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BACKGROUND: Metabolic engineering enables the sustainable and cost-efficient production of complex chemicals. Efficient production of terpenes in Saccharomyces cerevisiae can be achieved by recruiting an intermediate of the mevalonate pathway. The present study aimed to evaluate the engineering strategies of S. cerevisiae for the production of taxadiene, a precursor of taxol, an antineoplastic drug. RESULT: SCIGS22a, a previously engineered strain with modifications in the mevalonate pathway (MVA), was used as a background strain. This strain was engineered to enable a high flux towards farnesyl diphosphate (FPP) and the availability of NADPH. The strain MVA was generated from SCIGS22a by overexpressing all mevalonate pathway genes. Combining the background strains with 16 different episomal plasmids, which included the combination of 4 genes: tHMGR (3-hydroxy-3-methylglutaryl-CoA reductase), ERG20 (farnesyl pyrophosphate synthase), GGPPS (geranyl diphosphate synthase) and TS (taxadiene synthase) resulted in the highest taxadiene production in S. cerevisiae of 528 mg/L. CONCLUSION: Our study highlights the critical role of pathway balance in metabolic engineering, mainly when dealing with toxic molecules like taxadiene. We achieved significant improvements in taxadiene production by employing a combinatorial approach and focusing on balancing the downstream and upstream pathways. These findings emphasize the importance of minor gene expression modification levels to achieve a well-balanced pathway, ultimately leading to enhanced taxadiene accumulation.
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Ingeniería Metabólica , Ácido Mevalónico , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ingeniería Metabólica/métodos , Ácido Mevalónico/metabolismo , Alquenos/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Diterpenos/metabolismo , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , NADP/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , SesquiterpenosRESUMEN
Aurantiochytrium sp. 18W-13a, a marine heterotrophic protist belonging to the genus thraustochytrid, is known to accumulate high levels of squalene and carotenoids. Nowadays, the mutagenesis breeding of microorganisms is still widely practiced because the induced mutations of DNA do not involve the permanent integration of heterologous DNA sequences. Therefore, in this study, we focused on the improvement of squalene yield by mutagenesis breeding using Aurantiochytrium sp. 18W-13a. To bypass the massively laborious screening, we propose to use colony colors as the first criterion to screen mutants with high squalene accumulation, since the carotenoid and squalene synthetic pathways share an intermediate. We selected pale (white)-colored mutants after carbon ion irradiation. The white mutants exhibited larger squalene yields than twice as much of the original strain. The results clearly indicate that the present screening method with colony colors promises to obtain productive strains of squalene.
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Lactiplantibacillus plantarum is a food-grade lactic acid bacterium widely used in the food and beverage industry. Recently, this probiotic organism has been applied as a biofactory for the production of pharmaceutical and food-related compounds, but existing promoters and expression vectors for the genetic engineering of L. plantarum rely on inefficient cloning strategies and are usually not well-characterized. We therefore developed a modular and standardized Golden Gate Assembly-based toolbox for the de novo assembly of shuttle vectors from Escherichia coli to L. plantarum. A collection of the most relevant genetic parts, e.g., different origins of replication and promoters, was incorporated in our toolbox and thoroughly characterized by flow cytometry and the fluorescence assay. Standardized fusion sites allow combining the genetic part freely into a plasmid in one step. This approach allows for the high-throughput assembly of numerous constructs in a standardized genetic context, thus improving the efficiency and predictability of metabolic engineering in L. plantarum. Using our toolbox, we were able to produce the aroma compounds linalool and geraniol in L. plantarum by extending its native mevalonate pathway with plant-derived monoterpenoid synthases.
