newly synthesized proteins Dotaz Zobrazit nápovědu
Agonisté ß3-adrenergních receptorů jsou látky, které stimulují lipolýzu v bílé tukové tkáni a termogenezi v hnědé tukové tkáni, čehož by mohlo být využito při léčbě obezity a s ní spojeného metabolického syndromu. Cílem této práce bylo zhodnotit vliv šestitýdenního p.o. podávání látky B496 – ethylesteru (4-{2-[(2hydroxy-3-(4- -ethylkarbamoyl)fenoxypropyl)amino]ethyl}fenoxy) octové kyseliny ve formě ve vodě rozpustného hydrochloridu laboratorním potkanům kmene Wistar samčího pohlaví krmených hypertučnou dietou na koncentrace glukosy, triacylglyceridů, celkového cholesterolu a leptinu v séru a na váhový přírůstek. Zvířata byla rozdělena na skupinu léčenou látkou B496 a na skupinu kontrolní. Léčené skupině byla látka B496 rozpuštěna v destilované vodě v koncentraci 5 mg/1 kg vody. Testovaná látka B496 při dlouhodobém podávání laboratorním potkanům významně snižuje sérové koncentrace glukosy (-26 %, p<0,01), triacylglyceridů (-21 %, p<0,05) a leptinu (-43 %, p<0,01). Dále byl zkoumán vliv jednorázového i.p. podání (1 mg/kg hmotnosti zvířete) nově syntetizované látky B496 a látky BRL-37344 na koncentrace sérového leptinu u inbredních myší C57Bl/6J. Látka BRL-37344 vedla ke statisticky významnému snížení koncentrace leptinu v séru (-55 %, p<0,001). Takové snížení se nepodařilo prokázat u testované látky B496.
ß3-adrenoreceptor agonists can stimulate lipolysis in the white adipose tissue and thermogenesis in the brown adipose tissue. These activities could be useful in the treatment of obesity and the associated metabolic syndrome. The effects of six-week oral administration of the newly synthesized substance B496 (methyl-4-[2- -[2-hydroxy-3-(4-ethylcarbamoyl)phenoxyprophyl]amino]etyl)-phenoxyacetate hydrochloride) on serum glucose, triglycerides, total cholesterol, and leptin levels were studied in male Wistar rats fed with a high-fat diet. The animals were divided into a group treated with B496 (5 mg dissolved in 1 litre of water) and a control group. The results indicated a significant reduction in serum glucose levels (-26 %, p<0,01), triacylglyceride levels (-21 %, p<0,05) and leptin levels (-43 %, p<0,01). Further the effect of a single intraperitoneal dose (1 mg/kg) of B496 and BRL-37344 on serum leptin levels in the C57Bl/6J mouse was investigated. Administration of BRL-37344 resulted in a significant decrease in serum leptin levels (-55 %, p<0,001). This reduction was not demonstrated by newly synthesized substance B496.
- MeSH
- agonisté adrenergních beta-receptorů farmakologie chemická syntéza chemie MeSH
- finanční podpora výzkumu jako téma MeSH
- hypolipidemika farmakologie chemická syntéza chemie MeSH
- inbrední kmeny potkanů MeSH
- leptin biosyntéza fyziologie krev MeSH
- lidé MeSH
- modely u zvířat MeSH
- obezita farmakoterapie MeSH
- propanolaminy farmakologie chemická syntéza chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
INTRODUCTION: Protein synthesis is the outcome of tightly regulated gene expression which is responsive to a variety of conditions. Efforts are ongoing to monitor individual stages of protein synthesis to ensure maximum efficiency and accuracy. Due to post-transcriptional regulation mechanisms, the correlation between translatome and proteome is higher than between transcriptome and proteome. However, the most accurate approach to assess the key modulators and final protein expression is directly by using proteomics. Areas covered: This review covers various proteomic strategies that were used to better understand post-transcriptional regulation, specifically during and early after translation. The methods that identify both regulatory proteins associated with translational components and newly synthesized proteins are discussed. Expert commentary: Emerging proteomic approaches make it possible to monitor protein dynamics in cells, tissues and whole animals. The ability to detect alteration in protein abundance soon after their synthesis enables earlier recognition of disease causing factors and candidates to prevent/rectify disease phenotype.
