RESUMEN

BACKGROUND: Dysregulated lipid oxidation occurs in several pathological processes characterized by cell proliferation and migration. Nonetheless, the molecular mechanism of lipid oxidation is not well appreciated in liver fibrosis, which is accompanied by enhanced fibroblast proliferation and migration. METHODS: We investigated the causes and consequences of lipid oxidation in liver fibrosis using cultured cells, animal models, and clinical samples. RESULTS: Increased ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP1) expression caused increased lipid oxidation, resulting in the proliferation and migration of hepatic stellate cells (HSCs) that lead to liver fibrosis, whereas fibroblast-specific ENPP1 knockout reversing these results. Elevated ENPP1 and N6-methyladenosine (m6A) levels were associated with high expression of Wilms tumor 1 associated protein (WTAP). Mechanistically, WTAP-mediated m6A methylation of the 3'UTR of ENPP1 mRNA and induces its translation dependent of YTH domain family proteins 1 (YTHDF1). Additionally, ENPP1 could interact with hypoxia inducible lipid droplet associated (HILPDA) directly; overexpression of ENPP1 further recruits HILPDA-mediated lipid oxidation, thereby promotes HSCs proliferation and migration, while inhibition of ENPP1 expression produced the opposite effect. Clinically, increased expression of WTAP, YTHDF1, ENPP1, and HILPDA, and increased m6A mRNA content, enhanced lipid oxidation, and increased collagen deposition in human liver fibrosis tissues. CONCLUSIONS: We describe a novel mechanism in which WTAP catalyzes m6A methylation of ENPP1 in a YTHDF1-dependent manner to enhance lipid oxidation, promoting HSCs proliferation and migration and liver fibrosis.

Asunto(s)

Adenosina , Proliferación Celular , Metabolismo de los Lípidos , Cirrosis Hepática , Oxidación-Reducción , Hidrolasas Diéster Fosfóricas , Pirofosfatasas , ARN Mensajero , Pirofosfatasas/metabolismo , Pirofosfatasas/genética , Humanos , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Animales , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Cirrosis Hepática/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proliferación Celular/genética , Metabolismo de los Lípidos/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Movimiento Celular/genética , Ratones Endogámicos C57BL , Masculino , Epigénesis Genética , Fibroblastos/metabolismo , Fibroblastos/patología , Metilación , Factores de Empalme de ARN , Proteínas de Ciclo Celular

RESUMEN

Lysophosphatidic acids (LPAs) evoke nociception and itch in mice and humans. In this study, we assessed the signaling paths. Hydroxychloroquine was injected intradermally to evoke itch in mice, which evoked an increase of LPAs in the skin and in the thalamus, suggesting that peripheral and central LPA receptors (LPARs) were involved in HCQ-evoked pruriception. To unravel the signaling paths, we assessed the localization of candidate genes and itching behavior in knockout models addressing LPAR5, LPAR2, autotaxin/ENPP2 and the lysophospholipid phosphatases, as well as the plasticity-related genes Prg1/LPPR4 and Prg2/LPPR3. LacZ reporter studies and RNAscope revealed LPAR5 in neurons of the dorsal root ganglia (DRGs) and in skin keratinocytes, LPAR2 in cortical and thalamic neurons, and Prg1 in neuronal structures of the dorsal horn, thalamus and SSC. HCQ-evoked scratching behavior was reduced in sensory neuron-specific Advillin-LPAR5-/- mice (peripheral) but increased in LPAR2-/- and Prg1-/- mice (central), and it was not affected by deficiency of glial autotaxin (GFAP-ENPP2-/-) or Prg2 (PRG2-/-). Heat and mechanical nociception were not affected by any of the genotypes. The behavior suggested that HCQ-mediated itch involves the activation of peripheral LPAR5, which was supported by reduced itch upon treatment with an LPAR5 antagonist and autotaxin inhibitor. Further, HCQ-evoked calcium fluxes were reduced in primary sensory neurons of Advillin-LPAR5-/- mice. The results suggest that LPA-mediated itch is primarily mediated via peripheral LPAR5, suggesting that a topical LPAR5 blocker might suppress "non-histaminergic" itch.

