ABSTRACT
A stable cell line based on HEK293 cells that expresses proteasome subunit PSMD14 fused to the fluorescent protein EGFP and HTBH tag has been selected. This chimera was shown to be incorporated completely into proteolitic active proteasomes. The created cell line can be used for further fluorescent studies of proteasomes localization in the cell.
Subject(s)
Founder Effect , Green Fluorescent Proteins/metabolism , Plasmids/chemistry , Proteasome Endopeptidase Complex/metabolism , Recombinant Fusion Proteins/metabolism , Trans-Activators/metabolism , Cell Engineering , Gene Expression , Green Fluorescent Proteins/genetics , HEK293 Cells , Humans , Microscopy, Fluorescence , Proteasome Endopeptidase Complex/genetics , Proteolysis , Recombinant Fusion Proteins/genetics , Staining and Labeling , Trans-Activators/genetics , TransfectionABSTRACT
In this paper, we present a comparative analysis of different methods of purification of proteasomes from the culture medium in which proerithroleukemia human K562 cells were grown. The results obtained allowed us to purify proteasomes from samples of conditioned cell culture medium and control the quality of the proteasome preparations at all stages of their separation. Extracellular proteasomes purified via different approaches possess all the three types of peptidase activity described for intracellular counterparts.
Subject(s)
Caspases/isolation & purification , Chymotrypsin/isolation & purification , Proteasome Endopeptidase Complex/isolation & purification , Trypsin/isolation & purification , Caspases/chemistry , Chymotrypsin/chemistry , Culture Media, Conditioned/chemistry , Humans , K562 Cells , Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Trypsin/chemistry , UbiquitinationABSTRACT
The analysis of the extracellular proteasomes by isobaric tagging for relative and absolute quantifications (iTRAQ) mass spectrometry has been carried out. Here we show a standard set of 26S proteasomal subunits in the composition of the extracellular proteasomes. Moreover, extracellular proteasomes have a number of PA200 activators, which, as previously thought, are localized in the cell nucleus. Posttranslational modifications (PTMs) of subunits of the extracellular proteasomes were revealed by iTRAQ mass spectrometry. For the first time we have identified several ubiquitination and acetylation sites on subunits alpha2 (K196), alpha4 (K189 and K234), alpha6 (K217), and Rpn6 (A2). We have revealed a large number of proteasome-interacting proteins that are involved in various cell processes, such as transcription, DNA repair, translation, cytoskeletal proteins and the proteins of the ubiquitin-proteasome system (UPS). Immunoblot analysis has confirmed the interactions between purified extracellular proteasomes and nine proteins which were randomly selected from the set of interacting proteins.
Subject(s)
Cell Nucleus/metabolism , Proteasome Endopeptidase Complex , Protein Processing, Post-Translational , Electrophoresis, Gel, Two-Dimensional , Mass Spectrometry , Nuclear Proteins/metabolism , Phosphorylation , Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/classification , Proteasome Endopeptidase Complex/metabolism , Ubiquitin/metabolism , UbiquitinationABSTRACT
The 26S proteasome is a multi-subunit protein complex that consists of the catalytic 20S and regulatory 19S sub-complexes. The most well studied function of proteasomes is specific degradation of proteins. There are several purification schemes for obtaining the preparations of 26S proteasomes. An important step in purification of 26S proteasomes is concentration of the purified material for subsequent analysis of its biochemical functions. In this report we showed that the subunits composition of 26S proteasomes that have been concentrated by the different modes at the latest stage of their preparation is identical. However, the concentrating mode differently affects the functional activity of these complexes.
Subject(s)
Liver/chemistry , Multiprotein Complexes/isolation & purification , Proteasome Endopeptidase Complex/isolation & purification , Animals , Cytoplasm/chemistry , Multiprotein Complexes/chemistry , Proteasome Endopeptidase Complex/chemistry , Proteolysis , RatsABSTRACT
The presented review concerns the intracellular proteasome and their possible functions. The ubiquitin-proteasome system (UPS) is responsible for the common regulated proteolysis in the cell. 26S proteasome is a central proteolytic unit of UPS and is a multisubunit protein complex consisting of a core catalytic complex, called 20S proteasome, capped at one or both ends by 19S regulatory complex. Proteasomes have been shown in the extracellular space: in alveolar and cerebrospinal fluids, blood plasma. Extracellular proteasomes are intact intracellular particles that exhibit three types of specific peptidase activity. Extracellular proteasomes have been detected in both healthy people and patients with different diseases. Its concentration has been found to be increased in patients suffering from autoimmune diseases, malignant tumors, trauma or sepsis and to correlate with the disease progression, which has both diagnostic and prognostic value.
Subject(s)
Autoimmune Diseases/diagnosis , Bronchial Diseases/diagnosis , Extracellular Space/metabolism , Neoplasms/diagnosis , Proteasome Endopeptidase Complex , Sepsis/diagnosis , Animals , Autoimmune Diseases/blood , Autoimmune Diseases/cerebrospinal fluid , Bronchial Diseases/blood , Bronchial Diseases/cerebrospinal fluid , Bronchoalveolar Lavage Fluid/chemistry , Disease Progression , Humans , Neoplasms/blood , Neoplasms/cerebrospinal fluid , Prognosis , Proteasome Endopeptidase Complex/blood , Proteasome Endopeptidase Complex/cerebrospinal fluid , Proteolysis , Sepsis/blood , Sepsis/cerebrospinal fluid , Ubiquitin/metabolismABSTRACT
The comparative analysis of peptidase activities of extra- and intracellular proteasomes was carried out. Here we have shown that excreted proteasomes exhibit higher chymotrypsin-type and lower tripsin-like peptidase activities that cytoplasmic particles. Posttranslational modifications (PTMs) of 20S proteasomal subunits were revealed by immunoblotting techniques. We have observed the difference in PTMs of associated with enzymatic activities subunits beta2, beta5 and beta5 of extracellular and cytoplasmic proteasomes. Proteasomal subunits alpha2, 4, 7 and beta7 also had a variety of PTMs. The phosphorylation level of excreted proteasomes was lower compared to that of the intracellular ones. This observation strongly suggests the involvement of this PTM in the regulation of proteasomes excretion from cells.
