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International Journal of Molecular... Jun 2024Arc (also known as Arg3.1) is an activity-dependent immediate early gene product enriched in neuronal dendrites. Arc plays essential roles in long-term potentiation,...
Arc (also known as Arg3.1) is an activity-dependent immediate early gene product enriched in neuronal dendrites. Arc plays essential roles in long-term potentiation, long-term depression, and synaptic scaling. Although its mechanisms of action in these forms of synaptic plasticity are not completely well established, the activities of Arc include the remodeling of the actin cytoskeleton, the facilitation of AMPA receptor (AMPAR) endocytosis, and the regulation of the transcription of AMPAR subunits. In addition, Arc has sequence and structural similarity to retroviral Gag proteins and self-associates into virus-like particles that encapsulate mRNA and perhaps other cargo for intercellular transport. Each of these activities is likely to be influenced by Arc's reversible self-association into multiple oligomeric species. Here, we used mass photometry to show that Arc exists predominantly as monomers, dimers, and trimers at approximately 20 nM concentration in vitro. Fluorescence fluctuation spectroscopy revealed that Arc is almost exclusively present as low-order (monomer to tetramer) oligomers in the cytoplasm of living cells, over a 200 nM to 5 μM concentration range. We also confirmed that an α-helical segment in the N-terminal domain contains essential determinants of Arc's self-association.
Topics: Protein Multimerization; Humans; Cytoskeletal Proteins; Nerve Tissue Proteins; Animals
PubMed: 38928159
DOI: 10.3390/ijms25126454 -
Genes May 2024Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma. The oncogene product Tax of HTLV-I is thought to play crucial roles...
Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma. The oncogene product Tax of HTLV-I is thought to play crucial roles in leukemogenesis by promoting proliferation of the virus-infected cells through activation of growth-promoting genes. These genes code for growth factors and their receptors, cytokines, cell adhesion molecules, growth signal transducers, transcription factors and cell cycle regulators. We show here that Tax activates the gene coding for coactivator-associated arginine methyltransferase 1 (CARM1), which epigenetically enhances gene expression through methylation of histones. Tax activated the gene and increased protein expression, not only in human T-cell lines but also in normal peripheral blood lymphocytes (PHA-PBLs). Tax increased R17-methylated histone H3 on the target gene , concomitant with increased expression of CARM1. Short hairpin RNA (shRNA)-mediated knockdown of CARM1 decreased Tax-mediated induction of and gene expression, reduced E2F activation and inhibited cell cycle progression. Tax acted via response elements in intron 1 of the gene, through the NF-κB pathway. These results suggest that Tax-mediated activation of the gene contributes to leukemogenic target-gene expression and cell cycle progression, identifying the first epigenetic target gene for Tax-mediated trans-activation in cell growth promotion.
Topics: Humans; Protein-Arginine N-Methyltransferases; Gene Products, tax; Human T-lymphotropic virus 1; Cyclin D2; Transcriptional Activation; Interleukin-2 Receptor alpha Subunit; NF-kappa B; Histones; Epigenesis, Genetic; Jurkat Cells
PubMed: 38927636
DOI: 10.3390/genes15060698 -
Genes May 2024A 23-month-old neutered male dog of unknown ancestry presented with a history of progressive neurological signs that included anxiety, cognitive impairment, tremors,...
A 23-month-old neutered male dog of unknown ancestry presented with a history of progressive neurological signs that included anxiety, cognitive impairment, tremors, seizure activity, ataxia, and pronounced visual impairment. The clinical signs were accompanied by global brain atrophy. Due to progression in the severity of disease signs, the dog was euthanized at 26 months of age. An examination of the tissues collected at necropsy revealed dramatic intracellular accumulations of autofluorescent inclusions in the brain, retina, and cardiac muscle. The inclusions were immunopositive for subunit c of mitochondrial ATP synthase, and their ultrastructural appearances were similar to those of lysosomal storage bodies that accumulate in some neuronal ceroid lipofuscinosis (NCL) diseases. The dog also exhibited widespread neuroinflammation. Based on these findings, the dog was deemed likely to have suffered from a form of NCL. A whole genome sequence analysis of the proband's DNA revealed a homozygous C to T substitution that altered the intron 3-exon 4 splice site of . Other mutations in cause NCL diseases in humans and animals, including dogs. The CLN6 protein was undetectable with immunolabeling in the tissues of the proband. Based on the clinical history, fluorescence and electron-microscopy, immunohistochemistry, and molecular genetic findings, the disorder in this dog was classified as an NCL resulting from the absence of the CLN6 protein. Screening the dog's genome for a panel of breed-specific polymorphisms indicated that its ancestry included numerous breeds, with no single breed predominating. This suggests that the disease variant is likely to be present in other mixed-breed dogs and at least some ancestral breeds, although it is likely to be rare since other cases have not been reported to date.