Asunto(s)

Hidroxicloroquina , Ratones Noqueados , Prurito , Receptores del Ácido Lisofosfatídico , Animales , Receptores del Ácido Lisofosfatídico/metabolismo , Receptores del Ácido Lisofosfatídico/genética , Prurito/inducido químicamente , Prurito/metabolismo , Prurito/genética , Prurito/tratamiento farmacológico , Ratones , Hidroxicloroquina/farmacología , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Masculino , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Lisofosfolípidos/metabolismo , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacos

RESUMEN

Topoisomerase 1 (Top1) controls DNA topology, relieves DNA supercoiling during replication and transcription, and is critical for mitotic progression to the G1 phase. Tyrosyl-DNA phosphodiesterase 1 (TDP1) mediates the removal of trapped Top1-DNA covalent complexes (Top1cc). Here, we identify CDK1-dependent phosphorylation of TDP1 at residue S61 during mitosis. A TDP1 variant defective for S61 phosphorylation (TDP1-S61A) is trapped on the mitotic chromosomes, triggering DNA damage and mitotic defects. Moreover, we show that Top1cc repair in mitosis occurs via a MUS81-dependent DNA repair mechanism. Replication stress induced by camptothecin or aphidicolin leads to TDP1-S61A enrichment at common fragile sites, which over-stimulates MUS81-dependent chromatid breaks, anaphase bridges, and micronuclei, ultimately culminating in the formation of 53BP1 nuclear bodies during G1 phase. Our findings provide new insights into the cell cycle-dependent regulation of TDP1 dynamics for the repair of trapped Top1-DNA covalent complexes during mitosis that prevents genomic instability following replication stress.

Asunto(s)

Proteína Quinasa CDC2 , Reparación del ADN , ADN-Topoisomerasas de Tipo I , Proteínas de Unión al ADN , Endonucleasas , Mitosis , Hidrolasas Diéster Fosfóricas , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Proteína Quinasa CDC2/metabolismo , Proteína Quinasa CDC2/genética , Fosforilación , Humanos , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo I/genética , Endonucleasas/metabolismo , Endonucleasas/genética , ADN/metabolismo , Células HeLa , Daño del ADN

RESUMEN

Yersinia pseudotuberculosis (Yptb) is a pathogenic gram-negative bacterium that can colonize the intestines of different animals. Its infection leads to the activation of the host's innate immunity. Both host and bacterial-derived cyclic dinucleotides (CDNs) could activate the innate immune response of host cells. In bacteria, CDNs like c-di-AMP, c-di-GMP, or 3'3'-cGAMP can be hydrolyzed by different hydrolases. Recent studies showed that the degradation of those second messengers helps the pathogen evade immune detection. In this study, we identified a hydrolase, YPK_3776, namely CpdB in Yptb. CpdB is predicted to bind bacterial-derived c-di-AMP, c-di-GMP, 3'3'-cGAMP and host-derived 2'3'-cGAMP. Surprisingly, by using high-performance liquid chromatography (HPLC), we found that CpdB could only degrade bacterial-derived CDNs but not host-derived 2'3'-cGAMP. In addition, CpdB has 2'3'-cNMP activity. Consistently, the Yptb mutant lacking the cpdB gene exhibited a higher level of intracellular c-di-GMP. Furthermore, the ∆cpdB mutant elicited stronger innate immune responses during Yptb infection in macrophages, suggesting CpdB enables Yptb to evade host immune surveillance. Furthermore, CpdB inhibited the Yptb-induced innate immune response in a STING-dependent manner. Finally, we showed the ∆cpdB infection in mice model exhibited in lower bacterial burden, as compared to wild-type strain infection, indicating CpdB is important for bacterial survival in the host. Together, we identified a cyclic dinucleotide hydrolase CpdB in Yptb that could degrade bacterial-derived CDNs which help the pathogen to evade immune detection via the STING pathway.

Asunto(s)

Inmunidad Innata , Hidrolasas Diéster Fosfóricas , Infecciones por Yersinia pseudotuberculosis , Yersinia pseudotuberculosis , Yersinia pseudotuberculosis/inmunología , Yersinia pseudotuberculosis/genética , Animales , Ratones , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Infecciones por Yersinia pseudotuberculosis/inmunología , Infecciones por Yersinia pseudotuberculosis/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Nucleótidos Cíclicos/metabolismo , Macrófagos/inmunología , Macrófagos/microbiología , Fosfatos de Dinucleósidos/metabolismo , Femenino , GMP Cíclico/análogos & derivados

