Abstract
Background
Pancreatic cancer is one of the most lethal malignancies and has an extremely poor diagnosis and prognosis. The development of resistance to gemcitabine is still a major ...challenge. The long noncoding RNA PVT1 was reported to be involved in carcinogenesis and chemoresistance; however, the mechanism by which PVT1 regulates the sensitivity of pancreatic cancer to gemcitabine remains poorly understood.
Methods
The viability of pancreatic cancer cells was assessed by MTT assay in vitro and xenograft tumor formation assay in vivo. The expression levels of PVT1 and miR-619-5p were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Western blotting analysis and qRT-PCR were performed to assess the protein and mRNA levels of Pygo2 and ATG14, respectively. Autophagy was explored via autophagic flux detection under confocal microscopy and autophagic vacuole investigation under transmission electron microscopy (TEM). The functional role and mechanism of PVT1 were further investigated by gain- and loss-of-function assays in vitro.
Results
In the present study, we demonstrated that PVT1 was up-regulated in gemcitabine-resistant pancreatic cancer cell lines. Gain- and loss-of-function assays revealed that PVT1 impaired sensitivity to gemcitabine in vitro and in vivo. We further found that PVT1 up-regulated the expression of both Pygo2 and ATG14 and thus regulated Wnt/β-catenin signaling and autophagic activity to overcome gemcitabine resistance through sponging miR-619-5p. Moreover, we discovered three TCF/LEF binding elements (TBEs) in the promoter region of PVT1, and activation of Wnt/β-catenin signaling mediated by the up-regulation of Pygo2 increased PVT1 expression by direct binding to the TBE region. Furthermore, PVT1 was discovered to interact with ATG14, thus promoting assembly of the autophagy specific complex I (PtdIns3K-C1) and ATG14-dependent class III PtdIns3K activity.
Conclusions
These data indicate that PVT1 plays a critical role in the sensitivity of pancreatic cancer to gemcitabine and highlight its potential as a valuable target for pancreatic cancer therapy.
Summary
Advanced structures of food proteins are maintained by many forces such as hydrophobic activities, electrostatic interaction, and disulphide bond interaction, which can affect their ...functional characteristics to a certain extent. Therefore, many approaches have been utilised to improve functional characteristics of food proteins. pH‐shift process refers to the method that food proteins are treated under extreme alkaline or acid conditions followed by adjusting pH to isoelectric point or neutral pH. Many studies have showed that pH‐shift process can significantly affect the functional characteristics of food proteins, for example emulsifying activities, forming property, solubility and water/oil adsorption ability. pH‐shift process has been utilised to recover protein isolates from many sources including fish, fish by‐products, meat processing products. Many researches have indicated that the functional and physicochemical characteristics of recovered protein isolates are significantly influenced by pH‐shift process. In this paper, the latest studies regarding the functional and physicochemical characteristics of proteins treated or recovered through pH‐shift process, and potential applications of obtained protein isolates in the production of the hydrolysates or used as a delivery system were reviewed.
Protein properties treated by pH‐shift process and its applications.
Bacterial pollution is a great risk for human health. Nanotechnology offers a way to develop new inorganic antibacterial agents. Nano-inorganic metal oxide has a potential to reduce bacterial ...contamination. MgO is an important inorganic oxide and has been widely used in many fields. Many studies have shown that MgO nanoparticles have good antibacterial activity. Therefore, in this paper, the main synthesis methods, antibacterial activity and antibacterial mechanisms of MgO nanoparticles are reviewed.
Regulation of PKD2 channel function by TACAN Liu, Xiong; Zhang, Rui; Fatehi, Mohammad ...
The Journal of physiology,
1 January 2023, 2023-01-00, 20230101, Volume:
601, Issue:
1
Journal Article
Peer reviewed
Autosomal dominant polycystic kidney disease is caused by mutations in the membrane receptor PKD1 or the cation channel PKD2. TACAN (also termed TMEM120A), recently reported as an ion channel in ...neurons for mechanosensing and pain sensing, is also distributed in diverse non‐neuronal tissues, such as kidney, heart and intestine, suggesting its involvement in other functions. In this study, we found that TACAN is in a complex with PKD2 in native renal cell lines. Using the two‐electrode voltage clamp in Xenopus oocytes, we found that TACAN inhibits the channel activity of PKD2 gain‐of‐function mutant F604P. TACAN fragments containing the first and last transmembrane domains interacted with the PKD2 C‐ and N‐terminal fragments, respectively. The TACAN N‐terminus acted as a blocking peptide, and TACAN inhibited the function of PKD2 by the binding of PKD2 with TACAN. By patch clamping in mammalian cells, we found that TACAN inhibits both the single‐channel conductance and the open probability of PKD2 and mutant F604P. PKD2 co‐expressed with TACAN, but not PKD2 alone, exhibited pressure sensitivity. Furthermore, we found that TACAN aggravates PKD2‐dependent tail curvature and pronephric cysts in larval zebrafish. In summary, this study revealed that TACAN acts as a PKD2 inhibitor and mediates mechanosensitivity of the PKD2–TACAN channel complex.
Key points
TACAN inhibits the function of PKD2 in vitro and in vivo.
TACAN N‐terminal S1‐containing fragment T160X interacts with the PKD2 C‐terminal fragment N580–L700, and its C‐terminal S6‐containing fragment L296–D343 interacts with the PKD2 N‐terminal A594X.
TACAN inhibits the function of the PKD2 channel by physical interaction.
The complex of PKD2 with TACAN, but not PKD2 alone, confers mechanosensitivity.
figure legend PKD2 is a Ca2+‐permeable cation channel and is mutated in ∼15% of cases of human autosomal dominant polycystic kidney disease. Using the two‐electrode voltage clamp with Xenopus oocytes, patch clamp with mammalian cells, the zebrafish model and molecular biology techniques, we have found that the membrane protein TACAN forms a complex with PKD2 on the primary cilia of renal epithelial cell lines and acts as an inhibitor of the function of PKD2 in vitro and in vivo.
