Pannexin 1 (PANX1) is an ATP-permeable channel with critical roles in a variety of physiological functions such as blood pressure regulation
, apoptotic cell clearance
and human oocyte development
. ...Here we present several structures of human PANX1 in a heptameric assembly at resolutions of up to 2.8 angström, including an apo state, a caspase-7-cleaved state and a carbenoxolone-bound state. We reveal a gating mechanism that involves two ion-conducting pathways. Under normal cellular conditions, the intracellular entry of the wide main pore is physically plugged by the C-terminal tail. Small anions are conducted through narrow tunnels in the intracellular domain. These tunnels connect to the main pore and are gated by a long linker between the N-terminal helix and the first transmembrane helix. During apoptosis, the C-terminal tail is cleaved by caspase, allowing the release of ATP through the main pore. We identified a carbenoxolone-binding site embraced by W74 in the extracellular entrance and a role for carbenoxolone as a channel blocker. We identified a gap-junction-like structure using a glycosylation-deficient mutant, N255A. Our studies provide a solid foundation for understanding the molecular mechanisms underlying the channel gating and inhibition of PANX1 and related large-pore channels.
Image-guided photothermal therapy (PTT) is an attractive strategy to improve the diagnosis accuracy and treatment outcomes by monitoring the accumulation of photothermal agents in tumors in real-time ...and determining the best treatment window. Taking advantage of the superior imaging quality of NIR-II fluorescence imaging and remote-controllable phototherapy modality of PTT, we developed a facile macromolecular fluorophore (PF) by conjugating a small-molecule NIR-II fluorophore (Flav7) with an amphiphilic polypeptide. The PF can form uniform micelles in aqueous solution, which exhibit a slight negative charge. In vitro experimental results showed that the PF nanoparticles showed satisfactory photophysical properties, prominent photothermal conversion efficiency (42.3%), excellent photothermal stability, negligible cytotoxicity, and photothermal toxicity. Meanwhile, the PF can visualize and feature the tumors by NIR-II fluorescence imaging owing to prolonged blood circulation time and enhanced accumulation in tumors. Moreover, in vivo studies revealed that the PF nanoparticles achieved an excellent photothermal ablation effect on tumors with a low dose of NIR-II dye and light irradiation, and the process can be traced by NIR fluorescence imaging.
Protein phosphorylation is one of the major molecular mechanisms regulating protein activity and function throughout the cell. Pannexin 1 (PANX1) is a large-pore channel permeable to ATP and other ...cellular metabolites. Its tyrosine phosphorylation and subsequent activation have been found to play critical roles in diverse cellular conditions, including neuronal cell death, acute inflammation, and smooth muscle contraction. Specifically, the non-receptor kinase Src has been reported to phosphorylate Tyr198 and Tyr308 of mouse PANX1 (equivalent to Tyr199 and Tyr309 of human PANX1), resulting in channel opening and ATP release. Although the Src-dependent PANX1 activation mechanism has been widely discussed in the literature, independent validation of the tyrosine phosphorylation of PANX1 has been lacking. Here, we show that commercially available antibodies against the two phosphorylation sites mentioned above-which were used to identify endogenous PANX1 phosphorylation at these two sites-are nonspecific and should not be used to interpret results related to PANX1 phosphorylation. We further provide evidence that neither tyrosine residue is a major phosphorylation site for Src kinase in heterologous expression systems. We call on the field to re-examine the existing paradigm of tyrosine phosphorylation-dependent activation of the PANX1 channel.
The proton-activated chloride channel (PAC) is active across a wide range of mammalian cells and is involved in acid-induced cell death and tissue injury
. PAC has recently been shown to represent a ...novel and evolutionarily conserved protein family
. Here we present two cryo-electron microscopy structures of human PAC in a high-pH resting closed state and a low-pH proton-bound non-conducting state. PAC is a trimer in which each subunit consists of a transmembrane domain (TMD), which is formed of two helices (TM1 and TM2), and an extracellular domain (ECD). Upon a decrease of pH from 8 to 4, we observed marked conformational changes in the ECD-TMD interface and the TMD. The rearrangement of the ECD-TMD interface is characterized by the movement of the histidine 98 residue, which is, after acidification, decoupled from the resting position and inserted into an acidic pocket that is about 5 Å away. Within the TMD, TM1 undergoes a rotational movement, switching its interaction partner from its cognate TM2 to the adjacent TM2. The anion selectivity of PAC is determined by the positively charged lysine 319 residue on TM2, and replacing lysine 319 with a glutamate residue converts PAC to a cation-selective channel. Our data provide a glimpse of the molecular assembly of PAC, and a basis for understanding the mechanism of proton-dependent activation.
