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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 -
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 -
ELife Jun 2024The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these...
The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these components are sorted to their specific locations remain unknown. While some motifs mediating subcellular protein localization have been identified, many membrane proteins and most membrane lipids lack known sorting determinants. A putative mechanism for sorting of membrane components is based on membrane domains known as lipid rafts, which are laterally segregated nanoscopic assemblies of specific lipids and proteins. To assess the role of such domains in the secretory pathway, we applied a robust tool for synchronized secretory protein traffic (RUSH, etention sing elective ooks) to protein constructs with defined affinity for raft phases. These constructs consist solely of single-pass transmembrane domains (TMDs) and, lacking other sorting determinants, constitute probes for membrane domain-mediated trafficking. We find that while raft affinity can be sufficient for steady-state PM localization, it is not sufficient for rapid exit from the endoplasmic reticulum (ER), which is instead mediated by a short cytosolic peptide motif. In contrast, we find that Golgi exit kinetics are highly dependent on raft affinity, with raft preferring probes exiting the Golgi ~2.5-fold faster than probes with minimal raft affinity. We rationalize these observations with a kinetic model of secretory trafficking, wherein Golgi export can be facilitated by protein association with raft domains. These observations support a role for raft-like membrane domains in the secretory pathway and establish an experimental paradigm for dissecting its underlying machinery.
Topics: Protein Transport; Endoplasmic Reticulum; Golgi Apparatus; Membrane Microdomains; Secretory Pathway; Humans; Kinetics; Cell Membrane; Membrane Proteins; HeLa Cells
PubMed: 38837189
DOI: 10.7554/eLife.89306 -
Frontiers in Immunology 2024Sialic acids as terminal sugar residues on cell surface or secreted proteins have many functional roles. In particular, the presence or absence of α2,6-linked sialic...
Sialic acids as terminal sugar residues on cell surface or secreted proteins have many functional roles. In particular, the presence or absence of α2,6-linked sialic acid residues at the immunoglobulin G (IgG) Fc fragment can switch IgG effector functions from pro- to anti-inflammatory activity. IgG glycosylation is considered to take place inside the plasma blast/plasma cell while the molecule travels through the endoplasmic reticulum and Golgi apparatus before being secreted. However, more recent studies have suggested that IgG sialylation may occur predominantly post-antibody secretion. To what extent this extracellular IgG sialylation process contributes to overall IgG sialylation remains unclear, however. By generating bone marrow chimeric mice with a B cell-specific deletion of ST6Gal1, the key enzyme required for IgG sialylation, we now show that sialylation of the IgG Fc fragment exclusively occurs within B cells pre-IgG secretion. We further demonstrate that B cells expressing ST6Gal1 have a developmental advantage over B cells lacking ST6Gal1 expression and thus dominate the plasma cell pool and the resulting serum IgG population in mouse models in which both ST6Gal1-sufficient and -deficient B cells are present.
Topics: Animals; Immunoglobulin G; Mice; Sialyltransferases; B-Lymphocytes; Mice, Knockout; Glycosylation; Mice, Inbred C57BL; Immunoglobulin Fc Fragments; beta-D-Galactoside alpha 2-6-Sialyltransferase; Plasma Cells; Antibody Formation
PubMed: 38827747
DOI: 10.3389/fimmu.2024.1402000 -
Frontiers in Neural Circuits 2024For neural circuit construction in the brain, coarse neuronal connections are assembled prenatally following genetic programs, being reorganized postnatally by... (Review)
Review
For neural circuit construction in the brain, coarse neuronal connections are assembled prenatally following genetic programs, being reorganized postnatally by activity-dependent mechanisms to implement area-specific computational functions. Activity-dependent dendrite patterning is a critical component of neural circuit reorganization, whereby individual neurons rearrange and optimize their presynaptic partners. In the rodent primary somatosensory cortex (barrel cortex), driven by thalamocortical inputs, layer 4 (L4) excitatory neurons extensively remodel their basal dendrites at neonatal stages to ensure specific responses of barrels to the corresponding individual whiskers. This feature of barrel cortex L4 neurons makes them an excellent model, significantly contributing to unveiling the activity-dependent nature of dendrite patterning and circuit reorganization. In this review, we summarize recent advances in our understanding of the activity-dependent mechanisms underlying dendrite patterning. Our focus lays on the mechanisms revealed by time-lapse imaging, and the role of activity-dependent Golgi apparatus polarity regulation in dendrite patterning. We also discuss the type of neuronal activity that could contribute to dendrite patterning and hence connectivity.
