Allosteric autoregulation of DNA binding via a DNA-mimicking protein domain: a biophysical study of ZNF410-DNA interaction using small angle X-ray scattering.

ZNF410 is a highly-conserved transcription factor, remarkable in that it recognizes a 15-base pair DNA element but has just a single responsive target gene in mammalian erythroid cells. ZNF410 includes a tandem array of five zinc-fingers (ZFs), surrounded by uncharacterized N- and C-terminal regions. Unexpectedly, full-length ZNF410 has reduced DNA binding affinity, compared to that of the isolated DNA binding ZF array, both in vitro and in cells. AlphaFold predicts a partially-folded N-terminal subdomain that includes a 30-residue long helix, preceded by a hairpin loop rich in acidic (aspartate/glutamate) and serine/threonine residues. This hairpin loop is predicted by AlphaFold to lie against the DNA binding interface of the ZF array. In solution, ZNF410 is a monomer and binds to DNA with 1:1 stoichiometry. Surprisingly, the single best-fit model for the experimental small angle X-ray scattering profile, in the absence of DNA, is the original AlphaFold model with the N-terminal long-helix and the hairpin loop occupying the ZF DNA binding surface. For DNA binding, the hairpin loop presumably must be displaced. After combining biophysical, biochemical, bioinformatic and artificial intelligence-based AlphaFold analyses, we suggest that the hairpin loop mimics the structure and electrostatics of DNA, and provides an additional mechanism, supplementary to sequence specificity, of regulating ZNF410 DNA binding.

Retinal protective effect of curcumin metabolite hexahydrocurcumin against blue light-induced RPE damage.

BACKGROUND: Age-related macular degeneration (AMD) is a disease of retinal pigment epithelium (RPE) cells. We have previously demonstrated that blue light can damage RPE cells and their underlying mechanisms. We found that hexahydrocurcumin (HHC), a metabolite of curcumin, had better retinal protection than curcumin. However, the involved mechanisms remain unclear. METHODS: By exposing ARPE-19 human RPE cells and mouse primary RPE cells to blue light, the intracellular mechanisms of HHC in cells were investigated, including the proliferation of RPE cells and the effects of HHC on activating intracellular protective mechanisms and related factors. Next-generation sequencing (NGS) RNA sequencing revealed the underlying mechanisms involved in the induction and regulation of HHC treatment following blue light exposure. RESULTS: HHC promoted autophagy by enhancing autophagic flux, reduced oxidative stress and endoplasmic reticulum (ER) stress, and effectively reversed blue light-induced cell death. RNA sequencing-based bioinformatics approaches comprehensively analyze HHC-mediated cellular processes. CONCLUSION: Our findings elucidate the mechanisms of HHC against blue light damage in RPE cells and are beneficial for the development of natural metabolite-based preventive drugs or functional foods.

Transmembrane protein KIRREL1 regulates Hippo signaling via a feedback loop and represents a therapeutic target in YAP/TAZ-active cancers.

The Hippo tumor-suppressor pathway is frequently dysregulated in human cancers and represents a therapeutic target. However, strategies targeting the mammalian Hippo pathway are limited because of the lack of a well-established cell-surface regulator. Here, we show that transmembrane protein KIRREL1, by interacting with both SAV1 and LATS1/2, promotes LATS1/2 activation by MST1/2 (Hippo kinases), and LATS1/2 activation, in turn, inhibits activity of YAP/TAZ oncoproteins. Conversely, YAP/TAZ directly induce the expression of KIRREL1 in a TEAD1-4-dependent manner. Indeed, KIRREL1 expression positively correlates with canonical YAP/TAZ target gene expression in clinical tumor specimens and predicts poor prognosis. Moreover, transgenic expression of KIRREL1 effectively blocks tumorigenesis in a mouse intrahepatic cholangiocarcinoma model, indicating a tumor-suppressor role of KIRREL1. Hence, KIRREL1 constitutes a negative feedback mechanism regulating the Hippo pathway and serves as a cell-surface marker and potential drug target in cancers with YAP/TAZ dependency.

The Role of Oxidative Stress and Autophagy in Blue-Light-Induced Damage to the Retinal Pigment Epithelium in Zebrafish In Vitro and In Vivo.

