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Science Advances May 2024Mutations in the gene encoding lamins A/C cause an array of tissue-selective diseases, with the heart being the most commonly affected organ. Despite progress in...
Mutations in the gene encoding lamins A/C cause an array of tissue-selective diseases, with the heart being the most commonly affected organ. Despite progress in understanding the perturbations emanating from mutations, an integrative understanding of the pathogenesis underlying cardiac dysfunction remains elusive. Using a novel conditional deletion model capable of translatome profiling, we observed that cardiomyocyte-specific deletion in adult mice led to rapid cardiomyopathy with pathological remodeling. Before cardiac dysfunction, -deleted cardiomyocytes displayed nuclear abnormalities, Golgi dilation/fragmentation, and CREB3-mediated stress activation. Translatome profiling identified MED25 activation, a transcriptional cofactor that regulates Golgi stress. Autophagy is disrupted in the hearts of these mice, which can be recapitulated by disrupting the Golgi. Systemic administration of modulators of autophagy or ER stress significantly delayed cardiac dysfunction and prolonged survival. These studies support a hypothesis wherein stress responses emanating from the perinuclear space contribute to the cardiomyopathy development.
Topics: Animals; Lamin Type A; Mice; Nuclear Envelope; Cardiomyopathies; Myocytes, Cardiac; Autophagy; Stress, Physiological; Disease Models, Animal; Endoplasmic Reticulum Stress; Golgi Apparatus; Mice, Knockout
PubMed: 38718107
DOI: 10.1126/sciadv.adh0798 -
BioRxiv : the Preprint Server For... Jul 2023TorsinA is an atypical ATPase that lacks intrinsic activity unless it is bound to its activators lamina-associated polypeptide 1 (LAP1) in the perinuclear space or...
TorsinA is an atypical ATPase that lacks intrinsic activity unless it is bound to its activators lamina-associated polypeptide 1 (LAP1) in the perinuclear space or luminal domain-like LAP1 (LULL1) throughout the endoplasmic reticulum. However, the interaction of torsinA with LAP1 and LULL1 has not yet been shown to modulate a defined physiological process in mammals . We previously demonstrated that depletion of torsinA from mouse hepatocytes leads to reduced liver triglyceride secretion and marked steatosis, whereas depletion of LAP1 had more modest similar effects. We now show that depletion of LULL1 alone does not significantly decrease liver triglyceride secretion or cause steatosis. However, simultaneous depletion of both LAP1 and LULL1 from hepatocytes leads to defective triglyceride secretion and marked steatosis similar to that observed with depletion of torsinA. Our results demonstrate that torsinA and its activators dynamically regulate a physiological process in mammals .
PubMed: 37547008
DOI: 10.1101/2023.06.21.545957 -
Molecular Biology of the Cell May 2022How nuclear pore complexes (NPCs) assemble in the intact nuclear envelope (NE) is only rudimentarily understood. Nucleoporins (Nups) accumulate at the inner nuclear...
How nuclear pore complexes (NPCs) assemble in the intact nuclear envelope (NE) is only rudimentarily understood. Nucleoporins (Nups) accumulate at the inner nuclear membrane (INM) and deform this membrane toward the outer nuclear membrane (ONM), and eventually INM and ONM fuse by an unclear mechanism. In budding yeast, the integral membrane protein Brl1 that transiently associates with NPC assembly intermediates is involved in INM/ONM fusion during NPC assembly but leaving the molecular mechanism open. AlphaFold predictions indicate that Brl1-like proteins carry as common motifs an α-helix with amphipathic features (AαH) and a disulfide-stabilized, anti-parallel helix bundle (DAH) in the perinuclear space. Mutants with defective AαH (, ) impair the essential function of . Overexpression of promotes the formation of INM and ONM enclosed petal-like structures that carry Nups at their base, suggesting that they are derived from an NPC assembly attempt with failed INM/ONM fusion. Accordingly, expression triggers mislocalization of Nup159 and Nup42 and to a lesser extent Nsp1, which localize on the cytoplasmic face of the NPC. The DAH also contributes to the function of Brl1, and AαH has functions independent of DAH. We propose that AαH and DAH in Brl1 promote INM/ONM fusion during NPC assembly.
Topics: Membrane Proteins; Nuclear Envelope; Nuclear Pore; Nuclear Pore Complex Proteins; Protein Conformation, alpha-Helical; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 35293775
DOI: 10.1091/mbc.E21-12-0616 -
Open Biology Nov 2021The integral membrane protein Apq12 is an important nuclear envelope (NE)/endoplasmic reticulum (ER) modulator that cooperates with the nuclear pore complex (NPC)...