Topics: Neuronal Ceroid-Lipofuscinoses; Animals; Dogs; Male; Dog Diseases; RNA Splice Sites; Membrane Proteins; Mitochondrial Proton-Translocating ATPases; Brain; Mutation
PubMed: 38927597
DOI: 10.3390/genes15060661 -
Biomedicines Jun 2024The complex regulation of traction forces (TF) produced during cellular migration remains poorly understood. We have previously found that calpain 4 (Capn4), the small...
The complex regulation of traction forces (TF) produced during cellular migration remains poorly understood. We have previously found that calpain 4 (Capn4), the small non-catalytic subunit of the calpain 1 and 2 proteases, regulates the production of TF independent of the proteolytic activity of the larger subunits. Capn4 was later found to facilitate tyrosine phosphorylation and secretion of the lectin-binding protein galectin-3 (Gal3). In this study, recombinant Gal3 (rGal3) was added to the media-enhanced TF generated by mouse embryonic fibroblasts (MEFs). Extracellular Gal3 also rescued defects in the distribution, morphology, and adhesive strength of focal adhesions present in MEF cells. Surprisingly, extracellular Gal3 does not influence mechanosensing. c-Abl kinase was found to affect Gal3 secretion and the production of TF through phosphorylation of Y107 on Gal3. Our study also suggests that Gal3-mediated regulation of TF occurs through signaling pathways triggered by β1 integrin but not by focal adhesion kinase (FAK) Y397 autophosphorylation. Our findings provide insights into the signaling mechanism by which Capn4 and secreted Gal3 regulate cell migration through the modulation of TF distinctly independent from a mechanosensing mechanism.
PubMed: 38927454
DOI: 10.3390/biomedicines12061247 -
Biomolecules Jun 2024Antithrombin (AT) is a critical regulator of the coagulation cascade by inhibiting multiple coagulation factors including thrombin and FXa. Binding of heparinoids to...
Molecular Mechanisms of the Impaired Heparin Pentasaccharide Interactions in 10 Antithrombin Heparin Binding Site Mutants Revealed by Enhanced Sampling Molecular Dynamics.
Antithrombin (AT) is a critical regulator of the coagulation cascade by inhibiting multiple coagulation factors including thrombin and FXa. Binding of heparinoids to this serpin enhances the inhibition considerably. Mutations located in the heparin binding site of AT result in thrombophilia in affected individuals. Our aim was to study 10 antithrombin mutations known to affect their heparin binding in a heparin pentasaccharide bound state using two molecular dynamics (MD) based methods providing enhanced sampling, GaMD and LiGaMD2. The latter provides an additional boost to the ligand and the most important binding site residues. From our GaMD simulations we were able to identify four variants (three affecting amino acid Arg47 and one affecting Lys114) that have a particularly large effect on binding. The additional acceleration provided by LiGaMD2 allowed us to study the consequences of several other mutants including those affecting Arg13 and Arg129. We were able to identify several conformational types by cluster analysis. Analysis of the simulation trajectories revealed the causes of the impaired pentasaccharide binding including pentasaccharide subunit conformational changes and altered allosteric pathways in the AT protein. Our results provide insights into the effects of AT mutations interfering with heparin binding at an atomic level and can facilitate the design or interpretation of in vitro experiments.
Topics: Heparin; Molecular Dynamics Simulation; Binding Sites; Humans; Antithrombins; Mutation; Protein Binding; Oligosaccharides
PubMed: 38927061
DOI: 10.3390/biom14060657 -
Biomolecules May 2024The exosome multiprotein complex plays a critical role in RNA processing and degradation. This system governs the regulation of mRNA quality, degradation in the...
