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1.
Nature ; 626(7999): 626-634, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38326614

RESUMO

Adoptive T cell therapies have produced exceptional responses in a subset of patients with cancer. However, therapeutic efficacy can be hindered by poor T cell persistence and function1. In human T cell cancers, evolution of the disease positively selects for mutations that improve fitness of T cells in challenging situations analogous to those faced by therapeutic T cells. Therefore, we reasoned that these mutations could be co-opted to improve T cell therapies. Here we systematically screened the effects of 71 mutations from T cell neoplasms on T cell signalling, cytokine production and in vivo persistence in tumours. We identify a gene fusion, CARD11-PIK3R3, found in a CD4+ cutaneous T cell lymphoma2, that augments CARD11-BCL10-MALT1 complex signalling and anti-tumour efficacy of therapeutic T cells in several immunotherapy-refractory models in an antigen-dependent manner. Underscoring its potential to be deployed safely, CARD11-PIK3R3-expressing cells were followed up to 418 days after T cell transfer in vivo without evidence of malignant transformation. Collectively, our results indicate that exploiting naturally occurring mutations represents a promising approach to explore the extremes of T cell biology and discover how solutions derived from evolution of malignant T cells can improve a broad range of T cell therapies.


Assuntos
Evolução Molecular , Imunoterapia Adotiva , Linfoma Cutâneo de Células T , Mutação , Linfócitos T , Humanos , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citocinas/biossíntese , Citocinas/imunologia , Citocinas/metabolismo , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Imunoterapia Adotiva/métodos , Linfoma Cutâneo de Células T/genética , Linfoma Cutâneo de Células T/imunologia , Linfoma Cutâneo de Células T/patologia , Linfoma Cutâneo de Células T/terapia , Fosfatidilinositol 3-Quinases , Transdução de Sinais/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplante
2.
Planta ; 259(3): 65, 2024 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-38329545

RESUMO

MAIN CONCLUSION: This study reveals that TRM21 acts as a positive regulator of flavonoid biosynthesis at the translational level in Arabidopsis, impacting both secondary metabolites and genes associated with root hair growth. TRM (TONNEAU1-recruiting motif) superfamily proteins are reported to be involved in microtubule assembly. However, the functions of this protein family are just beginning to be uncovered. Here, we provide metabolomic and genetic evidence that 1 of the 34 TRM members, TRM21, positively regulates the biosynthesis of flavonoids at the translational level in Arabidopsis thaliana. A loss-of-function mutation in TRM21 led to root hair growth defects and stunted plant growth, accompanied by significant alterations in secondary metabolites, particularly a marked reduction in flavonoid content. Interestingly, our study revealed that the transcription levels of genes involved in the flavonoid biosynthesis pathway remained unchanged in the trm21 mutants, but there was a significant downregulation in the translation levels of certain genes [flavanone 3-hydroxylase (F3H), dihydroflavonol-4-reductase (DFR), anthocyanidin reductase (ANR), flavanone 3'-hydroxylase (F3'H), flavonol synthase (FLS), chalcone synthase (CHS)]. Additionally, the translation levels of some genes related to root hair growth [RHO-related GTPases of plant 2 (ROP2), root hair defective 6 (RHD6), root hair defective 2 (RHD2)] were also reduced in the trm21 mutants. Taken together, these results indicate that TRM21 functions as a positive regulator of flavonoid biosynthesis at the translational level in Arabidopsis.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Flavonoides , Proteínas Associadas aos Microtúbulos , Antocianinas , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Regulação para Baixo , Flavonoides/biossíntese , Redes e Vias Metabólicas , Proteínas Associadas aos Microtúbulos/genética
3.
Int J Mol Sci ; 25(3)2024 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-38339093

RESUMO

Lactoferrin (LF) stands as one of the extensively investigated iron-binding glycoproteins within milk, exhibiting diverse biological functionalities. The global demand for LF has experienced consistent growth. Biotechnological strategies aimed at enhancing LF productivity through microbial expression systems offer substantial cost-effective advantages and exhibit fewer constraints compared to traditional animal bioreactor technologies. This study devised a novel recombinant plasmid, wherein the AOX1 promoter was replaced with a glucose-inducible G1 promoter (PG1) to govern the expression of recombinant porcine LF (rpLF) in Pichia pastoris GS115. High-copy-number PG1-rpLF yeast clones were meticulously selected, and subsequent induction with 0.05 g/L glucose demonstrated robust secretion of rpLF. Scaling up production transpired in a 5 L fermenter, yielding an estimated rpLF productivity of approximately 2.8 g/L by the conclusion of glycerol-fed fermentation. A three-step purification process involving tangential-flow ultrafiltration yielded approximately 6.55 g of rpLF crude (approximately 85% purity). Notably, exceptional purity of rpLF was achieved through sequential heparin and size-exclusion column purification. Comparatively, the present glucose-inducible system outperformed our previous methanol-induced system, which yielded a level of 87 mg/L of extracellular rpLF secretion. Furthermore, yeast-produced rpLF demonstrated affinity for ferric ions (Fe3+) and exhibited growth inhibition against various pathogenic microbes (E. coli, S. aureus, and C. albicans) and human cancer cells (A549, MDA-MB-231, and Hep3B), similar to commercial bovine LF (bLF). Intriguingly, the hydrolysate of rpLF (rpLFH) manifested heightened antimicrobial and anticancer effects compared to its intact form. In conclusion, this study presents an efficient glucose-inducible yeast expression system for large-scale production and purification of active rpLF protein with the potential for veterinary or medical applications.


