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Nature Cancer Jan 2024In vitro-transcribed (IVT) mRNA has arisen as a rapid method for the production of nucleic acid drugs. Here, we have constructed an oncolytic IVT mRNA that utilizes...
In vitro-transcribed (IVT) mRNA has arisen as a rapid method for the production of nucleic acid drugs. Here, we have constructed an oncolytic IVT mRNA that utilizes human rhinovirus type 2 (HRV2) internal ribosomal entry sites (IRESs) to selectively trigger translation in cancer cells with high expression of EIF4G2 and PTBP1. The oncolytic effect was provided by a long hGSDMD-F1L mutant mRNA sequence with mitochondrial inner membrane cardiolipin targeting toxicity that triggers mitophagy. Utilizing the permuted intron-exon (PIE) splicing circularization strategy and lipid nanoparticle (LNP) encapsulation reduced immunogenicity of the mRNA and enabled delivery to eukaryotic cells in vivo. Engineered HRV2 IRESs-GSDMD-F1L circRNA-LNPs (GSDMD circRNA) successfully inhibited EIF4G2/PTBP1 pan-adenocarcinoma xenografts growth. Importantly, in a spontaneous tumor model with abnormal EIF4G2 and PTBP1 caused by KRAS G12D mutation, GSDMD circRNA significantly prevented the occurrence of pancreatic, lung and colon adenocarcinoma, improved the survival rate and induced persistent KRAS G12D tumor antigen-specific cytotoxic T lymphocyte responses.
Topics: Humans; RNA, Circular; Cardiolipins; Adenocarcinoma; Proto-Oncogene Proteins p21(ras); Colonic Neoplasms; RNA, Messenger; Eukaryotic Initiation Factor-4G; Heterogeneous-Nuclear Ribonucleoproteins; Polypyrimidine Tract-Binding Protein
PubMed: 37845485
DOI: 10.1038/s43018-023-00650-8 -
Molecular Cell Aug 2023Solute carrier spinster homolog 2 (SPNS2), one of only four known major facilitator superfamily (MFS) lysolipid transporters in humans, exports sphingosine-1-phosphate...
Solute carrier spinster homolog 2 (SPNS2), one of only four known major facilitator superfamily (MFS) lysolipid transporters in humans, exports sphingosine-1-phosphate (S1P) across cell membranes. Here, we explore the synergistic effects of lipid binding and conformational dynamics on SPNS2's transport mechanism. Using mass spectrometry, we discovered that SPNS2 interacts preferentially with PI(4,5)P. Together with functional studies and molecular dynamics (MD) simulations, we identified potential PI(4,5)P binding sites. Mutagenesis of proposed lipid binding sites and inhibition of PI(4,5)P synthesis reduce S1P transport, whereas the absence of the N terminus renders the transporter essentially inactive. Probing the conformational dynamics of SPNS2, we show how synergistic binding of PI(4,5)P and S1P facilitates transport, increases dynamics of the extracellular gate, and stabilizes the intracellular gate. Given that SPNS2 transports a key signaling lipid, our results have implications for therapeutic targeting and also illustrate a regulatory mechanism for MFS transporters.
Topics: Humans; Lysophospholipids; Sphingosine; Anion Transport Proteins
PubMed: 37499662
DOI: 10.1016/j.molcel.2023.06.033 -
Clinical Immunology (Orlando, Fla.) Oct 2023The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the persistent presence of antibodies directed against phospholipids and... (Review)
Review
The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the persistent presence of antibodies directed against phospholipids and phospholipid-binding proteins that are associated with thrombosis and pregnancy-related morbidity. The latter includes fetal deaths, premature birth and maternal complications. In the early 1990s, a distinct set of autoantibodies, termed collectively antiphospholipid antibodies (aPL), were identified as the causative agents of this disorder. Subsequently histological analyses of the placenta from APS pregnancies revealed various abnormalities, including inflammation at maternal-fetal interface and poor placentation manifested by reduced trophoblast invasion and limited uterine spiral artery remodeling. Further preclinical investigations identified the molecular targets of aPL and the downstream intracellular pathways of key placental cell types. While these discoveries suggest potential therapeutics for this disorder, definitive clinical trials have not been completed. This concise review focuses on the recent developments in the field of basic and translational research pursuing novel mechanisms underlying obstetric APS.