Topics: Animals; Dendrites; Somatosensory Cortex; Vibrissae; Animals, Newborn
PubMed: 38827189
DOI: 10.3389/fncir.2024.1409993 -
The EMBO Journal May 2024The degradation of organelles by autophagy is essential for cellular homeostasis. The Golgi apparatus has recently been demonstrated to be degraded by autophagy, but...
The degradation of organelles by autophagy is essential for cellular homeostasis. The Golgi apparatus has recently been demonstrated to be degraded by autophagy, but little is known about how the Golgi is recognized by the forming autophagosome. Using quantitative proteomic analysis and two novel Golgiphagy reporter systems, we found that the five-pass transmembrane Golgi-resident proteins YIPF3 and YIPF4 constitute a Golgiphagy receptor. The interaction of this complex with LC3B, GABARAP, and GABARAPL1 is dependent on a LIR motif within YIPF3 and putative phosphorylation sites immediately upstream; the stability of the complex is governed by YIPF4. Expression of a YIPF3 protein containing a mutated LIR motif caused an elongated Golgi morphology, indicating the importance of Golgi turnover via selective autophagy. The reporter assays reported here may be readily adapted to different experimental contexts to help deepen our understanding of Golgiphagy.
PubMed: 38822137
DOI: 10.1038/s44318-024-00131-3 -
Nature Communications May 2024Knowledge on the distribution and dynamics of glycosylation enzymes in the Golgi is essential for better understanding this modification. Here, using a combination of...
Knowledge on the distribution and dynamics of glycosylation enzymes in the Golgi is essential for better understanding this modification. Here, using a combination of CRISPR/Cas9 knockin technology and super-resolution microscopy, we show that the Golgi complex is assembled by a number of small 'Golgi units' that have 1-3 μm in diameter. Each Golgi unit contains small domains of glycosylation enzymes which we call 'zones'. The zones of N- and O-glycosylation enzymes are colocalised. However, they are less colocalised with the zones of a glycosaminoglycan synthesizing enzyme. Golgi units change shapes dynamically and the zones of glycosylation enzymes rapidly move near the rim of the unit. Photobleaching analysis indicates that a glycosaminoglycan synthesizing enzyme moves between units. Depletion of giantin dissociates units and prevents the movement of glycosaminoglycan synthesizing enzymes, which leads to insufficient glycosaminoglycan synthesis. Thus, we show the structure-function relationship of the Golgi and its implications in human pathogenesis.
Topics: Golgi Apparatus; Glycosylation; Humans; Glycosaminoglycans; HeLa Cells; CRISPR-Cas Systems; Membrane Proteins; Golgi Matrix Proteins
PubMed: 38802491
DOI: 10.1038/s41467-024-48901-1 -
Nature Communications May 2024To facilitate inter-tissue communication and the exchange of proteins, lipoproteins, and metabolites with the circulation, hepatocytes have an intricate and efficient...
