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BioRxiv : the Preprint Server For... Jun 2024Osteosarcoma (OS) is the most common primary pediatric bone malignancy. One promising new therapeutic target is , encoding a substrate recognition factor of the SCF E3...
Comprehensive single cell transcriptomics analysis of murine osteosarcoma uncovers function in metastasis, genomic instability and immune activation and reveals additional target pathways.
Osteosarcoma (OS) is the most common primary pediatric bone malignancy. One promising new therapeutic target is , encoding a substrate recognition factor of the SCF E3 ubiquitin ligase responsible for ubiquitination and proteasome degradation of substrate p27, thus driving cellular proliferation. We have shown previously that knockout of in an immunocompetent transgenic mouse model of OS improved survival, drove apoptosis, and induced tumor inflammation. Here, we applied single-cell RNA-sequencing (scRNA-seq) to study primary OS tumors derived from Osx-Cre driven conditional knockout of and . We showed that murine OS models recapitulate the tumor heterogeneity and microenvironment complexity observed in patient tumors. We further compared this model with OS models with functional disruption of : one with knockout and the other with the Skp2-p27 interaction disrupted (resulting in p27 overexpression). We found reduction of T cell exhaustion and upregulation of interferon activation, along with evidence of replicative and endoplasmic reticulum-related stress in the disruption models, and showed that interferon induction was correlated with improved survival in OS patients. Additionally, our scRNA-seq analysis uncovered decreased activities of metastasis-related gene signatures in the -disrupted OS, which we validated by observation of a strong reduction in lung metastasis in the knockout mice. Finally, we report several potential mechanisms of escape from targeting in OS, including upregulation of targets, DNA copy number amplification and overexpression of alternative E3 ligase genes, and potential alternative lineage activation. These mechanistic insights into OS tumor biology and function suggest novel targets for new, synergistic therapies, while the data and our comprehensive analysis may serve as a public resource for further big data-driven OS research.
PubMed: 38895216
DOI: 10.1101/2024.06.04.597347 -
International Journal of Molecular... Jun 2024mutations cause X-linked amelogenesis imperfecta (AI), known as AI types IE, IIB, and IIC in Witkop's classification, characterized by hypoplastic (reduced thickness)...
mutations cause X-linked amelogenesis imperfecta (AI), known as AI types IE, IIB, and IIC in Witkop's classification, characterized by hypoplastic (reduced thickness) and/or hypomaturation (reduced hardness) enamel defects. In this study, we conducted whole exome analyses to unravel the disease-causing mutations for six AI families. Splicing assays, immunoblotting, and quantitative RT-PCR were conducted to investigate the molecular and cellular effects of the mutations. Four pathogenic variants (NM_182680.1:c.2T>C; c.29T>C; c.77del; c.145-1G>A) and a whole gene deletion (NG_012494.2:g.307534_403773del) were identified. The affected individuals exhibited enamel malformations, ranging from thin, poorly mineralized enamel with a "snow-capped" appearance to severe hypoplastic defects with minimal enamel. The c.145-1G>A mutation caused a -1 frameshift (NP_001133.1:p.Val35Cysfs*5). Overexpression of c.2T>C and c.29T>C demonstrated that mutant amelogenin proteins failed to be secreted, causing elevated endoplasmic reticulum stress and potential cell apoptosis. This study reveals a genotype-phenotype relationship for -associated AI: While amorphic mutations, including large deletions and 5' truncations, of cause hypoplastic-hypomaturation enamel with snow-capped teeth (AI types IIB and IIC) due to a complete loss of gene function, neomorphic variants, including signal peptide defects and 3' truncations, lead to severe hypoplastic/aplastic enamel (AI type IE) probably caused by "toxic" cellular effects of the mutant proteins.