PubMed: 37625670
DOI: 10.1016/j.clim.2023.109745 -
Nature Structural & Molecular Biology Nov 2023The fusion of mononucleated myoblasts produces multinucleated muscle fibers leading to the formation of skeletal muscle. Myomaker, a skeletal muscle-specific membrane...
The fusion of mononucleated myoblasts produces multinucleated muscle fibers leading to the formation of skeletal muscle. Myomaker, a skeletal muscle-specific membrane protein, is essential for myoblast fusion. Here we report the cryo-EM structures of mouse Myomaker (mMymk) and Ciona robusta Myomaker (cMymk). Myomaker contains seven transmembrane helices (TMs) that adopt a G-protein-coupled receptor-like fold. TMs 2-4 form a dimeric interface, while TMs 3 and 5-7 create a lipid-binding site that holds the polar head of a phospholipid and allows the alkyl tails to insert into Myomaker. The similarity of cMymk and mMymk suggests a conserved Myomaker-mediated cell fusion mechanism across evolutionarily distant species. Functional analyses demonstrate the essentiality of the dimeric interface and the lipid-binding site for fusogenic activity, and heterologous cell-cell fusion assays show the importance of transcellular interactions of Myomaker protomers for myoblast fusion. Together, our findings provide structural and functional insights into the process of myoblast fusion.
Topics: Animals; Mice; Cryoelectron Microscopy; Cell Differentiation; Muscle, Skeletal; Myoblasts; Lipids; Muscle Development
PubMed: 37770716
DOI: 10.1038/s41594-023-01110-8 -
Science Signaling Aug 2023TPC2 is a pathophysiologically relevant lysosomal ion channel that is activated directly by the phosphoinositide PI(3,5)P and indirectly by the calcium ion...
TPC2 is a pathophysiologically relevant lysosomal ion channel that is activated directly by the phosphoinositide PI(3,5)P and indirectly by the calcium ion (Ca)-mobilizing molecule NAADP through accessory proteins that associate with the channel. TPC2 toggles between PI(3,5)P-induced, sodium ion (Na)-selective and NAADP-induced, Ca-permeable states in response to these cues. To address the molecular basis of polymodal gating and ion-selectivity switching, we investigated the mechanism by which NAADP and its synthetic functional agonist, TPC2-A1-N, induced Ca release through TPC2 in human cells. Whereas NAADP required the NAADP-binding proteins JPT2 and LSM12 to evoke endogenous calcium ion signals, TPC2-A1-N did not. Residues in TPC2 that bind to PI(3,5)P were required for channel activation by NAADP but not for activation by TPC2-A1-N. The cryptic voltage-sensing region of TPC2 was required for the actions of TPC2-A1-N and PI(3,5)P but not for those of NAADP. These data mechanistically distinguish natural and synthetic agonist action at TPC2 despite convergent effects on Ca permeability and delineate a route for pharmacologically correcting impaired NAADP-evoked Ca signals.
Topics: Humans; Calcium; Permeability; Cues; Phosphatidylinositols; Research Personnel
PubMed: 37607219
DOI: 10.1126/scisignal.adg0661 -
Biomolecules Oct 2023Metazoan cell nuclei contain non-membrane pools of the phosphoinositide lipid PI(4,5)P2 (PIP2), but how this hydrophobic lipid exists within the aqueous nucleoplasm...
