protein sorting
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Plasma membrane proteins synthesised at the endoplasmic reticulum are delivered to the cell surface via sorting pathways. Hydrophobic mismatch theory based on the length of the transmembrane domain (TMD) dominates discussion about determinants required for protein sorting to the plasma membrane. Transmembrane adaptor proteins (TRAP) are involved in signalling events which take place at the plasma membrane. Members of this protein family have TMDs of varying length. We were interested in whether palmitoylation or other motifs contribute to the effective sorting of TRAP proteins. We found that palmitoylation is essential for some, but not all, TRAP proteins independent of their TMD length. We also provide evidence that palmitoylation and proximal sequences can modulate sorting of artificial proteins with TMDs of suboptimal length. Our observations point to a unique character of each TMD defined by its primary amino acid sequence and its impact on membrane protein localisation. We conclude that, in addition to the TMD length, secondary sorting determinants such as palmitoylation or flanking sequences have evolved for the localisation of membrane proteins.
- MeSH
- adaptorové proteiny signální transdukční chemie metabolismus MeSH
- buněčná membrána metabolismus MeSH
- extracelulární prostor chemie MeSH
- glykosylace MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- Jurkat buňky MeSH
- lidé MeSH
- lipoylace * MeSH
- membránové proteiny chemie metabolismus MeSH
- terciární struktura proteinů MeSH
- transport proteinů MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Many proteins are present in the nucleus; some are involved with its structural and functional organization, some with gene expression, and some with cell division. The plant nuclear proteome has not been well explored. Its characterization requires extraction methods which minimize both the artifactual alteration of the proteins and the extent of contamination with non-nuclear proteins. The conventional multi-step fractionation procedure is both laborious and prone to contamination. Here, we describe a single-step method based on flow sorting. The method allows the separation of G1, S and G2 phase nuclei and minimizes the risk of contamination by non-nuclear proteins. Preliminary results obtained using G1 phase cell nuclei from barley root tips indicate that flow sorting coupled with a protein/peptide separation and mass spectrometry will permit a comprehensive characterization of the plant nuclear proteome.
Specificity of membrane fusion in vesicular trafficking is dependent on proper subcellular distribution of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Although SNARE complexes are fairly promiscuous in vitro, substantial specificity is achieved in cells owing to the spatial segregation and shielding of SNARE motifs prior to association with cognate Q-SNAREs. In this study, we identified phosphatidylinositol 4-kinase IIα (PI4K2A) as a binding partner of vesicle-associated membrane protein 3 (VAMP3), a small R-SNARE involved in recycling and retrograde transport, and found that the two proteins co-reside on tubulo-vesicular endosomes. PI4K2A knockdown inhibited VAMP3 trafficking to perinuclear membranes and impaired the rate of VAMP3-mediated recycling of the transferrin receptor. Moreover, depletion of PI4K2A significantly decreased association of VAMP3 with its cognate Q-SNARE Vti1a. Although binding of VAMP3 to PI4K2A did not require kinase activity, acute depletion of phosphatidylinositol 4-phosphate (PtdIns4P) on endosomes significantly delayed VAMP3 trafficking. Modulation of SNARE function by phospholipids had previously been proposed based on in vitro studies, and our study provides mechanistic evidence in support of these claims by identifying PI4K2A and PtdIns4P as regulators of an R-SNARE in intact cells.
- MeSH
- buněčná membrána metabolismus MeSH
- Cercopithecus aethiops MeSH
- COS buňky MeSH
- endozomy metabolismus MeSH
- fosfotransferasy s alkoholovou skupinou jako akceptorem metabolismus MeSH
- fúze membrán fyziologie MeSH
- lidé MeSH
- membránový protein 3 asociovaný s vezikuly metabolismus MeSH
- proteiny SNARE metabolismus MeSH
- receptory transferinu metabolismus MeSH
- transport proteinů fyziologie MeSH
- vezikulární transportní proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Intramural MeSH
Translokace chromozómů jsou prokázány u 50–70 % případů lidské leukémie. Gen kódující protein PML (promyelocytární leukémie) se účastní přestavby chromozómů t(15;17) u akutní promyelocytární leukémie (APL). Gen PML kóduje protein, který se koncentruje v PML-jaderných tělíscích. Histonacetyltransferázy a histondeacetylázy, proteiny modifikující chromatin, se také hromadí v těchto nukleárních tělíscích v komplexech s proteinem PML a svědčí o úloze těchto komplexů v regulaci transkripce. Prokázané interakce proteinu PML s transkripčními faktory, koaktivátory a korepresory transkripce odpovídají účasti PML v regulaci transkripce. PML hraje důležitou úlohu v apoptóze, proliferaci a stárnutí buněk. Gen pro PML je genem potlačujícím vznik nádorů (tumour suppressor gene) a produkt jeho exprese ovlivňuje v negativním smyslu buněčné množení. Všechny tyto aktivity proteinu PML jsou připisovány jeho funkcím v jádře buněk. Cytoplazmatická forma PML (cPML) je také velmi důležitá a má významnou roli v přenosu signálu transformačního růstového faktoru-β (TGF-β). Cytoplazmatický PML reaguje s dvěma receptory pro TGF-β (TβRI a TβRII) na povrchu buňky a tvoří můstek mezi proteinem SARA (Smad anchor of receptor activation) a proteiny Smad a je důležitý pro dopravu celého komplexu do raných endozómů v přenosu signálu TGF-β. Ztráta funkčního cPML vede nejen k APL, ale přispívá obecně k rezistenci buněk na TGF-β a vzniku nádorů.