Topics: Amelogenesis Imperfecta; Humans; Amelogenin; Male; Female; Genetic Association Studies; Mutation; Pedigree; Phenotype; Child; Endoplasmic Reticulum Stress; Genotype; Exome Sequencing
PubMed: 38892321
DOI: 10.3390/ijms25116132 -
International Journal of Molecular... Jun 2024Glycerol-3-phosphoacyltransferase (GPAT) is an important rate-limiting enzyme in the biosynthesis of triacylglycerol (TAG), which is of great significance for plant...
Genome-Wide Identification, Characterization, Evolutionary Analysis, and Expression Pattern of the GPAT Gene Family in Barley and Functional Analysis of under Abiotic Stress.
Glycerol-3-phosphoacyltransferase (GPAT) is an important rate-limiting enzyme in the biosynthesis of triacylglycerol (TAG), which is of great significance for plant growth, development, and response to abiotic stress. Although the characteristics of GPAT have been studied in many model plants, little is known about its expression profile and function in barley, especially under abiotic stress. In this study, 22 genes were identified in the barley genome and divided into three groups (I, II, III), with the latter Group III subdivided further into three subgroups based on the phylogenetic analysis. The analyses of conserved motifs, gene structures, and the three-dimensional structure of HvGPAT proteins also support this classification. Through evolutionary analysis, we determined that HvGPATs in Group I were the earliest to diverge during 268.65 MYA, and the differentiation of other HvGPATs emerged during 86.83-169.84 MYA. The tissue expression profile showed that 22 genes were almost not expressed in INF1 (inflorescence 1). Many functional elements related to stress responses and hormones in cis-element analysis, as well as qRT-PCR results, confirm that these genes were involved in abiotic stress responses. The expression level of was significantly increased under abiotic stress and its subcellular localization indicated its function in the endoplasmic reticulum. Various physiological traits under abiotic stress were evaluated using transgenic Arabidopsis to gain further insight into the role of , and it was found that transgenic seedlings have stronger resistance under abiotic stress than to the wild-type (WT) plants. Overall, our results provide new insights into the evolution and function of the barley GPAT gene family and enable us to explore the molecular mechanism of functional diversity behind the evolutionary history of these genes.
Topics: Hordeum; Stress, Physiological; Gene Expression Regulation, Plant; Phylogeny; Evolution, Molecular; Plant Proteins; Multigene Family; Glycerol-3-Phosphate O-Acyltransferase; Genome, Plant; Gene Expression Profiling
PubMed: 38892304
DOI: 10.3390/ijms25116101 -
International Journal of Molecular... May 2024The study of the mechanisms by which melatonin protects against cadmium (Cd) toxicity in plants is still in its infancy, particularly at the molecular level. In this...
Melatonin-Regulated Chaperone Binding Protein Plays a Key Role in Cadmium Stress Tolerance in Rice, Revealed by the Functional Characterization of a Novel Serotonin -Acetyltransferase 3 () in Rice.
The study of the mechanisms by which melatonin protects against cadmium (Cd) toxicity in plants is still in its infancy, particularly at the molecular level. In this study, the gene encoding a novel serotonin -acetyltransferase 3 () in rice, a pivotal enzyme in the melatonin biosynthetic pathway, was cloned. Rice () is the first identified plant ortholog of archaeon . The purified recombinant OsSNAT3 catalyzed the conversion of serotonin and 5-methoxytryptamine to -acetylserotonin and melatonin, respectively. The suppression of by RNAi led to a decline in endogenous melatonin levels followed by a reduction in Cd tolerance in transgenic RNAi rice lines. In addition, the expression levels of genes encoding the endoplasmic reticulum (ER) chaperones , , and were much lower in RNAi lines than in the wild type. In transgenic rice plants overexpressing (SNAT3-OE), however, melatonin levels were higher than in wild-type plants. SNAT3-OE plants also tolerated Cd stress, as indicated by seedling growth, malondialdehyde, and chlorophyll levels. expression was much higher in the SNAT3-OE lines than in the wild type. These results indicate that melatonin engineering could help crops withstand Cd stress, resulting in high yields in Cd-contaminated fields.