Assuntos
Anti-Infecciosos , Lactoferrina , Proteínas Recombinantes , Animais , Bovinos , Humanos , Anti-Infecciosos/farmacologia , Escherichia coli/metabolismo , Fermentação , Glucose/metabolismo , Lactoferrina/biossíntese , Lactoferrina/genética , Lactoferrina/farmacologia , Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Saccharomycetales , Staphylococcus aureus/efeitos dos fármacos , Suínos
4.
J Cell Biol ; 223(4)2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38323936

RESUMO

Inosine monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in guanosine triphosphate (GTP) synthesis and assembles into filaments in cells, which desensitizes the enzyme to feedback inhibition and boosts nucleotide production. The vertebrate retina expresses two splice variants IMPDH1(546) and IMPDH1(595). In bovine retinas, residue S477 is preferentially phosphorylated in the dark, but the effects on IMPDH1 activity and regulation are unclear. Here, we generated phosphomimetic mutants to investigate structural and functional consequences of S477 phosphorylation. The S477D mutation resensitized both variants to GTP inhibition but only blocked assembly of IMPDH1(595) filaments. Cryo-EM structures of both variants showed that S477D specifically blocks assembly of a high-activity assembly interface, still allowing assembly of low-activity IMPDH1(546) filaments. Finally, we discovered that S477D exerts a dominant-negative effect in cells, preventing endogenous IMPDH filament assembly. By modulating the structure and higher-order assembly of IMPDH, S477 phosphorylation acts as a mechanism for downregulating retinal GTP synthesis in the dark when nucleotide turnover is decreased.


Assuntos
Citoesqueleto , Guanosina Trifosfato , IMP Desidrogenase , Retina , Animais , Bovinos , Guanosina Trifosfato/biossíntese , Nucleotídeos , Fosforilação , Retina/enzimologia , IMP Desidrogenase/metabolismo
5.
Nature ; 626(7999): 653-660, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38267580

RESUMO

Two newly duplicated copies of genomic DNA are held together by the ring-shaped cohesin complex to ensure faithful inheritance of the genome during cell division1-3. Cohesin mediates sister chromatid cohesion by topologically entrapping two sister DNAs during DNA replication4,5, but how cohesion is established at the replication fork is poorly understood. Here, we studied the interplay between cohesin and replication by reconstituting a functional replisome using purified proteins. Once DNA is encircled before replication, the cohesin ring accommodates replication in its entirety, from initiation to termination, leading to topological capture of newly synthesized DNA. This suggests that topological cohesin loading is a critical molecular prerequisite to cope with replication. Paradoxically, topological loading per se is highly rate limiting and hardly occurs under the replication-competent physiological salt concentration. This inconsistency is resolved by the replisome-associated cohesion establishment factors Chl1 helicase and Ctf4 (refs. 6,7), which promote cohesin loading specifically during continuing replication. Accordingly, we found that bubble DNA, which mimics the state of DNA unwinding, induces topological cohesin loading and this is further promoted by Chl1. Thus, we propose that cohesin converts the initial electrostatic DNA-binding mode to a topological embrace when it encounters unwound DNA structures driven by enzymatic activities including replication. Together, our results show how cohesin initially responds to replication, and provide a molecular model for the establishment of sister chromatid cohesion.