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Monoterpenos Acíclicos , Escherichia coli , Lactobacillus plantarum , Ingeniería Metabólica , Monoterpenos , Ingeniería Metabólica/métodos , Lactobacillus plantarum/genética , Lactobacillus plantarum/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Monoterpenos/metabolismo , Monoterpenos Acíclicos/metabolismo , Plásmidos/genética , Terpenos/metabolismo , Regiones Promotoras Genéticas/genética , Vectores Genéticos/genéticaRESUMEN
Porokeratoses (PK) are a group of uncommon dermatoses characterized by abnormal epidermal differentiation due to a disorder of the mevalonate metabolic pathway. Several clinical subtypes exist that can be associated with the same patient or affect different patients within a family and could, therefore, be different expressions of one disease. All PK subtypes share a common histopathologic finding, the cornoid lamella, a vertical stack of parakeratotic corneocytes embedded in an orthokeratotic horny layer. PK often affects immunosuppressed patients, in whom the course may parallel the level of immunosuppression. The pathogenesis of PK, which had long remained mysterious, has been recently unraveled after discovering pathogenic variants of genes involved in the mevalonate metabolic pathway. The disease is due to germline pathogenic variants of genes of this pathway but requires a second-hit event to manifest; therefore, PK is considered a dominantly inherited but recessively expressed condition. The prognosis of PK is usually favorable, even though the lesions progress to keratinocyte carcinomas in 7%-16% of patients. The treatment of PK was based on physical (ablative) procedures and various (topical or systemic) treatments, whose efficacy is nevertheless inconsistent and often temporary. The discovery of the metabolic pathway involved in the pathogenesis of PK paved the way for the elaboration of new topical treatments (combination of statins and cholesterol), which are more regularly efficacious compared with older treatments, even though the management of some patients with PK may still be challenging.
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We present a patient with a rare systemic autoinflammatory disease (mevalonate kinase deficiency -MKD-) with the identification of two heterozygous variants (c.1129G>A and c.32C>T) in the Mevalonate Kinase gene, detected by next generation sequencing and a highly prevalent glomerulonephritis (IgA nephropathy). The patient presents clinically with a monthly recurrent periodic fever from 12 days of age, accompanied by mucocutaneous lesions (maculopapular rash in extremities, aphthous stomatitis), joint (arthralgias in ankles, wrists and knees), lymphoid (cervical lymphadenopathy, splenomegaly), gastrointestinal (diarrhea, abdominal pain) and kidney (hematuria and proteinuria) with repeated biopsies showing IgA nephropathy alternating activity with chronicity. During follow-up. The patients presented a poor therapeutic response to multiple immunosuppressive regimens used for 7 years (corticosteroids, azathioprine, mycophenolate, cyclophosphamide, rituximab and tocilizumab), and finally a good response to canakinumab. Four years after starting canakinumab, during the course of an infection due to a muscle abscess, the clinical presentation is complicated by a severe renal microvascular event (renal cortical necrosis -RCN-) with acute kidney injury and dialysis requirement. Therecurrent episodes of inflammation due to MKD could act as triggers for the reactivation of glomerulonephritis (which would explain the poor response to immunosuppressants and the rapid progression to histological chronicity) and to generate a microenvironment that predisposes the development of RCN in the face of a non-serious infection. A defect in IgA molecules has been described in MKD, a phenomenon also observed in IgA nephropathy. This raises the challenging hypothesis of a common pathogenetic link between all the patient's clinical manifestations.
Presentamos un paciente con una rara enfermedad autoinflamatoria sistémica (deficiencia de mevalonato quinasa -DMQ-) con la identificación de dos variantes heterocigotas (c.1129G>A y c.32C>T) en el gen Mevalonato Quinasa, detectadas por secuenciación masiva en paralelo y una glomerulonefritis de alta prevalencia (nefropatía por IgA). El paciente presentó un cuadro de fiebre periódica recurrente mensual desde los 12 días de vida, acompañada de lesiones mucocutáneas (rash maculopapular en extremidades, estomatitis aftosa), compromiso articular (artralgias en tobillos, muñecas y rodillas), linfoideo (linfoadenopatía cervical, esplenomegalia), gastrointestinal (diarrea, dolor abdominal) y renal (hematuria y proteinuria) con repetidas biospias mostrando nefropatía por IgA alternando actividad y cronicidad. Durante el seguimiento, tuvo una pobre respuesta terapéutica a múltiples esquemas inmunosupresores utilizados durante 7 años (corticoides, azatrioprina, micofenolato, ciclofosfamida, rituximab y tocilizumab), y buena respuesta finalmente a canakinumab. Cuatro años posteriores al inicio de canakinumab, durante el curso de una infección por un absceso muscular, el cuadro clínico se complica con un evento microvascular renal grave (necrosis cortical renal -NCR-) con fallo renal agudo y necesidad de diálisis. Los episodios recurrentes de inflamación por la DMQ podrían actuar como gatillos para la reactivación de su glomerulonefritis (lo que explicaría la escasa respuesta a inmunosupresores y la progresión rápida a cronicidad histológica) y para generar un microambiente que predisponga el desarrollo de una NCR ante una infección no grave. En la DMQ se ha descripto un defecto en las moléculas de IgA, fenómeno también observado en la nefropatía por IgA. Esto plantea la desafiante hipótesis de un vínculo patogénico común entre todas las manifestaciones clínicas del paciente.
