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PLoS Pathogens Apr 2024African swine fever (ASF) is an acute, hemorrhagic, and severe infectious disease caused by the ASF virus (ASFV). ASFV has evolved multiple strategies to escape host...
African swine fever (ASF) is an acute, hemorrhagic, and severe infectious disease caused by the ASF virus (ASFV). ASFV has evolved multiple strategies to escape host antiviral immune responses. Here, we reported that ASFV pB318L, a trans-geranylgeranyl-diphosphate synthase, reduced the expression of type I interferon (IFN-I) and IFN-stimulated genes (ISGs). Mechanically, pB318L not only interacted with STING to reduce the translocation of STING from the endoplasmic reticulum to the Golgi apparatus but also interacted with IFN receptors to reduce the interaction of IFNAR1/TYK2 and IFNAR2/JAK1. Of note, ASFV with interruption of B318L gene (ASFV-intB318L) infected PAMs produces more IFN-I and ISGs than that in PAMs infected with its parental ASFV HLJ/18 at the late stage of infection. Consistently, the pathogenicity of ASFV-intB318L is attenuated in piglets compared with its parental virus. Taken together, our data reveal that B318L gene may partially affect ASFV pathogenicity by reducing the production of IFN-I and ISGs. This study provides a clue to design antiviral agents or live attenuated vaccines to prevent and control ASF.
Topics: Animals; Swine; African Swine Fever Virus; African Swine Fever; Farnesyltranstransferase; Viral Proteins; Nucleotidyltransferases; Interferon Type I; Signal Transduction
PubMed: 38620034
DOI: 10.1371/journal.ppat.1012136 -
International Journal of Molecular... Apr 2024The Golgi apparatus, long recognized for its roles in protein processing and vesicular trafficking, has recently been identified as a crucial contributor to innate... (Review)
Review
The Golgi apparatus, long recognized for its roles in protein processing and vesicular trafficking, has recently been identified as a crucial contributor to innate immune signaling pathways. This review discusses our expanding understanding of the Golgi apparatus's involvement in initiating and activating these pathways. It highlights the significance of membrane connections between the Golgi and other organelles, such as the endoplasmic reticulum, mitochondria, endosomes, and autophagosomes. These connections are vital for the efficient transmission of innate immune signals and the activation of effector responses. Furthermore, the article delves into the Golgi apparatus's roles in key immune pathways, including the inflammasome-mediated activation of caspase-1, the -STING pathway, and TLR/RLR signaling. Overall, this review aims to provide insights into the multifunctional nature of the Golgi apparatus and its impact on innate immunity.
Topics: Golgi Apparatus; Immunity, Innate; Inflammasomes; Autophagosomes; Caspase 1
PubMed: 38612929
DOI: 10.3390/ijms25074120 -
Cell Stress & Chaperones Jun 2024Anhydrobiotic species can survive virtually complete water loss by entering a reversible ametabolic glassy state that may persist for years in ambient conditions. The...
Anhydrobiotic species can survive virtually complete water loss by entering a reversible ametabolic glassy state that may persist for years in ambient conditions. The Pv11 cell line was derived from the egg mass of the anhydrobiotic midge, Polypedilum vanderplanki, and is currently the only available anhydrobiotic cell line. Our results demonstrate that the necessary preconditioning for Pv11 cells to enter anhydrobiosis causes autophagy and reduces mitochondrial respiration by over 70%. We speculate that reorganizing cellular bioenergetics to create and conserve energy stores may be valuable to successfully recover after rehydration. Furthermore, mitochondria in preconditioned cells lose their membrane potential during desiccation but rapidly restore it within 30 min upon rehydration, demonstrating that the inner mitochondrial membrane integrity is well-preserved. Strikingly, the nucleolus remains visible immediately upon rehydration in preconditioned cells while absent in control cells. In contrast, a preconditioning-induced membraneless organelle reformed after rehydration, demonstrating that membraneless organelles in Pv11 cells can be either stabilized or recovered. Staining the endoplasmic reticulum and the Golgi apparatus revealed that these organelles fragment during preconditioning. We hypothesize that this process reduces sheering stress caused by rapid changes in cellular volume during desiccation and rehydration. Additionally, preconditioning was found to cause the filamentous-actin (F-actin) network to disassemble significantly and reduce the fusion of adjacent plasma membranes. This study offers several exciting avenues for future studies in the animal model and Pv11 cell line that will further our understanding of anhydrobiosis and may lead to advancements in storing sensitive biologics at ambient temperatures for months or years.