Chromosome translocations are detected in 50-70 % of human leukaemia. The promyelocytic leukaemia (PML) gene is involved in the t(15;17) chromosomal translocation of acute promyelocytic leukaemia (APL). PML gene encodes a protein, which was shown to be concentrated in PML-nuclear bodies. Histone acetyltransferases and deacetylases, and chromatin-modifying proteins are accumulated in complexes with PML protein in these nuclear bodies giving the evidence of their role in transcription regulation. Physical interactions of PML protein with transcription factors, co-activators and co-repressors of transcription correspond with the role of PML in transcription regulation. PML plays an important role in apoptosis, proliferation and senescence of cells. PML gene is a tumour-suppressor gene and a product of its expression acts as a potent cell growth suppressor. All these activities of PML protein are ascribed to its nuclear functions. Cytoplasmic form of PML (cPML) is also very important and it is critical for transforming growth factor-β (TGF-β) signalling. Cytoplasmic PML interacts with two TGF-β receptors (TβRI and TβRII) and acts as a bridging factor between protein called Smad anchor of receptor activation (SARA) and Smad proteins and it plays a role in the transport of whole complex into the early endosomes in TGF-β signalling. The loss of functional cPML induces not only APL but it might influence behaviour of cancer cells and their resistance to TGF-β.
- MeSH
- akutní myeloidní leukemie genetika patologie MeSH
- cytoplazmatické struktury fyziologie MeSH
- finanční podpora výzkumu jako téma MeSH
- genetická transkripce MeSH
- geny MeSH
- lidé MeSH
- nádorové proteiny genetika imunologie MeSH
- proteiny - lokalizační signály genetika MeSH
- transformující růstový faktor beta genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- přehledy MeSH
- Klíčová slova
- klinický význam, volný kortizol,
- MeSH
- bolest MeSH
- estrogenní substituční terapie škodlivé účinky MeSH
- genetické nemoci vrozené genetika MeSH
- genetika MeSH
- glukokortikoidy metabolismus MeSH
- glukonát vápenatý * chemická syntéza krev metabolismus MeSH
- hydrokortison biosyntéza krev sekrece MeSH
- lidé MeSH
- mozek - chemie MeSH
- myši MeSH
- syndrom chronické únavy MeSH
- systém hypofýza - nadledviny patofyziologie MeSH
- těhotenství metabolismus MeSH
- transport proteinů fyziologie genetika MeSH
- zánět patofyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- těhotenství metabolismus MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
... -- 2.4 Expression of PrPc in red blood cells and development of antibodies against glycated prion protein ... ... Cellular prion protein in blood platelets associates with both lipid rafts and the cytoskeleton. ... ... Underestimation of the expression of cellular prion protein on human red blood cells. ... ... Development of monoclonal antibodies specific for glycated prion protein. ... ... Expression of prion protein in mouse erythroid progenitors and differentiating murine erythroleukemia ...
49 listů, 75 nečíslovaných listů příloh : ilustrace ; 30 cm
- MeSH
- biochemická analýza krve MeSH
- krevní buňky MeSH
- prionová bílkovina MeSH
- prionové nemoci MeSH
- priony MeSH
- průtoková cytometrie MeSH
- Publikační typ
- vysokoškolské kvalifikační práce MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- neurologie
The major organelles of the endomembrane system were in place by the time of the last eukaryotic common ancestor (LECA) (~1.5 billion years ago). Their acquisitions were defining milestones during eukaryogenesis. Comparative cell biology and evolutionary analyses show multiple instances of homology in the protein machinery controlling distinct interorganelle trafficking routes. Resolving these homologous relationships allows us to explore processes underlying the emergence of additional, distinct cellular compartments, infer ancestral states predating LECA, and explore the process of eukaryogenesis itself. Here, we undertake a molecular evolutionary analysis (including providing a transcriptome of the jakobid flagellate Reclinomonas americana), exploring the origins of the machinery responsible for the biogenesis of lysosome-related organelles (LROs), the Biogenesis of LRO Complexes (BLOCs 1,2, and 3). This pathway has been studied only in animals and is not considered a feature of the basic eukaryotic cell plan. We show that this machinery is present across the eukaryotic tree of life and was likely in place prior to LECA, making it an underappreciated facet of eukaryotic cellular organisation. Moreover, we resolve multiple points of ancient homology between all three BLOCs and other post-endosomal retrograde trafficking machinery (BORC, CCZ1 and MON1 proteins, and an unexpected relationship with the "homotypic fusion and vacuole protein sorting" (HOPS) and "Class C core vacuole/endosomal tethering" (CORVET) complexes), offering a mechanistic and evolutionary unification of these trafficking pathways. Overall, this study provides a comprehensive account of the rise of the LROs biogenesis machinery from before the LECA to current eukaryotic diversity, integrating it into the larger mechanistic framework describing endomembrane evolution.
