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Cellular and Molecular Gastroenterology... 2024The functional maturation of the liver largely occurs after birth. In the early stages of life, the liver of a newborn encounters enormous high-fat metabolic stress...
BACKGROUND & AIMS
The functional maturation of the liver largely occurs after birth. In the early stages of life, the liver of a newborn encounters enormous high-fat metabolic stress caused by the consumption of breast milk. It is unclear how the maturing liver adapts to high lipid metabolism. Liver sinusoidal endothelial cells (LSECs) play a fundamental role in establishing liver vasculature and are decorated with many glycoproteins on their surface. The Slc35a1 gene encodes a cytidine-5'-monophosphate (CMP)-sialic acid transporter responsible for transporting CMP-sialic acids between the cytoplasm and the Golgi apparatus for protein sialylation. This study aimed to determine whether endothelial sialylation plays a role in hepatic vasculogenesis and functional maturation.
METHODS
Endothelial-specific Slc35a1 knockout mice were generated. Liver tissues were collected for histologic analysis, lipidomic profiling, RNA sequencing, confocal immunofluorescence, and immunoblot analyses.
RESULTS
Endothelial Slc35a1-deficient mice exhibited excessive neonatal hepatic lipid deposition, severe liver damage, and high mortality. Endothelial deletion of Slc35a1 led to sinusoidal capillarization and disrupted hepatic zonation. Mechanistically, vascular endothelial growth factor receptor 2 (VEGFR2) in LSECs was desialylated and VEGFR2 signaling was enhanced in Slc35a1-deficient mice. Inhibition of VEGFR2 signaling by SU5416 alleviated lipid deposition and restored hepatic vasculature in Slc35a1-deficient mice.
CONCLUSIONS
Our findings suggest that sialylation of LSECs is critical for maintaining hepatic vascular development and lipid homeostasis. Targeting VEGFR2 signaling may be a new strategy to prevent liver disorders associated with abnormal vasculature and lipid deposition.
Topics: Animals; Mice; Animals, Newborn; Endothelial Cells; Lipid Metabolism; Liver; Mice, Knockout; Nucleotide Transport Proteins; Vascular Endothelial Growth Factor Receptor-2
PubMed: 38467191
DOI: 10.1016/j.jcmgh.2024.03.002 -
ELife Mar 2024Secretory proteins are sorted at the -Golgi network (TGN) for export into specific transport carriers. However, the molecular players involved in this fundamental...
Secretory proteins are sorted at the -Golgi network (TGN) for export into specific transport carriers. However, the molecular players involved in this fundamental process remain largely elusive. Here, we identified the human transmembrane protein TGN46 as a receptor for the export of secretory cargo protein PAUF in CARTS - a class of protein kinase D-dependent TGN-to-plasma membrane carriers. We show that TGN46 is necessary for cargo sorting and loading into nascent carriers at the TGN. By combining quantitative fluorescence microscopy and mutagenesis approaches, we further discovered that the lumenal domain of TGN46 encodes for its cargo sorting function. In summary, our results define a cellular function of TGN46 in sorting secretory proteins for export from the TGN.
Topics: Humans; Membrane Proteins; Protein Transport; trans-Golgi Network
PubMed: 38466628
DOI: 10.7554/eLife.91708 -
BioRxiv : the Preprint Server For... Feb 2024Proteins carrying a signal peptide and/or a transmembrane domain enter the intracellular secretory pathway at the endoplasmic reticulum (ER) and are transported to the...
Proteins carrying a signal peptide and/or a transmembrane domain enter the intracellular secretory pathway at the endoplasmic reticulum (ER) and are transported to the Golgi apparatus via COPII vesicles or tubules. SAR1 initiates COPII coat assembly by recruiting other coat proteins to the ER membrane. Mammalian genomes encode two paralogs, and . While these paralogs exhibit ~90% amino acid sequence identity, it is unknown whether they perform distinct or overlapping functions in vivo. We now report that genetic inactivation of in mice results in lethality during mid-embryogenesis. We also confirm previous reports that complete deficiency of murine results in perinatal lethality. In contrast, we demonstrate that deletion of restricted to hepatocytes is compatible with survival, though resulting in hypocholesterolemia that can be rescued by adenovirus-mediated overexpression of either SAR1A or SAR1B. To further examine the in vivo function of these 2 paralogs, we genetically engineered mice with the coding sequence replacing that of at the endogenous locus. Mice homozygous for this allele survive to adulthood and are phenotypically normal, demonstrating complete or near-complete overlap in function between the two SAR1 protein paralogs in mice. These data also suggest upregulation of gene expression as a potential approach for the treatment of SAR1B deficiency (chylomicron retention disease) in humans.