Topics: Animals; Cell Line; Desiccation; Mitochondria; Organelles; Autophagy; Endoplasmic Reticulum; Golgi Apparatus
PubMed: 38608858
DOI: 10.1016/j.cstres.2024.04.002 -
Nature Communications Apr 2024Autophagy is a conserved, catabolic process essential for maintaining cellular homeostasis. Malfunctional autophagy contributes to neurodevelopmental and...
Autophagy is a conserved, catabolic process essential for maintaining cellular homeostasis. Malfunctional autophagy contributes to neurodevelopmental and neurodegenerative diseases. However, the exact role and targets of autophagy in human neurons remain elusive. Here we report a systematic investigation of neuronal autophagy targets through integrated proteomics. Deep proteomic profiling of multiple autophagy-deficient lines of human induced neurons, mouse brains, and brain LC3-interactome reveals roles of neuronal autophagy in targeting proteins of multiple cellular organelles/pathways, including endoplasmic reticulum (ER), mitochondria, endosome, Golgi apparatus, synaptic vesicle (SV) for degradation. By combining phosphoproteomics and functional analysis in human and mouse neurons, we uncovered a function of neuronal autophagy in controlling cAMP-PKA and c-FOS-mediated neuronal activity through selective degradation of the protein kinase A - cAMP-binding regulatory (R)-subunit I (PKA-RI) complex. Lack of AKAP11 causes accumulation of the PKA-RI complex in the soma and neurites, demonstrating a constant clearance of PKA-RI complex through AKAP11-mediated degradation in neurons. Our study thus reveals the landscape of autophagy degradation in human neurons and identifies a physiological function of autophagy in controlling homeostasis of PKA-RI complex and specific PKA activity in neurons.
Topics: Mice; Animals; Humans; Proteomics; Neurons; Cyclic AMP-Dependent Protein Kinases; Autophagy; Homeostasis
PubMed: 38600097
DOI: 10.1038/s41467-024-47440-z -
Molecular & Cellular Proteomics : MCP May 2024Glycoproteins located on the cell surface play a pivotal role in nearly every extracellular activity. N-glycosylation is one of the most common and important protein...
Glycoproteins located on the cell surface play a pivotal role in nearly every extracellular activity. N-glycosylation is one of the most common and important protein modifications in eukaryotic cells, and it often regulates protein folding and trafficking. Glycosylation of cell-surface proteins undergoes meticulous regulation by various enzymes in the endoplasmic reticulum (ER) and the Golgi, ensuring their proper folding and trafficking to the cell surface. However, the impacts of protein N-glycosylation, N-glycan maturity, and protein folding status on the trafficking of cell-surface glycoproteins remain to be explored. In this work, we comprehensively and site-specifically studied the trafficking of cell-surface glycoproteins in human cells. Integrating metabolic labeling, bioorthogonal chemistry, and multiplexed proteomics, we investigated 706 N-glycosylation sites on 396 cell-surface glycoproteins in monocytes, either by inhibiting protein N-glycosylation, disturbing N-glycan maturation, or perturbing protein folding in the ER. The current results reveal their distinct impacts on the trafficking of surface glycoproteins. The inhibition of protein N-glycosylation dramatically suppresses the trafficking of many cell-surface glycoproteins. The N-glycan immaturity has more substantial effects on proteins with high N-glycosylation site densities, while the perturbation of protein folding in the ER exerts a more pronounced impact on surface glycoproteins with larger sizes. Furthermore, for N-glycosylated proteins, their trafficking to the cell surface is related to the secondary structures and adjacent amino acid residues of glycosylation sites. Systematic analysis of surface glycoprotein trafficking advances our understanding of the mechanisms underlying protein secretion and surface presentation.
Topics: Humans; Glycosylation; Protein Transport; Endoplasmic Reticulum; Polysaccharides; Glycoproteins; Cell Membrane; Protein Folding; Proteomics; Membrane Glycoproteins; Golgi Apparatus; Protein Processing, Post-Translational
PubMed: 38593903
DOI: 10.1016/j.mcpro.2024.100761 -
Cellular and Molecular Life Sciences :... Apr 2024Kinesin family member 3A (KIF3A) is a microtubule-oriented motor protein that belongs to the kinesin-2 family for regulating intracellular transport and microtubule...