To facilitate inter-tissue communication and the exchange of proteins, lipoproteins, and metabolites with the circulation, hepatocytes have an intricate and efficient intracellular trafficking system regulated by small Rab GTPases. Here, we show that Rab30 is induced in the mouse liver by fasting, which is amplified in liver-specific carnitine palmitoyltransferase 2 knockout mice (Cpt2) lacking the ability to oxidize fatty acids, in a Pparα-dependent manner. Live-cell super-resolution imaging and in vivo proximity labeling demonstrates that Rab30-marked vesicles are highly dynamic and interact with proteins throughout the secretory pathway. Rab30 whole-body, liver-specific, and Rab30; Cpt2 liver-specific double knockout (DKO) mice are viable with intact Golgi ultrastructure, although Rab30 deficiency in DKO mice suppresses the serum dyslipidemia observed in Cpt2 mice. Corresponding with decreased serum triglyceride and cholesterol levels, DKO mice exhibit decreased circulating but not hepatic ApoA4 protein, indicative of a trafficking defect. Together, these data suggest a role for Rab30 in the selective sorting of lipoproteins to influence hepatocyte and circulating triglyceride levels, particularly during times of excessive lipid burden.
Topics: Animals; Male; Mice; Carnitine O-Palmitoyltransferase; Cholesterol; Fasting; Golgi Apparatus; Hepatocytes; Homeostasis; Lipid Metabolism; Liver; Mice, Inbred C57BL; Mice, Knockout; rab GTP-Binding Proteins; Triglycerides
PubMed: 38796472
DOI: 10.1038/s41467-024-48959-x -
Life Science Alliance Aug 2024Complexes of ERLIN1 and ERLIN2 (ER lipid raft-associated 1 and 2) form large ring-like cup-shaped structures on the endoplasmic reticulum (ER) membrane and serve as...
Complexes of ERLIN1 and ERLIN2 (ER lipid raft-associated 1 and 2) form large ring-like cup-shaped structures on the endoplasmic reticulum (ER) membrane and serve as platforms to bind cholesterol and E3 ubiquitin ligases, potentially defining functional nanodomains. Here, we show that ERLIN scaffolds mediate the interaction between the full-length isoform of TMUB1 (transmembrane and ubiquitin-like domain-containing 1) and RNF170 (RING finger protein 170). We identify a luminal N-terminal conserved region in TMUB1 and RNF170, which is required for this interaction. Three-dimensional modelling shows that this conserved motif binds the stomatin/prohibitin/flotillin/HflKC domain of two adjacent ERLIN subunits at different interfaces. Protein variants that preclude these interactions have been previously linked to hereditary spastic paraplegia. Using omics-based approaches in combination with phenotypic characterization of HeLa cells lacking both ERLINs, we demonstrate a role of ERLIN scaffolds in limiting cholesterol esterification, thereby favouring cholesterol transport from the ER to the Golgi apparatus and regulating Golgi morphology and the secretory pathway.
Topics: Humans; Membrane Proteins; Cholesterol; Ubiquitin-Protein Ligases; Endoplasmic Reticulum; HeLa Cells; Golgi Apparatus; Secretory Pathway; Protein Binding; Nerve Tissue Proteins
PubMed: 38782601
DOI: 10.26508/lsa.202402620 -
Biophysics Reports Apr 2024In animal cells, the Golgi apparatus serves as the central hub of the endomembrane secretory pathway. It is responsible for the processing, modification, and sorting of...
In animal cells, the Golgi apparatus serves as the central hub of the endomembrane secretory pathway. It is responsible for the processing, modification, and sorting of proteins and lipids. The unique stacking and ribbon-like architecture of the Golgi apparatus forms the foundation for its precise functionality. Under cellular stress or pathological conditions, the structure of the Golgi and its important glycosylation modification function may change. It is crucial to employ suitable methodologies to study the structure and function of the Golgi apparatus, particularly when assessing the involvement of a target protein in Golgi regulation. This article provides a comprehensive overview of the diverse microscopy techniques used to determine the specific location of the target protein within the Golgi apparatus. Additionally, it outlines methods for assessing changes in the Golgi structure and its glycosylation modification function following the knockout of the target gene.
PubMed: 38774352
DOI: 10.52601/bpr.2024.240008