- MeSH
- biomedicínský výzkum metody MeSH
- proteomika * MeSH
- proteosyntéza genetika MeSH
- regulace genové exprese genetika MeSH
- ribozomy genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Mitochondrial DNA (mtDNA) is compacted in ribonucleoprotein complexes called nucleoids, which can divide or move within the mitochondrial network. Mitochondrial nucleoids are able to aggregate into clusters upon reaction with intercalators such as the mtDNA depletion agent Ethidium Bromide (EB) or anticancer drug Doxorobicin (DXR). However, the exact mechanism of nucleoid clusters formation remains unknown. Resolving these processes may help to elucidate the mechanisms of DXR-induced cardiotoxicity. Therefore, we addressed the role of two key nucleoid proteins; mitochondrial transcription factor A (TFAM) and mitochondrial single-stranded binding protein (mtSSB); in the formation of mitochondrial nucleoid clusters during the action of intercalators. We found that both intercalators cause numerous aberrations due to perturbing their native status. By blocking mtDNA replication, both agents also prevented mtDNA association with TFAM, consequently causing nucleoid aggregation into large nucleoid clusters enriched with TFAM, co-existing with the normal nucleoid population. In the later stages of intercalation (>48h), TFAM levels were reduced to 25%. In contrast, mtSSB was released from mtDNA and freely distributed within the mitochondrial network. Nucleoid clusters mostly contained nucleoids with newly replicated mtDNA, however the nucleoid population which was not in replication mode remained outside the clusters. Moreover, the nucleoid clusters were enriched with p53, an anti-oncogenic gatekeeper. We suggest that mitochondrial nucleoid clustering is a mechanism for protecting nucleoids with newly replicated DNA against intercalators mediating genotoxic stress. These results provide new insight into the common mitochondrial response to mtDNA stress and can be implied also on DXR-induced mitochondrial cytotoxicity.
- MeSH
- buňky Hep G2 MeSH
- DNA vazebné proteiny metabolismus MeSH
- doxorubicin MeSH
- ethidium MeSH
- GTP-fosfohydrolasy metabolismus MeSH
- jaterní mitochondrie metabolismus MeSH
- lidé MeSH
- mitochondriální DNA metabolismus MeSH
- mitochondriální proteiny metabolismus MeSH
- nádorový supresorový protein p53 metabolismus MeSH
- poškození DNA MeSH
- proteiny asociované s mikrotubuly metabolismus MeSH
- transkripční faktory metabolismus MeSH
- transportní proteiny mitochondriální membrány metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
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.
Bioorthogonal chemistry provides one of the possibilities to modify various biomolecules in their native environment. The combination of Click chemistry with the BONCAT method (bioorthogonal non-canonical amino acid tagging) is widely used for tagging and analysis of newly synthesized proteins, which are clearly distinguishable from the pre-existing protein pool. However, the commonly used procedure results in low quality 2D electrophoretic profiles. We put a lot of effort into obtaining clear results using a standard Click protocol, with a negligible effect. Here we describe a Click-on-membrane approach which we successfully used not only to monitor de novo protein synthesis but also to detect newly synthesized RNA.