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Glomerulonefritis por IGA , Necrosis de la Corteza Renal , Humanos , Glomerulonefritis por IGA/complicaciones , Glomerulonefritis por IGA/patología , Necrosis de la Corteza Renal/etiología , Necrosis de la Corteza Renal/patología , Masculino , Femenino , AdultoRESUMEN
The prognosis of HER2-positive breast cancer (BC) has improved with the development of anti-HER2 therapies; however, the problem remains that there are still cases where anti-HER2 therapies do not respond well. We found that the expression of SREBF2, a master transcriptional factor in the mevalonate pathway, was correlated with ERBB2 (HER2) expression and a poor prognosis in HER2-positive BC. The target gene expressions of SREBF2 were associated with higher expression of ERBB2 in HER2-positive BC cells. Statins, anti-hypercholesterolemia drugs that inhibit the mevalonate pathway, enhanced the efficacy of HER2-targeting agents with inducing apoptosis in a geranylgeranylation-dependent manner. Mechanistically, statins specifically inhibited membrane localization of Rac1, a target protein of geranylgeranylation, and suppressed the activation of HER2 downstreams AKT and ERK pathways. Consistently, retrospective analysis showed a longer recurrence-free survival in Rac1-high/HER2-positive BC patients treated with HER2-targeting agents with statins than without statins. Our findings thus suggest that Rac1 expression could be used as a biomarker to stratify HER2-positive BC patients that could benefit from dual blockade, i.e., targeting HER2 with inhibition of geranylgeranylation of Rac1 using statins, thereby opening avenues for precision medicine in a new subset of Rac1-high/HER2-positive BC.
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Neoplasias de la Mama , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Receptor ErbB-2 , Proteína de Unión al GTP rac1 , Humanos , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/mortalidad , Femenino , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Pronóstico , Línea Celular Tumoral , Persona de Mediana Edad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Apoptosis/efectos de los fármacosRESUMEN
Membrane-anchored proteins play critical roles in cell signaling, cellular architecture, and membrane biology. Hydrophilic proteins are post-translationally modified by a diverse range of lipid molecules such as phospholipids, glycosylphosphatidylinositol, and isoprenes, which allows their partition and anchorage to the cell membrane. In this review article, we discuss the biochemical basis of isoprenoid synthesis, the mechanisms of isoprene conjugation to proteins, and the functions of prenylated proteins in the neural retina. Recent discovery of novel prenyltransferases, prenylated protein chaperones, non-canonical prenylation-target motifs, and reversible prenylation is expected to increase the number of inherited systemic and blinding diseases with aberrant protein prenylation. Recent important investigations have also demonstrated the role of several unexpected regulators (such as protein charge, sequence/protein-chaperone interaction, light exposure history) in the photoreceptor trafficking of prenylated proteins. Technical advances in the investigation of the prenylated proteome and its application in vision research are discussed. Clinical updates and technical insights into known and putative prenylation-associated retinopathies are provided herein. Characterization of non-canonical prenylation mechanisms in the retina and retina-specific prenylated proteome is fundamental to the understanding of the pathogenesis of protein prenylation-associated inherited blinding disorders.