Assuntos
Replicação do DNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Cromátides/metabolismo , DNA Fúngico/biossíntese , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Eletricidade Estática
6.
Nature ; 626(7998): 411-418, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38297130

RESUMO

Ferroptosis, a form of regulated cell death that is driven by iron-dependent phospholipid peroxidation, has been implicated in multiple diseases, including cancer1-3, degenerative disorders4 and organ ischaemia-reperfusion injury (IRI)5,6. Here, using genome-wide CRISPR-Cas9 screening, we identified that the enzymes involved in distal cholesterol biosynthesis have pivotal yet opposing roles in regulating ferroptosis through dictating the level of 7-dehydrocholesterol (7-DHC)-an intermediate metabolite of distal cholesterol biosynthesis that is synthesized by sterol C5-desaturase (SC5D) and metabolized by 7-DHC reductase (DHCR7) for cholesterol synthesis. We found that the pathway components, including MSMO1, CYP51A1, EBP and SC5D, function as potential suppressors of ferroptosis, whereas DHCR7 functions as a pro-ferroptotic gene. Mechanistically, 7-DHC dictates ferroptosis surveillance by using the conjugated diene to exert its anti-phospholipid autoxidation function and shields plasma and mitochondria membranes from phospholipid autoxidation. Importantly, blocking the biosynthesis of endogenous 7-DHC by pharmacological targeting of EBP induces ferroptosis and inhibits tumour growth, whereas increasing the 7-DHC level by inhibiting DHCR7 effectively promotes cancer metastasis and attenuates the progression of kidney IRI, supporting a critical function of this axis in vivo. In conclusion, our data reveal a role of 7-DHC as a natural anti-ferroptotic metabolite and suggest that pharmacological manipulation of 7-DHC levels is a promising therapeutic strategy for cancer and IRI.


Assuntos
Desidrocolesteróis , Ferroptose , Humanos , Membrana Celular/metabolismo , Colesterol/biossíntese , Colesterol/metabolismo , Sistemas CRISPR-Cas/genética , Desidrocolesteróis/metabolismo , Genoma Humano , Nefropatias/metabolismo , Membranas Mitocondriais/metabolismo , Metástase Neoplásica , Neoplasias/metabolismo , Neoplasias/patologia , Fosfolipídeos/metabolismo , Traumatismo por Reperfusão/metabolismo
7.
Science ; 383(6683): 622-629, 2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38271490

RESUMO

Paclitaxel is a well known anticancer compound. Its biosynthesis involves the formation of a highly functionalized diterpenoid core skeleton (baccatin III) and the subsequent assembly of a phenylisoserinoyl side chain. Despite intensive investigation for half a century, the complete biosynthetic pathway of baccatin III remains unknown. In this work, we identified a bifunctional cytochrome P450 enzyme [taxane oxetanase 1 (TOT1)] in Taxus mairei that catalyzes an oxidative rearrangement in paclitaxel oxetane formation, which represents a previously unknown enzyme mechanism for oxetane ring formation. We created a screening strategy based on the taxusin biosynthesis pathway and uncovered the enzyme responsible for the taxane oxidation of the C9 position (T9αH1). Finally, we artificially reconstituted a biosynthetic pathway for the production of baccatin III in tobacco.


Assuntos
Alcaloides , Sistema Enzimático do Citocromo P-450 , Engenharia Metabólica , Paclitaxel , Proteínas de Plantas , Taxoides , Taxus , Alcaloides/biossíntese , Alcaloides/genética , Hidrocarbonetos Aromáticos com Pontes/química , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Éteres Cíclicos/química , Éteres Cíclicos/metabolismo , Paclitaxel/biossíntese , Taxoides/metabolismo , Taxus/enzimologia , Taxus/genética , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética
8.
Nature ; 625(7995): 611-617, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38123676

RESUMO

The respiratory syncytial virus (RSV) polymerase is a multifunctional RNA-dependent RNA polymerase composed of the large (L) protein and the phosphoprotein (P). It transcribes the RNA genome into ten viral mRNAs and replicates full-length viral genomic and antigenomic RNAs1. The RSV polymerase initiates RNA synthesis by binding to the conserved 3'-terminal RNA promoters of the genome or antigenome2. However, the lack of a structure of the RSV polymerase bound to the RNA promoter has impeded the mechanistic understanding of RSV RNA synthesis. Here we report cryogenic electron microscopy structures of the RSV polymerase bound to its genomic and antigenomic viral RNA promoters, representing two of the first structures of an RNA-dependent RNA polymerase in complex with its RNA promoters in non-segmented negative-sense RNA viruses. The overall structures of the promoter-bound RSV polymerases are similar to that of the unbound (apo) polymerase. Our structures illustrate the interactions between the RSV polymerase and the RNA promoters and provide the structural basis for the initiation of RNA synthesis at positions 1 and 3 of the RSV promoters. These structures offer a deeper understanding of the pre-initiation state of the RSV polymerase and could aid in antiviral research against RSV.


