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1.
Int J Mol Sci ; 25(10)2024 May 13.
Artículo en Inglés | MEDLINE | ID: mdl-38791363

RESUMEN

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This process often causes proteins to associate with the membrane and participate in signal transduction pathways. The most common substrates of FTase are proteins that have C-terminal tetrapeptide CaaX box sequences where the cysteine is the site of modification. However, recent work has shown that five amino acid sequences can also be recognized, including the pentapeptides CMIIM and CSLMQ. In this work, peptide libraries were initially used to systematically vary the residues in those two parental sequences using an assay based on Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). In addition, 192 pentapeptide sequences from the human proteome were screened using that assay to discover additional extended CaaaX-box motifs. Selected hits from that screening effort were rescreened using an in vivo yeast reporter protein assay. The X-ray crystal structure of CMIIM bound to FTase was also solved, showing that the C-terminal tripeptide of that sequence interacted with the enzyme in a similar manner as the C-terminal tripeptide of CVVM, suggesting that the tripeptide comprises a common structural element for substrate recognition in both tetrapeptide and pentapeptide sequences. Molecular dynamics simulation of CMIIM bound to FTase further shed light on the molecular interactions involved, showing that a putative catalytically competent Zn(II)-thiolate species was able to form. Bioinformatic predictions of tetrapeptide (CaaX-box) reactivity correlated well with the reactivity of pentapeptides obtained from in vivo analysis, reinforcing the importance of the C-terminal tripeptide motif. This analysis provides a structural framework for understanding the reactivity of extended CaaaX-box motifs and a method that may be useful for predicting the reactivity of additional FTase substrates bearing CaaaX-box sequences.


Asunto(s)
Biología Computacional , Biblioteca de Péptidos , Humanos , Biología Computacional/métodos , Especificidad por Sustrato , Farnesiltransferasa/metabolismo , Farnesiltransferasa/química , Oligopéptidos/química , Oligopéptidos/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Unión Proteica
2.
Nat Commun ; 15(1): 3422, 2024 Apr 23.
Artículo en Inglés | MEDLINE | ID: mdl-38653965

RESUMEN

Targeting Anaplastic lymphoma kinase (ALK) is a promising therapeutic strategy for aberrant ALK-expressing malignancies including neuroblastoma, but resistance to ALK tyrosine kinase inhibitors (ALK TKI) is a distinct possibility necessitating drug combination therapeutic approaches. Using high-throughput, genome-wide CRISPR-Cas9 knockout screens, we identify miR-1304-5p loss as a desensitizer to ALK TKIs in aberrant ALK-expressing neuroblastoma; inhibition of miR-1304-5p decreases, while mimics of this miRNA increase the sensitivity of neuroblastoma cells to ALK TKIs. We show that miR-1304-5p targets NRAS, decreasing cell viability via induction of apoptosis. It follows that the farnesyltransferase inhibitor (FTI) lonafarnib in addition to ALK TKIs act synergistically in neuroblastoma, inducing apoptosis in vitro. In particular, on combined treatment of neuroblastoma patient derived xenografts with an FTI and an ALK TKI complete regression of tumour growth is observed although tumours rapidly regrow on cessation of therapy. Overall, our data suggests that combined use of ALK TKIs and FTIs, constitutes a therapeutic approach to treat high risk neuroblastoma although prolonged therapy is likely required to prevent relapse.


Asunto(s)
Quinasa de Linfoma Anaplásico , Dibenzocicloheptenos , Farnesiltransferasa , GTP Fosfohidrolasas , MicroARNs , Neuroblastoma , Piperidinas , Inhibidores de Proteínas Quinasas , Piridinas , Animales , Femenino , Humanos , Ratones , Quinasa de Linfoma Anaplásico/genética , Quinasa de Linfoma Anaplásico/metabolismo , Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Farnesiltransferasa/antagonistas & inhibidores , Farnesiltransferasa/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , MicroARNs/genética , MicroARNs/metabolismo , Mutación , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/genética , Neuroblastoma/patología , Neuroblastoma/metabolismo , Piperidinas/farmacología , Piperidinas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/farmacología , Piridinas/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto
3.
PLoS Pathog ; 20(4): e1012136, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38620034