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The sterol regulatory element-binding protein (SREBP) pathway is an integral cellular mechanism that regulates lipid homeostasis, in which transcriptional activator SREBPs regulate the expression of various genes. In the carotenogenic yeast Xanthophyllomyces dendrorhous, Sre1 (the yeast SREBP homolog) regulates lipid biosynthesis and carotenogenesis, among other processes. Despite the characterization of several components of the SREBP pathway across various eukaryotes, the specific elements of this pathway in X. dendrorhous remain largely unknown. This study aimed to explore the potential regulatory mechanisms of the SREBP pathway in X. dendrorhous using the strain CBS.cyp61- as a model, which is known to have Sre1 in its active state under standard culture conditions, resulting in a carotenoid-overproducing phenotype. This strain was subjected to random mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (NTG), followed by a screening methodology that focused on identifying mutants with altered Sre1 activation phenotypes. Single-nucleotide polymorphism (SNP) analysis of 20 selected mutants detected 5439 single-nucleotide variants (SNVs), narrowing them down to 1327 SNPs of interest after a series of filters. Classification based on SNP impact identified 116 candidate genes, including 49 genes with high impact and 68 genes with deleterious moderate-impact mutations. BLAST, InterProScan, and gene ontology enrichment analyses highlighted 25 genes as potential participants in regulating Sre1 in X. dendrorhous. The key findings of this study include the identification of genes potentially encoding proteins involved in protein import/export to the nucleus, sterol biosynthesis, the ubiquitin-proteasome system, protein regulatory activities such as deacetylases, a subset of kinases and proteases, as well as transcription factors that could be influential in SREBP regulation. These findings are expected to significantly contribute to the current understanding of the intricate regulation of the transcription factor Sre1 in X. dendrorhous, providing valuable groundwork for future research and potential biotechnological applications.
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Basidiomycota , Proteínas de Unión a los Elementos Reguladores de Esteroles , Basidiomycota/genética , Basidiomycota/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/genética , Polimorfismo de Nucleótido Simple , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Carotenoides/metabolismo , MutaciónRESUMEN
Hyperimmunoglobulin D syndrome (HIDS) is a rare but severe autoinflammatory disease with a poor prognosis if not diagnosed and treated early. Here, we report three cases of HIDS in children with typical clinical manifestations and a clear genetic diagnosis. Patient 1 experienced recurrent fever flares with a maculo-papular skin rash. Patient 2 presented with periodic fever, cholestasis, lymphadenopathy, aphthous stomatitis, arthralgia, and abdominal pain and underwent surgery for intestinal obstruction. Patient 3, a sibling of patient 2, presented with periodic fever and underwent a surgical procedure for intussusception. All three patients were administered interleukin (IL)-6 receptor antagonist (tocilizumab). The results showed that tocilizumab effectively reduced inflammatory flares. Early diagnosis and tocilizumab treatment are effective at improving the prognosis of HIDS patients.
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Introduction: Blumea balsamifera L. (Ainaxiang) DC. is a perennial herb of the compositae family. It is also the primary source of natural borneol. Endo-borneol, the principal medical active element in B. balsamifera, is anti-inflammatory, antioxidant, and analgesic; enhances medicine absorption; refreshes; and is used as a spice and in cosmetic. Industrialization of B. balsamifera is limited by its low L-borneol concentration. Thus, understanding the accumulation pattern of the secondary metabolite endo-borneol and its synthesis process in secondary metabolism is critical for increasing B. balsamifera active ingredient content and cultivation efficiency. Methods: In this work, B. balsamifera was treated with varying concentrations (1.00 and 10.00 mmol/L) of methyl jasmonate (MeJA) as an exogenous foliar activator. The physiological parameters and L-borneol concentration were then assessed. Transcriptome sequencing of B. balsamifera-induced leaves was used to identify key genes for monoterpene synthesis. Results: The treatment effect of 1 mmol/L MeJA was the best, and the leaves of all three leaf positions accumulated the highest L-borneol after 120 h, correspondingly 3.043 mg·g-1 FW, 3.346 mg·g-1 FW, and 2.044 mg·g-1 FW, with significant differences from the control. The main assembly produced 509,285 transcripts with min and max lengths of 201 and 23,172, respectively. DEG analysis employing volcano blots revealed 593, 224, 612, 2,405, 1,353, and 921 upregulated genes and 4, 123, 573, 1,745, 766, and 763 downregulated genes in the treatments D1_1vsCK, D1_10vsCK, D2_1vsCK, D2_10vsCK, D5_1vsCK, and D5_10vsCK. Interestingly, when exposed to MeJA treatments, the MEP pathway's unigenes express themselves more than those of the MVA route. Finally, when treated with 1 mmol/L, the genes DXR, DXS, and GPS showed increased expression over time. At the same time, a 10 mmol/L therapy resulted in elevated levels of ispH and GGPS. Discussion: Our preliminary research indicates that exogenous phytohormones can raise the level of L borneol in B. balsamifera (L.) DC when given in the appropriate amounts. The most significant discovery made while analyzing the effects of different hormones and concentrations on B. balsamifera (L.) DC was the effect of 1 mmol/L MeJA treatment.