Assuntos
Regiões Promotoras Genéticas , RNA Polimerase Dependente de RNA , Vírus Sincicial Respiratório Humano , Regiões Promotoras Genéticas/genética , Vírus Sincicial Respiratório Humano/enzimologia , Vírus Sincicial Respiratório Humano/genética , RNA Viral/biossíntese , RNA Viral/genética , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , RNA Polimerase Dependente de RNA/ultraestrutura , Replicação Viral/genética , Microscopia Crioeletrônica , RNA Subgenômico/biossíntese , RNA Subgenômico/genética , RNA Subgenômico/metabolismo
9.
Nature ; 626(7997): 186-193, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38096901

RESUMO

The long interspersed element-1 (LINE-1, hereafter L1) retrotransposon has generated nearly one-third of the human genome and serves as an active source of genetic diversity and human disease1. L1 spreads through a mechanism termed target-primed reverse transcription, in which the encoded enzyme (ORF2p) nicks the target DNA to prime reverse transcription of its own or non-self RNAs2. Here we purified full-length L1 ORF2p and biochemically reconstituted robust target-primed reverse transcription with template RNA and target-site DNA. We report cryo-electron microscopy structures of the complete human L1 ORF2p bound to structured template RNAs and initiating cDNA synthesis. The template polyadenosine tract is recognized in a sequence-specific manner by five distinct domains. Among them, an RNA-binding domain bends the template backbone to allow engagement of an RNA hairpin stem with the L1 ORF2p C-terminal segment. Moreover, structure and biochemical reconstitutions demonstrate an unexpected target-site requirement: L1 ORF2p relies on upstream single-stranded DNA to position the adjacent duplex in the endonuclease active site for nicking of the longer DNA strand, with a single nick generating a staggered DNA break. Our research provides insights into the mechanism of ongoing transposition in the human genome and informs the engineering of retrotransposon proteins for gene therapy.


Assuntos
DNA Complementar , Elementos Nucleotídeos Longos e Dispersos , RNA , Retroelementos , Transcrição Reversa , Humanos , Microscopia Crioeletrônica , DNA Complementar/biossíntese , DNA Complementar/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Retroelementos/genética , RNA/química , RNA/genética , RNA/metabolismo , Domínio Catalítico , Endonucleases/química , Endonucleases/metabolismo , Endonucleases/ultraestrutura , Terapia Genética , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/metabolismo , DNA Polimerase Dirigida por RNA/ultraestrutura , DNA de Cadeia Simples/metabolismo , Quebras de DNA
10.
Nature ; 626(7997): 194-206, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38096902

RESUMO

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.


Assuntos
Endonucleases , Elementos Nucleotídeos Longos e Dispersos , DNA Polimerase Dirigida por RNA , Transcrição Reversa , Humanos , Microscopia Crioeletrônica , Endonucleases/química , Endonucleases/genética , Endonucleases/metabolismo , Elementos Nucleotídeos Longos e Dispersos/genética , RNA/genética , DNA Polimerase Dirigida por RNA/química , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Cristalografia por Raios X , DNA/biossíntese , DNA/genética , Imunidade Inata , Interferons/biossíntese
11.
Nature ; 626(7998): 385-391, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38096903

RESUMO

A limitation of current SARS-CoV-2 vaccines is that they provide minimal protection against infection with current Omicron subvariants1,2, although they still provide protection against severe disease. Enhanced mucosal immunity may be required to block infection and onward transmission. Intranasal administration of current vaccines has proven inconsistent3-7, suggesting that alternative immunization strategies may be required. Here we show that intratracheal boosting with a bivalent Ad26-based SARS-CoV-2 vaccine results in substantial induction of mucosal humoral and cellular immunity and near-complete protection against SARS-CoV-2 BQ.1.1 challenge. A total of 40 previously immunized rhesus macaques were boosted with a bivalent Ad26 vaccine by the intramuscular, intranasal and intratracheal routes, or with a bivalent mRNA vaccine by the intranasal route. Ad26 boosting by the intratracheal route led to a substantial expansion of mucosal neutralizing antibodies, IgG and IgA binding antibodies, and CD8+ and CD4+ T cell responses, which exceeded those induced by Ad26 boosting by the intramuscular and intranasal routes. Intratracheal Ad26 boosting also led to robust upregulation of cytokine, natural killer, and T and B cell pathways in the lungs. After challenge with a high dose of SARS-CoV-2 BQ.1.1, intratracheal Ad26 boosting provided near-complete protection, whereas the other boosting strategies proved less effective. Protective efficacy correlated best with mucosal humoral and cellular immune responses. These data demonstrate that these immunization strategies induce robust mucosal immunity, suggesting the feasibility of developing vaccines that block respiratory viral infections.