RESUMEN

African swine fever (ASF) is an acute, hemorrhagic, and severe infectious disease caused by the ASF virus (ASFV). ASFV has evolved multiple strategies to escape host antiviral immune responses. Here, we reported that ASFV pB318L, a trans-geranylgeranyl-diphosphate synthase, reduced the expression of type I interferon (IFN-I) and IFN-stimulated genes (ISGs). Mechanically, pB318L not only interacted with STING to reduce the translocation of STING from the endoplasmic reticulum to the Golgi apparatus but also interacted with IFN receptors to reduce the interaction of IFNAR1/TYK2 and IFNAR2/JAK1. Of note, ASFV with interruption of B318L gene (ASFV-intB318L) infected PAMs produces more IFN-I and ISGs than that in PAMs infected with its parental ASFV HLJ/18 at the late stage of infection. Consistently, the pathogenicity of ASFV-intB318L is attenuated in piglets compared with its parental virus. Taken together, our data reveal that B318L gene may partially affect ASFV pathogenicity by reducing the production of IFN-I and ISGs. This study provides a clue to design antiviral agents or live attenuated vaccines to prevent and control ASF.


Asunto(s)
Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Interferón Tipo I , Animales , Porcinos , Farnesiltransferasa/metabolismo , Proteínas Virales/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Transducción de Señal
4.
Aging Cell ; 23(5): e14105, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38504487

RESUMEN

Hutchinson-Gilford Progeria syndrome (HGPS) is a severe premature ageing disorder caused by a 50 amino acid truncated (Δ50AA) and permanently farnesylated lamin A (LA) mutant called progerin. On a cellular level, progerin expression leads to heterochromatin loss, impaired nucleocytoplasmic transport, telomeric DNA damage and a permanent growth arrest called cellular senescence. Although the genetic basis for HGPS has been elucidated 20 years ago, the question whether the Δ50AA or the permanent farnesylation causes cellular defects has not been addressed. Moreover, we currently lack mechanistic insight into how the only FDA-approved progeria drug Lonafarnib, a farnesyltransferase inhibitor (FTI), ameliorates HGPS phenotypes. By expressing a variety of LA mutants using a doxycycline-inducible system, and in conjunction with FTI, we demonstrate that the permanent farnesylation, and not the Δ50AA, is solely responsible for progerin-induced cellular defects, as well as its rapid accumulation and slow clearance. Importantly, FTI does not affect clearance of progerin post-farnesylation and we demonstrate that early, but not late FTI treatment prevents HGPS phenotypes. Collectively, our study unravels the precise contributions of progerin's permanent farnesylation to its turnover and HGPS cellular phenotypes, and how FTI treatment ameliorates these. These findings are applicable to other diseases associated with permanently farnesylated proteins, such as adult-onset autosomal dominant leukodystrophy.


Asunto(s)
Lamina Tipo A , Progeria , Lamina Tipo A/metabolismo , Lamina Tipo A/genética , Humanos , Progeria/metabolismo , Progeria/genética , Progeria/patología , Progeria/tratamiento farmacológico , Farnesiltransferasa/metabolismo , Farnesiltransferasa/antagonistas & inhibidores , Farnesiltransferasa/genética , Prenilación de Proteína , Dibenzocicloheptenos , Piperidinas , Piridinas
5.
Plant Cell ; 36(5): 1868-1891, 2024 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-38299382

RESUMEN

Carotenoids are essential for photosynthesis and photoprotection. Plants must evolve multifaceted regulatory mechanisms to control carotenoid biosynthesis. However, the regulatory mechanisms and the regulators conserved among plant species remain elusive. Phytoene synthase (PSY) catalyzes the highly regulated step of carotenogenesis and geranylgeranyl diphosphate synthase (GGPPS) acts as a hub to interact with GGPP-utilizing enzymes for the synthesis of specific downstream isoprenoids. Here, we report a function of Nudix hydrolase 23 (NUDX23), a Nudix domain-containing protein, in post-translational regulation of PSY and GGPPS for carotenoid biosynthesis. NUDX23 expresses highly in Arabidopsis (Arabidopsis thaliana) leaves. Overexpression of NUDX23 significantly increases PSY and GGPPS protein levels and carotenoid production, whereas knockout of NUDX23 dramatically reduces their abundances and carotenoid accumulation in Arabidopsis. NUDX23 regulates carotenoid biosynthesis via direct interactions with PSY and GGPPS in chloroplasts, which enhances PSY and GGPPS protein stability in a large PSY-GGPPS enzyme complex. NUDX23 was found to co-migrate with PSY and GGPPS proteins and to be required for the enzyme complex assembly. Our findings uncover a regulatory mechanism underlying carotenoid biosynthesis in plants and offer promising genetic tools for developing carotenoid-enriched food crops.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Carotenoides , Regulación de la Expresión Génica de las Plantas , Carotenoides/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Hidrolasas Nudix , Cloroplastos/metabolismo , Geranilgeranil-Difosfato Geranilgeraniltransferasa/metabolismo , Geranilgeranil-Difosfato Geranilgeraniltransferasa/genética , Farnesiltransferasa/metabolismo , Farnesiltransferasa/genética , Pirofosfatasas/metabolismo , Pirofosfatasas/genética , Procesamiento Proteico-Postraduccional , Plantas Modificadas Genéticamente , Hojas de la Planta/metabolismo , Hojas de la Planta/genética
6.
Insect Mol Biol ; 33(2): 147-156, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-37962063