Kinesin family member 3A (KIF3A) is a microtubule-oriented motor protein that belongs to the kinesin-2 family for regulating intracellular transport and microtubule movement. In this study, we characterized the critical roles of KIF3A during mouse oocyte meiosis. We found that KIF3A associated with microtubules during meiosis and depletion of KIF3A resulted in oocyte maturation defects. LC-MS data indicated that KIF3A associated with cell cycle regulation, cytoskeleton, mitochondrial function and intracellular transport-related molecules. Depletion of KIF3A activated the spindle assembly checkpoint, leading to metaphase I arrest of the first meiosis. In addition, KIF3A depletion caused aberrant spindle pole organization based on its association with KIFC1 to regulate expression and polar localization of NuMA and γ-tubulin; and KIF3A knockdown also reduced microtubule stability due to the altered microtubule deacetylation by histone deacetylase 6 (HDAC6). Exogenous Kif3a mRNA supplementation rescued the maturation defects caused by KIF3A depletion. Moreover, KIF3A was also essential for the distribution and function of mitochondria, Golgi apparatus and endoplasmic reticulum in oocytes. Conditional knockout of epithelial splicing regulatory protein 1 (ESRP1) disrupted the expression and localization of KIF3A in oocytes. Overall, our results suggest that KIF3A regulates cell cycle progression, spindle assembly and organelle distribution during mouse oocyte meiosis.
Topics: Animals; Mice; Biological Transport; Kinesins; Meiosis; Metaphase; Oocytes
PubMed: 38587639
DOI: 10.1007/s00018-024-05213-3 -
Cell Reports Apr 2024Nucleotide oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome hyperactivation contributes to many human chronic inflammatory diseases, and...
Nucleotide oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome hyperactivation contributes to many human chronic inflammatory diseases, and understanding how NLRP3 inflammasome is regulated can provide strategies to treat inflammatory diseases. Here, we demonstrate that NLRP3 Cys126 is palmitoylated by zinc finger DHHC-type palmitoyl transferase 7 (ZDHHC7), which is critical for NLRP3-mediated inflammasome activation. Perturbing NLRP3 Cys126 palmitoylation by ZDHHC7 knockout, pharmacological inhibition, or modification site mutation diminishes NLRP3 activation in macrophages. Furthermore, Cys126 palmitoylation is vital for inflammasome activation in vivo. Mechanistically, ZDHHC7-mediated NLRP3 Cys126 palmitoylation promotes resting NLRP3 localizing on the trans-Golgi network (TGN) and activated NLRP3 on the dispersed TGN, which is indispensable for recruitment and oligomerization of the adaptor ASC (apoptosis-associated speck-like protein containing a CARD). The activation of NLRP3 by ZDHHC7 is different from the termination effect mediated by ZDHHC12, highlighting versatile regulatory roles of S-palmitoylation. Our study identifies an important regulatory mechanism of NLRP3 activation that suggests targeting ZDHHC7 or the NLRP3 Cys126 residue as a potential therapeutic strategy to treat NLRP3-related human disorders.
Topics: Inflammasomes; NLR Family, Pyrin Domain-Containing 3 Protein; Lipoylation; Acyltransferases; Humans; Animals; Cysteine; Mice; HEK293 Cells; Mice, Inbred C57BL; trans-Golgi Network; Macrophages; Acetyltransferases
PubMed: 38583156
DOI: 10.1016/j.celrep.2024.114070 -
Acta Neuropathologica Apr 2024Transcription factor EB (TFEB) is a master regulator of genes involved in the maintenance of autophagic and lysosomal homeostasis, processes which have been implicated...