Assuntos
Vacinas contra COVID-19 , COVID-19 , Imunidade nas Mucosas , Imunização Secundária , Macaca mulatta , SARS-CoV-2 , Animais , Humanos , Administração Intranasal , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/imunologia , Citocinas/imunologia , Imunidade nas Mucosas/imunologia , Imunização Secundária/métodos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Injeções Intramusculares , Células Matadoras Naturais/imunologia , Pulmão/imunologia , Macaca mulatta/imunologia , Macaca mulatta/virologia , Vacinas de mRNA/administração & dosagem , Vacinas de mRNA/imunologia , SARS-CoV-2/classificação , SARS-CoV-2/imunologia , Traqueia/imunologia , Traqueia/virologia
12.
J Biol Chem ; 300(1): 105572, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38110032

RESUMO

Mutations in, or deficiency of, fragile X messenger ribonucleoprotein (FMRP) is responsible for the Fragile X syndrome (FXS), the most common cause for inherited intellectual disability. FMRP is a nucleocytoplasmic protein, primarily characterized as a translation repressor with poorly understood nuclear function(s). We recently reported that FXS patient cells lacking FMRP sustain higher level of DNA double-strand breaks (DSBs) than normal cells, specifically at sequences prone to forming R-loops, a phenotype further exacerbated by DNA replication stress. Moreover, expression of FMRP, and not an FMRPI304N mutant known to cause FXS, reduced R-loop-associated DSBs. We subsequently reported that recombinant FMRP directly binds R-loops, primarily through the carboxyl terminal intrinsically disordered region. Here, we show that FMRP directly interacts with an RNA helicase, DHX9. This interaction, which is mediated by the amino terminal structured domain of FMRP, is reduced with FMRPI304N. We also show that FMRP inhibits DHX9 helicase activity on RNA:DNA hybrids and the inhibition is also dependent on the amino terminus. Furthermore, the FMRPI304N mutation causes both FMRP and DHX9 to persist on the chromatin in replication stress. These results suggest an antagonistic relationship between FMRP and DHX9 at the chromatin, where their proper interaction leads to dissociation of both proteins from the fully resolved R-loop. We propose that the absence or the loss of function of FMRP leads to persistent presence of DHX9 or both proteins, respectively, on the unresolved R-loop, ultimately leading to DSBs. Our study sheds new light on our understanding of the genome functions of FMRP.


Assuntos
RNA Helicases DEAD-box , Replicação do DNA , Proteína do X Frágil de Retardo Mental , Proteínas de Neoplasias , Estresse Fisiológico , Humanos , Cromatina/genética , Cromatina/metabolismo , RNA Helicases DEAD-box/metabolismo , DNA/biossíntese , DNA/química , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Proteína do X Frágil de Retardo Mental/genética , Proteína do X Frágil de Retardo Mental/metabolismo , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Mutação , Proteínas de Neoplasias/metabolismo , Hibridização de Ácido Nucleico , Estruturas R-Loop , RNA/química , RNA/metabolismo
13.
J Biol Chem ; 300(1): 105587, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38141768

RESUMO

The Saccharomyces cerevisiae Nem1-Spo7 protein phosphatase complex dephosphorylates and thereby activates Pah1 at the nuclear/endoplasmic reticulum membrane. Pah1, a phosphatidate phosphatase catalyzing the dephosphorylation of phosphatidate to produce diacylglycerol, is one of the most highly regulated enzymes in lipid metabolism. The diacylglycerol produced in the lipid phosphatase reaction is utilized for the synthesis of triacylglycerol that is stored in lipid droplets. Disruptions of the Nem1-Spo7/Pah1 phosphatase cascade cause a plethora of physiological defects. Spo7, the regulatory subunit of the Nem1-Spo7 complex, is required for the Nem1 catalytic function and interacts with the acidic tail of Pah1. Spo7 contains three conserved homology regions (CR1-3) that are important for the interaction with Nem1, but its region for the interaction with Pah1 is unknown. Here, by deletion and site-specific mutational analyses of Spo7, we revealed that the C-terminal basic tail (residues 240-259) containing five arginine and two lysine residues is important for the Nem1-Spo7 complex-mediated dephosphorylation of Pah1 and its cellular function (triacylglycerol synthesis, lipid droplet formation, maintenance of nuclear/endoplasmic reticulum membrane morphology, and cell growth at elevated temperatures). The glutaraldehyde cross-linking analysis of synthetic peptides indicated that the Spo7 basic tail interacts with the Pah1 acidic tail. This work advances our understanding of the Spo7 function and the Nem1-Spo7/Pah1 phosphatase cascade in yeast lipid synthesis.


