Rhomboid proteases are intramembrane enzymes that hydrolyze peptide bonds of transmembrane proteins in the lipid bilayer. They play a variety of roles in key biological events and are linked to several disease states. Over the last decade a great deal of structural and functional knowledge has been generated on this fascinating class of proteases. Both structural and kinetic analyses require milligram amounts of protein, which may be challenging for membrane proteins such as rhomboids. Here, we present a detailed protocol for optimization of expression and purification of three rhomboid proteases from Escherichia coli (ecGlpG), Haemophilus influenzae (hiGlpG), and Providencia stuartii (AarA). We discuss the optimization of expression conditions, such as concentration of inducing agent, induction time, and temperature, as well as purification protocol with precise details for each step. The provided protocol yields 1-2.5mg of rhomboid enzyme per liter of bacterial culture and can assist in structural and functional studies of intramembrane proteases.
- MeSH
- DNA vazebné proteiny biosyntéza chemie genetika izolace a purifikace MeSH
- endopeptidasy biosyntéza chemie genetika izolace a purifikace MeSH
- Escherichia coli enzymologie MeSH
- Haemophilus influenzae enzymologie MeSH
- kinetika MeSH
- lipidové dvojvrstvy chemie MeSH
- membránové proteiny biosyntéza chemie genetika izolace a purifikace MeSH
- molekulární biologie metody MeSH
- proteiny z Escherichia coli biosyntéza chemie genetika izolace a purifikace MeSH
- Providencia enzymologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
Rhomboids are ubiquitous intramembrane serine proteases that are involved in various signaling pathways. This fascinating class of proteases harbors an active site buried within the lipid milieu. High-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed the catalytic mechanism for rhomboid-mediated proteolysis; however, a quantitative characterization was lacking. Assessing an enzyme's catalytic parameters is important for understanding the details of its proteolytic reaction and regulatory mechanisms. To assay rhomboid protease activity, many challenges exist such as the lipid environment and lack of known substrates. Here, we summarize various enzymatic assays developed over the last decade to study rhomboid protease activity. We present detailed protocols for gel-shift and FRET-based assays, and calculation of KM and Vmax to measure catalytic parameters, using detergent solubilized rhomboids with TatA, the only known substrate for bacterial rhomboids, and the model substrate fluorescently labeled casein.
- MeSH
- buněčná membrána enzymologie MeSH
- DNA vazebné proteiny chemie izolace a purifikace metabolismus MeSH
- endopeptidasy chemie izolace a purifikace metabolismus MeSH
- enzymatické testy metody MeSH
- Escherichia coli enzymologie MeSH
- katalytická doména MeSH
- membránové proteiny chemie izolace a purifikace metabolismus MeSH
- proteiny z Escherichia coli chemie izolace a purifikace metabolismus MeSH
- proteolýza * MeSH
- substrátová specifita MeSH
- vazba proteinů MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
Dehydrogenase/reductase SDR family member 7 (DHRS7, SDR34C1, retSDR4) is one of the many endoplasmic reticulum bound members of the SDR superfamily. Preliminary results indicate its potential significance in human metabolism. DHRS7 containing TEV-cleavable His10 and FLAG-tag expressed in the Sf9 cell line was solubilised, purified, and reconstituted into liposomes to enable the improved characterisation of this enzyme in the future. Igepal CA-630 was determined to be the best detergent for the solubilisation process. The solubilised DHRS7 was purified using affinity chromatography, and the purified enzyme was subjected to TEV cleavage of the affinity tags and then repurified using subtractive Ni-IMAC. The cleaved and uncleaved versions of DHRS7 were successfully reconstituted into liposomes. In addition, using tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as the substrate, the cleaved liposomal DHRS7 was found to be inactive, whereas the pure and uncleaved liposomal DHRS7 were confirmed as enzymes, which reduce carbonyl group of the substrates.
