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Journal of Lipid Research Jun 2024Sterol-regulatory element binding proteins (SREBPs) are a conserved transcription factor family governing lipid metabolism. When cellular cholesterol level is low,...
Sterol-regulatory element binding proteins (SREBPs) are a conserved transcription factor family governing lipid metabolism. When cellular cholesterol level is low, SREBP2 is transported from the endoplasmic reticulum to the Golgi apparatus where it undergoes proteolytic activation to generate a soluble N-terminal fragment, which drives the expression of lipid biosynthetic genes. Malfunctional SREBP activation is associated with various metabolic abnormalities. In this study, we find that overexpression of the active nuclear form SREBP2 (nSREBP2) causes caspase-dependent lytic cell death in various types of cells. These cells display typical pyroptotic and necrotic signatures, including plasma membrane ballooning and release of cellular contents. However, this phenotype is independent of the gasdermin family proteins or mixed lineage kinase domain-like (MLKL). Transcriptomic analysis identifies that nSREBP2 induces expression of p73, which further activates caspases. Through whole-genome CRISPR-Cas9 screening, we find that Pannexin-1 (PANX1) acts downstream of caspases to promote membrane rupture. Caspase-3 or 7 cleaves PANX1 at the C-terminal tail and increases permeability. Inhibition of pore-forming activity of PANX1 alleviates lytic cell death. PANX1 can mediate gasdermins and MLKL-independent cell lysis during TNF-induced or chemotherapeutic reagents (doxorubicin or cisplatin)-induced cell death. Together, this study uncovers a noncanonical function of SREBPs as a potentiator of programmed cell death and suggests that PANX1 can directly promote lytic cell death independent of gasdermins and MLKL.
PubMed: 38880128
DOI: 10.1016/j.jlr.2024.100579 -
Cell Death & Disease Jun 2024Chemotherapy is a crucial treatment for colorectal tumors. However, its efficacy is restricted by chemoresistance. Recently, Golgi dispersal has been suggested to be a...
Chemotherapy is a crucial treatment for colorectal tumors. However, its efficacy is restricted by chemoresistance. Recently, Golgi dispersal has been suggested to be a potential response to chemotherapy, particularly to drugs that induce DNA damage. However, the underlying mechanisms by which Golgi dispersal enhances the capacity to resist DNA-damaging agents remain unclear. Here, we demonstrated that DNA-damaging agents triggered Golgi dispersal in colorectal cancer (CRC), and cancer stem cells (CSCs) possessed a greater degree of Golgi dispersal compared with differentiated cancer cells (non-CSCs). We further revealed that Golgi dispersal conferred resistance against the lethal effects of DNA-damaging agents. Momentously, Golgi dispersal activated the Golgi stress response via the PKCα/GSK3α/TFE3 axis, resulting in enhanced protein and vesicle trafficking, which facilitated drug efflux through ABCG2. Identification of Golgi dispersal indicated an unexpected pathway regulating chemoresistance in CRC.
Topics: Neoplastic Stem Cells; Humans; Golgi Apparatus; Colorectal Neoplasms; Drug Resistance, Neoplasm; Animals; Cell Line, Tumor; ATP Binding Cassette Transporter, Subfamily G, Member 2; DNA Damage; Mice; Mice, Nude; Neoplasm Proteins; Antineoplastic Agents
PubMed: 38879509
DOI: 10.1038/s41419-024-06817-0 -
American Journal of Physiology. Renal... Jun 2024Ubiquitination influences the expression of the epithelial Na channel (ENaC). We assessed the mechanisms of selective ubiquitination of the mature, cleaved form of...
