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Cell Reports Jan 2024Fanconi anemia (FA) is characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. The central FA protein complex FANCI/FANCD2 (ID2) is...
Fanconi anemia (FA) is characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. The central FA protein complex FANCI/FANCD2 (ID2) is activated by monoubiquitination and recruits DNA repair proteins for interstrand crosslink (ICL) repair and replication fork protection. Defects in the FA pathway lead to R-loop accumulation, which contributes to genomic instability. Here, we report that the splicing factor SRSF1 and FANCD2 interact physically and act together to suppress R-loop formation via mRNA export regulation. We show that SRSF1 stimulates FANCD2 monoubiquitination in an RNA-dependent fashion. In turn, FANCD2 monoubiquitination proves crucial for the assembly of the SRSF1-NXF1 nuclear export complex and mRNA export. Importantly, several SRSF1 cancer-associated mutants fail to interact with FANCD2, leading to inefficient FANCD2 monoubiquitination, decreased mRNA export, and R-loop accumulation. We propose a model wherein SRSF1 and FANCD2 interaction links DNA damage response to the avoidance of pathogenic R-loops via regulation of mRNA export.
Topics: Humans; R-Loop Structures; Active Transport, Cell Nucleus; Fanconi Anemia; Fanconi Anemia Complementation Group Proteins; Fanconi Anemia Complementation Group D2 Protein; Ubiquitination; DNA Repair; Neoplasms; RNA, Messenger; DNA Damage; Serine-Arginine Splicing Factors
PubMed: 38165804
DOI: 10.1016/j.celrep.2023.113610 -
Cells Dec 2023Explaining changes at the gene level that occur during neurodegeneration in the CA3 area is crucial from the point of view of memory impairment and the development of...
Explaining changes at the gene level that occur during neurodegeneration in the CA3 area is crucial from the point of view of memory impairment and the development of post-ischemic dementia. An ischemic model of Alzheimer's disease was used to evaluate changes in the expression of genes related to amyloid transport in the CA3 region of the hippocampus after 10 min of brain ischemia with survival of 2, 7 and 30 days and 12, 18 and 24 months. The quantitative reverse transcriptase PCR assay revealed that the expression of the and genes involved in amyloid transport was dysregulated from 2 days to 24 months post-ischemia in the CA3 area of the hippocampus. gene expression 2 and 7 days after ischemia was below control values. However, its expression from day 30 to 24 months, survival after an ischemic episode was above control values. gene expression 2 days after ischemia was below control values, reaching a maximum increase 7 and 30 days post-ischemia. Then, after 12, 18 and 24 months, it was again below the control values. The data indicate that in the CA3 area of the hippocampus, an episode of brain ischemia causes the increased expression of the gene for 7-30 days during the acute phase and that of from 1 to 24 months after ischemia during the chronic stage. In other words, in the early post-ischemic stage, the expression of the gene that transport amyloid to the brain increases (7-30 days). Conversely, in the late post-ischemic stage, amyloid scavenging/cleaning gene activity increases, reducing and/or preventing further neuronal damage or facilitating the healing of damaged sites. This is how the new phenomenon of pyramidal neuronal damage in the CA3 area after ischemia is defined. In summary, post-ischemic modification of the and genes is useful in the study of the ischemic pathways and molecular factors involved in the development of Alzheimer's disease.
Topics: Humans; Alzheimer Disease; Amyloidogenic Proteins; Brain Ischemia; Hippocampus; Ischemia; Low Density Lipoprotein Receptor-Related Protein-1; tau Proteins; Protein Transport
PubMed: 38067191
DOI: 10.3390/cells12232763 -
FEBS Open Bio Dec 2023Epidemiological studies have shown that abnormalities of glucose metabolism are involved in leucine-rich repeat kinase 2 (LRRK2)-associated Parkinson's disease (PD)....
