- 标题
- 摘要
- 关键词
- 实验方案
- 产品
-
Aberrant Activity of TAK1 is Associated with Retinal Pathology
摘要： Transforming growth factor-β-activated kinase-1 (TAK1) is a mitogen activated protein kinase kinase kinase that is involved in diverse biological roles across species. Functioning downstream of TGF-β, TAK1 mediates the activation of the c-Jun N-terminal kinase (JNK) signaling pathway, serves as the target of pro-inflammatory cytokines, such as TNF-α, mediates NF-κβ activation, and plays a role in Wnt signaling in mesenchymal stem cells. Still, the expression of TAK1 in the retina has not been defined. In our study, pathological and immunohistochemical assessments indicate a link between retinal pathology and TAK1 phosphorylation. We observed similar TAK1 expression both in non-obvious and obvious retinal pathologies. However, the phosphorylated form of TAK1 in the segments of retina with obvious pathology was hardly detected compared to its expression in the segments with non-obvious pathology. This finding indicates, for the first time, a possible involvement of TAK1 in human retinal pathologies. Better understanding the expression pattern of TAK1 may serve as a new therapeutic avenue for retinal pathologies.
关键词： Phosphorylation,Retinal pigment epithelium (RPE),Transforming growth factor-β-activated kinase 1 (TAK1)
更新于2025-09-23 15:23:52
-
A fluorometric method for determination of the activity of T4 polynucleotide kinase by using a DNA-templated silver nanocluster probe
摘要： The authors describe a turn-off fluorometric method for the determination of the activity of the T4 polynucleotide kinase (T4 PNK). It is based on the use of DNA-templated silver nanoclusters (AgNCs). DNA probes with terminal 5′ hydroxy groups are used as substrates for DNA phosphatases. If subsequently treated with T4 PNK and Lambda exonuclease (λ exo), the AgNC DNA probes with a modified C-rich sequence and the G-rich sequence is separated. Upon their separation, the strong fluorescence (with excitation/emission maxima at 580/650 nm) that is caused by the proximity of the G-rich region and the C-rich region in the AgNCs decreases sharply. This enabled the fluorometric kinetic determination of the activity of T4 PNK. The assay is characterized by a wide linear range (from 0.01 to 12.5 U·mL?1), a low detection limit (0.01 U·mL?1) and short assay time (typically 60 min). This makes it a promising tool for use in studying processes related to DNA phosphorylation, in drug discovery and in diagnostics.
关键词： DNA-AgNCs,Cell extracts,Clinical diagnostics,Lambda exonuclease,Proximity effect,T4 PNK,Inhibitor,ATP,Na2HPO4,Phosphorylation
更新于2025-09-23 15:23:52
-
Phosphorylation at Serine 21 in G protein‐coupled receptor kinase 1 (GRK1) is required for normal kinetics of dark adaption in rod but not cone photoreceptors
摘要： Timely recovery of the light response in photoreceptors requires efficient inactivation of photoactivated rhodopsin. This process is initiated by phosphorylation of its carboxyl terminus by G protein-coupled receptor kinase 1 (GRK1). Previously, we showed that GRK1 is phosphorylated in the dark at Ser21 in a cAMP-dependent manner and dephosphorylated in the light. Results in vitro indicate that dephosphorylation of Ser21 increases GRK1 activity, leading to increased phosphorylation of rhodopsin. This creates the possibility of light-dependent regulation of GRK1 activity and its efficiency in inactivating the visual pigment. To address the functional role of GRK1 phosphorylation in rods and cones in vivo, we generated mutant mice in which Ser21 is substituted with alanine (GRK1-S21A), preventing dark-dependent phosphorylation of GRK1. GRK1-S21A mice had normal retinal morphology, without evidence of degeneration. The function of dark-adapted GRK1-S21A rods and cones was also unaffected, as demonstrated by the normal amplitude and kinetics of their responses obtained by ex vivo and in vivo ERG recordings. In contrast, rod dark adaptation following exposure to bright bleaching light was significantly delayed in GRK1-S21A mice, suggesting that the higher activity of this kinase results in enhanced rhodopsin phosphorylation and therefore delays its regeneration. In contrast, dark adaptation of cones was unaffected by the S21A mutation. Taken together, these data suggest that rhodopsin phosphorylation/dephosphorylation modulates the recovery of rhodopsin to the ground state and rod dark adaptation. They also reveal a novel role for cAMP-dependent phosphorylation of GRK1 in regulating the dark adaptation of rod but not cone photoreceptors.
关键词： vision,phototransduction,photoreceptor,protein phosphorylation,cAMP
更新于2025-09-16 10:30:52
-
Revealing Conformational Transitions in G-Protein Coupled Receptor Rhodopsin upon Phosphorylation
摘要： GPCRs have evolved as highly specialized cellular machinery that can dictate biological outcomes in response to diverse stimuli. Specifically, they induce multiple pathway responses upon structural perturbations induced at local protein sites. GPCRs utilize concurrent strategy involving a central transmembrane topology and biochemical modifications for precise functional implementation. However, the specific role of latter is not decomposed due to the lack of precise probing techniques that can characterize receptor dynamics upon biochemical modifications. Phosphorylation is known to be one of the critical biochemical modifications in GPCRs that aids in receptor desensitization via arrestin binding. Here, we carried out all-atom molecular dynamics (MD) simulations of rhodopsin in membrane environment to study its conformational dynamics induced upon phosphorylation. Interestingly, our comparative analysis of non-phosphorylated and phosphorylated rhodopsin structure demonstrated enhanced receptor stability upon phosphorylation at the C-terminal region that leads to the opening of the extracellular part of the transmembrane helices. In addition, monitoring of the distinct number of phosphorylation states showed that less number of phosphorylated residues does not bring about appropriate conformational changes in the extracellular region. Since phosphorylation results in receptor desensitization and recycling of ligand, our findings provide significant insights into the conformational dynamics of the mechanism of ligand exit from the receptor.
关键词： conformational dynamics,molecular dynamics simulations,GPCRs,phosphorylation,rhodopsin
更新于2025-09-12 10:27:22