Ubiquitination influences the expression of the epithelial Na channel (ENaC). We assessed the mechanisms of selective ubiquitination of the mature, cleaved form of γENaC in both native rodent kidneys and in Fisher Rat Thyroid (FRT) cells expressing the channel heterologously. In both models, singly cleaved and fully cleaved γENaC were both strongly ubiquitinated, implying that the second cleavage releasing an inhibitory peptide was not essential for the process. To see if location of the protein in or near the apical membrane rather than cleavage influences ubiquitination we studied mutants of γENaC in which cleavage sites are abolished. These subunits were ubiquitinated only when co-expressed with α and βENaC, facilitating trafficking through the Golgi apparatus. To test whether reaching the apical surface is necessary we performed surface biotinylation and measured ENaC ubiquitination in the apical membrane of rat kidney. Ubiquitination of cleaved γENaC was similar in whole-kidney and surface fractions, implying that both apical and subapical channels could be modified. In FRT cells, inhibiting clathrin-mediated endocytosis with Dyngo-4a increased both total the ubiquitinated γENaC at the cell surface. Finally, we tested the idea that increased intracellular Na could stimulate ubiquitination. Administration of amiloride to block Na entry through the channels did not affect ubiquitination of γENaC in either FRT cells or rat kidney. However, presumed large increases in cellular Na produced by monensin in FRT cells or acute Na repletion in rats increased ubiquitination and decreased overall ENaC expression.
PubMed: 38867672
DOI: 10.1152/ajprenal.00037.2024 -
Zhongguo Zhen Jiu = Chinese Acupuncture... Jun 2024To observe the effects of electroacupuncture (EA) on the autophagy of ovarian granulosa cells in rats with premature ovarian insufficiency (POI), and explore the...
OBJECTIVE
To observe the effects of electroacupuncture (EA) on the autophagy of ovarian granulosa cells in rats with premature ovarian insufficiency (POI), and explore the mechanism of EA in improving POI.
METHODS
Thirty-two female SD rats were randomly divided into a blank group (=8) and a model making group (=24). The rats in the model making group were injected intraperitoneally with cyclophosphamide for 15 days to establish the POI model (the dosage on the 1st day was 50 mg/kg, and 8 mg/kg from the 2nd day to 15th day). The successfully modeled rats were then randomly divided into a model group, an EA group, and an estradiol (E2) group, with 8 rats in each group. Rats in the EA group received EA at bilateral "Gongsun" (SP 4) with continuous wave, frequency of 2 Hz, and current intensity of 0.1 to 1 mA, 20 min per treatment, once daily for 14 days. Rats in the E2 group were administered with E2 (0.01 mg/mL) by gavage (10 mL/kg), once daily for 14 days. The changes in estrous cycle were observed by rapid Giemsa staining before and after modeling. After intervention, ovarian tissue morphology was observed by HE staining; serum levels of follicle-stimulating hormone (FSH), E2, anti-Mullerian hormone (AMH), and inhibin B (INHB) were detected by ELISA; immunofluorescence staining was used to observe the expression of p62 in ovarian granulosa cells; the ultrastructure of ovarian granulosa cells was observed by transmission electron microscopy, and the number of autophagosomes and autolysosomes was compared; Western blot and real-time fluorescence quantitative PCR were used to detect the protein and mRNA expression of p62, Beclin-1, and microtubule-associated protein 1A/1B-light chain 3 (LC3) in ovarian tissue.
RESULTS
The results of vaginal smears in the blank group showed regular cyclical changes; the rats in the model group showed prolonged estrous cycle or cycle arrest, mostly in proestrus or metestrus, with overall ovarian atrophy, disordered structure, and decreased granulosa cells. Compared with the blank group, rats in the model group showed increased serum FSH level (<0.01), decreased serum levels of E2, AMH, and INHB (<0.01), decreased positive expression of p62 in ovarian granulosa cells (<0.01), with obvious swelling of ovarian granulosa cells, mild to moderate swelling of mitochondria, slight expansion of rough endoplasmic reticulum, and hypertrophy of Golgi apparatus; the number of autophagosomes and autolysosomes in the ovaries was increased (<0.01), the expression of p62 protein and mRNA was decreased (<0.01), and the expression of Beclin-1 and LC3 protein and mRNA in ovarian tissue was increased (<0.01). Compared with the model group, rats in the EA group and the E2 group showed decreased serum FSH levels (<0.01), increased levels of E2, AMH, and INHB (<0.01), increased positive expression of p62 in ovarian granulosa cells (<0.01), alleviated degree of ovarian granulosa cell damage, with relatively intact organelle morphology, and decreased number of autophagosomes and autolysosomes in the ovaries (<0.01); the rats also showed increased expression of p62 protein and mRNA (<0.01), and decreased expression of Beclin-1 and LC3 protein and mRNA (<0.01) in ovarian tissue.
