RESUMO
The development of powerful and general methods to acquire ubiquitin (Ub) chains has prompted the deciphering of Ub-mediated processes. Herein, the cysteine-aminoethylation assisted chemical ubiquitination (CAACU) strategy is extended and improved to enable the efficient semi-synthesis of atypical Ub chain analogues and Ub-based probes. Combining the Cys aminoethylation and the auxiliary-mediated protein ligation, several linkage- and length-defined atypical Ub chains including di-Ubs, K27C-linked tri-Ub, K11/K48C-branched tri-Ub, and even the SUMOlated Ub are successfully prepared from recombinantly expressed starting materials at about a 9-20â mg L-1 expression level. In addition, the utility of this strategy is demonstrated with the synthesis of a novel non-hydrolyzable di-Ub PA probe, which may provide a new useful tool for the mechanistic studies of deubiquitinase (DUB) recognition.
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The breakdown of long-lived proteins (LLPs) is associated with aging, as well as disease; however, our understanding of the molecular processes involved is still limited. Of particular relevance, cross-linked proteins are often reported in aged tissues but the mechanisms for their formation are poorly understood. In the present study, sites of protein cross-linking in human ocular lenses were characterized using proteomic techniques. In long-lived lens proteins, several sites of cross-linking were found to involve the addition of Lys to Asp or Asn residues. Using model peptides containing Asp or Asn, a mechanism was elucidated that involves a succinimide intermediate. Succinimides formed readily from Asn at neutral pH, whereas a higher rate of formation from Asp peptides was observed at more acidic pHs. Succinimides were found to be relatively stable in the absence of nucleophiles. Since racemization of Asp residues, as well as deamidation of Asn, involves a succinimide intermediate, sites of d-Asp and isoAsp in LLPs should also be considered as potential sites of protein covalent cross-linking.
Assuntos
Asparagina/metabolismo , Ácido Aspártico/metabolismo , Cristalino/metabolismo , Succinimidas/metabolismo , Idoso , Sequência de Aminoácidos , Asparagina/genética , Ácido Aspártico/genética , HumanosRESUMO
Cellular factor XIII (cFXIII, FXIII-A2), a transglutaminase, has been demonstrated in a few cell types. Its main function is to cross-link proteins by isopeptide bonds. Here, we investigated the presence of cFXIII in cells of human cornea. Tissue sections of the cornea were immunostained for FXIII-A in combination with staining for CD34 antigen or isopeptide cross-links. Isolated corneal keratocytes were also evaluated by immunofluorescent microscopy and flow cytometry. FXIII-A in the corneal stroma was quantified by Western blotting. FXIII-A mRNA was detected by RT-qPCR. The cornea of FXIII-A-deficient patients was evaluated by cornea topography. FXIII-A was detected in 68 ± 13% of CD34+ keratocytes. Their distribution in the corneal stroma was unequal; they were most abundant in the subepithelial tertile. cFXIII was of cytoplasmic localization. In the stroma, 3.64 ng cFXIII/mg protein was measured. The synthesis of cFXIII by keratocytes was confirmed by RT-qPCR. Isopeptide cross-links were detected above, but not within the corneal stroma. Slight abnormality of the cornea was detected in six out of nine FXIII-A-deficient patients. The presence of cFXIII in human keratocytes was established for the first time. cFXIII might be involved in maintaining the stability of the cornea and in the corneal wound healing process.
Assuntos
Ceratócitos da Córnea/metabolismo , Substância Própria/metabolismo , Fator XIII/metabolismo , Transglutaminases/metabolismo , Testes de Coagulação Sanguínea/métodos , Lesões da Córnea/metabolismo , Humanos , RNA Mensageiro/metabolismo , Cicatrização/fisiologiaRESUMO
Streptococcus pyogenes and other Gram-positive bacterial pathogens present long macromolecular filaments known as pili on their surface that mediate adhesion and colonization. These pili are covalent polymers, assembled by sortases. Typically, they comprise a putative adhesin at their tip, a backbone subunit present in multiple copies and a basal subunit that is covalently anchored to the peptidoglycan layer of the cell surface. The crystal structures of pilin subunits revealed the presence of unusual covalent linkages in these proteins, including intramolecular isopeptide and internal thioester bonds. The intramolecular isopeptide bonds in backbone pilins are important for protein stability. Here, using both the wild-type protein and a set of mutants, we assessed the proteolytic and thermal stability of the S. pyogenes pilus tip adhesin Spy0125, in the presence and absence of its intramolecular isopeptide and internal thioester bonds. We also determined a crystal structure of the internal thioester bond variant Spy0125(Cys426Ala). We find that mutations in the intramolecular isopeptide bonds compromise the stability of Spy0125. Using limited proteolysis and thermal denaturation assays, we could separate the contribution of each intramolecular isopeptide bond to Spy0125 stability. In contrast, mutation in the internal thioester bond had a lesser effect on protein stability and the crystal structure is essentially identical to wild type. This work suggests that the internal thioester in Spy0125, although having a minor contributory role, is not required for protein stability and must have a different primary function, most likely mediating a covalent interaction with host cell ligands.