- MeSH
- buněčná membrána MeSH
- lidé MeSH
- membránové proteiny chemie genetika izolace a purifikace metabolismus MeSH
- oxidoreduktasy chemie genetika izolace a purifikace metabolismus MeSH
- rekombinantní proteiny chemie genetika izolace a purifikace metabolismus MeSH
- Sf9 buňky MeSH
- Spodoptera MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Nejzhoubnější gynekologický nádor, karcinom ovaria, je příčinou více než 50 % úmrtí v této skupině nádorů. Více než 60 % případů je diagnostikováno až v pokročilých stadiích s výrazně sníženou pravděpodobností přežití pacientek. Diagnostické nástroje pro efektivní časnou detekci nebo screening zatím neexistují. Terapeutické možnosti (chirurgická operace, chemoterapie) jsou nedostatečně efektivní, neboť karcinom ovaria se vyznačuje silnou tendencí k recidivě spojenou s chemorezistencí. Jedním z nejdéle a nejrozsáhleji používaným markerem pro diagnostiku primárních nádorů i recidivy je CA125/MUC16. Tento glykoprotein je znám již od 80. let 20. století, ale teprve nejnovější studie odhalují jeho biologické funkce v karcinogenezi a interakcích s buňkami imunitního systému. V tomto článku se blíže podíváme na jeho biologický význam a především na současný stav použití CA125 jako významného diagnostického markeru u karcinomu ovaria. V diagnostice primárních nádorů se totiž začínají objevovat nové markery, navíc je význam monitoringu CA125 z hlediska detekce recidivy zpochybňován rozsáhlými klinickými studiemi, které nenalezly přínos pro dlouhodobé přežití pacientek. Také potenciální význam CA125 v imunoterapii karcinomu ovaria se nezdá být tak velký, jak se předpokládalo.
The most fatal gynecologic malignancy, ovarian cancer, causes more than 50% deaths in this tumor group. Most of cases (>60%) are diagnosed in advanced stages with poor 5-year survival prognosis. Diagnostic tools for an effective early detection or screening have not been found yet. Treatment possibilities (surgery, chemotherapy) are insufficient while high tendency to recurrence and chemoresistance occurs. CA125/MUC16 has been one of the most extensively used markers for a detection of primary tumors, or recurrence for a long time since its discovery in 80´s. However, the structure and biological functions of CA125 have been discovered relatively recently. CA125 may play an important role in carcinogenesis and interactions with cells of immune system. We reviewed the known biological functions and particularly the current state of using this marker as the diagnostic tool in ovarian cancer. Recently, many new markers emerged ambitiously to replace CA125; moreover, the importance of monitoring CA125 for the recurrence detection has also been questioned in large clinical trials. These studies have not found an impact for overall survival/mortality of patients. Also a potential of using CA125 targeted antibodies has not brought previously expected results so far.
- Klíčová slova
- RECAF, HE4, léčba, diagnostika,
- MeSH
- alfa-fetoproteiny diagnostické užití izolace a purifikace MeSH
- analýza přežití MeSH
- antigen CA-125 izolace a purifikace MeSH
- diagnostické techniky porodnicko-gynekologické trendy využití MeSH
- epididymální sekreční proteiny diagnostické užití izolace a purifikace MeSH
- financování organizované MeSH
- imunoterapie metody využití MeSH
- lidé MeSH
- lokální recidiva nádoru diagnóza komplikace MeSH
- membránové proteiny diagnostické užití izolace a purifikace MeSH
- nádorové biomarkery izolace a purifikace MeSH
- nádory vaječníků diagnóza prevence a kontrola MeSH
- statistika jako téma MeSH
- Check Tag
- lidé MeSH
BACKGROUND: Hemojuvelin (HJV) is one of essential components for expression of hepcidin, a hormone which regulates iron transport. HJV is mainly expressed in muscle and liver, and processing of HJV in both tissues is similar. However, hepcidin is expressed in liver but not in muscle and the role of the muscle HJV is yet to be established. Our preliminary analyses of mouse tissue HJV showed that the apparent molecular masses of HJV peptides are different in liver (50 kDa monomer and 35 and 20 kDa heterodimer fragments) and in muscle (55 kDa monomer and a 34 kDa possible large fragment of heterodimer). One possible explanation is glycosylation which could lead to difference in molecular mass. RESULTS: We investigated glycosylation of HJV in both liver and muscle tissue from mice. PNGase F treatment revealed that the HJV large fragments of liver and muscle were digested to peptides with similar masses, 30 and 31 kDa, respectively, and the liver 20 kDa small fragment of heterodimer was digested to 16 kDa, while the 50 kDa liver and 55 kDa muscle monomers were reduced to 42 and 48 kDa, respectively. Endo H treatment produced distinct digestion profiles of the large fragment: a small fraction of the 35 kDa peptide was reduced to 33 kDa in liver, while the majority of the 34 kDa peptide was digested to 33 kDa and a very small fraction to 31 kDa in muscle. In addition, liver HJV was found to be neuraminidase-sensitive but its muscle counterpart was neuraminidase-resistant. CONCLUSIONS: Our results indicate that different oligosaccharides are attached to liver and muscle HJV peptides, which may contribute to different functions of HJV in the two tissues.
