6 research outputs found
Witamina D – składnik o wielostronnym działaniu
Biologicznie czynna forma witaminy D, czyli 1,25-dwuhydroksywitamina D3, przez lata była uważana jedynie za czynnik zapewniający odpowiedni rozwój układu kostnego i stosowana w profilaktyce zachorowań na krzywicę. Dziś coraz więcej badań wskazuje, że jest to związek mający wpływ na ponad 200 genów i większość narządów ludzkiego organizmu. Termin witamina D odnosi się zarówno do witaminy (substancja egzogenna dostarczana z zewnątrz wraz z pożywieniem), jak również prohormonu (związek endogenny produkowany w skórze pod wpływem promieni słonecznych i później przekształcanego w aktywną postać). Obecnie witamina D nie jest postrzegana wyłącznie jako czynnik zapobiegający rozwojowi krzywicy i osteoporozy. Zwiększa się liczba badań pokazujących jej udział w prawidłowej pracy układu sercowo-naczyniowego, profilaktyce cukrzycy oraz chorób nowotworowych, a także jej wpływu podczas terapii reumatoidalnego zapalenia stawów (RZS), zwyrodnienia plamki żółtej (AMD) czy łuszczycy. Celem artykułu jest przedstawienie wielokierunkowego działania witaminy D
NanoBiT ‐ and NanoBiT/BRET ‐based assays allow the analysis of binding kinetics of Wnt‐3a to endogenous Frizzled 7 in a colorectal cancer model
Background and Purpose
Wnt binding to Frizzleds (FZD) is a crucial step that leads to the initiation of signalling cascades governing multiple processes during embryonic development, stem cell regulation and adult tissue homeostasis. Recent efforts have enabled us to shed light on Wnt–FZD pharmacology using overexpressed HEK293 cells. However, assessing ligand binding at endogenous receptor expression levels is important due to differential binding behaviour in a native environment. Here, we study FZD paralogue, FZD, and analyse its interactions with Wnt-3a in live CRISPR-Cas9-edited SW480 cells typifying colorectal cancer.
Experimental Approach
SW480 cells were CRISPR-Cas9-edited to insert a HiBiT tag on the N-terminus of FZD, preserving the native signal peptide. These cells were used to study eGFP-Wnt-3a association with endogenous and overexpressed HiBiT-FZD using NanoBiT/bioluminescence resonance energy transfer (BRET) and NanoBiT to measure ligand binding and receptor internalization.
Key Results
With this new assay the binding of eGFP-Wnt-3a to endogenous HiBiT-FZD was compared with overexpressed receptors. Receptor overexpression results in increased membrane dynamics, leading to an apparent decrease in binding on-rate and consequently in higher, up to 10 times, calculated Kd. Thus, measurements of binding affinities to FZD obtained in overexpressed cells are suboptimal compared with the measurements from endogenously expressing cells.
Conclusions and Implications
Binding affinity measurements in the overexpressing cells fail to replicate ligand binding affinities assessed in a (patho)physiologically relevant context where receptor expression is lower. Therefore, future studies on Wnt–FZD binding should be performed using receptors expressed under endogenous promotion
Overexpression of orphan receptor GPR61 increases cAMP levels upon forskolin stimulation in HEK293 cells:in vitro and in silico validation of 5-(nonyloxy)tryptamine as a low-affinity inverse agonist
Geissospermiculatine, a new alkaloid from Geissospermum reticulatum bark
A new alkaloid, geissospermiculatine was characterized in Geissospermum reticulatum A. H. Gentry bark (Apocynaceae). Here, following a simplified isolation protocol, the structure of the alkaloid was elucidated through GC-MS, LC-MS/MS, 1D, and 2D NMR (COSY, ROESY, HSQC, HMBC, 1H-15N HMBC). Cytotoxic properties were evaluated in vitro on malignant THP-1 cells, and the results demonstrated that the cytotoxicity of the alkaloid (30 μg/mL) was comparable with staurosporine (10 μM). Additionally, the toxicity was tested on zebrafish (Danio rerio) embryos in vivo by monitoring their development (0-72 h); toxicity was not evident at 30 μg/mL
NanoBiT‐ and NanoBiT/BRET‐based assays allow the analysis of binding kinetics of Wnt‐3a to endogenous Frizzled 7 in a colorectal cancer model
Background and Purpose: Wnt binding to Frizzleds (FZD) is a crucial step that leads to the initiation of signalling cascades governing multiple processes during embryonic development, stem cell regulation and adult tissue homeostasis. Recent efforts have enabled us to shed light on Wnt–FZD pharmacology using overexpressed HEK293 cells. However, assessing ligand binding at endogenous receptor expression levels is important due to differential binding behaviour in a native environment. Here, we study FZD paralogue, FZD7, and analyse its interactions with Wnt-3a in live CRISPR-Cas9-edited SW480 cells typifying colorectal cancer. Experimental Approach: SW480 cells were CRISPR-Cas9-edited to insert a HiBiT tag on the N-terminus of FZD7, preserving the native signal peptide. These cells were used to study eGFP-Wnt-3a association with endogenous and overexpressed HiBiT-FZD7 using NanoBiT/bioluminescence resonance energy transfer (BRET) and NanoBiT to measure ligand binding and receptor internalization. Key Results: With this new assay the binding of eGFP-Wnt-3a to endogenous HiBiT-FZD7 was compared with overexpressed receptors. Receptor overexpression results in increased membrane dynamics, leading to an apparent decrease in binding on-rate and consequently in higher, up to 10 times, calculated Kd. Thus, measurements of binding affinities to FZD7 obtained in overexpressed cells are suboptimal compared with the measurements from endogenously expressing cells. Conclusions and Implications: Binding affinity measurements in the overexpressing cells fail to replicate ligand binding affinities assessed in a (patho)physiologically relevant context where receptor expression is lower. Therefore, future studies on Wnt–FZD7 binding should be performed using receptors expressed under endogenous promotion
RNA:RNA interaction in ternary complexes resolved by chemical probing
RNA regulation can be performed by a second targeting RNA molecule, such as in the microRNA regulation mechanism. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) probes the structure of RNA molecules and can resolve RNA:protein interactions, but RNA:RNA interactions have not yet been addressed with this technique. Here, we apply SHAPE to investigate RNA-mediated binding processes in RNA:RNA and RNA:RNA-RBP complexes. We use RNA:RNA binding by SHAPE (RABS) to investigate microRNA-34a (miR-34a) binding its mRNA target, the silent information regulator 1 (mSIRT1), both with and without the Argonaute protein, constituting the RNA-induced silencing complex (RISC). We show that the seed of the mRNA target must be bound to the microRNA loaded into RISC to enable further binding of the compensatory region by RISC, while the naked miR-34a is able to bind the compensatory region without seed interaction. The method presented here provides complementary structural evidence for the commonly performed luciferase-assay-based evaluation of microRNA binding-site efficiency and specificity on the mRNA target site and could therefore be used in conjunction with it. The method can be applied to any nucleic acid-mediated RNA- or RBP-binding process, such as splicing, antisense RNA binding, or regulation by RISC, providing important insight into the targeted RNA structure