Serum concentrations of free ubiquitin and multiubiquitin chains

Takada, Koji; Nasu, Hidekazu; Hibi, Nozomu; Tsutsumi, Yutaka; Shibasaki, Toshiaki; Fujise, Kiyotaka; Fujimuro, Masahiro; Sawada, Hitoshi; Yokosawa, Hideyoshi; Ohkawa, Kiyoshi

doi:10.1093/clinchem/43.7.1188

Cited by 57 publications

(60 citation statements)

References 35 publications

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“…The sequence of purified free Ub from secretions of stimulated chromaffin cells and Ub-derived peptide (65)(66)(67)(68)(69)(70)(71)(72)(73)(74)(75)(76), resulting from the digestion of Ub with endoproteinase Glu-C, was determined in our laboratory by automatic Edman degradation on an Applied Biosystems (Perkin Elmer, Boston, MA) 473A sequencer at the picomolar range. Samples purified by HPLC were loaded on polybrene-treated and precycled glass-fiber filters (18).…”

Section: N-terminal Sequence Analysismentioning

confidence: 99%

“…In addition, the synthetic peptide corresponding to Ub 65-76 is active against the yeast forms of C. albicans, C. tropicalis, C. neoformans, and C. glabrata with a MIC varying from 15 to 20 µM. These experiments indicate a lytic effect of Ub [65][66][67][68][69][70][71][72][73][74][75][76] against Gram-positive bacteria and fungi, because we could not obtain a restart of the microorganism growth after 48 h. Furthermore, as other antimicrobial peptides (36)(37)(38), 50 µM Ub 65-76 is unable to induce the lysis of sensitive mammalian cells like erythrocytes at a concentration up to 50 µM.…”

Section: Antimicrobial Activities Of Bovine Ub and Ub 65-76mentioning

confidence: 99%

“…As a typical example, the activities of various concentrations of Ub 65-76 without (control experiment) and in the presence of 10 µM Ub 1-34 were tested against T. mentagrophytes (a widespread dermatophyte associated with skin mycose) and A. fumigatus as illustrated in Figure 4A and 4B, respectively. Ub [65][66][67][68][69][70][71][72][73][74][75][76] was tested against T. mentagrophytes after incubation during 48 h at increasing concentrations from 7.5 to 35 µM, and the complete inhibition was obtained with 35 µM. Similarly, Ub 65-76 was tested against A. fumigatus after incubation for 21 h at increasing concentrations from 15 to 50 µM, and the complete inhibition was obtained with 50 µM Ub 65-76. By comparison, the complete inhibition with the mixture of the two peptides is obtained with 10 µM Ub 1-34 /20 µM Ub 65-76 against T. mentagrophytes (Fig.…”

Section: Synergistic Effect Of Ub 1-34 On the Antifungal Activity Of mentioning

confidence: 99%

“…5A4). However, if A. fumigatus was treated for 1 h with unlabeled Ub [65][66][67][68][69][70][71][72][73][74][75][76] (20 µM) and CGB 602-626R (10 µM), an intense fluorescence was observed indicating that Ub 65-76 destabilizes the cell wall, allowing the inactive peptide to penetrate into the fungi (Fig. 5A5).…”

Section: Confocal Laser Scan Analysis Of the Interaction Of Ub 65-76 mentioning

confidence: 99%

“…Confocal laser microscopy on filamentous fungi, using rhodaminelabeled synthetic peptides Ub 1-34R and Ub 65-76R , showed the ability of these peptides to interact with (Ub 1-34R , Ub 65-76R ) and to cross through the cell wall and accumulate in microorganisms (Ub 65-76R ). We demonstrated that the N-and C-terminal fragments (Ub and Ub [65][66][67][68][69][70][71][72][73][74][75][76] , respectively) act synergistically to kill fungi by destabilization of fungal cell wall and plasma membrane. Furthermore, this mechanism may be completed by the inhibition of intracellular CaM-dependent enzymes.…”

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The N‐ and C‐terminal fragments of ubiquitin are important for the antimicrobial activities

et al. 2003

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Secretory granules of chromaffin cells contain catecholamines and several antimicrobial peptides derived from chromogranins and proenkephalin-A. These peptides are secreted in the extracellular medium following exocytosis. Here, we show that ubiquitin is stored in secretory chromaffin granules and released into the circulation upon stimulation of chromaffin cells. We also show that the C-terminal fragment (residues 65-76) of ubiquitin displays, at the micromolar range, a lytic antifungal activity. Using confocal laser scan microscopy and rhodamine-labeled synthetic peptides, we could demonstrate that the C-terminal peptide (residues 65-76) is able to cross the cell wall and the plasma membrane of fungi and to accumulate in fungi, whereas the N-terminal peptide (residues 1-34) is stopped at the fungal wall level. Furthermore, these two peptides act synergistically to kill filamentous fungi. Because of the interaction of the C-terminal sequence of ubiquitin with calmodulin, the synthetic peptide (residues 65-76) was tested in vitro against calmodulin-dependent calcineurin, an enzyme crucial for fungal growth. This peptide was found to inhibit the phosphatase activity of calcineurin. Our data show a new property of ubiquitin C-terminal-derived peptide (65-76) that could be used with N-terminal peptide (1-34) as a new potent antifungal agent.

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Section: N-terminal Sequence Analysismentioning

confidence: 99%

Section: Antimicrobial Activities Of Bovine Ub and Ub 65-76mentioning

confidence: 99%

Section: Synergistic Effect Of Ub 1-34 On the Antifungal Activity Of mentioning

confidence: 99%

Section: Confocal Laser Scan Analysis Of the Interaction Of Ub 65-76 mentioning

confidence: 99%

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confidence: 99%

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The N‐ and C‐terminal fragments of ubiquitin are important for the antimicrobial activities

et al. 2003

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MET‐Activating Ubiquitin Multimers

et al. 2023

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Receptor tyrosine kinases (RTKs) are generally activated through their dimerization and/or oligomerization induced by their cognate ligands, and one such RTK hepatocyte growth factor (HGF) receptor, known as MET, plays an important role in tissue regeneration. Here we show the development of ubiquitin (Ub)‐based protein ligand multimers, referred to as U‐bodies, which act as surrogate agonists for MET and are derived from MET‐binding macrocyclic peptides. Monomeric Ub constructs (U‐body) were first generated by genetic implantation of a macrocyclic peptide pharmacophore into a structural loop of Ub (lasso‐grafting) and subsequent optimization of its flanking spacer sequences via mRNA display. Such U‐body constructs exhibit potent binding affinity to MET, thermal stability, and proteolytic stability. The U‐body constructs also partially/fully inhibited or enhanced HGF‐induced MET‐phosphorylation. Their multimerization to dimeric, tetrameric, and octameric U‐bodies linked by an appropriate peptide linker yielded potent MET activation activity and downstream cell proliferation‐promoting activity. This work suggests that lasso‐grafting of macrocycles to Ub is an effective approach to devising protein‐based artificial RTK agonists and it can be useful in the development of a new class of biologics for various therapeutic applications.

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