Assuntos
Proteínas de Membrana , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Diglicerídeos/biossíntese , Proteínas Nucleares/metabolismo , Fosfatidato Fosfatase/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Triglicerídeos/biossíntese , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo
14.
J Biol Chem ; 300(1): 105567, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38103641

RESUMO

The role of RNA G-quadruplexes (rG4s) in bacteria remains poorly understood. High G-quadruplex densities have been linked to organismal stress. Here we investigate rG4s in mycobacteria, which survive highly stressful conditions within the host. We show that rG4-enrichment is a unique feature exclusive to slow-growing pathogenic mycobacteria, and Mycobacterium tuberculosis (Mtb) transcripts contain an abundance of folded rG4s. Notably, the PE/PPE family of genes, unique to slow-growing pathogenic mycobacteria, contain over 50% of rG4s within Mtb transcripts. We found that RNA oligonucleotides of putative rG4s in PE/PPE genes form G-quadruplex structures in vitro, which are stabilized by the G-quadruplex ligand BRACO19. Furthermore, BRACO19 inhibits the transcription of PE/PPE genes and selectively suppresses the growth of Mtb but not Mycobacterium smegmatis or other rapidly growing bacteria. Importantly, the stabilization of rG4s inhibits the translation of Mtb PE/PPE genes (PPE56, PPE67, PPE68, PE_PGRS39, and PE_PGRS41) ectopically expressed in M. smegmatis or Escherichia coli. In addition, the rG4-mediated reduction in PE/PPE protein levels attenuates proinflammatory response upon infection of THP-1 cells. Our findings shed new light on the regulation of PE/PPE genes and highlight a pivotal role for rG4s in Mtb transcripts as regulators of post-transcriptional translational control. The rG4s in mycobacterial transcripts may represent potential drug targets for newer therapies.


Assuntos
Proteínas de Bactérias , Quadruplex G , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis , Biossíntese de Proteínas , RNA Bacteriano , RNA Mensageiro , Humanos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos/genética , Inflamação/microbiologia , Ligantes , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Estabilidade de RNA , RNA Bacteriano/genética , RNA Mensageiro/genética , Células THP-1 , Transcrição Gênica/efeitos dos fármacos
15.
J Biol Chem ; 300(1): 105566, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38103643

RESUMO

Macrophages play critical roles in inflammation and tissue homeostasis, and their functions are regulated by various autocrine, paracrine, and endocrine factors. We have previously shown that CTRP6, a secreted protein of the C1q family, targets both adipocytes and macrophages to promote obesity-linked inflammation. However, the gene programs and signaling pathways directly regulated by CTRP6 in macrophages remain unknown. Here, we combine transcriptomic and phosphoproteomic analyses to show that CTRP6 activates inflammatory gene programs and signaling pathways in mouse bone marrow-derived macrophages (BMDMs). Treatment of BMDMs with CTRP6 upregulated proinflammatory, and suppressed the antiinflammatory, gene expression. We also showed that CTRP6 activates p44/42-MAPK, p38-MAPK, and NF-κB signaling pathways to promote inflammatory cytokine secretion from BMDMs, and that pharmacologic inhibition of these signaling pathways markedly attenuated the effects of CTRP6. Pretreatment of BMDMs with CTRP6 also sensitized and potentiated the BMDMs response to lipopolysaccharide (LPS)-induced inflammatory signaling and cytokine secretion. Consistent with the metabolic phenotype of proinflammatory macrophages, CTRP6 treatment induced a shift toward aerobic glycolysis and lactate production, reduced oxidative metabolism, and elevated mitochondrial reactive oxygen species production in BMDMs. Importantly, in accordance with our in vitro findings, BMDMs from CTRP6-deficient mice were less inflammatory at baseline and showed a marked suppression of LPS-induced inflammatory gene expression and cytokine secretion. Finally, loss of CTRP6 in mice also dampened LPS-induced inflammation and hypothermia. Collectively, our findings suggest that CTRP6 regulates and primes the macrophage response to inflammatory stimuli and thus may have a role in modulating tissue inflammatory tone in different physiological and disease contexts.


Assuntos
Adipocinas , Perfilação da Expressão Gênica , Inflamação , Lipopolissacarídeos , Macrófagos , Fosfoproteínas , Proteômica , Animais , Camundongos , Adipocinas/deficiência , Adipocinas/genética , Adipocinas/metabolismo , Células da Medula Óssea/citologia , Citocinas/metabolismo , Glicólise , Hipotermia/complicações , Inflamação/complicações , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Ácido Láctico/biossíntese , Lipopolissacarídeos/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Transdução de Sinais , Espécies Reativas de Oxigênio/metabolismo
16.
J Biol Chem ; 299(12): 105407, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38152849