CONCLUSION
EA at "Gongsun" (SP 4) could improve ovarian reserve function in POI rats by reducing the number of autophagosomes and autolysosomes, up-regulating p62 expression, and down-regulating Beclin-1 and LC3 expression, thus inhibiting autophagy of ovarian granulosa cells, and regulating the serum levels of FSH, E2, AMH, and INHB.
Topics: Animals; Female; Electroacupuncture; Primary Ovarian Insufficiency; Rats; Autophagy; Rats, Sprague-Dawley; Humans; Granulosa Cells; Disease Models, Animal
PubMed: 38867630
DOI: 10.13703/j.0255-2930.20230930-k0001 -
Biological & Pharmaceutical Bulletin 2024Ceramide (Cer) is synthesized de novo in the bilayer of the endoplasmic reticulum and transported to the cytosolic leaflet of the trans-Golgi apparatus for sphingomyelin...
Ceramide (Cer) is synthesized de novo in the bilayer of the endoplasmic reticulum and transported to the cytosolic leaflet of the trans-Golgi apparatus for sphingomyelin (SM) synthesis. As the active site of SM synthase (SMS) is located on the luminal side of the Golgi membrane, Cer translocates to the lumen via transbilayer movement for SM synthesis. However, the mechanism of transbilayer movement is not fully understood. As the Cer-related translocases seem to localize near the SMS, the protein was identified using proximity-dependent biotin identification proteomics. Phospholipid scramblase 1 (PLSCR1), which is thought to act as a scramblase for phosphatidylserine and phosphatidylethanolamine, was identified as a protein proximal to the SMS isoforms SMS1 and SMS2. Although five isoforms of PLSCR have been reported in humans, only PLSCR1, PLSCR3, and PLSCR4 are expressed in HEK293T cells. Confocal microscopic analysis showed that PLSCR1 and PLSCR4 partially co-localized with p230, a trans-Golgi network marker, where SMS isoforms are localized. We established CRISPR/Cas9-mediated PLSCR1, PLSCR3, and PLSCR4 single-knockout cells and PLSCR1, 3, 4 triple knockout HEK293T cells. Liquid chromatography-tandem mass spectrometry revealed that the levels of species with distinct acyl chains in Cer and SM were not significantly different in single knockout cells or in the triple knockout cells compared to the wild-type cells. Our findings suggest that PLSCR1 is localized in the vicinity of SMS isoforms, however is not involved in the transbilayer movement of Cer for SM synthesis.
Topics: Humans; Phospholipid Transfer Proteins; Transferases (Other Substituted Phosphate Groups); HEK293 Cells; Sphingomyelins; Membrane Proteins; Isoenzymes; Golgi Apparatus
PubMed: 38866522
DOI: 10.1248/bpb.b24-00177 -
Journal of the American Chemical Society Jun 2024Photomodulable fluorescent probes are drawing increasing attention due to their applications in advanced bioimaging. Whereas photoconvertible probes can be...
Photomodulable fluorescent probes are drawing increasing attention due to their applications in advanced bioimaging. Whereas photoconvertible probes can be advantageously used in tracking, photoswitchable probes constitute key tools for single-molecule localization microscopy to perform super-resolution imaging. Herein, we shed light on a red and far-red BODIPY, namely, BDP-576 and BDP-650, which possess both properties of conversion and switching. Our study demonstrates that these pyrrolyl-BODIPYs convert into typical green- and red-emitting BODIPYs that are perfectly adapted to microscopy. We also showed that this pyrrolyl-BODIPYs undergo Directed Photooxidation Induced Conversion, a photoconversion mechanism that we recently introduced, where the pyrrole moiety plays a central role. These unique features were used to develop targeted photoconvertible probes toward different organelles or subcellular units (plasma membrane, mitochondria, nucleus, actin, Golgi apparatus, ) using chemical targeting moieties and a Halo tag. We notably showed that BDP-650 could be used to track intracellular vesicles over more than 20 min in two-color imagings with laser scanning confocal microscopy, demonstrating its robustness. The switching properties of these photoconverters were studied at the single-molecule level and were then successfully used in live single-molecule localization microscopy in epithelial cells and neurons. Both membrane- and mitochondria- targeted probes could be used to decipher membrane 3D architecture and mitochondrial dynamics at the nanoscale. This study builds a bridge between the photoconversion and photoswitching properties of probes undergoing directed photooxidation and shows the versatility and efficacy of this mechanism in advanced live imaging.
