by the number of guiding nucleotides. Based on the RNA substrate specificity of RET2 and the purine-rich nature of U insertion sites, we propose that the distributive + 1 insertion creates a substrate for the processive gap-filling reaction. Upon base-pairing of the + 1 extended 5′ cleavage fragment with a guiding nucleotide, this substrate is recognized by RET2 in a different mode compared to the product of the initial nucleolytic cleavage. Therefore, RET2 distinguishes base pairs in gapped RNA substrates which may constitute an additional checkpoint contributing to overall fidelity of the editing process.     

Structure of the Human Respiratory Syncytial Virus M2-1 Protein in Complex with a Short Positive-Sense Gene-End RNA

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Circular RNA YAP1 acts as the sponge of microRNA‐21‐5p to secure HK‐2 cells from ischaemia/reperfusion‐induced injury

Circular RNA YAP1 (circYAP1) was reported to participate in progression of gastric cancer. However, the role of circYAP1 in acute kidney injury (AKI) remains obscure. We attempted to examine the effects of circYAP1 on ischaemia/reperfusion‐stimulated renal injury. AKI model was established by treating HK‐2 cells in ischaemia/reperfusion (I/R) environment. CircYAP1 expression in blood of AKI patients and I/R‐treated HK‐2 cells was evaluated via RT‐qPCR. CCK‐8, flow cytometry, ELISA and ROS assay were executed to test the impact of circYAP1 on cell viability, apoptosis, inflammatory cytokines and ROS generation. Bioinformatic analysis was executed to explore miRNA targets. The relativity between circYAP1 and miR‐21‐5p was verified by RT‐qPCR and luciferase assay. The functions of miR‐21‐5p in I/R‐triggered injury were reassessed. PI3K/AKT/mTOR pathway was detected by Western blot. Down‐regulated circYAP1 was observed in AKI blood samples and I/R‐treated HK‐2 cells. CircYAP1 overexpression expedited cell growth and weakened secretion of inflammatory factors and ROS generation in I/R‐disposed cells. Besides, we found circYAP1 could sponge to miR‐21‐5p. Interestingly, miR‐21‐5p overexpression overturned the repressive effects of circYAP1 on cell injury. Moreover, PI3K/AKT/mTOR pathway was activated by circYAP1 via inhibiting miR‐21‐5p. We demonstrated that circYAP1 activated PI3K/AKT/mTOR pathway and secured HK‐2 cells from I/R injury via sponging miR‐21‐5p.     

Electrostatic interactions play an essential role in the binding of oleic acid with α‐lactalbumin in the HAMLET‐like complex: A study using charge‐specific chemical modifications

H uman α ‐lactalbumin m ade le thal to t umor cells (HAMLET) and its analogs are partially unfolded protein‐oleic acid (OA) complexes that exhibit selective tumoricidal activity normally absent in the native protein itself. To understand the nature of the interaction between protein and OA moieties, charge‐specific chemical modifications of lysine side chains involving citraconylation, acetylation, and guanidination were employed and the biophysical and biological properties were probed. Upon converting the original positively‐charged lysine residues to negatively‐charged citraconyl or neutral acetyl groups, the binding of OA to protein was eliminated, as were any cytotoxic activities towards osteosarcoma cells. Retention of the positive charges by converting lysine residues to homoarginine groups (guanidination); however, yielded unchanged binding of OA to protein and identical tumoricidal activity to that displayed by the wild‐type α‐lactalbumin‐oleic acid complex. With the addition of OA, the wild‐type and guanidinated α‐lactalbumin proteins underwent substantial conformational changes, such as partial unfolding, loss of tertiary structure, but retention of secondary structure. In contrast, no significant conformational changes were observed in the citraconylated and acetylated α‐lactalbumins, most likely because of the absence of OA binding. These results suggest that electrostatic interactions between the positively‐charged basic groups on α‐lactalbumin and the negatively‐charged carboxylate groups on OA molecules play an essential role in the binding of OA to α‐lactalbumin and that these interactions appear to be as important as hydrophobic interactions. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Glycan structure and site of glycosylation in the ER-resident glycoprotein,uridine 5′-diphosphate-glucose: glycoprotein glucosyltransferases 1 from rat,porcine, bovine,and human

Here we report glycan structures and their position of attachment to a carrier protein, uridine 5′-diphosphate-glucose: glycoprotein glucosyltransferase (UGGT1), as detected using tandem mass spectrometry. UGGT1 acts as a folding sensor of newly synthesized glycosylated polypeptides in the endoplasmic reticulum, and the transferase itself is known to be glycosylated. The structure of glycan attached to UGGT1, however, has not been investigated. In this study, we reveal the site of glycosylation (N269) and the glycan structures (Hex_5–8HexNAc₂) in UGGT1 obtained from rat (Rattus norvegicus), pig (Sus scrofa), cow (Bos taurus), and human (Homo sapiens).     