The integral membrane protein Apq12 is an important nuclear envelope (NE)/endoplasmic reticulum (ER) modulator that cooperates with the nuclear pore complex (NPC) biogenesis factors Brl1 and Brr6. How Apq12 executes these functions is unknown. Here, we identified a short amphipathic α-helix (AH) in Apq12 that links the two transmembrane domains in the perinuclear space and has liposome-binding properties. Cells expressing an () version in which AH is disrupted show NPC biogenesis and NE integrity defects, without impacting Apq12-ah topology or NE/ER localization. Overexpression of but not triggers striking over-proliferation of the outer nuclear membrane (ONM)/ER and promotes accumulation of phosphatidic acid (PA) at the NE. Apq12 and Apq12-ah both associate with NPC biogenesis intermediates and removal of AH increases both Brl1 levels and the interaction between Brl1 and Brr6. We conclude that the short amphipathic α-helix of Apq12 regulates the function of Brl1 and Brr6 and promotes PA accumulation at the NE possibly during NPC biogenesis.
Topics: Membrane Proteins; Mutation; Nuclear Envelope; Nuclear Pore; Phosphatidic Acids; Protein Conformation, alpha-Helical; Protein Domains; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 34814743
DOI: 10.1098/rsob.210250 -
Archives of Razi Institute Oct 2021The soft and delicate tissue of the brain, which is the center of our coordination, is protected by its surrounding layers. The disruption of these layers results in...
The soft and delicate tissue of the brain, which is the center of our coordination, is protected by its surrounding layers. The disruption of these layers results in complicated situations and serious health problems. The brain has three protective layers of bone or skull tissue, the blood tissue layer, and finally the meningeal layer. The layer of blood tissue contains the blood vessels that are located between the skull and the meningeal membranes. If germs or foreign matter enter the fluid through the blood vessels under any circumstances and cause infection, the bones that protect the meninges will break and cause tissue damage. The present study aimed to assess the histological and immunohistochemical characteristics of the brain of rats that underwent induced acute purulent pneumococcal meningitis after antibiotic therapy with Ceftriaxone. A number of 20 white adult male Wistar rats were assigned to three groups. The first group (n=5) regarded as the control were injected with a saline solution into the subarachnoid space in an equivalent amount. The second and third groups of rats (n=5 and 10, respectively) were infected with acute purulent meningitis by the injection of 10 μl of Streptococcus pneumoniae (S. pneumonia) suspension into the subarachnoid space of the brain using a 23-G needle. The various areas of the brains of rats after meningitis induced by S. pneumoniae were examined after the treatment with Ceftriaxone. The S. pneumoniae culture was injected into the subarachnoid space in the area of the rhomboid fossa. Treatment started 18 h after the injection. On day 10, a repeated puncture was performed with the analysis of cerebrospinal fluid in order to confirm the absence of meningitis; thereafter, the animals were taken out of the experiment. No signs of meningitis were found on histological examination. Mild perivascular and pericellular focal edema were revealed with signs of overload of the lymphatic system in the brain and focal ischemic changes in neurons. The investigation of expression with caspase-3 revealed a positive reaction of individual neurons. A positive reaction with antibodies to NeuN and Doublecortin was detected in most neurons; moreover, Glial fibrillary acidic protein (GFAP)-positive astrocytes and their processes were visualized in all layers of the brain substance. The reaction with neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP-2), CD 31, and CD 34 was negative. Typical structure and pictures pointed to an intact brain and purulent meningitis in the first and second groups. The microscopic image and the changes revealed during immunohistochemistry by dual corticosteroid antibodies and neuronal nuclear protein were characterized by predominantly cytoplasmic and perinuclear reactions, respectively. Some neurons are positive for caspase-3 and are related to changes in the characteristic of premature aging.
Topics: Animals; Male; Rats; Anti-Bacterial Agents; Brain; Ceftriaxone; Meningitis, Pneumococcal; Rats, Wistar
PubMed: 35096336
DOI: 10.22092/ari.2021.355885.1733 -
Journal of Virology Dec 2019The herpesvirus nuclear egress complex (NEC) is composed of two viral proteins. They play key roles in mediating the translocation of capsids from the nucleus to the...