Metazoan cell nuclei contain non-membrane pools of the phosphoinositide lipid PI(4,5)P2 (PIP2), but how this hydrophobic lipid exists within the aqueous nucleoplasm remains unclear. Steroidogenic Factor-1 (NR5A1, SF-1) is a nuclear receptor that binds PIP2 in vitro, and a co-crystal structure of the complex suggests the acyl chains of PIP2 are hidden in the hydrophobic core of the SF-1 protein while the PIP2 headgroup is solvent-exposed. This binding mode explains how SF-1 can solubilize nuclear PIP2; however, cellular evidence that SF-1 expression associates with nuclear PIP2 has been lacking. Here, we examined if tetracycline induction of SF-1 expression would associate with nuclear accumulation of PIP2, using antibodies directed against the PIP2 headgroup. Indeed, tetracycline induction of wild-type SF-1 induced a signal in the nucleus of HEK cells that cross-reacts with PIP2 antibodies, but did not cross-react with antibodies against the lower abundance phosphoinositide PI(3,4,5)P3 (PIP3). The nuclear PIP2 signal co-localized with FLAG-tagged SF-1 in the nuclear compartment. To determine if the nuclear PIP2 signal was dependent on the ability of SF-1 to bind PIP2, we examined a "pocket mutant" of SF-1 (A270W, L345F) shown to be deficient in phospholipid binding by mass spectrometry. Tetracycline induction of this pocket mutant SF-1 in HEK cells failed to induce a detectable PIP2 antibody cross-reactive signal, despite similar Tet-induced expression levels of the wild-type and pocket mutant SF-1 proteins in these cells. Together, these data are the first to suggest that expression of SF-1 induces a PIP2 antibody cross-reactive signal in the nucleus, consistent with X-ray crystallographic and biochemical evidence suggesting SF-1 binds PIP2 in human cells.
Topics: Animals; Humans; Cell Nucleus; Phosphatidylinositols; Receptors, Cytoplasmic and Nuclear; Tetracyclines; Steroidogenic Factor 1
PubMed: 37892191
DOI: 10.3390/biom13101509 -
Proteins Sep 2023The human genome codes for 12 annexins with highly homologous membrane-binding cores and unique amino termini, which endow each protein with its specific biological... (Review)
Review
The human genome codes for 12 annexins with highly homologous membrane-binding cores and unique amino termini, which endow each protein with its specific biological properties. Not unique to vertebrate biology, multiple annexin orthologs are present in almost all eukaryotes. Their ability to combine either dynamically or constitutively with membrane lipid bilayers is hypothetically the key property that has led to their retention and multiple adaptation in eukaryotic molecular cell biology. Annexin genes are differentially expressed in many cell types but their disparate functions are still being discovered after more than 40 years of international research. A picture is emerging from gene knock down and knock out studies of individual annexins that these are important supporters rather than critical players in organism development and normal cell and tissue function. However, they appear to be highly significant "early responders" toward challenges arising from cell and tissue abiotic or biotic stress. In humans, recent focus has been on involvement of the annexin family for its involvement in diverse pathologies, especially cancer. From what has become an exceedingly broad field of investigation, we have selected four annexins in particular: AnxA1, 2, 5, and 6. Present both within and external to cells, these annexins are currently under intensive investigation in translational research as biomarkers of cellular dysfunction and as potential therapeutic targets for inflammatory conditions, neoplasia, and tissue repair. Annexin expression and release in response to biotic stress appears to be a balancing act. Under- or over-expression in different circumstances appears to damage rather than restore a healthy homeostasis. This review reflects briefly on what is already known of the structures and molecular cell biology of these selected annexins and considers their actual and potential roles in human health and disease.
Topics: Humans; Annexin A1; Annexins; Eukaryota; Eukaryotic Cells; Lipid Bilayers
PubMed: 37218507
DOI: 10.1002/prot.26512 -
Frontiers in Immunology 2023Exosomes, bilaterally phospholipid-coated small vesicles, are produced and released by nearly all cells, which comprise diverse biological macromolecules, including... (Review)
Review
Exosomes, bilaterally phospholipid-coated small vesicles, are produced and released by nearly all cells, which comprise diverse biological macromolecules, including proteins, DNA, RNA, and others, that participate in the regulation of their biological functions. An increasing number of studies have revealed that the contents of exosomes, particularly microRNA(miRNA), play a significant role in the pathogenesis of various diseases, including autoimmune skin diseases. MiRNA is a class of single-stranded non-coding RNA molecules that possess approximately 22 nucleotides in length with the capability of binding to the untranslated as well as coding regions of target mRNA to regulate gene expression precisely at the post-transcriptional level. Various exosomal miRNAs have been found to be significantly expressed in some autoimmune skin diseases and involved in the pathogenesis of conditions via regulating the secretion of crucial pathogenic cytokines and the direction of immune cell differentiation. Thus, exosomal miRNAs might be promising biomarkers for monitoring disease progression, relapse and reflection to treatment based on their functions and changes. This review summarized the current studies on exosomal miRNAs in several common autoimmune skin diseases, aiming to dissect the underlying mechanism from a new perspective, seek novel biomarkers for disease monitoring and lay the foundation for developing innovative target therapy in the future.
