121 research outputs found

    N-terminal proteomics assisted profiling of the unexplored translation initiation landscape in Arabidopsis thaliana

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    Proteogenomics is an emerging research field yet lacking a uniform method of analysis. Proteogenomic studies in which N-terminal proteomics and ribosome profiling are combined, suggest that a high number of protein start sites are currently missing in genome annotations. We constructed a proteogenomic pipeline specific for the analysis of N-terminal proteomics data, with the aim of discovering novel translational start sites outside annotated protein coding regions. In summary, unidentified MS/MS spectra were matched to a specific N-terminal peptide library encompassing protein N termini encoded in the Arabidopsis thaliana genome. After a stringent false discovery rate filtering, 117 protein N termini compliant with N-terminal methionine excision specificity and indicative of translation initiation were found. These include N-terminal protein extensions and translation from transposable elements and pseudogenes. Gene prediction provided supporting protein-coding models for approximately half of the protein N termini. Besides the prediction of functional domains (partially) contained within the newly predicted ORFs, further supporting evidence of translation was found in the recently released Araport11 genome re-annotation of Arabidopsis and computational translations of sequences stored in public repositories. Most interestingly, complementary evidence by ribosome profiling was found for 23 protein N termini. Finally, by analyzing protein N-terminal peptides, an in silico analysis demonstrates the applicability of our N-terminal proteogenomics strategy in revealing protein-coding potential in species with well-and poorly-annotated genomes

    Anti-melanocortin-4 receptor autoantibodies in obesity

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    Background: The melanocortin-4 receptor (MC4R) is part of an important pathway regulating energy balance. Here we report the existence of autoantibodies (autoAbs) against the MC4R in sera of obese patients. Methods: The autoAbs were detected after screening of 216 patients' sera by using direct and inhibition ELISA with an N-terminal sequence of the MC4R. Binding to the native MC4R was evaluated by flow cytometry and pharmacological effects by measuring adenylyl cyclase activity. Results: Positive results in all tests were obtained in patients with overweight or obesity (prevalence: 3.6%) but not in normal weight patients. The selective binding properties of anti-MC4R autoAbs were confirmed by surface plasmon resonance and by immunoprecipitation with the native MC4R. Finally it was demonstrated that these autoAbs increased food intake in rats after passive transfer via intracerebroventricular injection. Conclusion: These observations suggest that inhibitory anti-MC4R autoAbs might contribute to the development of obesity in a small subpopulation of patients

    Integrin αvβ6-specific therapy for pancreatic cancer developed from foot-and-mouth-disease virus

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    GOALS OF INVESTIGATION: The 5-year survival rate for pancreatic ductal adenocarcinoma (PDAC) has remained at <5% for decades because no effective therapies have been identified. Integrin αvβ6 is overexpressed in most PDAC and represents a promising therapeutic target. Thus, we attempted to develop an αvβ6-specific peptide-drug conjugate (PDC) for therapy of PDAC. METHODOLOGY: We conjugated the DNA-binding pyrrolobenzodiazepine (PBD)-based payload SG3249 (tesirine) to an αvβ6-specific 20mer peptide from the VP1 coat protein of foot-and-mouth-disease virus (FMDV) (forming conjugate SG3299) or to a non-targeting peptide (forming conjugate SG3511). PDCs were tested for specificity and toxicity on αvβ6-negative versus-positive PDAC cells, patient-derived cell lines from tumor xenografts, and on two different in vivo models of PDAC. Immunohistochemical analyses were performed to establish therapeutic mechanism. RESULTS: The αvβ6-targeted PDC SG3299 was significantly more toxic (up to 78-fold) for αvβ6-expressing versus αvβ6-negative PDAC cell lines in vitro, and achieved significantly higher toxicity at equal dose than the non-targeted PDC SG3511 (up to 15-fold better). Moreover, SG3299 eliminated established (100mm3) Capan-1 PDAC human xenografts, extending the lifespan of mice significantly (P=0.005). Immunohistochemistry revealed SG3299 induced DNA damage and apoptosis (increased γH2AX and cleaved caspase 3, respectively) associated with significant reductions in proliferation (Ki67), β6 expression and PDAC tumour growth. CONCLUSION: The FMDV-peptide drug conjugate SG3299 showed αvβ6-selectivity in vitro and in vivo and can specifically eliminate αvβ6-positive cancers, providing a promising new molecular- specific therapy for pancreatic cancer

    Biomechanical modulation of collagen fragment-induced anabolic and catabolic activities in chondrocyte/agarose constructs

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    The present study examined the effect of collagen fragments on anabolic and catabolic activities by chondrocyte/agarose constructs subjected to dynamic compression..