Transcription factor EB (TFEB) is a master regulator of genes involved in the maintenance of autophagic and lysosomal homeostasis, processes which have been implicated in the pathogenesis of GBA-related and sporadic Parkinson's disease (PD), and dementia with Lewy bodies (DLB). TFEB activation results in its translocation from the cytosol to the nucleus. Here, we investigated TFEB subcellular localization and its relation to intracellular alpha-synuclein (aSyn) accumulation in post-mortem human brain of individuals with either incidental Lewy body disease (iLBD), GBA-related PD/DLB (GBA-PD/DLB) or sporadic PD/DLB (sPD/DLB), compared to control subjects. We analyzed nigral dopaminergic neurons using high-resolution confocal and stimulated emission depletion (STED) microscopy and semi-quantitatively scored the TFEB subcellular localization patterns. We observed reduced nuclear TFEB immunoreactivity in PD/DLB patients compared to controls, both in sporadic and GBA-related cases, as well as in iLBD cases. Nuclear depletion of TFEB was more pronounced in neurons with Ser129-phosphorylated (pSer129) aSyn accumulation in all groups. Importantly, we observed previously-unidentified TFEB-immunopositive perinuclear clusters in human dopaminergic neurons, which localized at the Golgi apparatus. These TFEB clusters were more frequently observed and more severe in iLBD, sPD/DLB and GBA-PD/DLB compared to controls, particularly in pSer129 aSyn-positive neurons, but also in neurons lacking detectable aSyn accumulation. In aSyn-negative cells, cytoplasmic TFEB clusters were more frequently observed in GBA-PD/DLB and iLBD patients, and correlated with reduced GBA enzymatic activity as well as increased Braak LB stage. Altered TFEB distribution was accompanied by a reduction in overall mRNA expression levels of selected TFEB-regulated genes, indicating a possible early dysfunction of lysosomal regulation. Overall, we observed cytoplasmic TFEB retention and accumulation at the Golgi in cells without apparent pSer129 aSyn accumulation in iLBD and PD/DLB patients. This suggests potential TFEB impairment at the early stages of cellular disease and underscores TFEB as a promising therapeutic target for synucleinopathies.
Topics: Humans; alpha-Synuclein; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Brain; Dopaminergic Neurons; Lewy Bodies; Lewy Body Disease
PubMed: 38581586
DOI: 10.1007/s00401-024-02707-z -
Proceedings of the National Academy of... Apr 2024Adjacent plant cells are connected by specialized cell wall regions, called middle lamellae, which influence critical agricultural characteristics, including fruit...
Adjacent plant cells are connected by specialized cell wall regions, called middle lamellae, which influence critical agricultural characteristics, including fruit ripening and organ abscission. Middle lamellae are enriched in pectin polysaccharides, specifically homogalacturonan (HG). Here, we identify a plant-specific DUF1068 protein, called NKS1/ELMO4, that is required for middle lamellae integrity and cell adhesion. NKS1 localizes to the Golgi apparatus and loss of NKS1 results in changes to Golgi structure and function. The mutants also display HG deficient phenotypes, including reduced seedling growth, changes to cell wall composition, and tissue integrity defects. These phenotypes are comparable to and mutants, which are defective in HG biosynthesis. Notably, genetic interactions indicate that NKS1 and the QUAs work in a common pathway. Protein interaction analyses and modeling corroborate that they work together in a stable protein complex with other pectin-related proteins. We propose that NKS1 is an integral part of a large pectin synthesis protein complex and that proper function of this complex is important to support Golgi structure and function.
Topics: Arabidopsis; Arabidopsis Proteins; Cell Adhesion; Pectins; Golgi Apparatus; Cell Wall
PubMed: 38579009
DOI: 10.1073/pnas.2321759121 -
The Journal of Cell Biology Jul 2024The AP-1 adaptor complex is found in all eukaryotes, but it has been implicated in different pathways in different organisms. To look directly at AP-1 function, we...
The AP-1 adaptor complex is found in all eukaryotes, but it has been implicated in different pathways in different organisms. To look directly at AP-1 function, we generated stably transduced HeLa cells coexpressing tagged AP-1 and various tagged membrane proteins. Live cell imaging showed that AP-1 is recruited onto tubular carriers trafficking from the Golgi apparatus to the plasma membrane, as well as onto transferrin-containing early/recycling endosomes. Analysis of single AP-1 vesicles showed that they are a heterogeneous population, which starts to sequester cargo 30 min after exit from the ER. Vesicle capture showed that AP-1 vesicles contain transmembrane proteins found at the TGN and early/recycling endosomes, as well as lysosomal hydrolases, but very little of the anterograde adaptor GGA2. Together, our results support a model in which AP-1 retrieves proteins from post-Golgi compartments back to the TGN, analogous to COPI's role in the early secretory pathway. We propose that this is the function of AP-1 in all eukaryotes.
Topics: Humans; Adaptor Proteins, Vesicular Transport; Cell Membrane; Endosomes; Golgi Apparatus; HeLa Cells; Membrane Proteins; Protein Transport; trans-Golgi Network; Transcription Factor AP-1
PubMed: 38578286
DOI: 10.1083/jcb.202310071