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The archaeal mevalonate pathway is a recently discovered modified version of the eukaryotic mevalonate pathway. This pathway is widely conserved in archaea, except for some archaeal lineages possessing the eukaryotic or other modified mevalonate pathways. Although the pathway seems almost exclusive to the domain Archaea, the whole set of homologous genes of the pathway is found in the metagenome-assembled genome sequence of an uncultivated bacterium, Candidatus Promineifilum breve, of the phylum Chloroflexota. To prove the existence of the archaea-specific pathway in the domain Bacteria, we confirmed the activities of the enzymes specific to the pathway, phosphomevalonate dehydratase and anhydromevalonate phosphate decarboxylase, because only these two enzymes are absent in closely related Chloroflexota bacteria that possess a different type of modified mevalonate pathway. The activity of anhydromevalonate phosphate decarboxylase was evaluated by carotenoid production via the archaeal mevalonate pathway reconstituted in Escherichia coli cells, whereas that of phosphomevalonate dehydratase was confirmed by an in vitro assay using the recombinant enzyme after purification and iron-sulfur cluster reconstruction. Phylogenetic analyses of some mevalonate pathway-related enzymes suggest an evolutionary route for the archaeal mevalonate pathway in Candidatus P. breve, which probably involves horizontal gene transfer events.IMPORTANCEThe recent discovery of various modified mevalonate pathways in microorganisms, such as archaea and Chloroflexota bacteria, has shed light on the complexity of the evolution of metabolic pathways, including those involved in primary metabolism. The fact that the archaeal mevalonate pathway, which is almost exclusive to the domain Archaea, exists in a Chloroflexota bacterium provides valuable insights into the molecular evolution of the mevalonate pathways and associated enzymes. Putative genes probably involved in the archaeal mevalonate pathway have also been found in the metagenome-assembled genomes of Chloroflexota bacteria. Such genes can contribute to metabolic engineering for the bioproduction of valuable isoprenoids because the archaeal mevalonate pathway is known to be an energy-saving metabolic pathway that consumes less ATP than other mevalonate pathways do.
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Ácido Mevalónico , Ácido Mevalónico/metabolismo , Archaea/genética , Archaea/metabolismo , Archaea/clasificación , Archaea/enzimología , Chloroflexi/genética , Chloroflexi/metabolismo , Chloroflexi/enzimología , Chloroflexi/clasificación , Redes y Vías Metabólicas/genética , Filogenia , Escherichia coli/genética , Escherichia coli/metabolismoRESUMEN
The infectivity of Leishmania is determined by its ability to invade and evade host and its thriving capacity within the macrophage. Our study revealed the role of Leishmania donovani mevalonate kinase (MVK), an enzyme of mevalonate pathway in visceral leishmaniasis pathogenesis. Peritoneal exudate cells (PEC)-derived macrophages from BALB/c mice were infected with wild type (WT), MVK over expressing (MVK OE) and knockdown (KD) parasites and MVK OE parasites were found to be more infective than WT and MVK KD parasites. Incubation of macrophages with MVK OE parasites declined inducible nitric oxide synthase (iNOS) expression as well as nitric oxide (NO) production, both by 2 times in comparison to WT parasites. Moreover, â¼3 fold increase in Arginase1 expression indicated that MVK might induce polarization of macrophage towards M2, favouring the survival of parasite within the macrophages. Post 24 h infection of the macrophages with mutant strains, the levels of different cytokines (TNF-α, IL-12, IL-10 and IFN-γ) were measured. Infection of macrophages with MVK OE parasites showed an increase in the level of anti-inflammatory cytokine: IL-10 while infection with MVK KD parasites exhibited an increase in the level of pro-inflammatory cytokines: TNF-α, IL-12, and IFN-γ. Hence, Leishmania donovani mevalonate kinase (LdMVK) modulates macrophage functions and has a significant role in pathogenesis.