BACKGROUND INFORMATION: Macarpine (MA) is a quaternary benzophenanthridine plant alkaloid isolated from Macleaya microcarpa or Stylophorum lasiocarpum. Benzophenanthridine alkaloids are interesting natural products that display antiproliferative, antimicrobial, antifungal and anti-inflammatory activities, and also fluorescence properties. In a previous study, we demonstrated that thanks to its ability to interact with DNA and its spectral properties MA could be used as a supravital DNA probe for fluorescence microscopy and flow cytometry including analyses of the cell cycle. In this study, we evaluated the suitability of MA as a DNA dye for time-lapse microscopy and flow-cytometric cell sorting. RESULTS: Living A-375 and MEF cells stained with MA were monitored by time-lapse microscopy for 24 h. Mitoses were observed at MA concentrations up to 0.5 μg/ml during the first 2-3 h. After this period of time, cells treated with MA at concentrations of 0.75 and 0.5 μg/ml underwent apoptosis. Cells cultivated with MA at concentration of 0.25 μg/ml or lower survived throughout the 24 h period. Toxicity of MA was dependent on light wavelength and frequency of image capturing. The intensity of MA fluorescence decreased during the incubation. MA concentration of 0.1 μg/ml was identified as the most suitable for live cell imaging with respect to fluorescence intensity and toxicity. MA at the concentration 10 μg/ml was used for sorting of enhanced green fluorescent protein (EGFP)-labelled neurons and fibroblasts yielding profiles similar to those obtained with DRAQ5. Contrary to DRAQ5, MA-stained cells survived in culture, and the sorted cells lost the MA signal suggesting reversible binding of the dye to the DNA. CONCLUSION: The results proved that MA may readily be used for chromosomes depicting and mitosis monitoring by time-lapse microscopy. In addition, MA has shown to be a suitable probe for sorting of EGFP-labelled cells, including neurons, that survived the labelling process. SIGNIFICANCE: In consideration of the results, we highly anticipate an onward use of MA in a broad range of applications based on live cell sorting and imaging, for example, cell synchronisation and monitoring of proliferation as an important experimental and/or diagnostic utility.
- MeSH
- benzofenantridiny analýza MeSH
- buněčné kultury MeSH
- buněčný cyklus fyziologie MeSH
- DNA analýza MeSH
- fluorescenční barviva analýza MeSH
- fluorescenční mikroskopie metody MeSH
- lidé MeSH
- průtoková cytometrie * metody MeSH
- separace buněk metody MeSH
- viabilita buněk MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The plant hormone gibberellic acid (GA) is a crucial regulator of growth and development. The main paradigm of GA signaling puts forward transcriptional regulation via the degradation of DELLA transcriptional repressors. GA has also been shown to regulate tropic responses by modulation of the plasma membrane incidence of PIN auxin transporters by an unclear mechanism. Here we uncovered the cellular and molecular mechanisms by which GA redirects protein trafficking and thus regulates cell surface functionality. Photoconvertible reporters revealed that GA balances the protein traffic between the vacuole degradation route and recycling back to the cell surface. Low GA levels promote vacuolar delivery and degradation of multiple cargos, including PIN proteins, whereas high GA levels promote their recycling to the plasma membrane. This GA effect requires components of the retromer complex, such as Sorting Nexin 1 (SNX1) and its interacting, microtubule (MT)-associated protein, the Cytoplasmic Linker-Associated Protein (CLASP1). Accordingly, GA regulates the subcellular distribution of SNX1 and CLASP1, and the intact MT cytoskeleton is essential for the GA effect on trafficking. This GA cellular action occurs through DELLA proteins that regulate the MT and retromer presumably via their interaction partners Prefoldins (PFDs). Our study identified a branching of the GA signaling pathway at the level of DELLA proteins, which, in parallel to regulating transcription, also target by a nontranscriptional mechanism the retromer complex acting at the intersection of the degradation and recycling trafficking routes. By this mechanism, GA can redirect receptors and transporters to the cell surface, thus coregulating multiple processes, including PIN-dependent auxin fluxes during tropic responses.
- MeSH
- Arabidopsis růst a vývoj metabolismus MeSH
- buněčná membrána metabolismus MeSH
- gibereliny farmakologie MeSH
- kyseliny indoloctové farmakologie MeSH
- mikrotubuly metabolismus MeSH
- proteiny huseníčku genetika metabolismus MeSH
- regulace genové exprese u rostlin účinky léků MeSH
- regulátory růstu rostlin farmakologie MeSH
- signální transdukce MeSH
- transport proteinů MeSH
- třídící nexiny genetika metabolismus MeSH
- vakuoly metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