RESUMEN

The Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) ectoenzyme regulates vascular intimal proliferation and mineralization of bone and soft tissues. ENPP1 variants cause Generalized Arterial Calcification of Infancy (GACI), a rare genetic disorder characterized by ectopic calcification, intimal proliferation, and stenosis of large- and medium-sized arteries. ENPP1 hydrolyzes extracellular ATP to pyrophosphate (PPi) and AMP. AMP is the precursor of adenosine, which has been implicated in the control of neointimal formation. Herein, we demonstrate that an ENPP1-Fc recombinant therapeutic inhibits proliferation of vascular smooth muscle cells (VSMCs) in vitro and in vivo. Addition of ENPP1 and ATP to cultured VSMCs generated AMP, which was metabolized to adenosine. It also significantly decreased cell proliferation. AMP or adenosine alone inhibited VSMC growth. Inhibition of ecto-5'-nucleotidase CD73 decreased adenosine accumulation and suppressed the anti-proliferative effects of ENPP1/ATP. Addition of AMP increased cAMP synthesis and phosphorylation of VASP at Ser157. This AMP-mediated cAMP increase was abrogated by CD73 inhibitors or by A2aR and A2bR antagonists. Ligation of the carotid artery promoted neointimal hyperplasia in wild-type mice, which was exacerbated in ENPP1-deficient ttw/ttw mice. Prophylactic or therapeutic treatments with ENPP1 significantly reduced intimal hyperplasia not only in ttw/ttw but also in wild-type mice. These findings provide the first insight into the mechanism of the anti-proliferative effect of ENPP1 and broaden its potential therapeutic applications beyond enzyme replacement therapy.

Asunto(s)

5'-Nucleotidasa , Adenosina , Proliferación Celular , Músculo Liso Vascular , Miocitos del Músculo Liso , Hidrolasas Diéster Fosfóricas , Pirofosfatasas , Transducción de Señal , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Pirofosfatasas/metabolismo , Pirofosfatasas/genética , 5'-Nucleotidasa/metabolismo , 5'-Nucleotidasa/genética , Animales , Proliferación Celular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Adenosina/metabolismo , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Miocitos del Músculo Liso/efectos de los fármacos , Ratones , Humanos , Adenosina Monofosfato/metabolismo , Ratones Endogámicos C57BL , AMP Cíclico/metabolismo , Masculino , Calcificación Vascular/metabolismo , Calcificación Vascular/patología , Calcificación Vascular/genética

RESUMEN

Autotaxin (ATX) is a member of the ectonucleotide pyrophosphate/phosphodiesterase (ENPP) family; it is encoded by the ENPP2 gene. ATX is a secreted glycoprotein and catalyzes the hydrolysis of lysophosphatidylcholine to lysophosphatidic acid (LPA). LPA is responsible for the transduction of various signal pathways through the interaction with at least six G protein-coupled receptors, LPA Receptors 1 to 6 (LPAR1-6). The ATX-LPA axis is involved in various physiological and pathological processes, such as angiogenesis, embryonic development, inflammation, fibrosis, and obesity. However, significant research also reported its connection to carcinogenesis, immune escape, metastasis, tumor microenvironment, cancer stem cells, and therapeutic resistance. Moreover, several studies suggested ATX and LPA as relevant biomarkers and/or therapeutic targets. In this review of the literature, we aimed to deepen knowledge about the role of the ATX-LPA axis as a promoter of cancer development, progression and invasion, and therapeutic resistance. Finally, we explored its potential application as a prognostic/predictive biomarker and therapeutic target for tumor treatment.

Asunto(s)

Lisofosfolípidos , Neoplasias , Hidrolasas Diéster Fosfóricas , Humanos , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Lisofosfolípidos/metabolismo , Animales , Transducción de Señal , Receptores del Ácido Lisofosfatídico/metabolismo , Receptores del Ácido Lisofosfatídico/genética , Carcinogénesis/genética , Carcinogénesis/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo

RESUMEN

Interaction of mast cells (MCs) with fibroblasts is essential for MC maturation within tissue microenvironments, although the underlying mechanism is incompletely understood. Through a phenotypic screening of >30 mouse lines deficient in lipid-related genes, we found that deletion of the lysophosphatidic acid (LPA) receptor LPA1, like that of the phospholipase PLA2G3, the prostaglandin D2 (PGD2) synthase L-PGDS, or the PGD2 receptor DP1, impairs MC maturation and thereby anaphylaxis. Mechanistically, MC-secreted PLA2G3 acts on extracellular vesicles (EVs) to supply lysophospholipids, which are converted by fibroblast-derived autotaxin (ATX) to LPA. Fibroblast LPA1 then integrates multiple pathways required for MC maturation by facilitating integrin-mediated MC-fibroblast adhesion, IL-33-ST2 signaling, L-PGDS-driven PGD2 generation, and feedforward ATX-LPA1 amplification. Defective MC maturation resulting from PLA2G3 deficiency is restored by supplementation with LPA1 agonists or PLA2G3-modified EVs. Thus, the lipid-orchestrated paracrine circuit involving PLA2G3-driven lysophospholipid, eicosanoid, integrin, and cytokine signaling fine-tunes MC-fibroblast communication, ensuring MC maturation.