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Citocinas , Leishmania donovani , Leishmaniasis Visceral , Macrófagos Peritoneales , Ratones Endogámicos BALB C , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico , Fosfotransferasas (Aceptor de Grupo Alcohol) , Leishmania donovani/enzimología , Leishmania donovani/patogenicidad , Leishmania donovani/genética , Leishmania donovani/fisiología , Animales , Ratones , Leishmaniasis Visceral/parasitología , Leishmaniasis Visceral/inmunología , Óxido Nítrico/metabolismo , Macrófagos Peritoneales/parasitología , Macrófagos Peritoneales/enzimología , Citocinas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Arginasa/metabolismo , Arginasa/genética , Femenino , Técnicas de Silenciamiento del GenRESUMEN
Porokeratoses are a heterogenous group of autoinflammatory keratinization disorders all characterized by the presence of a cornoid lamella. In addition to gene mutations affecting the mevalonate pathway, environmental factors such as UV radiation, immunosuppression, trauma, and infection are also thought to contribute to porokeratoses. To date, there are no management guidelines or levels of evidence for commonly used pharmacologic and non-pharmacologic treatment options for porokeratoses. Conventional treatment strategies encompass topical and systemic drugs (e.g., salicylic acid, topical glucocorticoids, and retinoids), phototherapy, laser, and surgical interventions. Better insights into the pathogenesis of porokeratoses have paved the way for the development of novel therapeutic approaches, such as topical statins or the use of monoclonal antibodies. This narrative review aims to summarize both conventional and novel treatment options, including their level of evidence, advantages, and disadvantages.
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Poroqueratosis , Humanos , Poroqueratosis/terapia , Fototerapia/métodos , Fármacos Dermatológicos/uso terapéutico , Terapia por Láser/métodos , Retinoides/uso terapéutico , Glucocorticoides/uso terapéuticoRESUMEN
The mevalonate (MVA) pathway plays a crucial role in the occurrence and progression of various diseases, such as osteoporosis, breast cancer, and lung cancer, etc. However, determining all the MVA pathway intermediates is still challenging due to their high polarity, low concentration, chelation effect with metal compartments, and poor mass spectrometric response. In this study, we established a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method coupled with N2, N2, N4, N4-tetramethyl-6-(4-(piperazin-1-ylsulfonyl) phenyl)-1,3,5-triazine-2,4-diamine (Tmt-PP) labeling for the simultaneous analysis of all MVA intermediates in biospecimens. Chemical derivatization significantly improved the chromatographic retention, peak shape, and detection sensitivity of the analytes. Moreover, we employed a method named mass spectrum calculation to achieve the absolute quantification of the isomers, i.e., isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The established method was fully qualified and applied to explore the difference of these metabolites in cisplatin-resistant non-small cell lung cancer (NSCLC) cells. Additionally, several MVA intermediate analogs, including isopentenyl monophosphate or dimethylallyl monophosphate (IMP/DMAMP), geranyl monophosphate (GMP), 5-triphosphomevalonate (MTP), and isopentenyl triphosphate or dimethylallyl triphosphate (ITP/DMATP), were identified for the first time using a knowledge-driven prediction strategy. We further explored the tissue distribution of these novel metabolites. Overall, this work developed a sensitive quantification method for all MVA intermediates, which will enhance our understanding of the role of this pathway in various health and disease conditions. The novel metabolites we discovered warrant further investigations into their biosynthesis and biological functions.
