Residue-specific incorporation of non-canonical amino acids (ncAAs) introduces bio-orthogonal functionalities into proteins. As such, this technique is applied in protein characterization and quantification. Here, we studied protein expression with three methionine analogs, namely photo-methionine (pMet), azidohomoalanine (Aha) and homopropargylglycine (Hpg), in prototrophic E. coli BL-21 and auxotrophic E. coli B834 to maximize ncAA content, thereby assessing the effect of ncAAs on bacterial growth and the expression of cytochrome b5 (b5M46), green fluorescence protein (MBP-GFP) and phage shock protein A. In auxotrophic E. coli, ncAA incorporation ranged from 50 to 70% for pMet and reached approximately 50% for Aha, after 26 h expression, with medium and low expression levels of MBP-GFP and b5M46, respectively. In the prototrophic strain, by contrast, the protein expression levels were higher, albeit with a sharp decrease in the ncAA content after the first hours of expression. Similar expression levels and 70-80% incorporation rates were achieved in both bacterial strains with Hpg. Our findings provide guidance for expressing proteins with a high content of ncAAs, highlight pitfalls in determining the levels of methionine replacement by ncAAs by MALDI-TOF mass spectrometry and indicate a possible systematic bias in metabolic labeling techniques using Aha or Hpg.
Biochemické děje jako fotosyntéza a respirace, při nichž dochází k redoxním reakcím a přenosu elektronů pomocí kaskád proteinových molekul, souhrnně je označujeme jako elektron‐transportní systémy, jsou pro život na Zemi klíčové a nezbytné. Vhodným modelovým proteinem pro studium strukturně funkčních vztahů elektron‐transportních systémů proteinové povahy a vlivu aminokyselinových zbytků či záměny centrálního kovu na redox procesy je azurin, který patří do rodiny malých rozpustných cupredoxinů a podílí se na přenosu elektronů při anaerobní respiraci bakterií rodu Pseudomonas.
Biochemical processes like photosynthesis and cellular respiration, which involve redox reactions and are facilitated by electron transfer through a cascade of protein molecules, commonly referred to as electron‐transfer systems, play a key role in all life on Earth. Azurin is a suitable model to study structural properties of such electron‐transfer systems and the role of individual amino acid residues or the central metal ion in redox reactions. This protein belongs to the family of small soluble electron transporters known as cupredoxines and has a physiological role during anaerobic respiration in Pseudomonas bacterial species.
- Klíčová slova
- cupredoxiny,
- MeSH
- azurin * chemie MeSH
- transport elektronů MeSH
- Publikační typ
- abstrakt z konference MeSH
Dihydromyricetin (DHM) is a natural flavonoid showing several health promoting effects such as protective activity during severe alcohol intoxication. The mechanism underlying the effects of DHM on alcohol metabolism is virtually unknown. The present paper is focused on clarifying the role of DHM in the liver alcohol elimination at its molecular level. First, impact of DHM on alcohol dehydrogenase (ADH) activity in vitro and the enzyme induction in vivo was examined. Neither the ADH activity nor the enzyme expression were influenced by DHM. Next, the effect of DHM during alcohol intoxication were studied on primary hepatocytes isolated from EtOH-premedicated and untreated rats. The viability of cells exposed to alcohol, estimated based on the released enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), was slightly affected by DHM. Although the expected hepatoprotective effect of DHM was not fully achieved, DHM (in a concentration manner) proved to reduce the level of ROS/RNS in hepatocytes. However, no change in the rate of alcohol metabolism in vivo was found when rats were administered with a single or repeated dose of ethanol supplemented with DHM. In conclusion, the proposed positive effect of DHM during alcohol intoxication has not been proven. Moreover, there is no effect of DHM on the alcohol metabolism. The "hoped-for" DHM hepatoprotective activity can be attributed to the reduction of ROS/RNS levels in cells.