Epidemiological studies have shown that abnormalities of glucose metabolism are involved in leucine-rich repeat kinase 2 (LRRK2)-associated Parkinson's disease (PD). However, the physiological significance of this association is unclear. In the present study, we investigated the effect of LRRK2 on high-fat diet (HFD)-induced glucose intolerance using Lrrk2-knockout (KO) mice. We found for the first time that HFD-fed KO mice display improved glucose tolerance compared with their wild-type (WT) counterparts. In addition, high serum insulin and leptin, as well as low serum adiponectin resulting from HFD in WT mice were improved in KO mice. Using western blotting, we found that Lrrk2 is highly expressed in adipose tissues compared with other insulin-related tissues that are thought to be important in glucose tolerance, including skeletal muscle, liver, and pancreas. Lrrk2 expression and phosphorylation of its kinase substrates Rab8a and Rab10 were significantly elevated after HFD treatment in WT mice. In cell culture experiments, treatment with a LRRK2 kinase inhibitor stimulated insulin-dependent membrane translocation of glucose transporter 4 (Glut4) and glucose uptake in mouse 3T3-L1 adipocytes. We conclude that increased LRRK2 kinase activity in adipose tissue exacerbates glucose tolerance by suppressing Rab8- and Rab10-mediated GLUT4 membrane translocation.
Topics: Animals; Mice; Adipocytes; Adipose Tissue; Biological Transport; Glucose; Insulin; Mice, Knockout
PubMed: 37845194
DOI: 10.1002/2211-5463.13717 -
Journal of Cell Science Oct 2023The malaria-causing parasite, Plasmodium falciparum completely remodels its host red blood cell (RBC) through the export of several hundred parasite proteins, including...
The malaria-causing parasite, Plasmodium falciparum completely remodels its host red blood cell (RBC) through the export of several hundred parasite proteins, including transmembrane proteins, across multiple membranes to the RBC. However, the process by which these exported membrane proteins are extracted from the parasite plasma membrane for export remains unknown. To address this question, we fused the exported membrane protein, skeleton binding protein 1 (SBP1), with TurboID, a rapid, efficient and promiscuous biotin ligase (SBP1TbID). Using time-resolved proximity biotinylation and label-free quantitative proteomics, we identified two groups of SBP1TbID interactors - early interactors (pre-export) and late interactors (post-export). Notably, two promising membrane-associated proteins were identified as pre-export interactors, one of which possesses a predicted translocon domain, that could facilitate the export of membrane proteins. Further investigation using conditional mutants of these candidate proteins showed that these proteins were essential for asexual growth and localize to the host-parasite interface during early stages of the intraerythrocytic cycle. These data suggest that they might play a role in ushering membrane proteins from the parasite plasma membrane for export to the host RBC.
Topics: Animals; Humans; Biotinylation; Erythrocytes; Malaria; Plasmodium falciparum; Porins; Protein Transport; Protozoan Proteins
PubMed: 37772444
DOI: 10.1242/jcs.260506 -
Cells Dec 2023Cellular nucleocytoplasmic trafficking is mediated by the importin family of nuclear transport proteins. The well-characterized importin alpha (IMPA) and importin beta... (Review)
Review
Cellular nucleocytoplasmic trafficking is mediated by the importin family of nuclear transport proteins. The well-characterized importin alpha (IMPA) and importin beta (IMPB) nuclear import pathway plays a crucial role in the innate immune response to viral infection by mediating the nuclear import of transcription factors such as IRF3, NFκB, and STAT1. The nuclear transport of these transcription factors ultimately leads to the upregulation of a wide range of antiviral genes, including IFN and IFN-stimulated genes (ISGs). To replicate efficiently in cells, viruses have developed mechanisms to block these signaling pathways. One strategy to evade host innate immune responses involves blocking the nuclear import of host antiviral transcription factors. By binding IMPA proteins, these viral proteins prevent the nuclear transport of key transcription factors and suppress the induction of antiviral gene expression. In this review, we describe examples of proteins encoded by viruses from several different families that utilize such a competitive inhibition strategy to suppress the induction of antiviral gene expression.
Topics: Active Transport, Cell Nucleus; alpha Karyopherins; Immunity, Innate; Antiviral Agents; Organophosphorus Compounds
PubMed: 38201275
DOI: 10.3390/cells13010071 -
The Journal of Cell Biology Jul 2023ADP-ribosylation factor (ARF) GTPases are major regulators of cellular membrane homeostasis. High sequence similarity and multiple, possibly redundant functions of the...