Fermentation-enabled wellness foods: A fresh perspective Xiang, Huan; Sun-Waterhouse, Dongxiao; Waterhouse, Geoffrey I.N. ...
Food science and human wellness,
September 2019, 2019-09-00, 2019-09-01, Letnik:
8, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Fermented foods represent an important segment of current food markets, especially traditional or ethnic food markets. The demand for efficient utilization of agrowastes, together with advancements ...in fermentation technologies (microbial- and enzyme-based processing), are stimulating rapid growth and innovation in the fermented food sector. In addition, the health-promoting benefits of fermented foods are attracting increasingly attention. The microorganisms contained in many common fermented foods can serve as “microfactories” to generate nutrients and bioactives with specific nutritional and health functionalities. Herein, recent research relating to the manufacture of fermented foods are critically reviewed, placing emphasis on the potential health benefits of fermentation-enabled wellness foods. The importance of the correct selection of microorganisms and raw materials and the need for precise control of fermentation processes are explored. Major knowledge gaps and obstacles to fermented food production and market penetration are discussed. The importance of integrating multidisciplinary knowledge, communicating with consumers, establishing regulatory frameworks specifically for fermentation-enabled wellness foods and functional fermented foods, are highlighted.
The Ca
-activated TRPM5 channel plays essential roles in taste perception and insulin secretion. However, the mechanism by which Ca
regulates TRPM5 activity remains elusive. We report cryo-EM ...structures of the zebrafish TRPM5 in an apo closed state, a Ca
-bound open state, and an antagonist-bound inhibited state. We define two novel ligand binding sites: a Ca
site (Ca
) in the intracellular domain and an antagonist site in the transmembrane domain (TMD). The Ca
site is unique to TRPM5 and has two roles: modulating the voltage dependence and promoting Ca
binding to the Ca
site, which is conserved throughout TRPM channels. Conformational changes initialized from both Ca
sites cooperatively open the ion-conducting pore. The antagonist NDNA wedges into the space between the S1-S4 domain and pore domain, stabilizing the transmembrane domain in an apo-like closed state. Our results lay the foundation for understanding the voltage-dependent TRPM channels and developing new therapeutic agents.
2,2’,4,4’-tetrabromodiphenyl ether (BDE-47)-induced nephrotoxicity is closely associated with oxidative stresses and mitochondrial abnormalities. Mitochondrial fusion and fission dynamics are crucial ...for maintaining mitochondrial and cellular physiological homeostasis. However, the detailed mechanisms through which BDE-47 disrupts this dynamic and contributes to renal injuries are still not fully understood. The porcine kidney-15 (PK15) cell line, a well-defined in vitro animal renal toxicological model, was exposed to BDE-47 with concentrations of 12.5, 25, 50, and 100 μM, respectively. Cell viability, the levels of reactive oxygen species (ROS) and adenosine triphosphate (ATP), the mitochondrial membrane potential (MMP), and the expression levels of key mitochondrial fusion and fission proteins were assessed. BDE-47 reduced cell viability and disrupted mitochondrial dynamics by inhibiting mitochondrial fusion and fission simultaneously, leading to MMP decreases, ROS overgeneration, ATP depletion, and cellular disintegration in a dose-dependent manner. Additionally, the mitochondrial division inhibitor (Mdivi-1) with the concentration of 20 μM observed to restore the downregulation of mitochondrial fusion and fission proteins, alleviate damages in mitochondrial morphology and functionality, correct ROS overproduction, and enable cell survival. The antioxidant N-acety-L-cysteine (NAC) with the concentration of 1 mM also simultaneously reversed the imbalance of mitochondrial dynamics, decreased ROS production, and restored mitochondrial morphology in PK15 cells exposed to BDE-47. Our data provide new insights indicating that BDE-47 disrupts mitochondrial fusion/fission dynamics to induce mitochondrial abnormalities, triggering oxidative stresses and thus contributing to PK15 cell dysfunction. ROS-dependent pathways in mitochondrial dynamics may provide a new avenue for developing effective strategies to protect cells against BDE-47-induced nephrotoxicity.
