Intermolecular Interactions between Cysteine and Aromatic Amino Acids with a Phenyl Moiety in the DNA-Binding Domain of Heat Shock Factor 1 Regulate Thermal Stress-Induced Trimerization.

In this study, we investigated the trimerization mechanism and structure of heat shock factor 1 (HSF1) using western blotting, tryptophan (Trp) fluorescence spectroscopy, and molecular modeling. First, we examined the DNA-binding domains of human (Homo sapiens), goldfish (Carassius auratus), and walleye pollock (Gadus chalcogrammus) HSF1s by mutating key residues (36 and 103) that are thought to directly affect trimer formation. Human, goldfish, and walleye pollock HSF1s contain cysteine at residue 36 but cysteine (C), tyrosine (Y), and phenylalanine (F), respectively, at residue 103. The optimal trimerization temperatures for the wild-type HSF1s of each species were found to be 42, 37, and 20 °C, respectively. Interestingly, a mutation experiment revealed that trimerization occurred at 42 °C when residue 103 was cysteine, at 37 °C when it was tyrosine, and at 20 °C when it was phenylalanine, regardless of the species. In addition, it was confirmed that when residue 103 of the three species was mutated to alanine, trimerization did not occur. This suggests that in addition to trimerization via disulfide bond formation between the cysteine residues in human HSF1, trimerization can also occur via the formation of a different type of bond between cysteine and aromatic ring residues such as tyrosine and phenylalanine. We also confirmed that at least one cysteine is required for the trimerization of HSF1s, regardless of its position (residue 36 or 103). Additionally, it was shown that the trimer formation temperature is related to growth and survival in fish.

pH Dependence of HSF1 trimerization is shaped by intramolecular interactions.

Heat shock factor 1 (HSF1) primarily regulates various cellular stress responses. Previous studies have shown that low pH within the physiological range directly activates HSF1 function in vitro. However, the detailed molecular mechanisms remain unclear. This study proposes a molecular mechanism based on the trimerization behavior of HSF1 at different pH values. Extensive mutagenesis of human and goldfish HSF1 revealed that the optimal pH for trimerization depended on the identity of residue 103. In particular, when residue 103 was occupied by tyrosine, a significant increase in the optimal pH was observed, regardless of the rest of the sequence. This behavior can be explained by the protonation state of the neighboring histidine residues, His101 and His110. Residue 103 plays a key role in trimerization by forming disulfide or non-covalent bonds with Cys36. If tyrosine resides at residue 103 in an acidic environment, its electrostatic interactions with positively charged histidine residues prevent effective trimerization. His101 and His110 are neutralized at a higher pH, which releases Tyr103 to interact with Cys36 and drives the effective trimerization of HSF1. This study showed that the protonation state of a histidine residue can regulate the intramolecular interactions, which consequently leads to a drastic change in the oligomerization behavior of the entire protein.

Revisiting the Analysis of Radiative Mid-Range Wireless Link for Pacemakers.

The development of a wireless link for biomedical applications requires an accurate estimation of the delivered power to implanted devices. In particular, a variety of mid-range applications in the biomedical area have gained significant attention. An appropriate method for the mid-range wireless link is required to implement a continuous wireless link through human tissue. Even though formulas used in this work are all based on previous works, this paper presents an implementation of the diverse formulas for the mid-range wireless link of an implanted antenna used for a pacemaker system based on the understanding on radiation properties varied with the distances from the antenna. The formulas based on input far-field data are successfully applied to compute the power transmission for the implanted devices, whose range includes radiative near-field and far-field regions. The wireless link for a pacemaker system is evaluated through using a patch antenna immersed with different depths of human tissue. A comparison of the computed and measured results shows an excellent agreement where the validity of the evaluation is demonstrated.

Interfering with Color Response by Porphyrin-Related Compounds in the MTT Tetrazolium-Based Colorimetric Assay.

Porphyrin compounds are widely distributed in various natural products and biological systems. In this study, effects of porphyrin-related compounds including zinc protoporphyrin (ZnPP), protoporphyrin IX (PPIX), cyanocobalamin (CBL), hemin, and zinc phthalocyanine (ZnPC) were analyzed on color response of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tetrazolium-based assay, a commonly-used method for analyzing cell viability. Color responses of MTT formazan formed in cells treated with ZnPP, PPIX, or ZnPC were significantly reduced even at submicromolar concentrations without affecting cell viability, whereas hemin and CBL did not. ZnPP, PPIX, and ZnPC rapidly induced degradation of MTT formazan already-produced by cells when exposed to light, but not under a dark condition. Photosensitizing properties of the three compounds were also verified through extensive generation of reactive oxygen species under light. The porphyrins did not affect the stability of water-soluble formazans including XTT, WST-1, WST-8, and MTS formazans. Several factors including different light sources and antioxidants modulated the degradation process of MTT formazan by the porphyrins. The results suggest that certain porphyrin compounds could cause a severe artifact in the MTT assay through rapid degradation of formazan dye due to their photosensitizing property, which needs to be considered carefully in the related assays.

The Role of Nuclear Receptor Subfamily 1 Group H Member 4 (NR1H4) in Colon Cancer Cell Survival through the Regulation of c-Myc Stability.

Nuclear receptor subfamily group H member 4 (NR1H4), also known as farnesoid X receptor, has been implicated in several cellular processes in the liver and intestine. Preclinical and clinical studies have suggested a role of NR1H4 in colon cancer development; however, how NR1H4 regulates colon cancer cell growth and survival remains unclear. We generated NR1H4 knockout (KO) colon cancer cells using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (CAS9) technology and explored the effects of NR1H4 KO in colon cancer cell proliferation, survival, and apoptosis. Interestingly, NR1H4 KO cells showed impaired cell proliferation, reduced colony formation, and increased apoptotic cell death compared to control colon cancer cells. We identified MYC as an important mediator of the signaling pathway alterations induced by NR1H4 KO. NR1H4 silencing in colon cancer cells resulted in reduced MYC protein levels, while NR1H4 activation using an NR1H4 ligand, chenodeoxycholic acid, resulted in time- and dose-dependent MYC induction. Moreover, NR1H4 KO enhanced the anti-cancer effects of doxorubicin and cisplatin, supporting the role of MYC in the enhanced apoptosis observed in NR1H4 KO cells. Taken together, our findings suggest that modulating NR1H4 activity in colon cancer cells might be a promising alternative approach to treat cancer using MYC-targeting agents.