"LM2015043" Dotaz Zobrazit nápovědu
In Part I, by using 31P-NMR spectroscopy, we have shown that isolated granum and stroma thylakoid membranes (TMs), in addition to the bilayer, display two isotropic phases and an inverted hexagonal (HII) phase; saturation transfer experiments and selective effects of lipase and thermal treatments have shown that these phases arise from distinct, yet interconnectable structural entities. To obtain information on the functional roles and origin of the different lipid phases, here we performed spectroscopic measurements and inspected the ultrastructure of these TM fragments. Circular dichroism, 77 K fluorescence emission spectroscopy, and variable chlorophyll-a fluorescence measurements revealed only minor lipase- or thermally induced changes in the photosynthetic machinery. Electrochromic absorbance transients showed that the TM fragments were re-sealed, and the vesicles largely retained their impermeabilities after lipase treatments-in line with the low susceptibility of the bilayer against the same treatment, as reflected by our 31P-NMR spectroscopy. Signatures of HII-phase could not be discerned with small-angle X-ray scattering-but traces of HII structures, without long-range order, were found by freeze-fracture electron microscopy (FF-EM) and cryo-electron tomography (CET). EM and CET images also revealed the presence of small vesicles and fusion of membrane particles, which might account for one of the isotropic phases. Interaction of VDE (violaxanthin de-epoxidase, detected by Western blot technique in both membrane fragments) with TM lipids might account for the other isotropic phase. In general, non-bilayer lipids are proposed to play role in the self-assembly of the highly organized yet dynamic TM network in chloroplasts.
Skyrin (SKR) is a plant bisanthraquinone secondary metabolite from the Hypericum genus with potential use in anticancer therapy. However, its effect and mechanism of action are still unknown. The negative effect of SKR on HCT 116 and HT-29 cancer cell lines in hypoxic and normoxic conditions was observed. HCT 116 cells were more responsive to SKR treatment as demonstrated by decreased metabolic activity, cellularity and accumulation of cells in the G1 phase. Moreover, an increasing number of apoptotic cells was observed after treatment with SKR. Based on the LC-MS comparative proteomic data from hypoxia and normoxia (data are available via ProteomeXchange with the identifier PXD019995), SKR significantly upregulated Death receptor 5 (DR5), which was confirmed by real-time qualitative PCR (RT-qPCR). Furthermore, multiple changes in the Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-activated cascade were observed. Moreover, the reversion of TRAIL resistance was observed in HCT 116, HT-29 and SW620 cell lines, even in hypoxia, which was linked to the upregulation of DR5. In conclusion, our results propose the use of SKR as a prospective anticancer drug, particularly as an adjuvant to TRAIL-targeting treatment to reverse TRAIL resistance in hypoxia.
- Publikační typ
- časopisecké články MeSH
NKp30 is one of the main human natural killer (NK) cell activating receptors used in directed immunotherapy. The oligomerization of the NKp30 ligand binding domain depends on the length of the C-terminal stalk region, but our structural knowledge of NKp30 oligomerization and its role in signal transduction remains limited. Moreover, ligand binding of NKp30 is affected by the presence and type of N-glycosylation. In this study, we assessed whether NKp30 oligomerization depends on its N-glycosylation. Our results show that NKp30 forms oligomers when expressed in HEK293S GnTI- cell lines with simple N-glycans. However, NKp30 was detected only as monomers after enzymatic deglycosylation. Furthermore, we characterized the interaction between NKp30 and its best-studied cognate ligand, B7-H6, with respect to glycosylation and oligomerization, and we solved the crystal structure of this complex with glycosylated NKp30, revealing a new glycosylation-induced mode of NKp30 dimerization. Overall, this study provides new insights into the structural basis of NKp30 oligomerization and explains how the stalk region and glycosylation of NKp30 affect its ligand affinity. This furthers our understanding of the molecular mechanisms involved in NK cell activation, which is crucial for the successful design of novel NK cell-based targeted immunotherapeutics.