The herpesvirus nuclear egress complex (NEC) is composed of two viral proteins. They play key roles in mediating the translocation of capsids from the nucleus to the cytoplasm by facilitating the budding of capsids into the perinuclear space (PNS). The NEC of alphaherpesvirus can induce the formation of virion-like vesicles from the nuclear membrane in the absence of other viral proteins. However, whether the NEC of gammaherpesvirus harbors the ability to do so in mammalian cells remains to be determined. In this study, we first constructed open reading frame 67 (ORF67)-null and ORF69-null mutants of murine gammaherpesvirus 68 (MHV-68) and demonstrated that both ORF67 and ORF69 play critical roles in nuclear egress and hence viral lytic replication. Biochemical and bioimaging analyses showed that ORF67 and ORF69 interacted with each other and were sufficient to induce the formation of virion-like vesicles from the nuclear membrane in mammalian cells. Thus, we designated ORF67 and ORF69 components of MHV-68 NEC. Furthermore, we identified amino acids critical for mediating the interaction between ORF67 and ORF69 through homology modeling and verified their function in nuclear egress, providing insights into the molecular basis of NEC formation in gammaherpesviruses. Increasing amounts of knowledge indicate that the nuclear egress complex (NEC) is critical for the nuclear egress of herpesvirus capsids, which can be viewed as a vesicle-mediated transport pathway through the nuclear membrane. In this study, we identified open reading frame 67 (ORF67) and ORF69 as components of the NEC in murine gammaherpesvirus 68 (MHV-68) and demonstrated that they efficiently induce virion-like vesicles from the nuclear membrane in mammalian cells. This is the first time that the NEC of a gammaherpesvirus has been found to demonstrate such an essential characteristic. In addition, we identified amino acids critical for mediating the interaction between ORF67 and ORF69 as well as nuclear egress. Notably, these amino acids are conserved in Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), providing a structural basis to design antigammaherpesvirus drugs.
Topics: Active Transport, Cell Nucleus; Animals; Capsid; Cell Nucleus; Cytoplasm; DNA, Viral; Gammaherpesvirinae; HEK293 Cells; HeLa Cells; Herpesviridae; Herpesviridae Infections; Herpesvirus 4, Human; Herpesvirus 8, Human; Humans; Loss of Function Mutation; Mice; Nuclear Envelope; Open Reading Frames; Viral Envelope Proteins; Viral Proteins; Virion; Virus Replication
PubMed: 31554685
DOI: 10.1128/JVI.01422-19 -
Biochimica Et Biophysica Acta.... Jan 2022Membraneless organelles have emerged during the evolution of eukaryotic cells as intracellular domains in which multiple proteins organize into complex structures to...
Membraneless organelles have emerged during the evolution of eukaryotic cells as intracellular domains in which multiple proteins organize into complex structures to perform specialized functions without the need of a lipid bilayer compartment. Here we describe the perinuclear space of eukaryotic cells as a highly organized network of cytoskeletal filaments that facilitates assembly of biomolecular condensates. Using bioinformatic analyses, we show that the perinuclear proteome is enriched in intrinsic disorder with several proteins predicted to undergo liquid-liquid phase separation. We also analyze immunofluorescence and transmission electron microscopy images showing the association between the nucleus and other organelles, such as mitochondria and lysosomes, or the labeling of specific proteins within the perinuclear region of cells. Altogether our data support the existence of a perinuclear dense sub-micron region formed by a well-organized three-dimensional network of structural and signaling proteins, including several proteins containing intrinsically disordered regions with phase behavior. This network of filamentous cytoskeletal proteins extends a few micrometers from the nucleus, contributes to local crowding, and organizes the movement of molecular complexes within the perinuclear space. Our findings take a key step towards understanding how membraneless regions within eukaryotic cells can serve as hubs for biomolecular condensates assembly, in particular the perinuclear space. Finally, evaluation of the disease context of the perinuclear proteins revealed that alterations in their expression can lead to several pathological conditions, and neurological disorders and cancer are among the most frequent.
Topics: Actin Cytoskeleton; Animals; Cells, Cultured; Chick Embryo; Intrinsically Disordered Proteins; Lysosomes; Microscopy, Electron, Transmission; Mitochondria; Nuclear Envelope; Proteome; Zebrafish
PubMed: 34655689
DOI: 10.1016/j.bbamcr.2021.119161 -
Genetics Jul 2021The chromatin landscape defines cellular identity in multicellular organisms with unique patterns of DNA accessibility and histone marks decorating the genome of each...