RESUMO

Cell proliferation requires metabolic reprogramming to accommodate biosynthesis of new cell components, and similar alterations occur in cancer cells. However, the mechanisms linking the cell cycle machinery to metabolism are not well defined. Cyclin D1, along with its main partner cyclin-dependent kinase 4 (Cdk4), is a pivotal cell cycle regulator and driver oncogene that is overexpressed in many cancers. Here, we examine hepatocyte proliferation to define novel effects of cyclin D1 on biosynthetic metabolism. Metabolomic studies reveal that cyclin D1 broadly promotes biosynthetic pathways including glycolysis, the pentose phosphate pathway, and the purine and pyrimidine nucleotide synthesis in hepatocytes. Proteomic analyses demonstrate that overexpressed cyclin D1 binds to numerous metabolic enzymes including those involved in glycolysis and pyrimidine synthesis. In the glycolysis pathway, cyclin D1 activates aldolase and GAPDH, and these proteins are phosphorylated by cyclin D1/Cdk4 in vitro. De novo pyrimidine synthesis is particularly dependent on cyclin D1. Cyclin D1/Cdk4 phosphorylates the initial enzyme of this pathway, carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and metabolomic analysis indicates that cyclin D1 depletion markedly reduces the activity of this enzyme. Pharmacologic inhibition of Cdk4 along with the downstream pyrimidine synthesis enzyme dihydroorotate dehydrogenase synergistically inhibits proliferation and survival of hepatocellular carcinoma cells. These studies demonstrate that cyclin D1 promotes a broad network of biosynthetic pathways in hepatocytes, and this model may provide insights into potential metabolic vulnerabilities in cancer cells.


Assuntos
Vias Biossintéticas , Ciclina D1 , Hepatócitos , Ciclina D1/genética , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Hepatócitos/metabolismo , Proteômica , Pirimidinas/biossíntese , Humanos , Animais , Camundongos , Linhagem Celular
17.
Aging (Albany NY) ; 16(1): 106-128, 2023 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-38157259

RESUMO

BACKGROUND: Patients with chronic kidney disease (CKD) lack efficacious treatment. Jian-Pi-Yi-Shen formula (JPYSF) has demonstrated significant clinical efficacy in treating CKD for decades. However, its renoprotective mechanism has not been fully elucidated. This study aimed to determine whether JPYSF could delay renal fibrosis progression in CKD by restoring nicotinamide adenine dinucleotide (NAD+) biosynthesis. METHODS: Adenine-diet feeding was used to model CKD in C57BL/6 mice. JPYSF was orally administered for 4 weeks. Human proximal tubular epithelial cells (HK-2) cells were stimulated with transforming growth factor-ß1 (TGF-ß1) with or without JPYSF treatment. Renal function of mice was assessed by serum creatinine and blood urea nitrogen levels. Renal histopathological changes were assessed using Periodic acid-Schiff and Masson's trichrome staining. Cell viability was assessed using a cell counting kit-8 assay. NAD+ concentrations were detected by a NAD+/NADH assay kit. Western blotting, immunohistochemistry, and immunofluorescence were employed to examine fibrosis-related proteins and key NAD+ biosynthesis enzymes expression in the CKD kidney and TGF-ß1-induced HK-2 cells. RESULTS: JPYSF treatment could not only improve renal function and pathological injury but also inhibit renal fibrosis in CKD mice. Additionally, JPYSF reversed fibrotic response in TGF-ß1-induced HK-2 cells. Moreover, JPYSF rescued the decreased NAD+ content in CKD mice and TGF-ß1-induced HK-2 cells through restoring expression of key enzymes in NAD+ biosynthesis, including quinolinate phosphoribosyltransferase, nicotinamide mononucleotide adenylyltransferase 1, and nicotinamide riboside kinase 1. CONCLUSIONS: JPYSF alleviated renal fibrosis in CKD mice and reversed fibrotic response in TGF-ß1-induced HK-2 cells, which may be related to the restoration of NAD+ biosynthesis.


Assuntos
NAD , Insuficiência Renal Crônica , Animais , Humanos , Camundongos , Fibrose , Rim/patologia , Camundongos Endogâmicos C57BL , NAD/biossíntese , Insuficiência Renal Crônica/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
18.
Science ; 382(6677): eadi5120, 2023 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-38127763

RESUMO

Transcription initiation is a complex process, and its mechanism is incompletely understood. We determined the structures of de novo transcribing complexes TC2 to TC17 with RNA polymerase II halted on G-less promoters when nascent RNAs reach 2 to 17 nucleotides in length, respectively. Connecting these structures generated a movie and a working model. As initially synthesized RNA grows, general transcription factors (GTFs) remain bound to the promoter and the transcription bubble expands. Nucleoside triphosphate (NTP)-driven RNA-DNA translocation and template-strand accumulation in a nearly sealed channel may promote the transition from initially transcribing complexes (ITCs) (TC2 to TC9) to early elongation complexes (EECs) (TC10 to TC17). Our study shows dynamic processes of transcription initiation and reveals why ITCs require GTFs and bubble expansion for initial RNA synthesis, whereas EECs need GTF dissociation from the promoter and bubble collapse for promoter escape.