RESUMEN

Geranylgeranyl pyrophosphate (diphosphate) synthase (GGPPS) plays an important role in various physiological processes in insects, such as isoprenoid biosynthesis and protein prenylation. Here, we functionally characterised the GGPPS from the major agricultural lepidopteran pests Spodoptera frugiperda and Helicoverpa armigera. Partial disruption of GGPPS by CRISPR in S. frugiperda decreased embryo hatching rate and larval survival, suggesting that this gene is essential. Functional expression in vitro of Helicoverpa armigera GGPPS in Escherichia coli revealed a catalytically active enzyme. Next, we developed and optimised an enzyme assay to screen for potential inhibitors, such as the zoledronate and the minodronate, which showed a dose-dependent inhibition. Phylogenetic analysis of GGPPS across insects showed that GGPPS is highly conserved but also revealed several residues likely to be involved in substrate binding, which were substantially different in bee pollinator and human GGPPS. Considering the essentiality of GGPPS and its putative binding residue variability qualifies a GGPPS as a novel pesticide target. The developed assay may contribute to the identification of novel insecticide leads.


Asunto(s)
Plaguicidas , Humanos , Animales , Abejas/genética , Farnesiltransferasa/genética , Farnesiltransferasa/metabolismo , Filogenia , Ácido Zoledrónico
7.
Mol Cancer Ther ; 23(1): 14-23, 2024 Jan 03.
Artículo en Inglés | MEDLINE | ID: mdl-37756579

RESUMEN

Geranylgeranyl diphosphate synthase (GGDPS), the source of the isoprenoid donor in protein geranylgeranylation reactions, has become an attractive target for anticancer therapy due to the reliance of cancers on geranylgeranylated proteins. Current GGDPS inhibitor development focuses on optimizing the drug-target enzyme interactions of nitrogen-containing bisphosphonate-based drugs. To advance GGDPS inhibitor development, understanding the enzyme structure, active site, and ligand/product interactions is essential. Here we provide a comprehensive structure-focused review of GGDPS. We reviewed available yeast and human GGDPS structures and then used AlphaFold modeling to complete unsolved structural aspects of these models. We delineate the elements of higher-order structure formation, product-substrate binding, the electrostatic surface, and small-molecule inhibitor binding. With the rise of structure-based drug design, the information provided here will serve as a valuable tool for rationally optimizing inhibitor selectivity and effectiveness.


Asunto(s)
Inhibidores Enzimáticos , Neoplasias , Humanos , Farnesiltransferasa/química , Farnesiltransferasa/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Inhibidores Enzimáticos/química , Terpenos/química , Terpenos/farmacología , Prenilación de Proteína , Neoplasias/tratamiento farmacológico
8.
J Immunol ; 211(4): 527-538, 2023 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-37449905

RESUMEN

IgE-mediated mast cell activation is a driving force in allergic disease in need of novel interventions. Statins, long used to lower serum cholesterol, have been shown in multiple large-cohort studies to reduce asthma severity. We previously found that statins inhibit IgE-induced mast cell function, but these effects varied widely among mouse strains and human donors, likely due to the upregulation of the statin target, 3-hydroxy-3-methylgutaryl-CoA reductase. Statin inhibition of mast cell function appeared to be mediated not by cholesterol reduction but by suppressing protein isoprenylation events that use cholesterol pathway intermediates. Therefore, we sought to circumvent statin resistance by targeting isoprenylation. Using genetic depletion of the isoprenylation enzymes farnesyltransferase and geranylgeranyl transferase 1 or their substrate K-Ras, we show a significant reduction in FcεRI-mediated degranulation and cytokine production. Furthermore, similar effects were observed with pharmacological inhibition with the dual farnesyltransferase and geranylgeranyl transferase 1 inhibitor FGTI-2734. Our data indicate that both transferases must be inhibited to reduce mast cell function and that K-Ras is a critical isoprenylation target. Importantly, FGTI-2734 was effective in vivo, suppressing mast cell-dependent anaphylaxis, allergic pulmonary inflammation, and airway hyperresponsiveness. Collectively, these findings suggest that K-Ras is among the isoprenylation substrates critical for FcεRI-induced mast cell function and reveal isoprenylation as a new means of targeting allergic disease.