Recent advances in perovskite ferroelectrics have fostered a host of exciting sensors and actuators. Defect engineering provides critical control of the performance of ferroelectric materials, ...especially lead‐free ones. However, it remains a challenge to quantitatively study the concentration of defects due to the complexity of measurement techniques. Here, a feasible approach to analyzing the A‐site defect and electron in alkali metal niobate is demonstrated. The theoretical relationships among defect concentration, conductivity, and oxygen partial pressure can be established based on the defect chemistry equilibria. The type and concentration of defects are reflected through the conductivity variation with oxygen partial pressure. As a result, the variation of defect concentration gives rise to defect‐driven interfacial polarization, which further leads to distinct properties of the ceramics. e.g., abnormal dielectric behavior. Furthermore, this study also suggests a strategy to manipulate defects and charges in perovskite oxides for performance optimization.
For perovskites, it is challenging to quantitatively study the concentration of defects. A theoretical relationship between defect concentration, conductivity, and oxygen partial pressure can be established based on the defect chemistry equilibria. The type and concentration of defects are reflected through the conductivity variation with oxygen partial pressure.
PKD2 and PKD1 genes are mutated in human autosomal dominant polycystic kidney disease. PKD2 can form either a homomeric cation channel or a heteromeric complex with the PKD1 receptor, presumed to ...respond to ligand(s) and/or mechanical stimuli. Here, we identify a two-residue hydrophobic gate in PKD2L1, and a single-residue hydrophobic gate in PKD2. We find that a PKD2 gain-of-function gate mutant effectively rescues PKD2 knockdown-induced phenotypes in embryonic zebrafish. The structure of a PKD2 activating mutant F604P by cryo-electron microscopy reveals a π- to α-helix transition within the pore-lining helix S6 that leads to repositioning of the gate residue and channel activation. Overall the results identify hydrophobic gates and a gating mechanism of PKD2 and PKD2L1.
Transient receptor potential melastatin member 8 (TRPM8), a Ca2+‐permeable nonselective cation channel activated by cold and cooling agents, mediates allodynia. Dysfunction or abnormal expression of ...TRPM8 has been found in several human cancers. The role of ubiquitination in the regulation of TRPM8 function remains poorly understood. Here, we identified the ubiquitin (Ub)‐ligase E3, tripartite motif‐containing 4 (TRIM4), as a novel interaction partner of TRPM8 and confirmed that the TRIM4–TRPM8 interaction was mediated through the SPRY domain of TRIM4. Patch‐clamp assays showed that TRIM4 negatively regulates TRPM8‐mediated currents in HEK293 cells. Moreover, TRIM4 reduced the expression of TRPM8 on the cell surface by promoting the K63‐linked ubiquitination of TRPM8. Further analyses revealed that the TRPM8 N‐terminal lysine residue at 423 was the major ubiquitination site that mediates its functional regulation by TRIM4. A Ub‐activating enzyme E1, Ub‐like modifier‐activating enzyme 1 (UBA1), was also found to interact with TRPM8, thereby regulating its channel function and ubiquitination. In addition, knockdown of UBA1 impaired the regulation of TRPM8 ubiquitination and function by TRIM4. Thus, this study demonstrates that TRIM4 downregulates TRPM8 via K423‐mediated TRPM8 ubiquitination and requires UBA1 to regulate TRPM8.
Tripartite motif‐containing 4 (TRIM4), an ubiquitin (Ub)‐ligase E3, downregulates transient receptor potential melastatin member 8 (TRPM8) via K423‐mediated TRPM8 ubiquitination and that it requires UBA1 to regulate TRPM8.
Transient receptor potential (TRP) channels, subdivided into 6 subfamilies in mammals, have essential roles in sensory physiology. They respond to remarkably diverse stimuli, comprising thermal, ...chemical, and mechanical modalities, through opening or closing of channel gates. In this study, we systematically substituted the hydrophobic residues within the distal fragment of pore‐lining helix S6 with hydrophilic residues and, based on Xenopus oocyte and mammalian cell electrophysiology and a hydrophobic gate theory, identified hydrophobic gates in TRPV6/V5/V4/C4/ M8. We found that channel activity drastically increased when TRPV6Ala616 or Met617 or TRPV5Ala576 or Met577, but not any of their adjacent residues, was substituted with hydrophilic residues. Channel activity strongly correlated with the hydrophilicity of the residues at those sites, suggesting that consecutive hydrophobic residues TRPV6Ala616‐Met617 and TRPV5Ala576‐Met577 form a double‐residue gate in each channel. By the same strategy, we identified a hydrophobic single‐residue gate in TRPV4Iso715, TRPC4Iso617, and TRPM8Val976. In support of the hydrophobic gate theory, hydrophilic substitution at the gate site, which removes the hydrophobic gate seal, substantially increased the activity of TRP channels in low‐activity states but had little effect on the function of activated channels. The double‐residue gate channels were more sensitive to small changes in the gate's hydrophobicity or size than single‐residue gate channels. The unconventional double‐reside gating mechanism in TRP channels may have been evolved to respond especially to physiologic stimuli that trigger relatively small gate conformational changes.—Zheng, W., Hu, R., Cai, R., Hofmann, L., Hu, Q., Fatehi, M., Long, W., Kong, T., Tang, J., Light, P., Flockerzi, V., Cao, Y., Chen, X.‐Z. Identification and characterization of hydrophobic gate residues in TRP channels. FASEB J. 32, 639–653 (2018). www.fasebj.org
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