- Publikační typ
- časopisecké články MeSH
The nascent polypeptide-associated (NAC) complex was described in yeast as a heterodimer composed of two subunits, α and β, and was shown to bind to the nascent polypeptides newly emerging from the ribosomes. NAC function was widely described in yeast and several information are also available about its role in plants. The knock down of individual NAC subunit(s) led usually to a higher sensitivity to stress. In Arabidopsis thaliana genome, there are five genes encoding NACα subunit, and two genes encoding NACβ. Double homozygous mutant in both genes coding for NACβ was acquired, which showed a delayed development compared to the wild type, had abnormal number of flower organs, shorter siliques and greatly reduced seed set. Both NACβ genes were characterized in more detail-the phenotype of the double homozygous mutant was complemented by a functional NACβ copy. Then, both NACβ genes were localized to nuclei and cytoplasm and their promoters were active in many organs (leaves, cauline leaves, flowers, pollen grains, and siliques together with seeds). Since flowers were the most affected organs by nacβ mutation, the flower buds' transcriptome was identified by RNA sequencing, and their proteome by gel-free approach. The differential expression analyses of transcriptomic and proteomic datasets suggest the involvement of NACβ subunits in stress responses, male gametophyte development, and photosynthesis.
- MeSH
- alely MeSH
- Arabidopsis fyziologie MeSH
- fenotyp MeSH
- geneticky modifikované rostliny MeSH
- homozygot MeSH
- klíčení MeSH
- květy fyziologie MeSH
- molekulární chaperony genetika metabolismus MeSH
- mutace MeSH
- proteiny huseníčku genetika metabolismus MeSH
- proteomika metody MeSH
- regulace genové exprese u rostlin MeSH
- semena rostlinná MeSH
- transkriptom MeSH
- vývoj rostlin * genetika MeSH
- Publikační typ
- časopisecké články MeSH
Lytic polysaccharide monooxygenases (LPMOs) are industrially important oxidoreductases employed in lignocellulose saccharification. Using advanced time-resolved mass spectrometric techniques, we elucidated the structural determinants for substrate-mediated stabilization of the fungal LPMO9C from Neurosporacrassa during catalysis. LPMOs require a reduction in the active-site copper for catalytic activity. We show that copper reduction in NcLPMO9C leads to structural rearrangements and compaction around the active site. However, longer exposure to the reducing agent ascorbic acid also initiated an uncoupling reaction of the bound oxygen species, leading to oxidative damage, partial unfolding, and even fragmentation of NcLPMO9C. Interestingly, no changes in the hydrogen/deuterium exchange rate were detected upon incubation of oxidized or reduced LPMO with crystalline cellulose, indicating that the LPMO-substrate interactions are mainly side-chain mediated and neither affect intraprotein hydrogen bonding nor induce significant shielding of the protein surface. On the other hand, we observed a protective effect of the substrate, which slowed down the autooxidative damage induced by the uncoupling reaction. These observations further complement the picture of structural changes during LPMO catalysis.
- MeSH
- celulosa chemie MeSH
- fungální proteiny chemie MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- hmotnostní spektrometrie MeSH
- katalytická doména MeSH
- katalýza MeSH
- koncentrace vodíkových iontů MeSH
- konformace proteinů MeSH
- kyslík chemie MeSH
- lignin chemie MeSH
- měď chemie MeSH
- Neurospora crassa enzymologie MeSH
- oxidační stres MeSH
- oxidoreduktasy chemie MeSH
- oxygenasy se smíšenou funkcí chemie MeSH
- polysacharidy chemie MeSH
- reaktivní formy kyslíku chemie MeSH
- substrátová specifita MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The Photorhabdus species is a Gram-negative bacteria of the family Morganellaceae that is known for its mutualistic relationship with Heterorhabditis nematodes and pathogenicity toward insects. This study is focused on the characterization of the recombinant lectin PLL3 with an origin in P. laumondii subsp. laumondii. PLL3 belongs to the PLL family of lectins with a seven-bladed β-propeller fold. The binding properties of PLL3 were tested by hemagglutination assay, glycan array, isothermal titration calorimetry, and surface plasmon resonance, and its structure was determined by X-ray crystallography. Obtained data revealed that PLL3 binds similar carbohydrates to those that the other PLL family members bind, with some differences in the binding properties. PLL3 exhibited the highest affinity toward l-fucose and its derivatives but was also able to interact with O-methylated glycans and other ligands. Unlike the other members of this family, PLL3 was discovered to be a monomer, which might correspond to a weaker avidity effect compared to homologous lectins. Based on the similarity to the related lectins and their proposed biological function, PLL3 might accompany them during the interaction of P. laumondii with both the nematode partner and the insect host.
- MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- fruktosa metabolismus MeSH
- kalorimetrie MeSH
- krystalografie rentgenová MeSH
- lektiny chemie genetika metabolismus MeSH
- Photorhabdus metabolismus MeSH
- povrchová plasmonová rezonance MeSH
- rekombinantní proteiny chemie metabolismus MeSH
- sekundární struktura proteinů MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
This work aims to find the most suitable method that is practically applicable for the calculation of 31P NMR chemical shifts of Pt(II) complexes. The influence of various all-electron and ECP basis sets, DFT functionals, and solvent effects on the optimized geometry was tested. A variety of combinations of DFT functionals BP86, B3LYP, PBE0, TPSSh, CAM-B3LYP, and ωB97XD with all-electron basis sets 6-31G, 6-31G(d), 6-31G(d,p), 6-311G(d,p), and TZVP and ECP basis sets SDD, LanL2DZ, and CEP-31G were used. Chemical shielding constants were then calculated using BP86, PBE0, and B3LYP functionals in combination with the TZ2P basis. The magnitude of spin-orbit interactions was also evaluated.
- Publikační typ
- časopisecké články MeSH
Laser-induced breakdown spectroscopy (LIBS) was examined as a novel method for readout of microtiter plate immunoassays involving nanoparticles (NP). The so-called Tag-LIBS technique is a sensitive method for the detection of specific biomarkers. It was applied to the determination of NP labels using nanosecond ablation sampling. The NP labels were examined from the bottom of a standard 96-well microtiter plate. Thanks to the flexibility of LIBS instrumentation, both the plasma emission collection and the focusing optics arrangements can be collinearly arranged. The experiments showed that silver NPs and gold NPs can be readily quantified on the bottom of the microtiter plate. Utilizing this technique, a sandwich immunoassay for human serum albumin using streptavidin-coated AgNP labels was developed. The assay has a 10 ng·mL-1 detection limit which is comparable to the sensitivity of fluorometric readout. The main advantage of this LIBS technique is its wide scope in which it enables a detection of almost any type of NP labels, irrespective to any fluorescence or catalytic properties. Owing to the immediate signal response, the relatively simple instrumentation also enables assay automation. The LIBS capability of multi-elemental analyses makes it a promising and fast alternative to other readout techniques, in particular with respect to multiplexed detection of biomarkers. Graphical abstract Laser-induced breakdown spectroscopy (LIBS) is used as a novel readout method of nanoparticle-based immunoassays in microtiter plates. After formation of sandwich immunocomplex, the analyte concentration is quantified as the signal of Ag nanoparticle labels determined by LIBS.
- MeSH
- biologické markery krev MeSH
- imunoanalýza metody MeSH
- kovové nanočástice chemie MeSH
- lasery * MeSH
- lidé MeSH
- lidský sérový albumin analýza MeSH
- povrchové vlastnosti MeSH
- stříbro chemie MeSH
- velikost částic MeSH
- zlato chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Interleukin 24 (IL-24) is a cytokine with the potential to be an effective treatment for autoimmune diseases and cancer. However, its instability and difficulties in its production have hampered detailed biological and biophysical studies. We approached the challenges of IL-24 production by using the PROSS algorithm to design more stable variants of IL-24. We used homology models built from the sequences and known structures of IL-20 and IL-19 and predicted and produced several extensively mutated IL-24 variants that were highly stable and produced in large yields; one of them was crystallized (IL-24B, PDB ID 6GG1; 3D Interactive at http://proteopedia.org/w/Journal: FEBS_Journal:1). The mutated variants, however, lost most of their binding capacity to the extracellular parts of cognate receptors. While the affinity to the receptor 2 (IL-20R2) was preserved, the variants lost affinity to IL-20R1 and IL-22R1 (shared receptors 1). Back engineering of the variants revealed that reintroduction of a single IL-24 wild-type residue (T198) to the patch interacting with receptors 1 restored 80% of the binding affinity and signaling capacity, accompanied by an acceptable drop in the protein stability by 9 °C. Multiple sequence alignment explains the stabilizing effect of the mutated residues in the IL-24 variants by their presence in the related and more stable cytokines IL-20 and IL-19. Our homology-based approach can enhance existing methods for protein engineering and represents a viable alternative to study and produce difficult proteins for which only in silico structural information is available, estimated as >40% of all important drug targets.