    Fluorogenic Tagging of Peptide and Protein 3-Nitrotyrosine with 4-(Aminomethyl)-benzenesulfonic Acid for Quantitative Analysis of Protein Tyrosine Nitration

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    Protein 3-nitrotyrosine (3-NT) has been recognized as an important biomarker of nitroxidative stress associated with inflammatory and degenerative diseases, and biological aging. Analysis of protein-bound 3-NT continues to represent a challenge since in vivo it frequently does not accumulate on proteins in amounts detectable by quantitative analytical methods. Here, we describe a novel approach of fluorescent tagging and quantitation of peptide-bound 3-NT residues based on the selective reduction to 3-AT followed by reaction with 4-(amino-methyl)benzenesulfonic acid (ABS) in the presence of K3Fe(CN)6 to form a highly fluorescent 2-phenylbenzoxazole product. Synthetic 3-NT peptide (0.005–1 μM) upon reduction with 10 mM sodium dithionite and tagging with 2 mM ABS and 5 μM K3Fe(CN)6 in 0.1 M Na2HPO4 buffer (pH 9.0) was converted with yields >95% to a single fluorescent product incorporating two ABS molecules per 3-NT residue, with fluorescence excitation and emission maxima at 360 ± 2 and 490 ± 2 nm, respectively, and a quantum yield of 0.77 ± 0.08, based on reverse-phase LC with UV and fluorescence detection, fluorescence spectroscopy and LC–MS–MS analysis. This protocol was successfully tested for quantitative analysis of in vitro Tyr nitration in a model protein, rabbit muscle phosphorylase b, and in a complex mixture of proteins from C2C12 cultured cells exposed to peroxynitrite, with a detection limit of ca. 1 pmol 3-NT by fluorescence spectrometry, and an apparent LOD of 12 and 40 pmol for nitropeptides alone or in the presence of 100 μg digested cell proteins, respectively. LC–MS–MS analysis of ABS tagged peptides revealed that the fluorescent derivatives undergo efficient backbone fragmentations, allowing for sequence-specific characterization of protein Tyr nitration in proteomic studies. Fluorogenic tagging with ABS also can be instrumental for detection and visualization of protein 3-NT in LC and gel-based protein separations

    N-terminal acetyltransferase Naa40p whereabouts put into N-terminal proteoform perspective

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    The evolutionary conserved N-alpha acetyltransferase Naa40p is among the most selective N-terminal acetyltransferases (NATs) identified to date. Here we identified a conserved N-terminally truncated Naa40p proteoform named Naa40p25 or short Naa40p (Naa40S). Intriguingly, although upon ectopic expression in yeast, both Naa40p proteoforms were capable of restoring N-terminal acetylation of the characterized yeast histone H2A Naa40p substrate, the Naa40p histone H4 substrate remained N-terminally free in human haploid cells specifically deleted for canonical Naa40p27 or 237 amino acid long Naa40p (Naa40L), but expressing Naa40S. Interestingly, human Naa40L and Naa40S displayed differential expression and subcellular localization patterns by exhibiting a principal nuclear and cytoplasmic localization, respectively. Furthermore, Naa40L was shown to be N-terminally myristoylated and to interact with N-myristoyltransferase 1 (NMT1), implicating NMT1 in steering Naa40L nuclear import. Differential interactomics data obtained by biotin-dependent proximity labeling (BioID) further hints to context-dependent roles of Naa40p proteoforms. More specifically, with Naa40S representing the main co-translationally acting actor, the interactome of Naa40L was enriched for nucleolar proteins implicated in ribosome biogenesis and the assembly of ribonucleoprotein particles, overall indicating a proteoform-specific segregation of previously reported Naa40p activities. Finally, the yeast histone variant H2A.Z and the transcriptionally regulatory protein Lge1 were identified as novel Naa40p substrates, expanding the restricted substrate repertoire of Naa40p with two additional members and further confirming Lge1 as being the first redundant yNatA and yNatD substrate identified to date

    Enhancement of cell membrane poration by the antimicrobial peptide Melp5

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    Melittin, a natural antimicrobial peptide comprising 26 amino acid residues, can kill bacteria by inducing pores in cell membranes. Clinical applications of melittin as an antibiotic require a thorough understanding of its poration mechanism and mutations that enhance its antimicrobial activity. Previous experiments showed Melp5, a variant of melittin with five mutations, exhibits a higher poration ability. However, the mechanism of the enhanced poration ability is not fully understood. Here, we investigated the mechanism by comparing the poration of melittin and Melp5 using coarse-grained (CG) and all-atom (AA) molecular dynamics (MD) simulations. We observe that Melp5 is likely to form a pore with 5 peptides (pentameric), while melittin is likely to form a pore with 4 peptides (tetrameric). Our atomistic MD simulations show that the pentameric pore of Melp5 has a higher water permeability than the tetrameric pore of melittin. We also analyze the stability of the pores of melittin and Melp5 by calculating the interaction energies of the pores. In particular, we investigate the effects of mutant residues on pore stability by calculating electrostatic and LJ interactions. These results should provide insights on the enhanced poration ability of Melp5 and push it toward applications
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