Topics: Oryza; Melatonin; Cadmium; Plant Proteins; Plants, Genetically Modified; Gene Expression Regulation, Plant; Stress, Physiological; Arylalkylamine N-Acetyltransferase; Molecular Chaperones; Serotonin
PubMed: 38892140
DOI: 10.3390/ijms25115952 -
International Journal of Molecular... May 2024Podophyllotoxin (PPT) is an active pharmaceutical ingredient (API) with established antitumor potential. However, due to its systemic toxicity, its use is restricted to...
Cellular Distribution and Ultrastructural Changes in HaCaT Cells, Induced by Podophyllotoxin and Its Novel Fluorescent Derivative, Supported by the Molecular Docking Studies.
Podophyllotoxin (PPT) is an active pharmaceutical ingredient (API) with established antitumor potential. However, due to its systemic toxicity, its use is restricted to topical treatment of anogenital warts. Less toxic PPT derivatives (e.g., etoposide and teniposide) are used intravenously as anticancer agents. PPT has been exploited as a scaffold of new potential therapeutic agents; however, fewer studies have been conducted on the parent molecule than on its derivatives. We have undertaken a study of ultrastructural changes induced by PPT on HaCaT keratinocytes. We have also tracked the intracellular localization of PPT using its fluorescent derivative (PPT-FL). Moreover, we performed molecular docking of both PPT and PPT-FL to compare their affinity to various binding sites of tubulin. Using the Presto blue viability assay, we established working concentrations of PPT in HaCaT cells. Subsequently, we have used selected concentrations to determine PPT effects at the ultrastructural level. Dynamics of PPT distribution by confocal microscopy was performed using PPT-FL. Molecular docking calculations were conducted using Glide. PPT induces a time-dependent cytotoxic effect on HaCaT cells. Within 24 h, we observed the elongation of cytoplasmic processes, formation of cytoplasmic vacuoles, progressive ER stress, and shortening of the mitochondrial long axis. After 48 h, we noticed disintegration of the cell membrane, progressive vacuolization, apoptotic/necrotic vesicles, and a change in the cell nucleus's appearance. PPT-FL was detected within HaCaT cells after ~10 min of incubation and remained within cells in the following measurements. Molecular docking confirmed the formation of a stable complex between tubulin and both PPT and PPT-FL. However, it was formed at different binding sites. PPT is highly toxic to normal human keratinocytes, even at low concentrations. It promptly enters the cells, probably via endocytosis. At lower concentrations, PPT causes disruptions in both ER and mitochondria, while at higher concentrations, it leads to massive vacuolization with subsequent cell death. The novel derivative of PPT, PPT-FL, forms a stable complex with tubulin, and therefore, it is a useful tracker of intracellular PPT binding and trafficking.
Topics: Humans; Molecular Docking Simulation; Podophyllotoxin; HaCaT Cells; Tubulin; Keratinocytes; Cell Survival; Mitochondria; Fluorescent Dyes; Binding Sites; Endoplasmic Reticulum Stress
PubMed: 38892135
DOI: 10.3390/ijms25115948 -
International Journal of Molecular... May 2024Amino acid permeases (AAPs) transporters are crucial for the long-distance transport of amino acids in plants, from source to sink. While and rice have been extensively...
Amino acid permeases (AAPs) transporters are crucial for the long-distance transport of amino acids in plants, from source to sink. While and rice have been extensively studied, research on foxtail millet is limited. This study identified two transcripts of , both of which were induced by NO and showed similar expression patterns. The overexpression of and in inhibited plant growth and seed size, although SiAAP9 was found to transport more amino acids into seeds. Furthermore, transgenic showed increased tolerance to high concentrations of glutamate (Glu) and histidine (His). The high overexpression level of suggested its protein was not only located on the plasma membrane but potentially on other organelles, as well. Interestingly, sequence deletion reduced SiAAP9's sensitivity to Brefeldin A (BFA), and SiAAP9 had ectopic localization on the endoplasmic reticulum (ER). Protoplast amino acid uptake experiments indicated that SiAAP9 enhanced Glu transport into foxtail millet cells. Overall, the two transcripts of have similar functions, but SiAAP9L shows a higher colocalization with BFA compartments compared to SiAAP9S. Our research identifies a potential candidate gene for enhancing the nutritional quality of foxtail millet through breeding.