Asunto(s)

Anafilaxia , Fibroblastos , Lisofosfolípidos , Mastocitos , Ratones Noqueados , Comunicación Paracrina , Hidrolasas Diéster Fosfóricas , Receptores del Ácido Lisofosfatídico , Transducción de Señal , Animales , Mastocitos/inmunología , Mastocitos/metabolismo , Anafilaxia/inmunología , Anafilaxia/metabolismo , Ratones , Fibroblastos/metabolismo , Lisofosfolípidos/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Receptores del Ácido Lisofosfatídico/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Prostaglandina D2/metabolismo , Vesículas Extracelulares/metabolismo , Interleucina-33/metabolismo , Oxidorreductasas Intramoleculares/metabolismo , Oxidorreductasas Intramoleculares/genética , Receptores de Prostaglandina/metabolismo , Receptores de Prostaglandina/genética , Diferenciación Celular , Ratones Endogámicos C57BL , Proteína 1 Similar al Receptor de Interleucina-1 , Lipocalinas

RESUMEN

Nuclear exclusion of the RNA- and DNA-binding protein TDP-43 can induce neurodegeneration in different diseases. Diverse processes have been implicated to influence TDP-43 mislocalization, including disrupted nucleocytoplasmic transport (NCT); however, the physiological pathways that normally ensure TDP-43 nuclear localization are unclear. The six-transmembrane enzyme glycerophosphodiester phosphodiesterase 2 (GDE2 or GDPD5) cleaves the glycosylphosphatidylinositol (GPI) anchor that tethers some proteins to the membrane. Here we show that GDE2 maintains TDP-43 nuclear localization by regulating the dynamics of canonical Wnt signaling. Ablation of GDE2 causes aberrantly sustained Wnt activation in adult neurons, which is sufficient to cause NCT deficits, nuclear pore abnormalities, and TDP-43 nuclear exclusion. Disruption of GDE2 coincides with TDP-43 abnormalities in postmortem tissue from patients with amyotrophic lateral sclerosis (ALS). Further, GDE2 deficits are evident in human neural cell models of ALS, which display erroneous Wnt activation that, when inhibited, increases mRNA levels of genes regulated by TDP-43. Our study identifies GDE2 as a critical physiological regulator of Wnt signaling in adult neurons and highlights Wnt pathway activation as an unappreciated mechanism contributing to nucleocytoplasmic transport and TDP-43 abnormalities in disease.

Asunto(s)

Proteínas de Unión al ADN , Neuronas , Hidrolasas Diéster Fosfóricas , Vía de Señalización Wnt , Humanos , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Animales , Neuronas/metabolismo , Ratones , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo

RESUMEN

Rapid information processing in the central nervous system requires the myelination of axons by oligodendrocytes. The transcription factor Sox2 and its close relative Sox3 redundantly regulate the development of myelin-forming oligodendrocytes, but little is known about the underlying molecular mechanisms. Here, we characterized the expression profile of cultured oligodendroglial cells during early differentiation and identified Bcas1, Enpp6, Zfp488 and Nkx2.2 as major downregulated genes upon Sox2 and Sox3 deletion. An analysis of mice with oligodendrocyte-specific deletion of Sox2 and Sox3 validated all four genes as downstream targets in vivo. Additional functional assays identified regulatory regions in the vicinity of each gene that are responsive to and bind both Sox proteins. Bcas1, Enpp6, Zfp488 and Nkx2.2 therefore likely represent direct target genes and major effectors of Sox2 and Sox3. Considering the preferential expression and role of these genes in premyelinating oligodendrocytes, our findings suggest that Sox2 and Sox3 impact oligodendroglial development at the premyelinating stage with Bcas1, Enpp6, Zfp488 and Nkx2.2 as their major effectors.

Asunto(s)

Diferenciación Celular , Proteína Homeobox Nkx-2.2 , Oligodendroglía , Factores de Transcripción SOXB1 , Factores de Transcripción , Animales , Ratones , Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Oligodendroglía/metabolismo , Oligodendroglía/citología , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXC/metabolismo , Factores de Transcripción SOXC/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética

RESUMEN

Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger in bacteria that regulates multiple biological functions, including biofilm formation, virulence, and intercellular communication. However, c-di-GMP signaling is virtually unknown in economically important filamentous cyanobacteria, Arthrospira. In this study, we predicted 31 genes encoding GGDEF-domain proteins from A. platensis NIES39 as potential diguanylate cyclases (DGCs). Phylogenetic distribution analysis showed five genes (RS09460, RS04865, RS26155, M01840, and E02220) with highly conserved distribution across 25 Arthrospira strains. Adc1 encoded by RS09460 was further characterized as a typical DGC. By establishing the genetic transformation system of Arthrospira, we demonstrated that the overexpression of Adc1 promoted the production of extracellular polymeric substances (EPS), which in turn caused the aggregation of filaments. We also confirmed that RS04865 and RS26155 may encode active DGCs, while enzymatic activity assays showed that proteins encoded by M01840 and E02220 have phosphodiesterase (PDE) activity. Meta-analysis revealed that the expression profiles of RS09460 and RS04865 were unaffected under 31 conditions, suggesting that they may function as conserved genes in maintaining the basal level of c-di-GMP in Arthrospira. In summary, this report will provide the basis for further studies of c-di-GMP signal in Arthrospira.