Age-related macular degeneration (AMD) is the progressive degeneration of the retinal pigment epithelium (RPE), retina, and choriocapillaris among elderly individuals and is the leading cause of blindness worldwide. Thus, a better understanding of the underlying mechanisms in retinal tissue activated by blue light exposure is important for developing novel treatment and intervention strategies. In this study, blue-light-emitting diodes with a wavelength of 440 nm were applied to RPE cells at a dose of 3.7 ± 0.75 mW/cm2 for 24 h. ARPE-19 cells were used to investigate the underlying mechanism induced by blue light exposure. A trypan blue exclusion assay was used for the cell viability determination. Flow cytometry was used for apoptosis rate detection and autophagy analysis. An immunofluorescence microscopy analysis was used to investigate cellular oxidative stress and DNA damage using DCFDA fluorescence staining and an anti-γH2AX antibody. Blue light exposure of zebrafish larvae was established to investigate the effect on retinal tissue development in vivo. To further demonstrate the comprehensive effect of blue light on ARPE-19 cells, next-generation sequencing (NGS) was performed for an ingenuity pathway analysis (IPA) to reveal additional related mechanisms. The results showed that blue light exposure caused a decrease in cell proliferation and an increase in apoptosis in ARPE-19 cells in a time-dependent manner. Oxidative stress increased during the early stage of 2 h of exposure and activated DNA damage in ARPE-19 cells after 8 h. Furthermore, autophagy was activated in response to blue light exposure at 24-48 h. The zebrafish larvae model showed the unfavorable effect of blue light in prohibiting retinal tissue development. The RNA-Seq results confirmed that blue light induced cell death and participated in tissue growth inhibition and maturation. The current study reveals the mechanisms by which blue light induces cell death in a time-dependent manner. Moreover, both the in vivo and NGS data uncovered blue light's effect on retinal tissue development, suggesting that exposing children to blue light could be relatively dangerous. These results could benefit the development of preventive strategies utilizing herbal medicine-based treatments for eye diseases or degeneration in the future.

Nonvisual and visual object shape representations in occipitotemporal cortex: evidence from congenitally blind and sighted adults.

Knowledge of object shape is primarily acquired through the visual modality but can also be acquired through other sensory modalities. In the present study, we investigated the representation of object shape in humans without visual experience. Congenitally blind and sighted participants rated the shape similarity of pairs of 33 familiar objects, referred to by their names. The resulting shape similarity matrices were highly similar for the two groups, indicating that knowledge of the objects' shapes was largely independent of visual experience. Using fMRI, we tested for brain regions that represented object shape knowledge in blind and sighted participants. Multivoxel activity patterns were established for each of the 33 aurally presented object names. Sighted participants additionally viewed pictures of these objects. Using representational similarity analysis, neural similarity matrices were related to the behavioral shape similarity matrices. Results showed that activity patterns in occipitotemporal cortex (OTC) regions, including inferior temporal (IT) cortex and functionally defined object-selective cortex (OSC), reflected the behavioral shape similarity ratings in both blind and sighted groups, also when controlling for the objects' tactile and semantic similarity. Furthermore, neural similarity matrices of IT and OSC showed similarities across blind and sighted groups (within the auditory modality) and across modality (within the sighted group), but not across both modality and group (blind auditory-sighted visual). Together, these findings provide evidence that OTC not only represents objects visually (requiring visual experience) but also represents objects nonvisually, reflecting knowledge of object shape independently of the modality through which this knowledge was acquired.

Tool selectivity in left occipitotemporal cortex develops without vision.

Previous studies have provided evidence for a tool-selective region in left lateral occipitotemporal cortex (LOTC). This region responds selectively to pictures of tools and to characteristic visual tool motion. The present human fMRI study tested whether visual experience is required for the development of tool-selective responses in left LOTC. Words referring to tools, animals, and nonmanipulable objects were presented auditorily to 14 congenitally blind and 16 sighted participants. Sighted participants additionally viewed pictures of these objects. In whole-brain group analyses, sighted participants showed tool-selective activity in left LOTC in both visual and auditory tasks. Importantly, virtually identical tool-selective LOTC activity was found in the congenitally blind group performing the auditory task. Furthermore, both groups showed equally strong tool-selective activity for auditory stimuli in a tool-selective LOTC region defined by the picture-viewing task in the sighted group. Detailed analyses in individual participants showed significant tool-selective LOTC activity in 13 of 14 blind participants and 14 of 16 sighted participants. The strength and anatomical location of this activity were indistinguishable across groups. Finally, both blind and sighted groups showed significant resting state functional connectivity between left LOTC and a bilateral frontoparietal network. Together, these results indicate that tool-selective activity in left LOTC develops without ever having seen a tool or its motion. This finding puts constraints on the possible role that this region could have in tool processing and, more generally, provides new insights into the principles shaping the functional organization of OTC.