ADP-ribosylation factor (ARF) GTPases are major regulators of cellular membrane homeostasis. High sequence similarity and multiple, possibly redundant functions of the five human ARFs make investigating their function a challenging task. To shed light on the roles of the different Golgi-localized ARF members in membrane trafficking, we generated CRISPR-Cas9 knockins (KIs) of type I (ARF1 and ARF3) and type II ARFs (ARF4 and ARF5) and mapped their nanoscale localization with stimulated emission depletion (STED) super-resolution microscopy. We find ARF1, ARF4, and ARF5 on segregated nanodomains on the cis-Golgi and ER-Golgi intermediate compartments (ERGIC), revealing distinct roles in COPI recruitment on early secretory membranes. Interestingly, ARF4 and ARF5 define Golgi-tethered ERGIC elements decorated by COPI and devoid of ARF1. Differential localization of ARF1 and ARF4 on peripheral ERGICs suggests the presence of functionally different classes of intermediate compartments that could regulate bi-directional transport between the ER and the Golgi. Furthermore, ARF1 and ARF3 localize to segregated nanodomains on the trans-Golgi network (TGN) and are found on TGN-derived post-Golgi tubules, strengthening the idea of distinct roles in post-Golgi sorting. This work provides the first map of the nanoscale organization of human ARF GTPases on cellular membranes and sets the stage to dissect their numerous cellular roles.
Topics: Humans; ADP-Ribosylation Factors; Golgi Apparatus; trans-Golgi Network; Protein Transport; Biological Transport; ADP-Ribosylation Factor 1
PubMed: 37102998
DOI: 10.1083/jcb.202205107 -
The Journal of Biological Chemistry Nov 2023T-cell receptor stimulation triggers cytosolic Ca signaling by inositol-1,4,5-trisphosphate (IP)-mediated Ca release from the endoplasmic reticulum (ER) and Ca entry...
T-cell receptor stimulation triggers cytosolic Ca signaling by inositol-1,4,5-trisphosphate (IP)-mediated Ca release from the endoplasmic reticulum (ER) and Ca entry through Ca release-activated Ca (CRAC) channels gated by ER-located stromal-interacting molecules (STIM1/2). Physiologically, cytosolic Ca signaling manifests as regenerative Ca oscillations, which are critical for nuclear factor of activated T-cells-mediated transcription. In most cells, Ca oscillations are thought to originate from IP receptor-mediated Ca release, with CRAC channels indirectly sustaining them through ER refilling. Here, experimental and computational evidence support a multiple-oscillator mechanism in Jurkat T-cells whereby both IP receptor and CRAC channel activities oscillate and directly fuel antigen-evoked Ca oscillations, with the CRAC channel being the major contributor. KO of either STIM1 or STIM2 significantly reduces CRAC channel activity. As such, STIM1 and STIM2 synergize for optimal Ca oscillations and activation of nuclear factor of activated T-cells 1 and are essential for ER refilling. The loss of both STIM proteins abrogates CRAC channel activity, drastically reduces ER Ca content, severely hampers cell proliferation and enhances cell death. These results clarify the mechanism and the contribution of STIM proteins to Ca oscillations in T-cells.
Topics: Humans; Calcium; Calcium Release Activated Calcium Channels; Calcium Signaling; Jurkat Cells; Stromal Interaction Molecule 1; Stromal Interaction Molecule 2; Gene Knockout Techniques; Models, Biological; Protein Isoforms; Protein Transport; Cell Proliferation; Cell Survival
PubMed: 37778728
DOI: 10.1016/j.jbc.2023.105310 -
Nature Communications Nov 2023Ligand-induced epidermal growth factor receptor (EGFR) endocytosis followed by endosomal EGFR signaling and lysosomal degradation plays important roles in controlling...
Ligand-induced epidermal growth factor receptor (EGFR) endocytosis followed by endosomal EGFR signaling and lysosomal degradation plays important roles in controlling multiple biological processes. ADP-ribosylation factor (Arf)-like protein 4 A (Arl4A) functions at the plasma membrane to mediate cytoskeletal remodeling and cell migration, whereas its localization at endosomal compartments remains functionally unknown. Here, we report that Arl4A attenuates EGFR degradation by binding to the endosomal sorting complex required for transport (ESCRT)-II component VPS36. Arl4A plays a role in prolonging the duration of EGFR ubiquitinylation and deterring endocytosed EGFR transport from endosomes to lysosomes under EGF stimulation. Mechanistically, the Arl4A-VPS36 direct interaction stabilizes VPS36 and ESCRT-III association, affecting subsequent recruitment of deubiquitinating-enzyme USP8 by CHMP2A. Impaired Arl4A-VPS36 interaction enhances EGFR degradation and clearance of EGFR ubiquitinylation. Together, we discover that Arl4A negatively regulates EGFR degradation by binding to VPS36 and attenuating ESCRT-mediated late endosomal EGFR sorting.