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•BDE-47 could cause nephrocyte lesion after exposure to PK15 cells.•BDE-47 disrupts mitochondrial fusion/fission dynamics related to the PK15 cell lesion•BDE-47 induces mitochondrial abnormalities, triggers oxidative stresses and thus contributes to PK15 cell dysfunction.
Proton-activated chloride (PAC) channel is a ubiquitously expressed pH-sensing ion channel, encoded by
PACC1
(
TMEM206
). PAC regulates endosomal acidification and macropinosome shrinkage by ...releasing chloride from the organelle lumens. It is also found at the cell surface, where it is activated under pathological conditions related to acidosis and contributes to acid-induced cell death. However, the pharmacology of the PAC channel is poorly understood. Here, we report that phosphatidylinositol (4,5)-bisphosphate (PIP
2
) potently inhibits PAC channel activity. We solved the cryo-electron microscopy structure of PAC with PIP
2
at pH 4.0 and identified its putative binding site, which, surprisingly, locates on the extracellular side of the transmembrane domain (TMD). While the overall conformation resembles the previously resolved PAC structure in the desensitized state, the TMD undergoes remodeling upon PIP
2
-binding. Structural and electrophysiological analyses suggest that PIP
2
inhibits the PAC channel by stabilizing the channel in a desensitized-like conformation. Our findings identify PIP
2
as a new pharmacological tool for the PAC channel and lay the foundation for future drug discovery targeting this channel.
The Tribbles family of pseudokinases recruits substrates to the ubiquitin ligase COP1 to facilitate ubiquitylation. CCAAT/enhancer-binding protein (C/EBP) family transcription factors are crucial ...Tribbles substrates in adipocyte and myeloid cell development. We found that the TRIB1 pseudokinase was able to recruit various C/EBP family members and that the binding of C/EBPβ was attenuated by phosphorylation. To explain the mechanism of C/EBP recruitment, we solved the crystal structure of TRIB1 in complex with C/EBPα, which revealed that TRIB1 underwent a substantial conformational change relative to its substrate-free structure and bound C/EBPα in a pseudosubstrate-like manner. Crystallographic analysis and molecular dynamics and subsequent biochemical assays showed that C/EBP binding triggered allosteric changes that link substrate recruitment to COP1 binding. These findings offer a view of pseudokinase regulation with striking parallels to bona fide kinase regulation-by means of the activation loop and αC helix-and raise the possibility of small molecules targeting either the activation "loop-in" or "loop-out" conformations of Tribbles pseudokinases.
Dysbiosis of gut microbiota and metabolic pathway disorders are closely related to the ulcerative colitis. Through network pharmacology, we found that puerarin is a potential ingredient that can ...improve the crypt deformation and inflammatory infiltration in mice, and decrease the levels of IL-1β, IL-6 and TNF-α significantly.
Listeria
,
Alistipes
and
P. copri
gradually became dominant bacteria in UC mice, which were positively correlated with inflammatory factors. Puerarin effectively improved dysbiosis by reducing the abundance of
Alistipes
,
P. copri
and
Veillonella
, and increasing the level of
Desulfovibrionacea
. Correlation network and metabolic function prediction analysis of the microbiota showed that they formed a tightly connected network and were widely involved in carbohydrate metabolism and amino acid metabolism. Specifically, we observed significant changes in the tryptophan metabolism pathway in DSS mice, with an increase in the abundance of
Bacteroidetes
and
Enterobacteriaceae
involved in tryptophan metabolism. However, this metabolic disorder was alleviated after puerarin treatment, including the reversal of 3-HAA levels and an increase in the abundance of
Rhodobacteraceae
and
Halomonadaceae
involved in kynurenine metabolism, as well as a significant increase in the purine metabolite guanosine. In conclusion, our study suggests that puerarin has a good therapeutic effect on UC, which is partially achieved by restoring the composition and abundance of gut microbiota and their metabolism.
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