Asunto(s)

Proteínas Bacterianas , GMP Cíclico , Liasas de Fósforo-Oxígeno , Filogenia , GMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Liasas de Fósforo-Oxígeno/genética , Liasas de Fósforo-Oxígeno/metabolismo , Spirulina/genética , Spirulina/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Regulación Bacteriana de la Expresión Génica , Cianobacterias/genética , Cianobacterias/metabolismo , Biopelículas/crecimiento & desarrollo , Proteínas de Escherichia coli

RESUMEN

Meiosis reduces ploidy through two rounds of chromosome segregation preceded by one round of DNA replication. In meiosis I, homologous chromosomes segregate, while in meiosis II, sister chromatids separate from each other. Topoisomerase II (Topo II) is a conserved enzyme that alters DNA structure by introducing transient double-strand breaks. During mitosis, Topo II relieves topological stress associated with unwinding DNA during replication, recombination, and sister chromatid segregation. Topo II also plays a role in maintaining mitotic chromosome structure. However, the role and regulation of Topo II during meiosis is not well-defined. Previously, we found an allele of Topo II, top-2(it7), disrupts homologous chromosome segregation during meiosis I of Caenorhabditis elegans spermatogenesis. In a genetic screen, we identified different point mutations in 5'-tyrosyl-DNA phosphodiesterase two (Tdp2, C. elegans tdpt-1) that suppress top-2(it7) embryonic lethality. Tdp2 removes trapped Top-2-DNA complexes. The tdpt-1 suppressing mutations rescue embryonic lethality, ameliorate chromosome segregation defects, and restore TOP-2 protein levels of top-2(it7). Here, we show that both TOP-2 and TDPT-1 are expressed in germ line nuclei but occupy different compartments until late meiotic prophase. We also demonstrate that tdpt-1 suppression is due to loss of function of the protein and that the tdpt-1 mutations do not have a phenotype independent of top-2(it7) in meiosis. Lastly, we found that the tdpt-1 suppressing mutations either impair the phosphodiesterase activity, affect the stability of TDPT-1, or disrupt protein interactions. This suggests that the WT TDPT-1 protein is inhibiting chromosome biological functions of an impaired TOP-2 during meiosis.

Asunto(s)

Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Segregación Cromosómica , ADN-Topoisomerasas de Tipo II , Espermatogénesis , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Espermatogénesis/genética , Masculino , ADN-Topoisomerasas de Tipo II/metabolismo , ADN-Topoisomerasas de Tipo II/genética , Meiosis , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Mutación

RESUMEN

Though rod and cone photoreceptors use similar phototransduction mechanisms, previous model calculations have indicated that the most important differences in their light responses are likely to be differences in amplification of the G-protein cascade, different decay rates of phosphodiesterase (PDE) and pigment phosphorylation, and different rates of turnover of cGMP in darkness. To test this hypothesis, we constructed TrUx;GapOx rods by crossing mice with decreased transduction gain from decreased transducin expression, with mice displaying an increased rate of PDE decay from increased expression of GTPase-activating proteins (GAPs). These two manipulations brought the sensitivity of TrUx;GapOx rods to within a factor of 2 of WT cone sensitivity, after correcting for outer-segment dimensions. These alterations did not, however, change photoreceptor adaptation: rods continued to show increment saturation though at a higher background intensity. These experiments confirm model calculations that rod responses can mimic some (though not all) of the features of cone responses after only a few changes in the properties of transduction proteins.

