Human cyclophilin D is a mitochondrial peptidyl-prolyl isomerase that plays a role in regulating the opening of the mitochondrial permeability transition pore. It is considered a viable and promising molecular target for the treatment of diseases for which disease development is associated with pore opening, e.g., Alzheimer's disease or ischemia/reperfusion injury. Currently available and widely used in vitro methods based on Kofron's assay for determining cyclophilin D activity suffer from serious drawbacks and limitations. In this study, a completely novel approach for an in vitro assay of cyclophilin D activity using RNase T1 refolding is introduced. The method is simple and is more in line with the presumed physiological role of cyclophilin D in protein folding than Kofron's assay, which relies on a peptide substrate. The method is applicable for identifying novel inhibitors of cyclophilin D as potential drugs for the treatment of the diseases mentioned above. Moreover, the description of CypD activity in the in vitro RNase T1 refolding assay reveals new possibilities for investigating the role of cyclophilin D in protein folding in cells and may lead to a better understanding of its pathological and physiological roles.
- MeSH
- Aspergillus oryzae enzymologie MeSH
- guanyloribonukleasa chemie MeSH
- konformace proteinů MeSH
- lidé MeSH
- mitochondrie metabolismus MeSH
- molekulární modely MeSH
- objevování léků * MeSH
- peptidylprolylisomerasa F chemie metabolismus MeSH
- refolding proteinů * MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Maintenance of cellular proteostasis is achieved by a multi-layered quality control network, which counteracts the accumulation of misfolded proteins by refolding and degradation pathways. The organized sequestration of misfolded proteins, actively promoted by cellular sequestrases, represents a third strategy of quality control. Here we determine the role of sequestration within the proteostasis network in Saccharomyces cerevisiae and the mechanism by which it occurs. The Hsp42 and Btn2 sequestrases are functionally intertwined with the refolding activity of the Hsp70 system. Sequestration of misfolded proteins by Hsp42 and Btn2 prevents proteostasis collapse and viability loss in cells with limited Hsp70 capacity, likely by shielding Hsp70 from misfolded protein overload. Btn2 has chaperone and sequestrase activity and shares features with small heat shock proteins. During stress recovery Btn2 recruits the Hsp70-Hsp104 disaggregase by directly interacting with the Hsp70 co-chaperone Sis1, thereby shunting sequestered proteins to the refolding pathway.
- MeSH
- homeostáze proteinů * MeSH
- proteiny tepelného šoku HSP40 metabolismus MeSH
- proteiny tepelného šoku HSP70 metabolismus MeSH
- proteiny teplotního šoku metabolismus MeSH
- refolding proteinů MeSH
- Saccharomyces cerevisiae - proteiny metabolismus MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- transportní systémy aminokyselin metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mouse activating Nkrp1 proteins are commonly described as type II transmembrane receptors with disulfide-linked homodimeric structure. Their function and the manner in which Nkrp1 proteins of mouse strain (C57BL/6) oligomerize are still poorly understood. To assess the oligomerization state of Nkrp1 proteins, mouse activating EGFP-Nkrp1s were expressed in mammalian lymphoid cells and their oligomerization evaluated by Förster resonance energy transfer (FRET). Alternatively, Nkrp1s oligomers were detected by Western blotting to specify the ratio between monomeric and dimeric forms. We also performed structural characterization of recombinant ectodomains of activating Nkrp1 receptors. Nkrp1 isoforms c1, c2 and f were expressed prevalently as homodimers, whereas the Nkrp1a displays larger proportion of monomers on the cell surface. Cysteine-to-serine mutants revealed the importance of all stalk cysteines for protein dimerization in living cells with a major influence of cysteine at position 74 in two Nkrp1 protein isoforms. Our results represent a new insight into the oligomerization of Nkrp1 receptors on lymphoid cells, which will help to determine their function.
- MeSH
- antigeny Ly analýza MeSH
- Cercopithecus aethiops MeSH
- COS buňky MeSH
- Jurkat buňky MeSH
- lektinové receptory NK-buněk - podrodina B analýza MeSH
- lidé MeSH
- multimerizace proteinu MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- receptory imunologické analýza MeSH
- refolding proteinů MeSH
- rezonanční přenos fluorescenční energie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
As a part of the innate immunity, NK (Natural Killer) cells provide an early immune response to different stimuli, e.g. viral infections and tumor growths. However, their functions are more complex; they play an important role in reproduction, alloimmunity, autoimmunity and allergic diseases. NK cell activities require an intricate system of regulation that is ensured by many different receptors on a cell surface which integrate signals from interacting cells and soluble factors. One way to understand NK cell biology is through the structure of NK receptors, which can reveal ligand binding conditions. We present a modified protocol for recombinant expression in Escherichia coli and in vitro refolding of the ligand-binding domain of the inhibitory Nkrp1b (SJL/J) protein. Nkrp1b identity and folding was confirmed using mass spectrometry (accurate mass of the intact protein and evaluation of disulfide bonds) and one-dimensional nuclear magnetic resonance spectroscopy. The intention is to provide the basis for conducting structural studies of the inhibitory Nkrp1b protein, since only the activating Nkrp1a receptor structure is known.