Chemical synthesis of the 3-sulfooxy-7-N-acetylglucosaminyl-24-amidated conjugates of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, and related compounds: unusual, major metabolites of bile acid in a patient with Niemann-Pick disease type C1

The chemical synthesis of 3beta,7beta-dihydroxy-5-cholen-24-oic acid, triply conjugated by sulfuric acid at C-3, by N-acetylglucosamine (GlcNAc) at C-7, and by glycine or taurine at C-24, is described. These are unusual, major metabolites of bile acid found to be excreted in the urine of a patient with Niemann-Pick disease type C1. Analogous double-conjugates of 3beta-hydroxy-7-oxo-5-cholen-24-oic acid were also prepared. The principal reactions involved were: (1) beta-d-N-acetylglucosaminidation at C-7 of methyl 3beta-tert-butyldimethylsilyloxy (TBDMSi)-7beta-hydroxy-5-cholen-24-oate with 2-acetamido-1alpha-chloro-1,2-dideoxy-3,4,6-tri-O-acetyl-d-glucopyranose in the presence of CdCO(3) in boiling toluene; (2) sulfation at C-3 of the resulting 3beta-TBDMSi-7beta-GlcNAc with sulfur trioxide-trimethylamine complex in pyridine; and (3) direct amidation at C-24 of the 3beta-sulfooxy-7beta-GlcNAc conjugate with glycine methyl ester hydrochloride (or taurine) using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride as a coupling agent in DMF. The structures of the multi-conjugated bile acids were characterized by liquid chromatography-mass spectrometry with an electrospray ionization probe under the positive and negative ionization modes.     

Isoform identification, recombinant production and characterization of the allergen lipid transfer protein 1 from pear (Pyr c 3)

Non-specific lipid transfer proteins belonging to LTP1 family represent the most important allergens for non pollen-related allergies to Rosaceae fruits in the Mediterranean area. Peach LTP1 (Pru p 3) is a major allergen and is considered the prototypic allergenic LTP. On the contrary, pear allergy without pollinosis seems to be under-reported when compared to other Rosaceae fruits suggesting that the as-yet-uncharacterized pear LTP1 (Pyr c 3) has in vivo a low allergenicity. We report here on the identification of four cDNAs encoding for LTP1 in pear fruits. The two isoforms exhibiting amino acid sequences most similar to those of peach and apple homologues were obtained as recombinant proteins. Such isoforms exhibited CD spectra and lipid binding ability typical of LTP1 family. Moreover, pear LTP1 mRNA was mainly found in the peel, as previously shown for other Rosaceae fruits. By means of IgE ELISA assays a considerable immunoreactivity of these proteins to LTP-sensitive patient sera was detected, even though allergic reactions after ingestion of pear were not reported in the clinical history of the patients. Finally, the abundance of LTP1 in protein extracts from pear peel, in which LTP1 from Rosaceae fruits is mainly confined, was estimated to be much lower as compared to peach peel. Our data suggest that the two isoforms of pear LTP1 characterized in this study possess biochemical features and IgE-binding ability similar to allergenic LTPs. Their low concentrations in pear might be the cause of the low frequency of LTP-mediated pear allergy.     

Recombinant PNPLA1 catalyzes the synthesis of acylceramides and acyl acids with selective incorporation of linoleic acid

Loss-of-function mutations in patatin-like phospholipase domain-containing protein 1 (PNPLA1) cause autosomal recessive congenital ichthyosis, and altered PNPLA1 activity is implicated in the pathogenesis of atopic dermatitis and other common skin diseases. To examine the hypothesis that PNPLA1 catalyzes the synthesis of acylceramides and acyl acids, we expressed and partially purified a soluble, truncated form of PNPLA1 in Escherichia coli, (PNPLA1_trun) along with the related protein PNPLA2 (ATGL, adipose triglyceride lipase) and coactivator CGI-58. Liposomal substrates were incubated with recombinant enzymes for 0.5–24 h and products analyzed by HPLC-UV and LC-MS. Using trilinolein or dilinolein substrates, PNPLA1_trun, like ATGL_trun, catalyzed lipolysis and acyltransferase reactions with 2–30% conversion into linoleic acid, monolinolein, and trilinolein. CGI-58 enhanced ATGL-catalyzed lipolysis as previously reported, but transacylase activity was not enhanced with ATGL or PNPLA1. In matching the proposed activity in vivo, PNPLA1 catalyzed acyl transfer from trilinolein and dilinolein donors to omega-hydroxy ceramide, omega-hydroxy glucosylceramide, and omega-hydroxy acid acceptors to form acylceramide, glucosyl-acylceramide, and acyl acid, respectively, albeit with only ∼0.05% conversion of the substrates. Notably, in experiments comparing dilinolein vs. diolein acyl donors, PNPLA1 transferred linoleate with 3:1 selectivity over oleate into acylceramide. These results support the role for PNPLA1 in the synthesis of acylceramides and acyl acids in epidermis and suggest that the enrichment of these lipids with linoleic acid could result from the substrate selectivity of PNPLA1.