PubMed: 38463989
DOI: 10.1101/2024.02.27.582310 -
Aging Mar 2024The Golgi apparatus (GA) is crucial for protein synthesis and modification, and regulates various cellular processes. Dysregulation of GA can lead to pathological...
BACKGROUND
The Golgi apparatus (GA) is crucial for protein synthesis and modification, and regulates various cellular processes. Dysregulation of GA can lead to pathological conditions like neoplastic growth. GA-related genes (GARGs) mutations are commonly found in cancer, contributing to tumor metastasis. However, the expression and prognostic significance of GARGs in osteosarcoma are yet to be understood.
METHODS
Gene expression and clinical data of osteosarcoma patients were obtained from the TARGET and GEO databases. A consensus clustering analysis identified distinct molecular subtypes based on GARGs. Discrepancies in biological processes and immunological features among the subtypes were explored using GSVA, ssGSEA, and Metascape analysis. A GARGs signature was constructed using Cox regression. The prognostic value of the GARGs signature in osteosarcoma was evaluated using Kaplan-Meier curves and a nomogram.
RESULTS
Two GARG subtypes were identified, with Cluster A showing better prognosis, immunogenicity, and immune cell infiltration than Cluster B. A novel risk model of 3 GARGs was established using the TARGET dataset and validated with independent datasets. High-risk patients had poorer overall survival, and the GARGs signature independently predicted osteosarcoma prognosis. Combining risk scores and clinical characteristics in a nomogram improved prediction performance. Additionally, we discovered Stanniocalcin-2 (STC2) as a significant prognostic gene highly expressed in osteosarcoma and potential disease biomarker.
CONCLUSIONS
Our study revealed that patients with osteosarcoma can be divided into two GARGs subgroups. Furthermore, we have developed a GARGs prognostic signature that can accurately forecast the prognosis of osteosarcoma patients.
Topics: Humans; Prognosis; Osteosarcoma; Nomograms; Golgi Apparatus; Bone Neoplasms
PubMed: 38460960
DOI: 10.18632/aging.205645 -
Nature Methods Apr 2024We developed a system for optogenetic release of single molecules in cells. We confined soluble and transmembrane proteins to the Golgi apparatus via a photocleavable...
We developed a system for optogenetic release of single molecules in cells. We confined soluble and transmembrane proteins to the Golgi apparatus via a photocleavable protein and released them by short pulses of light. Our method allows for a light dose-dependent delivery of functional proteins to the cytosol and plasma membrane in amounts compatible with single-molecule imaging, greatly simplifying access to single-molecule microscopy of any protein in live cells. We were able to reconstitute ion conductance by delivering BK and LRRC8/volume-regulated anion channels to the plasma membrane. Finally we were able to induce NF-kB signaling in T lymphoblasts stimulated by interleukin-1 by controlled release of a signaling protein that had been knocked out. We observed light-induced formation of functional inflammatory signaling complexes that triggered phosphorylation of the inhibitor of nuclear factor kappa-B kinase only in activated cells. We thus developed an optogenetic method for the reconstitution and investigation of cellular function at the single-molecule level.
Topics: Delayed-Action Preparations; Optogenetics; Signal Transduction; NF-kappa B; Phosphorylation
PubMed: 38459384
DOI: 10.1038/s41592-024-02204-x -
Physiological Reviews Jul 2024The endomembrane system consists of organellar membranes in the biosynthetic pathway [endoplasmic reticulum (ER), Golgi apparatus, and secretory vesicles] as well as... (Review)
Review
The endomembrane system consists of organellar membranes in the biosynthetic pathway [endoplasmic reticulum (ER), Golgi apparatus, and secretory vesicles] as well as those in the degradative pathway (early endosomes, macropinosomes, phagosomes, autophagosomes, late endosomes, and lysosomes). These endomembrane organelles/vesicles work together to synthesize, modify, package, transport, and degrade proteins, carbohydrates, and lipids, regulating the balance between cellular anabolism and catabolism. Large ion concentration gradients exist across endomembranes: Ca gradients for most endomembrane organelles and H gradients for the acidic compartments. Ion (Na, K, H, Ca, and Cl) channels on the organellar membranes control ion flux in response to cellular cues, allowing rapid informational exchange between the cytosol and organelle lumen. Recent advances in organelle proteomics, organellar electrophysiology, and luminal and juxtaorganellar ion imaging have led to molecular identification and functional characterization of about two dozen endomembrane ion channels. For example, whereas IP3R1-3 channels mediate Ca release from the ER in response to neurotransmitter and hormone stimulation, TRPML1-3 and TMEM175 channels mediate lysosomal Ca and H release, respectively, in response to nutritional and trafficking cues. This review aims to summarize the current understanding of these endomembrane channels, with a focus on their subcellular localizations, ion permeation properties, gating mechanisms, cell biological functions, and disease relevance.