Topics: Humans; MicroRNAs; Exosomes; Biomarkers; Autoimmune Diseases
PubMed: 38106405
DOI: 10.3389/fimmu.2023.1307455 -
Nature Communications Aug 2023The dual functions of TMEM16F as Ca-activated ion channel and lipid scramblase raise intriguing questions regarding their molecular basis. Intrigued by the ability of...
The dual functions of TMEM16F as Ca-activated ion channel and lipid scramblase raise intriguing questions regarding their molecular basis. Intrigued by the ability of the FDA-approved drug niclosamide to inhibit TMEM16F-dependent syncytia formation induced by SARS-CoV-2, we examined cryo-EM structures of TMEM16F with or without bound niclosamide or 1PBC, a known blocker of TMEM16A Ca-activated Cl channel. Here, we report evidence for a lipid scrambling pathway along a groove harboring a lipid trail outside the ion permeation pore. This groove contains the binding pocket for niclosamide and 1PBC. Mutations of two residues in this groove specifically affect lipid scrambling. Whereas mutations of some residues in the binding pocket of niclosamide and 1PBC reduce their inhibition of TMEM16F-mediated Ca influx and PS exposure, other mutations preferentially affect the ability of niclosamide and/or 1PBC to inhibit TMEM16F-mediated PS exposure, providing further support for separate pathways for ion permeation and lipid scrambling.
Topics: Humans; Anoctamins; Calcium; Calcium Channels; Niclosamide; COVID-19; SARS-CoV-2; Lipids; Phospholipid Transfer Proteins
PubMed: 37573365
DOI: 10.1038/s41467-023-40410-x -
Nature Communications Nov 2023Asymmetric distribution of phospholipids in eukaryotic membranes is essential for cell integrity, signaling pathways, and vesicular trafficking. P4-ATPases, also known...
Asymmetric distribution of phospholipids in eukaryotic membranes is essential for cell integrity, signaling pathways, and vesicular trafficking. P4-ATPases, also known as flippases, participate in creating and maintaining this asymmetry through active transport of phospholipids from the exoplasmic to the cytosolic leaflet. Here, we present a total of nine cryo-electron microscopy structures of the human flippase ATP8B1-CDC50A complex at 2.4 to 3.1 Å overall resolution, along with functional and computational studies, addressing the autophosphorylation steps from ATP, substrate recognition and occlusion, as well as a phosphoinositide binding site. We find that the P4-ATPase transport site is occupied by water upon phosphorylation from ATP. Additionally, we identify two different autoinhibited states, a closed and an outward-open conformation. Furthermore, we identify and characterize the PI(3,4,5)P binding site of ATP8B1 in an electropositive pocket between transmembrane segments 5, 7, 8, and 10. Our study also highlights the structural basis of a broad lipid specificity of ATP8B1 and adds phosphatidylinositol as a transport substrate for ATP8B1. We report a critical role of the sn-2 ester bond of glycerophospholipids in substrate recognition by ATP8B1 through conserved S403. These findings provide fundamental insights into ATP8B1 catalytic cycle and regulation, and substrate recognition in P4-ATPases.
Topics: Humans; Adenosine Triphosphatases; Substrate Specificity; Cryoelectron Microscopy; Phospholipid Transfer Proteins; Phospholipids; Adenosine Triphosphate; Cell Membrane
PubMed: 37980352
DOI: 10.1038/s41467-023-42828-9