Asunto(s)

Células Fotorreceptoras Retinianas Conos , Células Fotorreceptoras Retinianas Bastones , Transducina , Animales , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Bastones/metabolismo , Ratones , Transducina/metabolismo , Transducina/genética , Retina/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética

RESUMEN

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) codes for a type 2 transmembrane glycoprotein which hydrolyzes extracellular phosphoanhydrides into bio-active molecules that regulate, inter alia, ectopic mineralization, bone formation, vascular endothelial proliferation, and the innate immune response. The clinical phenotypes produced by ENPP1 deficiency are disparate, ranging from life-threatening arterial calcifications to cutaneous hypopigmentation. To investigate associations between disease phenotype and enzyme activity we quantified the enzyme velocities of 29 unique ENPP1 pathogenic variants in 41 patients enrolled in an NIH study along with 33 other variants reported in literature. We correlated the relative enzyme velocities with the presenting clinical diagnoses, performing the catalytic velocity measurements simultaneously in triplicate using a high-throughput assay to reduce experimental variation. We found that ENPP1 variants associated with autosomal dominant phenotypes reduced enzyme velocities by 50 % or more, whereas variants associated with insulin resistance had non-significant effects on enzyme velocity. In Cole disease the catalytic velocities of ENPP1 variants associated with AD forms trended to lower values than those associated with autosomal recessive forms - 8-32 % vs. 33 % of WT, respectively. Additionally, ENPP1 variants leading to life-threatening vascular calcifications in GACI patients had widely variable enzyme activities, ranging from no significant differences compared to WT to the complete abolishment of enzyme velocity. Finally, disease severity in GACI did not correlate with the mean enzyme velocity of the variants present in affected compound heterozygotes but did correlate with the more severely damaging variant. In summary, correlation of ENPP1 enzyme velocity with disease phenotypes demonstrate that enzyme velocities below 50 % of WT levels are likely to occur in the context of autosomal dominant disease (due to a monoallelic variant), and that disease severity in GACI infants correlates with the more severely damaging ENPP1 variant in compound heterozygotes, not the mean velocity of the pathogenic variants present.

Asunto(s)

Fenotipo , Hidrolasas Diéster Fosfóricas , Pirofosfatasas , Pirofosfatasas/genética , Humanos , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Femenino , Variación Genética , Masculino , Mutación/genética

RESUMEN

Activating signal co-integrator complex 1 (ASCC1) acts with ASCC-ALKBH3 complex in alkylation damage responses. ASCC1 uniquely combines two evolutionarily ancient domains: nucleotide-binding K-Homology (KH) (associated with regulating splicing, transcriptional, and translation) and two-histidine phosphodiesterase (PDE; associated with hydrolysis of cyclic nucleotide phosphate bonds). Germline mutations link loss of ASCC1 function to spinal muscular atrophy with congenital bone fractures 2 (SMABF2). Herein analysis of The Cancer Genome Atlas (TCGA) suggests ASCC1 RNA overexpression in certain tumors correlates with poor survival, Signatures 29 and 3 mutations, and genetic instability markers. We determined crystal structures of Alvinella pompejana (Ap) ASCC1 and Human (Hs) PDE domain revealing high-resolution details and features conserved over 500 million years of evolution. Extending our understanding of the KH domain Gly-X-X-Gly sequence motif, we define a novel structural Helix-Clasp-Helix (HCH) nucleotide binding motif and show ASCC1 sequence-specific binding to CGCG-containing RNA. The V-shaped PDE nucleotide binding channel has two His-Φ-Ser/Thr-Φ (HXT) motifs (Φ being hydrophobic) positioned to initiate cyclic phosphate bond hydrolysis. A conserved atypical active-site histidine torsion angle implies a novel PDE substrate. Flexible active site loop and arginine-rich domain linker appear regulatory. Small-angle X-ray scattering (SAXS) revealed aligned KH-PDE RNA binding sites with limited flexibility in solution. Quantitative evolutionary bioinformatic analyses of disease and cancer-associated mutations support implied functional roles for RNA binding, phosphodiesterase activity, and regulation. Collective results inform ASCC1's roles in transactivation and alkylation damage responses, its targeting by structure-based inhibitors, and how ASCC1 mutations may impact inherited disease and cancer.

Asunto(s)

Hidrolasas Diéster Fosfóricas , Humanos , Biología Computacional/métodos , Cristalografía por Rayos X , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/genética , Motivos de Unión al ARN/genética

RESUMEN

2'3'-Cyclic guanosine monophosphate (GMP)-AMP (cGAMP) is a second messenger synthesized upon detection of cytosolic double-stranded DNA (dsDNA) and passed between cells to facilitate downstream immune signaling. Ectonucleotide pyrophosphatase phosphodiesterase I (ENPP1), an extracellular enzyme, was the only metazoan hydrolase known to regulate cGAMP levels to dampen anti-cancer immunity. Here, we uncover ENPP3 as the second and likely the only other metazoan cGAMP hydrolase under homeostatic conditions. ENPP3 has a tissue expression pattern distinct from ENPP1's and accounts for all cGAMP hydrolysis activity in ENPP1-deficient mice. Importantly, we also show that, as with ENPP1, selectively abolishing ENPP3's cGAMP hydrolysis activity results in diminished cancer growth and metastasis of certain tumor types in a stimulator of interferon genes (STING)-dependent manner. Both ENPP1 and ENPP3 are extracellular enzymes, suggesting the dominant role that extracellular cGAMP must play as a mediator of cell-cell innate immune communication. Our work demonstrates that ENPP1 and ENPP3 non-redundantly dampen extracellular cGAMP-STING signaling, pointing to ENPP3 as a target for cancer immunotherapy.