Topics: Arabidopsis; Plants, Genetically Modified; Gene Expression Regulation, Plant; Plant Proteins; Endoplasmic Reticulum; Seeds; Setaria Plant; Amino Acid Transport Systems; Protein Transport; Brefeldin A; Amino Acids; Glutamic Acid
PubMed: 38892028
DOI: 10.3390/ijms25115840 -
International Journal of Molecular... May 2024Childhood glaucoma encompasses congenital and juvenile primary glaucoma, which are heterogeneous, uncommon, and irreversible optic neuropathies leading to visual...
Childhood glaucoma encompasses congenital and juvenile primary glaucoma, which are heterogeneous, uncommon, and irreversible optic neuropathies leading to visual impairment with a poorly understood genetic basis. Our goal was to identify gene variants associated with these glaucoma types by assessing the mutational burden in 76 matrix metalloproteinase-related genes. We studied 101 childhood glaucoma patients with no identified monogenic alterations using next-generation sequencing. Gene expression was assessed through immunohistochemistry. Functional analysis of selected gene variants was conducted in cultured cells and in zebrafish. Patients presented a higher proportion of rare variants in four metalloproteinase-related genes, including and , compared to controls. ADAMTSL4 protein expression was observed in the anterior segment of both the adult human and zebrafish larvae's eye, including tissues associated with glaucoma. In HEK-293T cells, expression of four ADAMTSL4 variants identified in this study showed that two variants (p.Arg774Trp and p.Arg98Trp) accumulated intracellularly, inducing endoplasmic reticulum stress. Additionally, overexpressing these ADAMTSL4 variants in zebrafish embryos confirmed partial loss-of-function effects for p.Ser719Leu and p.Arg1083His. Double heterozygous functional suppression of and zebrafish orthologs resulted in reduced volume of both the anterior eye chamber and lens within the chamber, supporting a genetic interaction between these genes. Our findings suggest that accumulation of partial functional defects in matrix metalloproteinase-related genes may contribute to increased susceptibility to early-onset glaucoma and provide further evidence supporting the notion of a complex genetic inheritance pattern underlying the disease.
Topics: Humans; Animals; Zebrafish; Glaucoma; Child; Male; Female; Child, Preschool; HEK293 Cells; Genetic Predisposition to Disease; Mutation; Matrix Metalloproteinases; ADAMTS Proteins; Adolescent; Infant; Zebrafish Proteins; Endoplasmic Reticulum Stress
PubMed: 38891949
DOI: 10.3390/ijms25115757 -
International Journal of Molecular... May 2024In this review we examine the functionally diverse ATPase associated with various cellular activities (AAA-ATPase), valosin-containing protein (VCP/p97), its molecular... (Review)
Review
In this review we examine the functionally diverse ATPase associated with various cellular activities (AAA-ATPase), valosin-containing protein (VCP/p97), its molecular functions, the mutational landscape of VCP and the phenotypic manifestation of VCP disease. VCP is crucial to a multitude of cellular functions including protein quality control, endoplasmic reticulum-associated degradation (ERAD), autophagy, mitophagy, lysophagy, stress granule formation and clearance, DNA replication and mitosis, DNA damage response including nucleotide excision repair, ATM- and ATR-mediated damage response, homologous repair and non-homologous end joining. VCP variants cause multisystem proteinopathy, and pathology can arise in several tissue types such as skeletal muscle, bone, brain, motor neurons, sensory neurons and possibly cardiac muscle, with the disease course being challenging to predict.
Topics: Valosin Containing Protein; Humans; Phenotype; Animals; Mutation; Autophagy; DNA Repair
PubMed: 38891822
DOI: 10.3390/ijms25115633 -
International Journal of Molecular... May 2024Neural tube defects (NTDs), which are caused by impaired embryonic neural tube closure, are one of the most serious and common birth defects. Peptidyl-prolyl cis/trans...
