RESUMO
Peroxisomal matrix proteins contain either a peroxisomal targeting sequence 1 (PTS1) or a PTS2 that are recognized by the import receptors PEX5 and PEX7, respectively. PEX5 transports the PTS1 proteins and the PEX7/PTS2 complex to the docking translocation module (DTM) at the peroxisomal membrane. After cargo release PEX5 is monoubiquitinated and extracted from the peroxisomal membrane by the receptor export machinery (REM) comprising PEX26 and the AAA ATPases PEX1 and PEX6. Here, we investigated the protein interactions of monoubiquitinated PEX5 with the docking proteins PEX13, PEX14 and the REM. "Click" chemistry was used to synthesise monoubiquitinated recombinant PEX5. We found that monoubiquitinated PEX5 binds the PEX7/PTS2 complex and restores PTS2 protein import in vivo in ΔPEX5 fibroblasts. In vitro pull-down assays revealed an interaction of recombinant PEX5 and monoubiquitinated PEX5 with PEX13, PEX14 and with the REM components PEX1, PEX6 and PEX26. The interactions with the docking proteins were independent of the PEX5 ubiquitination status whereas the interactions with the REM components were increased when PEX5 is ubiquitinated.
Assuntos
Receptor 1 de Sinal de Orientação para Peroxissomos/química , Peroxissomos/química , Mapas de Interação de Proteínas/genética , Transporte Proteico/genética , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/genética , Sequência de Aminoácidos/genética , Animais , Química Click , Citosol/química , Citosol/metabolismo , Fibroblastos/química , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Simulação de Acoplamento Molecular , Mutação , Receptor 2 de Sinal de Orientação para Peroxissomos/química , Receptor 2 de Sinal de Orientação para Peroxissomos/genética , Sinais de Orientação para Peroxissomos/genética , Receptor 1 de Sinal de Orientação para Peroxissomos/genética , Peroxissomos/genética , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitinação/genéticaRESUMO
Capillary zone electrophoresis (CZE) provides an alternative means of separating native proteins on the basis of their inherent electrophoretic mobilities. The major advantage of CZE is the quantification by UV detection, circumventing the drawbacks of staining and densitometry in the case of gel electrophoresis methods. The data of this validation study showed that CZE is a reliable assay for the determination of protein composition in therapeutic preparations of human albumin and human polyclonal immunoglobulins. Data obtained by CZE are in line with "historical" data obtained by the compendial method, provided that peak integration is performed without time correction. The focus here was to establish a rapid and reliable test to substitute the current gel based zone electrophoresis techniques for the control of protein composition of human immunoglobulins or albumins in the European Pharmacopoeia. We believe that the more advanced and modern CZE method described here is a very good alternative to the procedures currently described in the relevant monographs.
Assuntos
Eletroforese Capilar/instrumentação , Eletroforese Capilar/métodos , Imunoglobulinas/química , Albumina Sérica/química , Humanos , Imunoglobulinas/uso terapêutico , Reprodutibilidade dos Testes , Albumina Sérica/uso terapêuticoRESUMO
Ubiquitin and ubiquitin-like proteins such as SUMO represent important and abundant post-translational modifications involved in many cellular processes. These modifiers are reversibly attached via an isopeptide bond to lysine side chains of their target proteins by the action of specific E1, E2, and E3 enzymes. A significant challenge in studying ubiquitylation and SUMOylation is the frequently encountered inability to access desired conjugates at a defined position of the target protein and in homogenous form by using enzymatic preparation. In recent years, several chemical conjugation approaches have been developed to overcome this limitation. In this study, we aimed to selectively SUMOylate a 189-amino acid fragment of human RanGAP1 (amino acids 398-587) at the position of Lys524 by applying two recently reported approaches based on the Cu(I)-catalyzed alkyne-azide cycloaddition. Because of low yields observed for the incorporation of an unnatural amino acid with an azide moiety by the tRNA suppression technology, this route was abandoned. However, installing a single cysteine at position 524 and its selective alkylation was successful to introduce the azide group. The triazole-linked SUMO1**RanGAP1 conjugate could be obtained in good yields, purified, and was shown to specifically interact with RanBP2/Ubc9. Thus, we expand the scope of proteins accessible to chemical conjugation with ubiquitin-like proteins and underline the importance of having alternative approaches to do so.