Asunto(s)

Inmunidad Innata , Proteínas de la Membrana , Nucleótidos Cíclicos , Hidrolasas Diéster Fosfóricas , Pirofosfatasas , Animales , Nucleótidos Cíclicos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Ratones , Proteínas de la Membrana/metabolismo , Pirofosfatasas/metabolismo , Pirofosfatasas/genética , Humanos , Ratones Endogámicos C57BL , Hidrólisis , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patología , Transducción de Señal

RESUMEN

TDP1 removes transcription-blocking topoisomerase I cleavage complexes (TOP1ccs), and its inactivating H493R mutation causes the neurodegenerative syndrome SCAN1. However, the molecular mechanism underlying the SCAN1 phenotype is unclear. Here, we generate human SCAN1 cell models using CRISPR-Cas9 and show that they accumulate TOP1ccs along with changes in gene expression and genomic distribution of R-loops. SCAN1 cells also accumulate transcriptional DNA double-strand breaks (DSBs) specifically in the G1 cell population due to increased DSB formation and lack of repair, both resulting from abortive removal of transcription-blocking TOP1ccs. Deficient TDP1 activity causes increased DSB production, and the presence of mutated TDP1 protein hampers DSB repair by a TDP2-dependent backup pathway. This study provides powerful models to study TDP1 functions under physiological and pathological conditions and unravels that a gain of function of the mutated TDP1 protein, which prevents DSB repair, rather than a loss of TDP1 activity itself, could contribute to SCAN1 pathogenesis.

Asunto(s)

Roturas del ADN de Doble Cadena , Reparación del ADN , Mutación , Enfermedades Neurodegenerativas , Hidrolasas Diéster Fosfóricas , Humanos , Hidrolasas Diéster Fosfóricas/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Mutación/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , ADN-Topoisomerasas de Tipo I/metabolismo , ADN-Topoisomerasas de Tipo I/genética , Transcripción Genética , Estructuras R-Loop , Sistemas CRISPR-Cas/genética

RESUMEN

The complex mechanism of neuropathic pain involves various aspects of both central and peripheral pain conduction pathways. An effective cure for neuropathic pain therefore remains elusive. We found that deficiency of the gene Gdpd3, encoding a lysophospholipase D enzyme, alleviates the inflammatory responses in dorsal root ganglia (DRG) of mice under neuropathic pain and reduces PE (20:4) and PGE2 in DRG. Gdpd3 deficiency had a stronger analgesic effect on neuropathic pain than Celecoxib, a nonsteroidal anti-inflammatory drug. Gdpd3 deficiency also interferes with the polarization of macrophages, switching from M1 towards M2 phenotype. The PPARγ/ FABP4 pathway was screened by RNA sequencing as functional related with Gdpd3 deficient BMDMs stimulated with LPS. Both protein and mRNA levels of PPARγ in GDPD3 deficient BMDMs were higher than those of the litter control mice. However, GW9962 (inhibitor of PPARγ) could reverse the reprogramming polarization of macrophages caused by GDPD3 deficiency. Therefore, our study suggests that GDPD3 deficiency exerts a relieving effect on neuropathic pain and alleviates neuroinflammation in DRG by switching the phenotype of macrophages from M1 to M2, which was mediated through PGE2 and PPARγ/ FABP4 pathway.

Asunto(s)

Dinoprostona , Macrófagos , Ratones Endogámicos C57BL , Neuralgia , PPAR gamma , Animales , Masculino , Ratones , Polaridad Celular/fisiología , Dinoprostona/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Proteínas de Unión a Ácidos Grasos/deficiencia , Ganglios Espinales/metabolismo , Macrófagos/metabolismo , Ratones Noqueados , Neuralgia/metabolismo , Neuralgia/tratamiento farmacológico , PPAR gamma/metabolismo , Transducción de Señal/fisiología , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo

RESUMEN

Glioblastoma is universally fatal and characterized by frequent chromosomal copy number alterations harboring oncogenes and tumor suppressors. In this study, we analyzed exome-wide human glioblastoma copy number data and found that cytoband 6q27 is an independent poor prognostic marker in multiple data sets. We then combined CRISPR-Cas9 data, human spatial transcriptomic data, and human and mouse RNA sequencing data to nominate PDE10A as a potential haploinsufficient tumor suppressor in the 6q27 region. Mouse glioblastoma modeling using the RCAS/tv-a system confirmed that Pde10a suppression induced an aggressive glioma phenotype in vivo and resistance to temozolomide and radiation therapy in vitro. Cell culture analysis showed that decreased Pde10a expression led to increased PI3K/AKT signaling in a Pten-independent manner, a response blocked by selective PI3K inhibitors. Single-nucleus RNA sequencing from our mouse gliomas in vivo, in combination with cell culture validation, further showed that Pde10a suppression was associated with a proneural-to-mesenchymal transition that exhibited increased cell adhesion and decreased cell migration. Our results indicate that glioblastoma patients harboring PDE10A loss have worse outcomes and potentially increased sensitivity to PI3K inhibition.

Asunto(s)

Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Animales , Ratones , Glioblastoma/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Haploinsuficiencia , Glioma/genética , Fosfohidrolasa PTEN/genética , Hidrolasas Diéster Fosfóricas/genética , Línea Celular Tumoral , Neoplasias Encefálicas/genética

RESUMEN

BACKGROUND: Dysregulated ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP) family occurs in metabolic reprogramming pathological processes. Nonetheless, the epigenetic mechanisms by which ENPP family impacts NAFLD, also known as metabolic dysfunction-associated steatotic liver disease (MASLD), is poorly appreciated. METHODS: We investigated the causes and consequences of ENPP1 promoter hypomethylation may boost NAFLD using NAFLD clinical samples, as well as revealed the underlying mechanisms using high-fat diet (HFD) + carbon tetrachloride (CCl4) induced mouse model of NAFLD and FFA treatment of cultured hepatocyte. RESULTS: Herein, we report that the expression level of ENPP1 are increased in patients with NAFLD liver tissue and in mouse model of NAFLD. Hypomethylation of ENPP1, is associated with the perpetuation of hepatocyte autophagy and liver fibrosis in the NAFLD. ENPP1 hypomethylation is mediated by the DNA demethylase TET3 in NAFLD liver fibrosis and hepatocyte autophagy. Additionally, knockdown of TET3 methylated ENPP1 promoter, reduced the ENPP1 expression, ameliorated the experimental NAFLD. Mechanistically, TET3 epigenetically promoted ENPP1 expression via hypomethylation of the promoter. Knocking down TET3 can inhibit the hepatocyte autophagy but an overexpression of ENPP1 showing rescue effect. CONCLUSIONS: We describe a novel epigenetic mechanism wherein TET3 promoted ENPP1 expression through promoter hypomethylation is a critical mediator of NAFLD. Our findings provide new insight into the development of preventative measures for NAFLD.

Asunto(s)

Autofagia , Metilación de ADN , Dioxigenasas , Modelos Animales de Enfermedad , Epigénesis Genética , Hepatocitos , Enfermedad del Hígado Graso no Alcohólico , Hidrolasas Diéster Fosfóricas , Regiones Promotoras Genéticas , Pirofosfatasas , Animales , Humanos , Masculino , Ratones , Autofagia/genética , Tetracloruro de Carbono/toxicidad , Dieta Alta en Grasa/efectos adversos , Dioxigenasas/genética , Dioxigenasas/metabolismo , Hepatocitos/metabolismo , Hepatocitos/patología , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Cirrosis Hepática/metabolismo , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Pirofosfatasas/genética , Pirofosfatasas/metabolismo

RESUMEN

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a human DNA repair protein. It is a member of the phospholipase D family based on structural similarity. TDP1 is a key enzyme of the repair of stalled topoisomerase 1 (TOP1)-DNA complexes. Previously, with the CRISPR/Cas9 method, we obtained HEK293A cells with a homozygous knockout of the TDP1 gene and used the TDP1 knockout cells as a cellular model for studying mechanisms of action of an anticancer therapy. In the present work, we hypothesized that the TDP1 knockout would alter the expression of DNA repair-related genes. By transcriptomic analysis, we investigated for the first time the effect of the TDP1 gene knockout on genes' expression changes in the human HEK293A cell line. We obtained original data implying a role of TDP1 in other processes besides the repair of the DNA-TOP1 complex. Differentially expressed gene analysis revealed that TDP1 may participate in cell adhesion and communication, spermatogenesis, mitochondrial function, neurodegeneration, a cytokine response, and the MAPK signaling pathway.

Asunto(s)

Sistemas CRISPR-Cas , Hidrolasas Diéster Fosfóricas , Humanos , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Células HEK293 , Técnicas de Inactivación de Genes/métodos , Transcriptoma/genética , Perfilación de la Expresión Génica , Reparación del ADN/genética