Assuntos
Adesinas Bacterianas/química , Fímbrias Bacterianas/química , Streptococcus pyogenes/genética , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Dicroísmo Circular , Cristalografia por Raios X , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Modelos Moleculares , Estabilidade Proteica , Streptococcus pyogenes/química , Streptococcus pyogenes/enzimologiaRESUMO
Sortase-dependent pili are long surface appendages that mediate attachment, colonization and biofilm formation in certain genera and species of Gram-positive bacteria. Ligilactobacillus ruminis is an autochthonous gut commensal that relies on sortase-dependent LrpCBA pili for host adherence and persistence. X-ray crystal structure snapshots of the backbone pilin LrpA were captured in two atypical bent conformations leading to a zigzag morphology in the LrpCBA pilus structure. Small-angle X-ray scattering and structural analysis revealed that LrpA also adopts the typical linear conformation, resulting in an elongated pilus morphology. Various conformational analyses and biophysical experiments helped to demonstrate that a hinge region located at the end of the flexible N-terminal domain of LrpA facilitates a new closure-and-twist motion for assembling dynamic pili during the assembly process and host attachment. Further, the incongruent combination of flexible domain-driven conformational dynamics and rigid isopeptide bond-driven stability observed in the LrpCBA pilus might also extend to the sortase-dependent pili of other bacteria colonizing a host.
Assuntos
Proteínas de Fímbrias , Fímbrias Bacterianas , Fímbrias Bacterianas/química , Cristalografia por Raios X , Proteínas de Fímbrias/química , Proteínas de Fímbrias/metabolismo , Modelos Moleculares , Domínios Proteicos , Bacillaceae , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Conformação ProteicaRESUMO
To reduce the waste from yak hair and introduce resource recycling into the yak-related industry, an eco-friendly yak keratin-based bioplastic film was developed. We employed yak keratin (USYK) from yak hair, soy protein isolate (SPI) from soybean meal as a film-forming agent, transglutaminase (EC 2.3.2.13, TGase) as a catalytic crosslinker, and glycerol as a plasticizer for USYK-SPI bioplastic film production. The structures of the USYK-SPI bioplastic film were characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and X-Ray diffraction (XRD). The mechanical properties, the thermal behavior, light transmittance performance, and water vapor permeability (WVP) were measured. The results revealed that the added SPI possibly acted as a reinforcement. The formation of Gln-Lys isopeptide bonds and hydrophobic interactions led to a stable crosslinking structure of USYK-SPI bioplastic film. The thermal and the mechanical behaviors of the USYK-SPI bioplastic film were improved. The enhanced dispersion and formation of co-continuous protein matrices possibly produced denser networks that limited the diffusion of water vapor and volatile compounds in the USYK-SPI bioplastic films. Moreover, the introduction of SPI prompted the relocation of hydrophobic groups on USYK molecules, which gave the USYK-SPI bioplastic film stronger surface hydrophobicity. The SPI and USYK molecules possess aromatic amino residuals (tyrosine, phenylalanine, tryptophan), which can absorb ultraviolet radiation. Thus, the USYK-SPI bioplastic films were shown to have an excellent UV barrier. The synergy effect between USYK and SPI is not only able to improve rigidity and the application performance of keratin-based composite film but can also reduce the cost of the keratin-based composite film through the low-cost of the SPI alternative which partially replaces the high-cost of keratin. The data obtained from this research can provide basic information for further research and practical applications of USYK-SPI bioplastic films. There is an increasing demand for the novel USYK-SPI bioplastic film in exploit packaging material, biomedical materials, eco-friendly wearable electronics, and humidity sensors.
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The internal isopeptide bonds are amide bonds formed autocatalytically between the side chains of Lys and Asn/Asp residues and have been discovered recently. These bonds are well conserved in Gram-positive bacterial pilin proteins and are also observed over a wide range of Gram-positive bacterial surface proteins. The presence of these bonds confers the pilus subunits with remarkable properties in terms of thermal stability and resistance to proteases. Like pili, microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) are also surface proteins found only in Gram-positive bacteria. They specifically interact with the extracellular matrix (ECM) molecules like collagen, fibrinogen, fibronectin, laminin, etc. Many biophysical and biochemical studies have been carried out to characterize the isopeptide bonds in pili proteins from Gram-positive bacteria, but no attempts have been made to study the isopeptide bonds in MSCRAMMs. This short review aims to study the significance of the isopeptide bonds in relation to their function, by analyzing the crystal structures of collagen- and fibrinogen-binding MSCRAMMs. In this analysis, interestingly, we observed that the putative isopeptide bonds are restricted to the collagen-binding MSCRAMMs. Based on analogy with bacterial pilus subunits, we hypothesize that the collagen-binding MSCRAMMs possessing putative isopeptide bonds exhibit similar structural properties, which could help the bacteria in colonizing the host and provide resistance against host-defense mechanisms.