Topics: Humans; Animals; Ion Channels; Intracellular Membranes; Organelles
PubMed: 38451235
DOI: 10.1152/physrev.00025.2023 -
Journal of Translational Medicine Mar 2024In diabetic retinopathy (DR), hypoxia-inducible factor (HIF-1α) induces oxidative stress by upregulating glycolysis. This process leads to neurodegeneration,...
BACKGROUND
In diabetic retinopathy (DR), hypoxia-inducible factor (HIF-1α) induces oxidative stress by upregulating glycolysis. This process leads to neurodegeneration, particularly photoreceptor cell damage, which further contributes to retinal microvascular deterioration. Further, the regulation of Wnt-inhibitory factor 1 (WIF1), a secreted Wnt signaling antagonist, has not been fully characterized in neurodegenerative eye diseases. We aimed to explore the impact of WIF1 on photoreceptor function within the context of DR.
METHOD
Twelve-week-old C57BL/KsJ-db/db mice were intravitreally injected with WIF1 overexpression lentivirus. After 4 weeks, optical coherence tomography (OCT), transmission electron microscopy (TEM), H&E staining, and electroretinography (ERG) were used to assess the retinal tissue and function. The potential mechanism of action of WIF1 in photoreceptor cells was explored using single-cell RNA sequencing. Under high-glucose conditions, 661 W cells were used as an in vitro DR model. WIF1-mediated signaling pathway components were assessed using quantitative real-time PCR, immunostaining, and western blotting.
RESULT
Typical diabetic manifestations were observed in db/db mice. Notably, the expression of WIF1 was decreased at the mRNA and protein levels. These pathological manifestations and visual function improved after WIF1 overexpression in db/db mice. TEM demonstrated that WIF1 restored damaged mitochondria, the Golgi apparatus, and photoreceptor outer segments. Moreover, ERG indicated the recovery of a-wave potential amplitude. Single-cell RNA sequencing and in vitro experiments suggested that WIF1 overexpression prevented the expression of glycolytic enzymes and lactate production by inhibiting the canonical Wnt signaling pathway, HIF-1α, and Glut1, thereby reducing retinal and cellular reactive oxygen species levels and maintaining 661 W cell viability.
CONCLUSIONS
WIF1 exerts an inhibitory effect on the Wnt/β-catenin-HIF-1α-Glut1 glycolytic pathway, thereby alleviating oxidative stress levels and mitigating pathological structural characteristics in retinal photoreceptor cells. This mechanism helps preserve the function of photoreceptor cells in DR and indicates that WIF1 holds promise as a potential therapeutic candidate for DR and other neurodegenerative ocular disorders.
Topics: Animals; Mice; Diabetes Mellitus; Diabetic Retinopathy; Glucose Transporter Type 1; Mice, Inbred C57BL; Photoreceptor Cells; Retina
PubMed: 38448948
DOI: 10.1186/s12967-024-05046-5 -
Molecular Biology of the Cell May 2024Neurons are polarized cells that require accurate membrane trafficking to maintain distinct protein complements at dendritic and axonal membranes. The Kinesin-3 family...
Neurons are polarized cells that require accurate membrane trafficking to maintain distinct protein complements at dendritic and axonal membranes. The Kinesin-3 family members KIF13A and KIF13B are thought to mediate dendrite-selective transport, but the mechanism by which they are recruited to polarized vesicles and the differences in the specific trafficking role of each KIF13 have not been defined. We performed live-cell imaging in cultured hippocampal neurons and found that KIF13A is a dedicated dendrite-selective kinesin. KIF13B confers two different transport modes, dendrite- and axon-selective transport. Both KIF13s are maintained at the trans-Golgi network by interactions with the heterotetrameric adaptor protein complex AP-1. Interference with KIF13 binding to AP-1 resulted in disruptions to both dendrite- and axon-selective trafficking. We propose that AP-1 is the molecular link between the sorting of polarized cargoes into vesicles and the recruitment of kinesins that confer polarized transport.