The exosome multiprotein complex plays a critical role in RNA processing and degradation. This system governs the regulation of mRNA quality, degradation in the cytoplasm, the processing of short noncoding RNA, and the breakdown of RNA fragments. We determined two crystal structures of exosome components from (): one with a resolution of 2.3 Å that reveals the central components (Rrp41 and Rrp42), and another with a resolution of 3.5 Å that displays the whole exosome (Rrp41, Rrp42, and Rrp4). The fundamental exosome structure revealed the presence of a heterodimeric complex consisting of Rrp41 and Rrp42. The structure comprises nine subunits, with Rrp41 and Rrp42 arranged in a circular configuration, while Rrp4 is located at the apex. The RNA degradation capabilities of the Rrp4:41:42 complex were verified by RNA degradation assays, consistent with prior findings in other archaeal exosomes. The resemblance between archaeal exosomes and bacterial PNPase suggests a common mechanism for RNA degradation. Despite sharing comparable topologies, the surface charge distributions of Rrp4 and other archaea structures are surprisingly distinct. Different RNA breakdown substrates may be responsible for this variation. These newfound structural findings enhance our comprehension of RNA processing and degradation in biological systems.
Topics: Thermoplasma; Exosomes; Crystallography, X-Ray; Archaeal Proteins; Models, Molecular; Protein Subunits; Exosome Multienzyme Ribonuclease Complex; RNA Stability
PubMed: 38927025
DOI: 10.3390/biom14060621 -
Zhongguo Shi Yan Xue Ye Xue Za Zhi Jun 2024To analyze the factors affecting overall survival (OS) of adult patients with core-binding factor acute myeloid leukemia (CBF-AML) and establish a prediction model.
OBJECTIVE
To analyze the factors affecting overall survival (OS) of adult patients with core-binding factor acute myeloid leukemia (CBF-AML) and establish a prediction model.
METHODS
A total of 216 newly diagnosed patients with CBF-AML in the First Affiliated Hospital of Zhengzhou University from May 2015 to July 2021 were retrospectively analyzed. The 216 CBF-AML patients were divided into the training and the validation cohort at 7∶3 ratio. The Cox regression model was used to analyze the clinical factors affecting OS. Stepwise regression was used to establish the optimal model and the nomogram. Receiver operating characteristic (ROC) curve, calibration curve and decision curve analysis (DCA) were used to evaluate the model performance.
RESULTS
Age(≥55 years old), peripheral blood blast(≥80%), fusion gene (), mutations were identified as independent adverse factors for OS. The area under the ROC curve at 3-year was 0.772 and 0.722 in the training cohort and validation cohort, respectively. The predicted value of the calibration curve is in good agreement with the measured value. DCA shows that this model performs better than a single factor.
CONCLUSION
This prediction model is simple and feasible, and can effectively predict the OS of CBF-AML, and provide a basis for treatment decision.
Topics: Humans; Leukemia, Myeloid, Acute; Prognosis; Retrospective Studies; Middle Aged; Female; Male; Mutation; ROC Curve; Core Binding Factors; Nomograms; Adult; RUNX1 Translocation Partner 1 Protein; Proto-Oncogene Proteins c-kit; Proportional Hazards Models; Oncogene Proteins, Fusion; Core Binding Factor Alpha 2 Subunit
PubMed: 38926955
DOI: 10.19746/j.cnki.issn.1009-2137.2024.03.007 -
Nature Jun 2024Transposases drive chromosomal rearrangements and the dissemination of drug-resistance genes and toxins. Although some transposases act alone, many rely on dedicated...