Topics: Humans; Endosomal Sorting Complexes Required for Transport; HeLa Cells; ErbB Receptors; Endosomes; Signal Transduction; Protein Transport
PubMed: 38030597
DOI: 10.1038/s41467-023-42979-9 -
Plant Physiology Jul 2023Thylakoid membrane protein quality control (PQC), which requires the coordination of membrane protein translocation and degradation of unassembled proteins, determines...
Thylakoid membrane protein quality control (PQC), which requires the coordination of membrane protein translocation and degradation of unassembled proteins, determines chloroplast development during de-etiolation. Despite numerous efforts, the regulation of this process in land plants is largely unknown. Here, we report the isolation and characterization of pale green Arabidopsis4 (pga4) mutants in Arabidopsis (Arabidopsis thaliana) with defects in chloroplast development during de-etiolation. Map-based cloning and complementation assays confirmed that PGA4 encodes the chloroplast Signal Recognition Particle 54 kDa (cpSRP54) protein. A heterogeneous Light-Harvesting Chlorophyll a/b Binding-Green Fluorescent Protein (LhcB2-GFP) fusion protein was generated as an indicative reporter for cpSRP54-mediated thylakoid translocation. LhcB2-GFP was dysfunctional and degraded to a short-form dLhcB2-GFP during de-etiolation through an N-terminal degradation initiated on thylakoid membranes. Further biochemical and genetic evidence demonstrated that the degradation of LhcB2-GFP to dLhcB2-GFP was disrupted in pga4 and yellow variegated2 (var2) mutants caused by mutations in the Filamentous Temperature-Sensitive H2 (VAR2/AtFtsH2) subunit of thylakoid FtsH. The yeast two-hybrid assay showed that the N-terminus of LhcB2-GFP interacts with the protease domain of VAR2/AtFtsH2. Moreover, the over-accumulated LhcB2-GFP in pga4 and var2 formed protein aggregates, which were insoluble in mild nonionic detergents. Genetically, cpSRP54 is a suppressor locus for the leaf variegation phenotype of var2. Together, these results demonstrate the coordination of cpSRP54 and thylakoid FtsH in maintaining thylakoid membrane PQC during the assembly of photosynthetic complexes and provide a trackable substrate and product for monitoring cpSRP54-dependent protein translocation and FtsH-dependent protein degradation.
Topics: Arabidopsis; Thylakoids; Arabidopsis Proteins; Peptide Hydrolases; Proteostasis; Chlorophyll A; Chloroplasts; Mutation; Metalloendopeptidases
PubMed: 36994815
DOI: 10.1093/plphys/kiad199 -
Nature Cell Biology Feb 2024The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth, metabolism and autophagy. Multiple pathways modulate mTORC1 in response to...
The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth, metabolism and autophagy. Multiple pathways modulate mTORC1 in response to nutrients. Here we describe that nucleus-cytoplasmic shuttling of p300/EP300 regulates mTORC1 activity in response to amino acid or glucose levels. Depletion of these nutrients causes cytoplasm-to-nucleus relocalization of p300 that decreases acetylation of the mTORC1 component raptor, thereby reducing mTORC1 activity and activating autophagy. This is mediated by AMP-activated protein kinase-dependent phosphorylation of p300 at serine 89. Nutrient addition to starved cells results in protein phosphatase 2A-dependent dephosphorylation of nuclear p300, enabling its CRM1-dependent export to the cytoplasm to mediate mTORC1 reactivation. p300 shuttling regulates mTORC1 in most cell types and occurs in response to altered nutrients in diverse mouse tissues. Interestingly, p300 cytoplasm-nucleus shuttling is altered in cells from patients with Hutchinson-Gilford progeria syndrome. p300 mislocalization by the disease-causing protein, progerin, activates mTORC1 and inhibits autophagy, phenotypes that are normalized by modulating p300 shuttling. These results reveal how nutrients regulate mTORC1, a cytoplasmic complex, by shuttling its positive regulator p300 in and out of the nucleus, and how this pathway is misregulated in Hutchinson-Gilford progeria syndrome, causing mTORC1 hyperactivation and defective autophagy.
Topics: Humans; Mice; Animals; Mechanistic Target of Rapamycin Complex 1; Progeria; Active Transport, Cell Nucleus; Regulatory-Associated Protein of mTOR; Amino Acids; Lamin Type A
PubMed: 38267537
DOI: 10.1038/s41556-023-01338-y