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Ácido Mevalónico , Espectrometría de Masas en Tándem , Humanos , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Hemiterpenos/análisis , Hemiterpenos/metabolismo , Límite de Detección , Cromatografía Líquida con Espectrometría de Masas/métodos , Neoplasias Pulmonares/metabolismo , Ácido Mevalónico/metabolismo , Ácido Mevalónico/análogos & derivados , Compuestos Organofosforados/química , Compuestos Organofosforados/análisis , Compuestos Organofosforados/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
Hepatocellular carcinoma (HCC) is a malignant tumor that occurs in the liver, with a high degree of malignancy and relatively poor prognosis. Gypenoside L has inhibitory effects on liver cancer cells. However, its mechanism of action is still unclear. This study aims to investigate the inhibitory effects of gypenoside L on HCC in vitro and in vivo, and explore its potential mechanisms. The results showed that gypenoside L reduced the cholesterol and triglyceride content in HepG2 and Huh-7 cells, inhibited cell proliferation, invasion and metastasis, arrested cell cycle at G0/G1 phase, promoted cell apoptosis. Mechanistically, it targeted the transcription factor SREPB2 to inhibit the expression of HMGCS1 protein and inhibited the downstream proteins HMGCR and MVK, thereby regulating the mevalonate (MVA) pathway. Overexpression HMGCS1 led to significant alterations in the cholesterol metabolism pathway of HCC, which mediated HCC cell proliferation and conferred resistance to the therapeutic effect of gypenoside L. In vivo, gypenoside L effectively suppressed HCC growth in tumor-bearing mice by reducing cholesterol production, exhibiting favorable safety profiles and minimal toxic side effects. Gypenoside L modulated cholesterol homeostasis, enhanced expression of inflammatory factors by regulating MHC I pathway-related proteins to augment anticancer immune responses. Clinical samples from HCC patients also exhibited high expression levels of MVA pathway-related genes in tumor tissues. These findings highlight gypenoside L as a promising agent for targeting cholesterol metabolism in HCC while emphasizing the effectiveness of regulating the SREBP2-HMGCS1 axis as a therapeutic strategy.
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Carcinoma Hepatocelular , Proliferación Celular , Gynostemma , Neoplasias Hepáticas , Proteína 2 de Unión a Elementos Reguladores de Esteroles , Humanos , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , Gynostemma/química , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Animales , Ratones , Relación Dosis-Respuesta a Droga , Estructura Molecular , Ensayos de Selección de Medicamentos Antitumorales , Apoptosis/efectos de los fármacos , Relación Estructura-Actividad , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/química , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/patología , Neoplasias Hepáticas Experimentales/metabolismo , Extractos VegetalesRESUMEN
INTRODUCTION: A neurotrophic tropomyosin receptor kinase (NTRK)-tyrosine kinase inhibitor (TKI) has shown dramatic efficacy against malignant tumors harboring an NTRK fusion gene. However, almost all tumors eventually acquire resistance to NTRK-TKIs. METHOD: To investigate the mechanism of resistance to NTRK-TKIs, we established cells resistant to three types of NTRK-TKIs (larotrectinib, entrectinib, and selitrectinib) using KM12 colon cancer cells with a TPM3-NTRK1 rearrangement. RESULT: Overexpression of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) was observed in three resistant cells (KM12-LR, KM12-ER, and KM12-SR) by microarray analysis. Lower expression of sterol regulatory element-binding protein 2 (SREBP2) and peroxisome proliferator activated receptor α (PPARα) was found in two cells (KM12-ER and KM12-SR) in which HMGCS2 was overexpressed compared to the parental KM12 and KM12-LR cells. In resistant cells, knockdown of HMGCS2 using small interfering RNA improved the sensitivity to NTRK-TKI. Further treatment with mevalonolactone after HMGCS2 knockdown reintroduced the NTRK-TKI resistance. In addition, simvastatin and silibinin had a synergistic effect with NTRK-TKIs in resistant cells, and delayed tolerance was observed after sustained exposure to clinical concentrations of NTRK-TKI and simvastatin in KM12 cells. In xenograft mouse models, combination treatment with entrectinib and simvastatin reduced resistant tumor growth compared with entrectinib alone. CONCLUSION: These results suggest that HMGCS2 overexpression induces resistance to NTRK-TKIs via the mevalonate pathway in colon cancer cells. Statin inhibition of the mevalonate pathway may be useful for overcoming this mechanistic resistance.
