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Background Korean pine nuts (Semen Pinus koraiensis ( P. koraiensis)) have been a valuable ingredient in Korean cuisine since ancient times, and the classical literature on traditional medicine states that pine nuts can moisturize skin. Aims To confirm the therapeutic efficacy of the ethanol extract of these pine nuts in an animal contact dermatitis (CD) model. Materials and Methods Korean pine nuts were extracted using 70% ethanol, and the crude extract obtained was condensed and lyophilized to produce EEPK (the 70% ethanol extract of P. koraiensis). A DNFB (1-fluoro-2,4-dinitrofluorobenzene)-induced BALB/c mouse model of CD was used to investigate the effects of EEPK on skin symptoms, histopathological abnormalities, and cytokine secretion in inflamed tissues. Results EEPK reduced skin color changes, lesion severities, and dorsal skin thickening and inhibited epidermis hyperplasia and immune cell infiltration into inflamed tissues. In addition, EEPK suppressed the productions of tumor necrosis factor-α, interferon-γ, interleukin-6, and monocyte chemoattractant protein-1 but did not affect body weights or spleen/body weight ratios. Conclusion P. koraiensis has potential as a therapeutic agent for CD and as a treatment for inflammatory skin disease management products. Furthermore, the mechanism underlying its therapeutic effects differs from that of corticosteroids.

Site-specific ubiquitination can regulate the functions of Rab proteins in membrane trafficking. Previously we showed that site-specific monoubiquitination on Rab5 downregulates its function. Rab7 acts in the downstream of Rab5. Although site-specific ubiquitination of Rab7 can affect its function, it remains elusive how the ubiquitination is involved in modulation of the function of Rab7 at molecular level. Here, we report molecular basis for the regulation of Rab7 by site-specific monoubiquitination. Rab7 was predominantly monoubiquitinated at multiple sites in the membrane fraction of cultured cells. Two major ubiquitination sites (K191 and K194), identified by mutational analysis with single K mutants, were responsible for membrane localization of monoubiquitinated Rab7. Using small-angle X-ray scattering, we derived structural models of site-specifically monoubiquitinated Rab7 in solution. Structural analysis combined with molecular dynamics simulation corroborated that the ubiquitin moieties on K191 and K194 are key determinants for exclusion of Rab7 from the endosomal membrane. Ubiquitination on the two major sites apparently mitigated colocalization of Rab7 with ORF3a of SARS-CoV-2, potentially deterring the egression of SARS-CoV-2. Our results establish that the regulatory effects of a Rab protein through site-specific monoubiquitination are commonly observed among Rab GTPases while the ubiquitination sites differ in each Rab protein.

The cannabinoid receptor 1 (CB1) is a class A G-protein coupled receptor (GPCR) that can exert various effects on the human body through the endocannabinoid system. Understanding CB1 activation has many benefits for the medical use of cannabinoids. A previous study reported that CB1 has two notable residues referred to as the toggle switch, F3.36 and W6.48, which are important for its activation mechanism. We performed a molecular dynamics simulation with a mutation in the toggle switch to examine its role in active and inactive states. We also examined structural changes, the residue–residue interaction network, and the interaction network among helices and loops of wildtype and mutant CB1 for both activation states. As a result, we found that the energetic changes in the hydrogen-bond network of the Na+ pocket, extracellular N-terminus–TM2–ECL1–TM3 interface including D2.63–K3.28 salt-bridge, and extracellular ECL2–TM5–ECL3–TM6 interface directly linked to the toggle switch contribute to the stability of CB1 by the broken aromatic interaction of the toggle switch. It makes the conformation of inactive CB1 receptor to be unstable. Our study explained the role of the toggle switch regarding the energetic interactions related to the Na+ pocket and extracellular loop interfaces, which could contribute to a better understanding of the activation mechanism of CB1.

Monomer dissociation and subsequent misfolding of the transthyretin (TTR) is one of the most critical causative factors of TTR amyloidosis. TTR amyloidosis causes several human diseases, such as senile systemic amyloidosis and familial amyloid cardiomyopathy/polyneuropathy; therefore, it is important to understand the molecular details of the structural deformation and aggregation mechanisms of TTR. However, such molecular characteristics are still elusive because of the complicated structural heterogeneity of TTR and its highly sensitive nature to various environmental factors. Several nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) studies of TTR variants have recently reported evidence of transient aggregation-prone structural states of TTR. According to these studies, the stability of the DAGH β-sheet, one of the two main β-sheets in TTR, is a crucial determinant of the TTR amyloidosis mechanism. In addition, its conformational perturbation and possible involvement of nearby structural motifs facilitates TTR aggregation. This study proposes aggregation-prone structural ensembles of TTR obtained by MD simulation with enhanced sampling and a multiple linear regression approach. This method provides plausible structural models that are composed of ensemble structures consistent with NMR chemical shift data. This study validated the ensemble models with experimental data obtained from circular dichroism (CD) spectroscopy and NMR order parameter analysis. In addition, our results suggest that the structural deformation of the DAGH β-sheet and the AB loop regions may correlate with the manifestation of the aggregation-prone conformational states of TTR. In summary, our method employing MD techniques to extend the structural ensembles from NMR experimental data analysis may provide new opportunities to investigate various transient yet important structural states of amyloidogenic proteins.

Aggregation of intrinsically disordered amyloid β (Aβ) is a hallmark of Alzheimer’s disease. Although complex aggregation mechanisms have been increasingly revealed, structural ensembles of Aβ monomers with heterogeneous and transient properties still hamper detailed experimental accesses to early events of amyloidogenesis. We herein developed a new mathematical tool based on multiple linear regression to obtain the reasonable ensemble structures of Aβ monomer by using the solution nuclear magnetic resonance (NMR) and molecular dynamics simulation data. Our approach provided the best-fit ensemble to two-dimensional NMR chemical shifts, also consistent with circular dichroism and dynamic light scattering analyses. The major monomeric structures of Aβ including β-sheets in both terminal and central hydrophobic core regions and the minor partially-helical structures suggested initial structure-based explanation on possible mechanisms of early molecular association and nucleation for amyloid generation. A wide-spectrum application of the current approach was also indicated by showing a successful utilization for ensemble structures of folded proteins. We propose that multiple linear regression in combination to experimental results will be highly promising for studies on protein misfolding diseases and functions by providing a convincing template structure.Graphic abstract