The chromatin landscape defines cellular identity in multicellular organisms with unique patterns of DNA accessibility and histone marks decorating the genome of each cell type. Thus, profiling the chromatin state of different cell types in an intact organism under disease or physiological conditions can provide insight into how chromatin regulates cell homeostasis in vivo. To overcome the many challenges associated with characterizing chromatin state in specific cell types, we developed an improved approach to isolate Drosophila melanogaster nuclei tagged with a GFPKASH protein. The perinuclear space-localized KASH domain anchors GFP to the outer nuclear membrane, and expression of UAS-GFPKASH can be controlled by tissue-specific Gal4 drivers. Using this protocol, we profiled chromatin accessibility using an improved version of Assay for Transposable Accessible Chromatin followed by sequencing (ATAC-seq), called Omni-ATAC. In addition, we examined the distribution of histone marks using Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and Cleavage Under Targets and Tagmentation (CUT&Tag) in adult photoreceptor neurons. We show that the chromatin landscape of photoreceptors reflects the transcriptional state of these cells, demonstrating the quality and reproducibility of our approach for profiling the transcriptome and epigenome of specific cell types in Drosophila.
Topics: Animals; Cell Nucleus; Chromatin; Chromatin Immunoprecipitation Sequencing; Drosophila melanogaster; Green Fluorescent Proteins; Histone Code; Organ Specificity
PubMed: 34022041
DOI: 10.1093/genetics/iyab079 -
Atg39 links and deforms the outer and inner nuclear membranes in selective autophagy of the nucleus.The Journal of Cell Biology Feb 2022In selective autophagy of the nucleus (hereafter nucleophagy), nucleus-derived double-membrane vesicles (NDVs) are formed, sequestered within autophagosomes, and...
In selective autophagy of the nucleus (hereafter nucleophagy), nucleus-derived double-membrane vesicles (NDVs) are formed, sequestered within autophagosomes, and delivered to lysosomes or vacuoles for degradation. In Saccharomyces cerevisiae, the nuclear envelope (NE) protein Atg39 acts as a nucleophagy receptor, which interacts with Atg8 to target NDVs to the forming autophagosomal membranes. In this study, we revealed that Atg39 is anchored to the outer nuclear membrane via its transmembrane domain and also associated with the inner nuclear membrane via membrane-binding amphipathic helices (APHs) in its perinuclear space region, thereby linking these membranes. We also revealed that autophagosome formation-coupled Atg39 crowding causes the NE to protrude toward the cytoplasm, and the tips of the protrusions are pinched off to generate NDVs. The APHs of Atg39 are crucial for Atg39 crowding in the NE and subsequent NE protrusion. These findings suggest that the nucleophagy receptor Atg39 plays pivotal roles in NE deformation during the generation of NDVs to be degraded by nucleophagy.
Topics: Autophagy; Autophagy-Related Proteins; Chromosomes, Fungal; Nuclear Envelope; Receptors, Cytoplasmic and Nuclear; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 35061008
DOI: 10.1083/jcb.202103178 -
Pharmaceuticals (Basel, Switzerland) Jun 2022Linearolactone (LL) is a -clerodane type diterpene that has been shown to exert giardicidal effects; however, its mechanism of action is unknown. This work analyzes the...
Linearolactone (LL) is a -clerodane type diterpene that has been shown to exert giardicidal effects; however, its mechanism of action is unknown. This work analyzes the cytotoxic effect of LL on trophozoites and identifies proteins that could be targeted by this active natural product. Increasing concentrations of LL and albendazole (ABZ) were used as test and reference drugs, respectively. Cell cycle progression, determination of reactive oxygen species (ROS) and apoptosis/necrosis events were evaluated by flow cytometry (FCM). Ultrastructural alterations were analyzed by transmission electron microscopy (TEM). Ligand-protein docking analyses were carried out using the LL structure raised from a drug library and the crystal structure of an aldose reductase homologue (GdAldRed) from . LL induced partial arrest at the S phase of trophozoite cell cycle without evidence of ROS production. LL induced pronecrotic death in addition to inducing ultrastructural alterations as changes in vacuole abundances, appearance of perinuclear and periplasmic spaces, and deposition of glycogen granules. On the other hand, the in silico study predicted that GdAldRed is a likely target of LL because it showed a favored change in Gibbs free energy for this complex.
PubMed: 35890108
DOI: 10.3390/ph15070809