Topics: Oxidation-Reduction; Boron Compounds; Humans; Fluorescent Dyes; Photochemical Processes; HeLa Cells; Animals; Optical Imaging; Molecular Structure
PubMed: 38861358
DOI: 10.1021/jacs.4c05231 -
American Journal of Medical Genetics.... Jun 2024Dyggve-Melchior-Clausen dysplasia (DMC) and Smith-McCort dysplasia (SMC types 1 and 2) are rare spondylo-epi-metaphyseal dysplasias with identical radiological and...
Dyggve-Melchior-Clausen dysplasia (DMC) and Smith-McCort dysplasia (SMC types 1 and 2) are rare spondylo-epi-metaphyseal dysplasias with identical radiological and clinical findings. DMC and SMC type 1 are allelic disorders caused by homozygous or compound heterozygous variants in DYM, while biallelic causative variants in RAB33B lead to SMC type 2. The terminology "skeletal golgipathies" has been recently used to describe these conditions, highlighting the pivotal role of these two genes in the organization and intracellular trafficking of the Golgi apparatus. In this study, we investigated 17 affected individuals (8 males, 9 females) from 10 unrelated consanguineous families, 10 diagnosed with DMC and seven with SMC type 2. The mean age at diagnosis was 9.61 ± 9.72 years, ranging from 20 months to 34 years, and the average height at diagnosis was 92.85 ± 15.50 cm. All patients exhibited variable degrees of short trunk with a barrel chest, protruding abdomen, hyperlordosis, and decreased joint mobility. A total of nine different biallelic variants were identified, with six being located in the DYM gene and the remaining three detected in RAB33B. Notably, five variants were classified as novel, four in the DYM gene and one in the RAB33B gene. This study aims to comprehensively assess clinical, radiological, and molecular findings along with the long-term follow-up findings in 17 patients with DMC and SMC type 2. Our results suggest that clinical symptoms of the disorder typically appear from infancy to early childhood. The central notches of the vertebral bodies were identified as early as 20 months and tended to become rectangular, particularly around 15 years of age. Pseudoepiphysis was observed in five patients; we believe this finding should be taken into consideration when evaluating hand radiographs in clinical assessments. Furthermore, our research contributes to an enhanced understanding of clinical and molecular aspects in these rare "skeletal golgipathies," expanding the mutational spectrum and offering insights into long-term disease outcomes.
PubMed: 38860472
DOI: 10.1002/ajmg.a.63785 -
International Journal of Nanomedicine 2024Reducing the first-pass hepatic effect via intestinal lymphatic transport is an effective way to increase the oral absorption of drugs. 2-Monoacylglycerol (2-MAG) as a...
PURPOSE
Reducing the first-pass hepatic effect via intestinal lymphatic transport is an effective way to increase the oral absorption of drugs. 2-Monoacylglycerol (2-MAG) as a primary digestive product of dietary lipids triglyceride, can be assembled in chylomicrons and then transported from the intestine into the lymphatic system. Herein, we propose a biomimetic strategy and report a 2-MAG mimetic nanocarrier to target the intestinal lymphatic system via the lipid absorption pathway and improve oral bioavailability.
METHODS
The 2-MAG mimetic liposomes were designed by covalently bonding serinol (SER) on the surface of liposomes named SER-LPs to simulate the structure of 2-MAG. Dihydroartemisinin (DHA) was chosen as the model drug because of its disadvantages such as poor solubility and high first-pass effect. The endocytosis and exocytosis mechanisms were investigated in Caco-2 cells and Caco-2 cell monolayers. The capacity of intestinal lymphatic transport was evaluated by ex vivo biodistribution and in vivo pharmacokinetic experiments.
RESULTS
DHA loaded SER-LPs (SER-LPs-DHA) had a particle size of 70 nm and a desirable entrapment efficiency of 93%. SER-LPs showed sustained release for DHA in the simulated gastrointestinal environment. In vitro cell studies demonstrated that the cellular uptake of SER-LPs primarily relied on the caveolae- rather than clathrin-mediated endocytosis pathway and preferred to integrate into the chylomicron assembly process through the endoplasmic reticulum/Golgi apparatus route. After oral administration, SER-LPs efficiently promoted drug accumulation in mesenteric lymphatic nodes. The oral bioavailability of DHA from SER-LPs was 10.40-fold and 1.17-fold larger than that of free DHA and unmodified liposomes at the same dose, respectively.