- MeSH
- extracelulární prostor metabolismus MeSH
- genový knockout MeSH
- glykopeptidasa metabolismus MeSH
- glykosylace MeSH
- játra cytologie metabolismus MeSH
- membránové proteiny nedostatek genetika izolace a purifikace metabolismus MeSH
- myši MeSH
- neuraminidasa metabolismus MeSH
- orgánová specificita MeSH
- svaly cytologie metabolismus MeSH
- transport proteinů MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- hepatocelulární karcinom diagnóza klasifikace patologie MeSH
- imunohistochemie metody využití MeSH
- lidé MeSH
- membránové proteiny imunologie izolace a purifikace MeSH
- nádorové biomarkery MeSH
- nemoci jater diagnóza etiologie imunologie MeSH
- onkogenní proteiny imunologie izolace a purifikace MeSH
- Check Tag
- lidé MeSH
Membrane rafts and signaling molecules associated with them are thought to play important roles in immunoreceptor signaling. Rafts differ in their lipid and protein compositions from the rest of the membrane and are relatively resistant to solubilization by Triton X-100 or similar detergents, producing buoyant, detergent-resistant membranes (DRMs) that can be isolated by density gradient ultracentrifugation. One of the key signaling molecules present in T cell DRMs is the transmembrane adaptor protein LAT (linker for activation of T cells). In contrast to previous results, a recent study demonstrated that a LAT construct not present in the buoyant DRMs is fully able to support TCR signaling and development of T cells in vivo. This finding caused doubts about the real physiological role of rafts in TCR signaling. In this study, we demonstrate that these results can be explained by the existence of a novel type of membrane raft-like microdomains, producing upon detergent solubilization "heavy DRMs" containing a number of membrane molecules. At a moderate level of expression, LAT supported TCR signaling more efficiently than constructs targeted to the microdomains producing heavy DRMs or to nonraft membrane. We suggest that different types of membrane microdomains provide environments regulating the functional efficiencies of signaling molecules present therein.
- MeSH
- adaptorové proteiny signální transdukční imunologie metabolismus MeSH
- aktivace lymfocytů imunologie MeSH
- Jurkat buňky MeSH
- lidé MeSH
- membránové mikrodomény chemie imunologie MeSH
- membránové proteiny chemie imunologie izolace a purifikace metabolismus MeSH
- receptory antigenů T-buněk imunologie metabolismus MeSH
- signální transdukce imunologie MeSH
- T-lymfocyty chemie imunologie metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Membránové proteiny jsou lokalizovány na rozhranícli různých prostředí, a proto je jejich izolace a identifikace nezbytným předpoldadem pro pochopení řady fyziologických procesů. Extrémní hydrofobicita proteinů s transmembránovými doménami činí jejich analýzu klasickými proteomickými technikami velmi komplikovanou nebo téměř nemožnou. Proto je třeba hledat pro jejich analýzu alternativní přístupy. Technika využívající chromatografii na reverzní fázi pro redukci kontaminace rozpustnými proteiny a technika kombinující jednorozměrnou elektroforézu s kapalinovou chromatografii spojenou s hmotnostní spektrometrií se jeví jako slibné přístupy.
Membrane proteins are located on the interface between different environments. That is reason why their isolation and identification is essential for understanding of number of physiological processes. The extremely hydrophobic nature of proteins with transmembrane domains makes their analysis by classical techniques used in proteomics very complicated or nearly impossible. Thus it is necessary to seek for the alternative approaches for their analysis. Technique using chromatography on reversed phase for reduction of soluble contamination and technique using combination 1 D-gel electrophoresis and liquid chromatography on reversed phase coupled to mass spectrometry seems to be very promising.
- MeSH
- biologické markery krev MeSH
- diagnostické techniky a postupy využití MeSH
- erytropoéza fyziologie imunologie MeSH
- infekce metabolismus patofyziologie MeSH
- kationické antimikrobiální peptidy izolace a purifikace metabolismus MeSH
- lidé MeSH
- membránové proteiny izolace a purifikace metabolismus MeSH
- nádory metabolismus patofyziologie MeSH
- poruchy metabolismu železa etiologie komplikace metabolismus MeSH
- proteiny vázající transferin izolace a purifikace metabolismus MeSH
- receptory transferinu izolace a purifikace krev metabolismus MeSH
- transferin izolace a purifikace metabolismus MeSH
- zánět metabolismus patofyziologie MeSH
- železo izolace a purifikace metabolismus nedostatek MeSH
- Check Tag
- lidé MeSH
- MeSH
- diagnostické techniky a postupy využití MeSH
- hemochromatóza etiologie genetika MeSH
- kationické antimikrobiální peptidy izolace a purifikace metabolismus MeSH
- lidé MeSH
- membránové proteiny izolace a purifikace metabolismus MeSH
- poruchy metabolismu železa diagnóza etiologie komplikace MeSH
- volné radikály izolace a purifikace metabolismus toxicita MeSH
- železo izolace a purifikace krev metabolismus MeSH
- Check Tag
- lidé MeSH