- MeSH
- alkoholdehydrogenasa metabolismus MeSH
- antioxidancia farmakologie MeSH
- cytochrom P-450 CYP2E1 metabolismus MeSH
- ethanol metabolismus MeSH
- flavonoly farmakologie MeSH
- hepatocyty účinky léků metabolismus MeSH
- játra účinky léků metabolismus MeSH
- kultivované buňky MeSH
- metabolická inaktivace MeSH
- nitrosativní stres účinky léků MeSH
- oxidační stres účinky léků MeSH
- potkani Wistar MeSH
- reaktivní formy dusíku metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
Studium proteinů a zvláště jejich 3D struktury či protein -proteinových interakcí hraje v dnešním biochemickém výzkumu nezanedbatelnou roli. Řada alternativních metod tohoto výzkumu (např. PIXL, FRET) využívá biotechnologické postupy pro zavedení nepřirozených aminokyselin či jejich strukturních analogů do proteinové sekvence během jejich rekombinantní přípravy. Předkládaná práce uvádí několik biotechnologických přístupů inkorporace foto -methioninu (pMet, L-2-amino-5,5-azi -hexanová kyselina) do sekvence dvou modelových savčích proteinů.
The study of proteins and especially their 3D structure or protein -protein interactions plays significant role in contemporary biochemical research. Many alternative methods of the research (e.g. PIXL, FRET) employing different biotechnology techniques to introduce the non -natural amino acids or their structural analogues within protein sequence during its recombinant expression. This study presents several biotechnology approaches to introduce photo -methionine (pMet, L-2-amino-5,5-azi -hexanoic acid) into the sequence of two model mammalian proteins.
- Klíčová slova
- světlem iniciované síťování, foto-methionin,
- MeSH
- aminoacyl-tRNA-synthetasy metabolismus MeSH
- aminokyseliny chemie metabolismus MeSH
- biotechnologie MeSH
- diazomethan MeSH
- konformace proteinů * MeSH
- mapování interakce mezi proteiny * MeSH
- methionin MeSH
- posttranslační úpravy proteinů * MeSH
- proteiny chemie MeSH
- proteosyntéza MeSH
- techniky in vitro MeSH
- zobrazování trojrozměrné MeSH
- Publikační typ
- práce podpořená grantem MeSH
Chromophobe renal cell carcinoma (CRCC) with neuroendocrine differentiation (CRCCND) has only recently been described. Eighteen cases of CRCC with morphologic features suggestive of neuroendocrine differentiation were selected from among 624 CRCCs in our registry. The tissues were fixed in neutral formalin, embedded in paraffin, cut into 4- to 5-μm-thick sections, and stained with hematoxylin and eosin. As CRCC with neuroendocrine features, tumors with following morphology were suggested: (1) trabecular/palisading/ribbon-like, gyriform, insular, glandular, and solid pattern; (2) uniform polygonal cells formed in small islets; and (3) cribriform pattern in combination with palisading. Selected cases were further analyzed using immunohistochemistry, electron microscopy, array comparative genomic hybridization, and fluorescence in situ hybridization. Cases were classified as CRCCND or CRCC with neuroendocrine-like features (CRCCND-L) based on the immunohistochemical expression of neuroendocrine markers: CRCCND, 4 cases, age range 49 to 79 years, size ranged from 2.2 to 22 cm, and CRCCND-L, 14 cases, age range 34 to 74 years, size range 3.8 to 16.5 cm. Follow-up information was available for 11 of 18 patients aged 0.5 to 12 years. Two of 4 CRCCNDs showed aggressive clinical course with metastatic spreading. Chromophobe renal cell carcinomas with neuroendocrine differentiation were focally positive for CD56 (4/4), synaptophysin (4/4), chromogranin A (1/4), and neuron-specific enolase (3/4). All 14 CRCCND-Ls were mostly negative or very weakly focally positive for some of the aforementioned markers. All 18 tumors were positive for cytokeratin 7 and CD117. Ultrastructural analysis showed poorly preserved neuroendocrine granules only in 2 of 4 analyzed CRCCNDs. Losses of chromosomes 1, 2, 6, and 10 were found in all analyzable CRCCNDs, whereas multiple losses (chromosomes 1, 2, 6, 10, 13, 17, and 21) and gains (chromosomes 4, 11, 12, 14, 15, 16, 19, and 20) were found in CRCCND-L.
- MeSH
- diferenciální diagnóza MeSH
- dospělí MeSH
- imunohistochemie MeSH
- karcinom z renálních buněk genetika metabolismus patologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- metastázy nádorů MeSH
- nádory ledvin genetika metabolismus patologie MeSH
- neuroendokrinní nádory genetika metabolismus patologie MeSH
- senioři MeSH
- srovnávací genomová hybridizace MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