Neural tube defects (NTDs), which are caused by impaired embryonic neural tube closure, are one of the most serious and common birth defects. Peptidyl-prolyl cis/trans isomerase 1 (Pin1) is a prolyl isomerase that uniquely regulates cell signaling by manipulating protein conformation following phosphorylation, although its involvement in neuronal development remains unknown. In this study, we explored the involvement of Pin1 in NTDs and its potential mechanisms both in vitro and in vivo. The levels of Pin1 expression were reduced in NTD models induced by all-trans retinoic acid (Atra). Pin1 plays a significant role in regulating the apoptosis, proliferation, differentiation, and migration of neurons. Moreover, Pin1 knockdown significantly was found to exacerbate oxidative stress (OS) and endoplasmic reticulum stress (ERs) in neuronal cells. Further studies showed that the Notch1-Nrf2 signaling pathway may participate in Pin1 regulation of NTDs, as evidenced by the inhibition and overexpression of the Notch1-Nrf2 pathway. In addition, immunofluorescence (IF), co-immunoprecipitation (Co-IP), and GST pull-down experiments also showed that Pin1 interacts directly with Notch1 and Nrf2. Thus, our study suggested that the knocking down of Pin1 promotes NTD progression by inhibiting the activation of the Notch1-Nrf2 signaling pathway, and it is possible that this effect is achieved by disrupting the interaction of Pin1 with Notch1 and Nrf2, affecting their proteostasis. Our research identified that the regulation of Pin1 by retinoic acid (RA) and its involvement in the development of NTDs through the Notch1-Nrf2 axis could enhance our comprehension of the mechanism behind RA-induced brain abnormalities.
Topics: Tretinoin; NIMA-Interacting Peptidylprolyl Isomerase; Animals; Mice; Neural Tube Defects; Receptor, Notch1; NF-E2-Related Factor 2; Signal Transduction; Down-Regulation; Apoptosis; Oxidative Stress; Neurons; Female; Neural Tube; Endoplasmic Reticulum Stress; Cell Proliferation; Cell Differentiation; Cell Movement; Humans
PubMed: 38891776
DOI: 10.3390/ijms25115588 -
International Journal of Molecular... May 2024Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disorder. While there are five FDA-approved drugs for treating this disease, each has only modest... (Review)
Review
Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disorder. While there are five FDA-approved drugs for treating this disease, each has only modest benefits. To design new and more effective therapies for ALS, particularly for sporadic ALS of unknown and diverse etiologies, we must identify key, convergent mechanisms of disease pathogenesis. This review focuses on the origin and effects of glutamate-mediated excitotoxicity in ALS (the cortical hyperexcitability hypothesis), in which increased glutamatergic signaling causes motor neurons to become hyperexcitable and eventually die. We characterize both primary and secondary contributions to excitotoxicity, referring to processes taking place at the synapse and within the cell, respectively. '' include upregulation of calcium-permeable AMPA receptors, dysfunction of the EAAT2 astrocytic glutamate transporter, increased release of glutamate from the presynaptic terminal, and reduced inhibition by cortical interneurons-all of which have been observed in ALS patients and model systems. '' include changes to mitochondrial morphology and function, increased production of reactive oxygen species, and endoplasmic reticulum (ER) stress. By identifying key targets in the excitotoxicity cascade, we emphasize the importance of this pathway in the pathogenesis of ALS and suggest that intervening in this pathway could be effective for developing therapies for this disease.
Topics: Amyotrophic Lateral Sclerosis; Humans; Glutamic Acid; Animals; Motor Neurons; Aging; Receptors, AMPA; Endoplasmic Reticulum Stress; Mitochondria; Excitatory Amino Acid Transporter 2; Astrocytes; Reactive Oxygen Species
PubMed: 38891774
DOI: 10.3390/ijms25115587