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Resistencia a Antineoplásicos , Ácido Mevalónico , Inhibidores de Proteínas Quinasas , Animales , Humanos , Ratones , Benzamidas/farmacología , Benzamidas/uso terapéutico , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Neoplasias del Colon/genética , Hidroximetilglutaril-CoA Sintasa/metabolismo , Hidroximetilglutaril-CoA Sintasa/genética , Indazoles/farmacología , Indazoles/uso terapéutico , Ácido Mevalónico/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , Receptor trkA/metabolismo , Receptor trkA/genética , Receptor trkA/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mevalonate kinase is a key regulator of the mevalonate pathway, subject to feedback inhibition by the downstream metabolite farnesyl pyrophosphate. In this study, we validated the hypothesis that monophosphonate compounds mimicking farnesyl pyrophosphate can inhibit mevalonate kinase. Exploring compounds originally synthesized as allosteric inhibitors of farnesyl pyrophosphate synthase, we discovered mevalonate kinase inhibitors with nanomolar activity. Kinetic characterization of the two most potent inhibitors demonstrated Ki values of 3.1 and 22 nm. Structural comparison suggested features of these inhibitors likely responsible for their potency. Our findings introduce the first class of nanomolar inhibitors of human mevalonate kinase, opening avenues for future research. These compounds might prove useful as molecular tools to study mevalonate pathway regulation and evaluate mevalonate kinase as a potential therapeutic target.
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Inhibidores Enzimáticos , Fosfotransferasas (Aceptor de Grupo Alcohol) , Humanos , Regulación Alostérica/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Cinética , Geraniltranstransferasa/antagonistas & inhibidores , Geraniltranstransferasa/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Sesquiterpenos/farmacología , Sesquiterpenos/metabolismo , Sesquiterpenos/químicaRESUMEN
There is an urgent need to provide immediate and effective options for the treatment of prostate cancer (PCa) to prevent progression to lethal castration-resistant PCa (CRPC). The mevalonate (MVA) pathway is dysregulated in PCa, and statin drugs commonly prescribed for hypercholesterolemia, effectively target this pathway. Statins exhibit anti-PCa activity, however the resulting intracellular depletion of cholesterol triggers a feedback loop that restores MVA pathway activity, thus diminishing statin efficacy and contributing to resistance. To identify drugs that block this feedback response and enhance the pro-apoptotic activity of statins, we performed a high-content image-based screen of a 1508 drug library, enriched for FDA-approved compounds. Two of the validated hits, Galeterone (GAL) and Quinestrol, share the cholesterol-related tetracyclic structure, which is also evident in the FDA-approved CRPC drug Abiraterone (ABI). Molecular modeling revealed that GAL, Quinestrol and ABI not only share structural similarity with 25-hydroxy-cholesterol (25HC) but were also predicted to bind similarly to a known protein-binding site of 25HC. This suggested GAL, Quinestrol and ABI are sterol-mimetics and thereby inhibit the statin-induced feedback response. Cell-based assays demonstrated that these agents inhibit nuclear translocation of sterol-regulatory element binding protein 2 (SREBP2) and the transcription of MVA genes. Sensitivity was independent of androgen status and the Fluva-GAL combination significantly impeded CRPC tumor xenograft growth. By identifying cholesterol-mimetic drugs that inhibit SREBP2 activation upon statin treatment, we provide a potent "one-two punch" against CRPC progression and pave the way for innovative therapeutic strategies to combat additional diseases whose etiology is associated with SREBP2 dysregulation.