In recent years, the use of synthetic cannabinoids (SCs) as drugs of abuse has greatly increased. SCs are associated with a risk of severe poisoning or even death. Therefore, more rapid, cost effective and reliable methods are needed, especially for the screening of drivers after traffic accidents and for detailed toxicological analysis in forensic laboratories. In this study, we developed a lateral flow immunoassay (LFIA) and an enzyme linked immunosorbent assay (ELISA) for the detection of JWH-200 in oral fluids. For this purpose a new hapten was prepared using a ten-step synthetic route. The developed immuno methods are based on antibodies obtained from rabbit immunized with synthesized hapten conjugated to carrier protein. The proposed methods are highly sensitive (LODLFIA = 0.08 ± 0.04 ng mL-1; LODELISA = 0.04 ± 0.02 ng mL-1). They were applied to the quantification of JHW-200 in spiked oral fluids. The recoveries ranged from 82 to 134% for both methods. The results correlated excellently with results obtained using UHPLC-MS/MS (R2LFIA = 0.99; R2ELISA = 0.99). Our developed methods could be an important tool for analyses of JWH-200 in human oral fluids. The one-step LFIA is particularly suitable for roadside and on-site monitoring due to the rapid qualitative results it delivers, while the ELISA is especially useful for laboratory quantitative analyses of positive samples captured by LFIA.
- Publikační typ
- časopisecké články MeSH
Many newly synthesized proteins must be translocated across one or more membranes to reach their destination in the individual organelles or membrane systems. Translocation, mostly requiring an energy source, a signal on the protein itself, loose conformation of the protein and the presence of cytosolic and/or membrane receptor-like proteins, is often accompanied by covalent modifications of transported proteins. In this review I discuss these aspects of protein transport via the classical secretory pathway and/or special translocation mechanisms in the unicellular eukaryotic organism Saccharomyces cerevisiae.
- MeSH
- aktivní transport MeSH
- fungální proteiny genetika metabolismus MeSH
- glykoproteiny genetika metabolismus MeSH
- kompartmentace buňky MeSH
- molekulární sekvence - údaje MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- sacharidové sekvence MeSH
- sekvence aminokyselin MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Efficient assembly and repair of the oxygen-evolving photosystem II (PSII) complex is vital for maintaining photosynthetic activity in plants, algae, and cyanobacteria. How chlorophyll is delivered to PSII during assembly and how vulnerable assembly complexes are protected from photodamage are unknown. Here, we identify a chlorophyll and β-carotene binding protein complex in the cyanobacterium Synechocystis PCC 6803 important for formation of the D1/D2 reaction center assembly complex. It is composed of putative short-chain dehydrogenase/reductase Ycf39, encoded by the slr0399 gene, and two members of the high-light-inducible protein (Hlip) family, HliC and HliD, which are small membrane proteins related to the light-harvesting chlorophyll binding complexes found in plants. Perturbed chlorophyll recycling in a Ycf39-null mutant and copurification of chlorophyll synthase and unassembled D1 with the Ycf39-Hlip complex indicate a role in the delivery of chlorophyll to newly synthesized D1. Sequence similarities suggest the presence of a related complex in chloroplasts.
Robust photosynthesis in chloroplasts and cyanobacteria requires the participation of accessory proteins to facilitate the assembly and maintenance of the photosynthetic apparatus located within the thylakoid membranes. The highly conserved Ycf48 protein acts early in the biogenesis of the oxygen-evolving photosystem II (PSII) complex by binding to newly synthesized precursor D1 subunit and by promoting efficient association with the D2 protein to form a PSII reaction center (PSII RC) assembly intermediate. Ycf48 is also required for efficient replacement of damaged D1 during the repair of PSII. However, the structural features underpinning Ycf48 function remain unclear. Here we show that Ycf48 proteins encoded by the thermophilic cyanobacterium Thermosynechococcus elongatus and the red alga Cyanidioschyzon merolae form seven-bladed beta-propellers with the 19-aa insertion characteristic of eukaryotic Ycf48 located at the junction of blades 3 and 4. Knowledge of these structures has allowed us to identify a conserved "Arg patch" on the surface of Ycf48 that is important for binding of Ycf48 to PSII RCs but also to larger complexes, including trimeric photosystem I (PSI). Reduced accumulation of chlorophyll in the absence of Ycf48 and the association of Ycf48 with PSI provide evidence of a more wide-ranging role for Ycf48 in the biogenesis of the photosynthetic apparatus than previously thought. Copurification of Ycf48 with the cyanobacterial YidC protein insertase supports the involvement of Ycf48 during the cotranslational insertion of chlorophyll-binding apopolypeptides into the membrane.