Transposases drive chromosomal rearrangements and the dissemination of drug-resistance genes and toxins. Although some transposases act alone, many rely on dedicated AAA+ ATPase subunits that regulate site selectivity and catalytic function through poorly understood mechanisms. Using IS21 as a model transposase system, we show how an ATPase regulator uses nucleotide-controlled assembly and DNA deformation to enable structure-based site selectivity, transposase recruitment, and activation and integration. Solution and cryogenic electron microscopy studies show that the IstB ATPase self-assembles into an autoinhibited pentamer of dimers that tightly curves target DNA into a half-coil. Two of these decamers dimerize, which stabilizes the target nucleic acid into a kinked S-shaped configuration that engages the IstA transposase at the interface between the two IstB oligomers to form an approximately 1 MDa transpososome complex. Specific interactions stimulate regulator ATPase activity and trigger a large conformational change on the transposase that positions the catalytic site to perform DNA strand transfer. These studies help explain how AAA+ ATPase regulators-which are used by classical transposition systems such as Tn7, Mu and CRISPR-associated elements-can remodel their substrate DNA and cognate transposases to promote function.
Topics: AAA Domain; Adenosine Triphosphatases; Catalytic Domain; Cryoelectron Microscopy; DNA; DNA Transposable Elements; Enzyme Activation; Models, Molecular; Protein Multimerization; Transposases
PubMed: 38926614
DOI: 10.1038/s41586-024-07550-6 -
Scientific Reports Jun 2024The oxygen-labile transcription factor called hypoxia-inducible factor (HIF) is responsible for the cellular and organismal adaptive response to reduced oxygen...
The oxygen-labile transcription factor called hypoxia-inducible factor (HIF) is responsible for the cellular and organismal adaptive response to reduced oxygen availability. Deregulation of HIF is associated with the pathogenesis of major human diseases including cardiovascular disease and cancer. Under normoxia, the HIFα subunit is hydroxylated on conserved proline residues within the oxygen-dependent degradation domain (ODD) that labels HIFα for proteasome-mediated degradation. Despite similar oxygen-dependent degradation machinery acting on HIF1α and HIF2α, these two paralogs have been shown to exhibit unique kinetics under hypoxia, which suggests that other regulatory processes may be at play. Here, we characterize the protease activity found in rabbit reticulocytes that specifically cleaves the ODD of HIF1α but not HIF2α. Notably, the cleavage product is observed irrespective of the oxygen-dependent prolyl-hydroxylation potential of HIF1α, suggesting independence from oxygen. HIF1α M561T substitution, which mimics an evolutionary substitution that occurred during the duplication and divergence of HIF1α and HIF2α, diminished the cleavage of HIF1α. Protease inhibitor screening suggests that cysteine proteases cathepsins L and B preferentially cleave HIF1αODD, thereby revealing an additional layer of differential HIF regulation.
Topics: Hypoxia-Inducible Factor 1, alpha Subunit; Animals; Cathepsin L; Proteolysis; Rabbits; Oxygen; Humans; Reticulocytes; Basic Helix-Loop-Helix Transcription Factors; Hydroxylation
PubMed: 38926538
DOI: 10.1038/s41598-024-65537-9 -
Nature Communications Jun 2024METTL3 is the catalytic subunit of the methyltransferase complex, which mediates mA modification to regulate gene expression. In addition, METTL3 regulates transcription...
METTL3 is the catalytic subunit of the methyltransferase complex, which mediates mA modification to regulate gene expression. In addition, METTL3 regulates transcription in an enzymatic activity-independent manner by driving changes in high-order chromatin structure. However, how these functions of the methyltransferase complex are coordinated remains unknown. Here we show that the methyltransferase complex coordinates its enzymatic activity-dependent and independent functions to regulate cellular senescence, a state of stable cell growth arrest. Specifically, METTL3-mediated chromatin loops induce Hexokinase 2 expression through the three-dimensional chromatin organization during senescence. Elevated Hexokinase 2 expression subsequently promotes liquid-liquid phase separation, manifesting as stress granule phase separation, by driving metabolic reprogramming. This correlates with an impairment of translation of cell-cycle related mRNAs harboring polymethylated mA sites. In summary, our results report a coordination of mA-dependent and -independent function of the methyltransferase complex in regulating senescence through phase separation driven by metabolic reprogramming.
Topics: Methyltransferases; Chromatin; Cellular Senescence; Humans; Stress Granules; Hexokinase; RNA, Messenger; Adenosine; HEK293 Cells; Metabolic Reprogramming; Phase Separation
PubMed: 38926365
DOI: 10.1038/s41467-024-49745-5