- MeSH
- buněčná inkluze MeSH
- disulfidy chemie MeSH
- Escherichia coli metabolismus MeSH
- hmotnostní spektrometrie MeSH
- lektinové receptory NK-buněk - podrodina B biosyntéza chemie genetika MeSH
- magnetická rezonanční spektroskopie MeSH
- molekulární sekvence - údaje MeSH
- myši MeSH
- refolding proteinů MeSH
- rekombinantní proteiny biosyntéza MeSH
- sekvence aminokyselin MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Proteins MPT63 and MPT83 which are common for both Mycobacterium tuberculosis and Mycobacterium bovis, due to their high immunogenicity, are thought to play a promising role in the development of immunodiagnostic reagents and vaccines. To enhance the antigenic and immunogenic properties of these proteins, fragments of the mpt83 and mpt63 genes were fused in tandem. In this article we present an effective method for the MPT63-MPT83 fusion product purification by metal-affinity chromatography and in vitro refolding. Our results demonstrate that the antigenic properties of the recombinant proteins obtained are comparable to their native analogues. The anti-rMPT63 and anti-rMPT83 sera were found to be highly reactive against the rMPT63-MPT83 fusion protein, which suggests that the fusion protein retains the antigenic properties of the parent proteins. Our results may potentially contribute to the development of improved diagnostic tools or vaccines against human and/or cattle tuberculosis.
- MeSH
- antigeny bakteriální * analýza genetika imunologie MeSH
- bakteriální proteiny * analýza genetika imunologie MeSH
- buněčná inkluze MeSH
- chromatografie MeSH
- epitopy imunologie MeSH
- Mycobacterium tuberculosis * genetika imunologie MeSH
- rekombinantní fúzní proteiny analýza biosyntéza MeSH
- rekombinantní proteiny biosyntéza terapeutické užití MeSH
- renaturace proteinů MeSH
- sbalování proteinů MeSH
- syntetické vakcíny genetika imunologie MeSH
- techniky amplifikace nukleových kyselin MeSH
- techniky in vitro MeSH
- western blotting MeSH
Mouse NKR-P1C(B6) receptor corresponding to NK1.1 alloantigen is one of the most widespread surface markers of mouse NK and NKT cells in C57BL/6 mice detected by monoclonal antibody PK136. Although functional studies revealed the ability of this receptor to activate both natural killing and production of cytokines upon antibody crosslinking, the ligand for NKR-P1C(B6) remains unknown. In order to initiate ligand identification, structural studies, and epitope mapping experiments, we developed a simple and efficient expression and purification protocol allowing to produce large amounts of pure soluble monomeric mouse NKR-P1C(B6). Our protein encompassed approximately half of the stalk region and the entire C-terminal globular ligand binding domain. The identity of protein that was devoid of N-terminal initiation methionine and had all three expected disulfides closed was confirmed using high resolution ion cyclotron resonance mass spectrometry. Protein produced into inclusion bodies in Escherichia coli was efficiently refolded into a unique three dimensional structure as confirmed by NMR using (1)H-(15)N-HSQC spectra of uniformly labeled protein. The exceptional purity of the protein should allow its crystallization and detailed structural investigations, and is a prerequisite for its use as a probe in ligand identification and antibody epitope mapping experiments.
- MeSH
- antigeny povrchové genetika imunologie izolace a purifikace metabolismus MeSH
- buněčná inkluze genetika metabolismus MeSH
- buňky NK imunologie metabolismus MeSH
- Escherichia coli MeSH
- exprese genu MeSH
- hmotnostní spektrometrie MeSH
- klonování DNA MeSH
- lektinové receptory NK-buněk - podrodina B genetika imunologie izolace a purifikace metabolismus MeSH
- ligandy MeSH
- molekulární sekvence - údaje MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- refolding proteinů MeSH
- rekombinantní proteiny genetika imunologie izolace a purifikace metabolismus MeSH
- rozpustnost MeSH
- sekundární struktura proteinů MeSH
- sekvence aminokyselin MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