Topics: Cells, Cultured; Golgi Apparatus; Kinesins; Neurons; Protein Transport; Adaptor Protein Complex 1; trans-Golgi Network
PubMed: 38446634
DOI: 10.1091/mbc.E23-10-0401 -
ELife Mar 2024Cell motility processes highly depend on the membrane distribution of Phosphoinositides, giving rise to cytoskeleton reshaping and membrane trafficking events. Membrane...
Cell motility processes highly depend on the membrane distribution of Phosphoinositides, giving rise to cytoskeleton reshaping and membrane trafficking events. Membrane contact sites serve as platforms for direct lipid exchange and calcium fluxes between two organelles. Here, we show that VAPA, an ER transmembrane contact site tether, plays a crucial role during cell motility. CaCo2 adenocarcinoma epithelial cells depleted for VAPA exhibit several collective and individual motility defects, disorganized actin cytoskeleton and altered protrusive activity. During migration, VAPA is required for the maintenance of PI(4)P and PI(4,5)P2 levels at the plasma membrane, but not for PI(4)P homeostasis in the Golgi and endosomal compartments. Importantly, we show that VAPA regulates the dynamics of focal adhesions (FA) through its MSP domain, is essential to stabilize and anchor ventral ER-PM contact sites to FA, and mediates microtubule-dependent FA disassembly. To conclude, our results reveal unknown functions for VAPA-mediated membrane contact sites during cell motility and provide a dynamic picture of ER-PM contact sites connection with FA mediated by VAPA.
Topics: Humans; Focal Adhesions; Caco-2 Cells; Golgi Apparatus; Actin Cytoskeleton; Cell Movement; Vesicular Transport Proteins
PubMed: 38446032
DOI: 10.7554/eLife.85962 -
MBio Apr 2024We compared the growth characteristics of a virulent strain (Sheila Smith) to an attenuated stain (Iowa) and a non-pathogenic species () in primary human dermal...
We compared the growth characteristics of a virulent strain (Sheila Smith) to an attenuated stain (Iowa) and a non-pathogenic species () in primary human dermal microvascular endothelial cells (HDMEC). All replicated in Vero cells, however, only the Sheila Smith strain productively replicated in HDMECs. The Iowa strain showed minimal replication over a 24-h period, while lost viability and induced lysis of the HDMECs via a rapid programmed cell death response. Both the virulent and attenuated strains, but not , induced an interferon-1 response, although the response was of lesser magnitude and delayed in the Sheila Smith strain. IFN-β secretion correlated with increased host cell lysis, and treatment with anti-IFNAR2 antibody decreased lysis from Iowa-infected but not Sheila Smith-infected cells. Both Sheila Smith- and Iowa-infected cells eventually lysed, although the response from Sheila Smith was delayed and showed characteristics of apoptosis. We, therefore, examined whether reconstitution of the Iowa strain with two recently described putative virulence determinants might enhance survival of Iowa within HDMECs. Reconstitution with RARP2, which is inhibitory to anterograde trafficking through the Golgi apparatus, reduced IFN-β secretion but had no effect on cell lysis. RapL, which proteolytically processes surface exposed autotransporters and enhances replication of Iowa in Guinea pigs, suppressed both IFN-β production and host cell lysis. These findings suggest distinct mechanisms by which virulent spotted fever group rickettsiae may enhance intracellular survival and replication.IMPORTANCEWe examined a naturally occurring non-pathogenic rickettsial species, , a laboratory-attenuated strain (Iowa), and a fully virulent strain (Sheila Smith) for growth in human dermal microvascular endothelial cells. The two avirulent strains replicated poorly or not at all. Only the virulent Sheila Smith strain replicated. IFN-β production correlated with the inhibition of Iowa. Reconstitution of Iowa with either of two recently described putative virulence determinants altered the IFN-β response. A rickettsial ankyrin repeat protein, RARP2, disrupts the Golgi network and inhibits IFN-β secretion. An autotransporter peptidase, RapL, restores proteolytic maturation of outer membrane autotransporters and diminishes the IFN-β response to enhance cell survival and permit replication of the recombinant strain. These studies point the way toward discovery of mechanisms for innate immune response avoidance by virulent rickettsia.
Topics: Animals; Guinea Pigs; Humans; Chlorocebus aethiops; Endothelial Cells; Rickettsia; Rickettsia rickettsii; Rocky Mountain Spotted Fever; Type V Secretion Systems; Vero Cells; Virulence; Virulence Factors; Interferon-beta
PubMed: 38445878
DOI: 10.1128/mbio.03450-23