CONCLUSION
SER-LPs improved oral bioavailability through efficient intestinal lymphatic transport. These findings of the current study provide a good alternative strategy for oral delivery of drugs with high first-pass hepatic metabolism.
Topics: Animals; Liposomes; Biological Availability; Caco-2 Cells; Humans; Administration, Oral; Artemisinins; Intestinal Absorption; Male; Tissue Distribution; Particle Size; Mice; Lymphatic System; Rats, Sprague-Dawley; Rats; Biomimetic Materials; Intestinal Mucosa
PubMed: 38859952
DOI: 10.2147/IJN.S462374 -
Biochemical and Biophysical Research... Sep 2024The primary cilium is a hair-like projection that controls cell development and tissue homeostasis. Although accumulated studies identify the molecular link between...
The primary cilium is a hair-like projection that controls cell development and tissue homeostasis. Although accumulated studies identify the molecular link between cilia and cilia-related diseases, the underlying etiology of ciliopathies has not been fully understood. In this paper, we determine the function of Rab34, a small GTPase, as a key regulator for controlling ciliogenesis and type I collagen trafficking in craniofacial development. Mechanistically, Rab34 is required to form cilia that control osteogenic proliferation, survival, and differentiation via cilia-mediated Hedgehog signaling. In addition, Rab34 is indispensable for regulating type I collagen trafficking from the ER to the Golgi. These results demonstrate that Rab34 has both ciliary and non-ciliary functions to regulate osteogenesis. Our study highlights the critical function of Rab34, which may contribute to understanding the novel etiology of ciliopathies that are associated with the dysfunction of RAB34 in humans.
Topics: Cilia; rab GTP-Binding Proteins; Animals; Mice; Osteogenesis; Humans; Skull; Hedgehog Proteins; Cell Differentiation; Collagen Type I; Signal Transduction; Bone Development; Facial Bones; Cell Proliferation; Protein Transport; Golgi Apparatus
PubMed: 38852507
DOI: 10.1016/j.bbrc.2024.150174 -
Journal of Photochemistry and... Jun 2024Hepatic fibrosis (HF) is caused by persistent inflammation, which is closely associated with hepatic oxidative stress. Peroxynitrite (ONOO) is significantly elevated in...
Visulization of peroxynitrite variation for accurate diagnosis and assessing treatment response of hepatic fibrosis using a Golgi-targetable ratiometric fluorescent probe.
Hepatic fibrosis (HF) is caused by persistent inflammation, which is closely associated with hepatic oxidative stress. Peroxynitrite (ONOO) is significantly elevated in HF, which would be regarded as a potential biomarker for the diagnosis of HF. Research has shown that ONOO in the Golgi apparatus can be overproduced in HF, and it can induce hepatocyte injury by triggering Golgi oxidative stress. Meanwhile, the ONOO inhibitors could effectively relieve HF by inhibiting Golgi ONOO, but as yet, no Golgi-targetable fluorescent probe available for diagnosis and assessing treatment response of HF through sensing Golgi ONOO. To this end, we reported a ratiometric fluorescent probe, Golgi-PER, for diagnosis and assessing treatment response of HF through monitoring the Golgi ONOO. Golgi-PER displayed satisfactory sensitivity, low detection limit, and exceptional selectivity to ONOO. Combined with excellent biocompatibility and good Golgi-targeting ability, Golgi-PER was further used for ratiometric monitoring the Golgi ONOO fluctuations and screening of ONOO inhibitors from polyphenols in living cells. Meanwhile, using Golgi-PER as a probe, the overexpression of Golgi ONOO in HF and the treatment response of HF to the screened rosmarinic acid were precisely visualized for the first time. Furthermore, the screened RosA has a remarkable therapeutic effect on HF, which may be a new strategy for HF treatment. These results demonstrated the practicability of Golgi-PER for monitoring the occurrence, development, and personalized treatment response of HF.
PubMed: 38851042
DOI: 10.1016/j.jphotobiol.2024.112950