Assuntos
RNA , Fatores Genéricos de Transcrição , Iniciação da Transcrição Genética , RNA Polimerases Dirigidas por DNA/química , RNA/biossíntese , RNA Polimerase II/química , Fatores Genéricos de Transcrição/metabolismo , Humanos , Animais , Sus scrofa , Microscopia Crioeletrônica , Filmes Cinematográficos
19.
JAMA ; 330(21): 2075-2083, 2023 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-37952254

RESUMO

Importance: Epidemiological and genetic data have implicated lipoprotein(a) as a potentially modifiable risk factor for atherosclerotic disease and aortic stenosis, but there are no approved pharmacological treatments. Objectives: To assess the safety, tolerability, pharmacokinetics, and effects of lepodisiran on lipoprotein(a) concentrations after single doses of the drug; lepodisiran is a short interfering RNA directed at hepatic synthesis of apolipoprotein(a), an essential component necessary for assembly of lipoprotein(a) particles. Design, Setting, and Participants: A single ascending-dose trial conducted at 5 clinical research sites in the US and Singapore that enrolled 48 adults without cardiovascular disease and with lipoprotein(a) serum concentrations of 75 nmol/L or greater (or ≥30 mg/dL) between November 18, 2020, and December 7, 2021; the last follow-up visit occurred on November 9, 2022. Interventions: Participants were randomized to receive placebo or a single dose of lepodisiran (4 mg, 12 mg, 32 mg, 96 mg, 304 mg, or 608 mg) administered subcutaneously. Main Outcomes and Measures: The primary outcome was the safety and tolerability of the single ascending doses of lepodisiran. The secondary outcomes included plasma levels of lepodisiran for 168 days after dose administration and changes in fasting lipoprotein(a) serum concentrations through a maximum follow-up of 336 days (48 weeks). Results: Of the 48 participants enrolled (mean age, 46.8 [SD, 11.6] years; 35% were women), 1 serious adverse event occurred. The plasma concentrations of lepodisiran reached peak levels within 10.5 hours and were undetectable by 48 hours. The median baseline lipoprotein(a) concentration was 111 nmol/L (IQR, 78 to 134 nmol/L) in the placebo group, 78 nmol/L (IQR, 50 to 152 nmol/L) in the 4 mg of lepodisiran group, 97 nmol/L (IQR, 86 to 107 nmol/L) in the 12-mg dose group, 120 nmol/L (IQR, 110 to 188 nmol/L) in the 32-mg dose group, 167 nmol/L (IQR, 124 to 189 nmol/L) in the 96-mg dose group, 96 nmol/L (IQR, 72 to 132 nmol/L) in the 304-mg dose group, and 130 nmol/L (IQR, 87 to 151 nmol/L) in the 608-mg dose group. The maximal median change in lipoprotein(a) concentration was -5% (IQR, -16% to 11%) in the placebo group, -41% (IQR, -47% to -20%) in the 4 mg of lepodisiran group, -59% (IQR, -66% to -53%) in the 12-mg dose group, -76% (IQR, -76% to -75%) in the 32-mg dose group, -90% (IQR, -94% to -85%) in the 96-mg dose group, -96% (IQR, -98% to -95%) in the 304-mg dose group, and -97% (IQR, -98% to -96%) in the 608-mg dose group. At day 337, the median change in lipoprotein(a) concentration was -94% (IQR, -94% to -85%) in the 608 mg of lepodisiran group. Conclusions and Relevance: In this phase 1 study of 48 participants with elevated lipoprotein(a) levels, lepodisiran was well tolerated and produced dose-dependent, long-duration reductions in serum lipoprotein(a) concentrations. The findings support further study of lepodisiran. Trial Registration: ClinicalTrials.gov Identifier: NCT04914546.


Assuntos
Apolipoproteínas A , Lipoproteína(a) , RNA Interferente Pequeno , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Método Duplo-Cego , Lipoproteína(a)/antagonistas & inibidores , Lipoproteína(a)/sangue , Fatores de Risco , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/efeitos adversos , RNA Interferente Pequeno/uso terapêutico , Singapura , Apolipoproteínas A/biossíntese , Fígado/metabolismo , Administração Cutânea , Estados Unidos
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