Death associated protein like 1 acts as a novel tumor suppressor in melanoma by increasing the stability of P21 protein.

Melanoma is a primary malignant tumor with high lethality, which occurs in the skin and eye tissues, while the molecular mechanisms of melanomagenesis remain largely unknown. Here, we show that death-associated protein-like 1 (DAPL1) expression is lower in melanoma tissues than in paracancerous tissues or nevus tissues, and Uveal melanoma patients with lower DAPL1 expression have a poorer survival rate than those with higher expression of DAPL1. Overexpression of DAPL1 inhibits proliferation of cultured melanoma cells, whereas knockdown of DAPL1 increases cell proliferation. Tumor transplantation experiment results also demonstrate that DAPL1 inhibits tumorigenesis of melanoma cells both in subretinal and subcutaneous tissues of nude mice in vivo. Finally, DAPL1 inhibits proliferation of melanoma cells by increasing the protein level of P21 via decreasing the ubiquitin mediated degradation of P21 and promoting its stability. Conversely, knockdown of P21 neutralizes the effects of inhibition of DAPL1 on melanoma cell proliferation and enhances the severity of melanoma tumorigenesis. These results suggest that DAPL1 is a novel melanoma tumor suppressor gene and thus a potential therapeutic target for melanoma.

Black carp ATG16L1 negatively regulates STING-mediated antiviral innate immune response.

The precise control of interferon (IFN) production is indispensable for the host to eliminate invading viruses and maintain a homeostatic state. In mammals, stimulator of interferon genes (STING) is a prominent adaptor involved in antiviral immune signaling pathways. However, the regulatory mechanism of piscine STING has not been thoroughly investigated. Here, we report that autophagy related 16 like 1 (bcATG16L1) of black carp (Mylopharyngodon piceus) is a negative regulator in black carp STING (bcSTING)-mediated signaling pathway. Initially, we substantiated that knockdown of bcATG16L1 increased the transcription of IFN and ISGs and enhanced the antiviral activity of the host cells. Subsequently, we identified that bcATG16L1 inhibited the bcSTING-mediated IFN promoter activation and proved that bcATG16L1 suppressed bcSTING-mediated antiviral ability. Furthermore, we revealed that bcATG16L1 interacted with bcSTING and the two proteins shared a similar subcellular distribution. Mechanically, we found that bcATG16L1 attenuated the oligomerization of bcSTING, which was a key step for bcSTING activation. Taken together, our results indicate that bcATG16L1 interacts with bcSTING, dampens the oligomerization of bcSTING, and negatively regulates bcSTING-mediated antiviral activity.

MiR-223 inhibits hyperosmolarity-induced inflammation through downregulating NLRP3 activation in human corneal epithelial cells and dry eye patients.

We previously showed that increases in reactive oxygen species (ROS) generation upregulate NLRP3 inflammasome and inflammation through increases in both caspase-1 activity and rises in IL-1ß expression levels in animal models of dry eye (DE). As changes in microRNA (miRNAs) expression levels can modulate inflammasome function, we determine here if there is a relationship in DE between changes in miR-223 expression levels and NLRP3 activation induced in an intelligent controlled environmental system (ICES) in mice. In parallel, ROS, miR-223 and NLRP3 expression levels were assessed in conjunctival impression cytology and tear fluid samples obtained from DE patients and normal subjects. MiR-223 expression levels were modulated by transfection of either a mimic or its negative control (NC) in a human corneal epithelial cell line (HCECs) exposed to a 500 mOsm hyperosmotic medium for 4 h. The dual-luciferase reporter assay confirmed that miR-223 controls NLRP3 gene expression readout through directly interacting with the 3' UTR of its mRNA. Hyperosmolarity-induced NLRP3 activation was confirmed based on recruitment and colocalization of NLRP3 with ASC as well as increases in IL-1ß expression. The miR-223 expression level decreased by 55% in the conjunctiva and cornea of the murine DE model from the level in the control group (P ≤ 0.047), while NLRP3 protein expression rose by 30% (P ≤ 0.017). In DE patients, miR-223 expression decreased in conjunctival impression cytology samples (P = 0.002), whereas IL-1ß tear content rose significantly (P < 0.001).The relevance of this decline was confirmed by showing that exposure to a 500 mOsm stress decreased the miR-223 expression level whereas ROS generation as well as the NLRP3, and IL-1ß expression levels rose in HCECs (P ≤ 0.037). In contrast, miR-223 mimic transfection reduced the NLRP3 protein expression level by 30% (P = 0.037), whereas both ROS generation and IL-1ß secretion rose compared to their corresponding levels in the control group (P ≤ 0.043). Thus, miR-223 negatively regulates NLRP3 inflammasome activity via suppressing NLRP3 translation in DE. This inverse regulation between miR-223 and NLRP3 expression levels suggests that selective upregulation of miR-223 expression may be a novel option to suppress chronic inflammation in DE.

DAPL1 prevents epithelial-mesenchymal transition in the retinal pigment epithelium and experimental proliferative vitreoretinopathy.

Epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is a hallmark of the pathogenesis of proliferative vitreoretinopathy (PVR) that can lead to severe vision loss. Nevertheless, the precise regulatory mechanisms underlying the pathogenesis of PVR remain largely unknown. Here, we show that the expression of death-associated protein-like 1 (DAPL1) is downregulated in PVR membranes and that DAPL1 deficiency promotes EMT in RPE cells in mice. In fact, adeno-associated virus (AAV)-mediated DAPL1 overexpression in RPE cells of Dapl1-deficient mice inhibited EMT in physiological and retinal-detachment states. In a rabbit model of PVR, ARPE-19 cells overexpressing DAPL1 showed reduced ability to induce experimental PVR, and AAV-mediated DAPL1 delivery attenuated the severity of experimental PVR. Furthermore, a mechanistic study revealed that DAPL1 promotes P21 phosphorylation and its stabilization partially through NFκB (RelA) in RPE cells, whereas the knockdown of P21 led to neutralizing effects on DAPL1-dependent EMT inhibition and enhanced the severity of experimental PVR. These results suggest that DAPL1 acts as a novel suppressor of RPE-EMT and has an important role in antagonizing the pathogenesis of experimental PVR. Hence, this finding has implications for understanding the mechanism of and potential therapeutic applications for PVR.

DAPL1 deficiency in mice impairs antioxidant defenses in the RPE and leads to retinal degeneration with AMD-like features.

The decreased antioxidant capacity in the retinal pigment epithelium (RPE) is the hallmark of retinal degenerative diseases including age-related macular degeneration (AMD). Nevertheless, the exact regulatory mechanisms underlying the pathogenesis of retinal degenerations remain largely unknown. Here we show in mice that deficiencies in Dapl1, a susceptibility gene for human AMD, impair the antioxidant capacity of the RPE and lead to age-related retinal degeneration in the 18-month-old mice homozygous for a partial deletion of Dapl1. Dapl1-deficiency is associated with a reduction of the RPE's antioxidant capacity, and experimental re-expression of Dapl1 reverses this reduction and protects the retina from oxidative damage. Mechanistically, DAPL1 directly binds the transcription factor E2F4 and inhibits the expression of MYC, leading to upregulation of the transcription factor MITF and its targets NRF2 and PGC1α, both of which regulate the RPE's antioxidant function. When MITF is experimentally overexpressed in the RPE of DAPL1 deficient mice, antioxidation is restored and retinas are protected from degeneration. These findings suggest that the DAPL1-MITF axis functions as a novel regulator of the antioxidant defense system of the RPE and may play a critical role in the pathogenesis of age-related retinal degenerative diseases.

A radical approach to radicals.

Nitroxide radicals are characterized by a long-lived spin-unpaired electronic ground state and are strongly sensitive to their chemical surroundings. Combined with electron paramagnetic resonance spectroscopy, these electronic features have led to the widespread application of nitroxide derivatives as spin labels for use in studying protein structure and dynamics. Site-directed spin labelling requires the incorporation of nitroxides into the protein structure, leading to a new protein-ligand molecular model. However, in protein crystallographic refinement nitroxides are highly unusual molecules with an atypical chemical composition. Because macromolecular crystallography is almost entirely agnostic to chemical radicals, their structural information is generally less accurate or even erroneous. In this work, proteins that contain an example of a radical compound (Chemical Component Dictionary ID MTN) from the nitroxide family were re-refined by defining its ideal structural parameters based on quantum-chemical calculations. The refinement results show that this procedure improves the MTN ligand geometries, while at the same time retaining higher agreement with experimental data.

Effects of Debaryomyces hansenii treatment on intestinal microorganisms in mice with antibiotics-induced diarrhea.

To investigate the influence of Debaryomyces hansenii treatment on intestinal microorganisms in mice with antibiotics-induced diarrhea, mouse model of antibiotics-induced diarrhea was created by gavaging mice with mixed antibiotics (23.33 mL/kg/days) composed of gentamycin sulfate and cefradine for 5 days. Mice with the symptom of diarrhea were then treated with D. hansenii by intragastric administration. The control group mice were given with sterile water. After 4 day treatment, total DNA of intestinal microflora of treated and control mice was extracted, and their quantities were measured by sequencing the V4 region of 16S rDNA. The results showed that when compared to the control (sterile water), treatment with D. hansenii increased the operational taxonomic units (OTUs) of intestinal bacteria. The Chao index in diarrhea treated group was higher than diarrhea control group and was similar to healthy control group, while all differences had no significance (P > 0.05). D. hansenii treatment increased the Shannon index but not significantly (P > 0.05). Moreover, there was not significant impact on density and diversity of intestinal bacterial population at phylum and genus levels (P > 0.05). Interestingly, D. hansenii treatment recovered the population density of certain bacterium species, such as Bacteroidaceae (in family level) (P < 0.05). Our results indicate that D. hansenii has potency of adjusting the density and diversity of intestinal bacteria and recovering the population density of Bacteroidaceae in family level.