Asunto(s)
Anafilaxia , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Ratones , Humanos , Animales , Receptores de IgE/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Farnesiltransferasa/metabolismo , Mastocitos/metabolismo , Anafilaxia/metabolismo , Transducción de Señal , Degranulación de la Célula , Inmunoglobulina E/metabolismo , Inflamación/metabolismo , Colesterol/metabolismo , Prenilación
9.
Front Biosci (Landmark Ed) ; 28(3): 55, 2023 03 20.
Artículo en Inglés | MEDLINE | ID: mdl-37005749

RESUMEN

BACKGROUND: Pseudoxanthoma elasticum (PXE) is a rare autosomal recessive disorder caused by mutations in the ATP-binding cassette sub-family C member 6 (ABCC6) gene. Patients with PXE show molecular and clinical characteristics of known premature aging syndromes, such as Hutchinson-Gilford progeria syndrome (HGPS). Nevertheless, PXE has only barely been discussed against the background of premature aging, although a detailed characterization of aging processes in PXE could contribute to a better understanding of its pathogenesis. Thus, this study was performed to evaluate whether relevant factors which are known to play a role in accelerated aging processes in HGPS pathogenesis are also dysregulated in PXE. METHODS: Primary human dermal fibroblasts from healthy donors (n = 3) and PXE patients (n = 3) and were cultivated under different culture conditions as our previous studies point towards effects of nutrient depletion on PXE phenotype. Gene expression of lamin A, lamin C, nucleolin, farnesyltransferase and zinc metallopeptidase STE24 were determined by quantitative real-time polymerase chain reaction. Additionally, protein levels of lamin A, C and nucleolin were evaluated by immunofluorescence and the telomere length was analyzed. RESULTS: We could show a significant decrease of lamin A and C gene expression in PXE fibroblasts under nutrient depletion compared to controls. The gene expression of progerin and farnesyltransferase showed a significant increase in PXE fibroblasts when cultivated in 10% fetal calf serum (FCS) compared to controls. Immunofluorescence microscopy of lamin A/C and nucleolin and mRNA expression of zinc metallopeptidase STE24 and nucleolin showed no significant changes in any case. The determination of the relative telomere length showed significantly longer telomeres for PXE fibroblasts compared to controls when cultivated in 10% FCS. CONCLUSIONS: These data indicate that PXE fibroblasts possibly undergo a kind of senescence which is independent of telomere damage and not triggered by defects of the nuclear envelope or nucleoli deformation.


Asunto(s)
Envejecimiento Prematuro , Progeria , Seudoxantoma Elástico , Humanos , Progeria/genética , Progeria/metabolismo , Progeria/patología , Envejecimiento Prematuro/genética , Envejecimiento Prematuro/metabolismo , Envejecimiento Prematuro/patología , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Seudoxantoma Elástico/genética , Seudoxantoma Elástico/metabolismo , Seudoxantoma Elástico/patología , Farnesiltransferasa/metabolismo , Metaloproteasas/metabolismo , Zinc/metabolismo , Fibroblastos/metabolismo
10.
G3 (Bethesda) ; 13(7)2023 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-37119806

RESUMEN

The current understanding of farnesyltransferase (FTase) specificity was pioneered through investigations of reporters like Ras and Ras-related proteins that possess a C-terminal CaaX motif that consists of 4 amino acid residues: cysteine-aliphatic1-aliphatic2-variable (X). These studies led to the finding that proteins with the CaaX motif are subject to a 3-step post-translational modification pathway involving farnesylation, proteolysis, and carboxylmethylation. Emerging evidence indicates, however, that FTase can farnesylate sequences outside the CaaX motif and that these sequences do not undergo the canonical 3-step pathway. In this work, we report a comprehensive evaluation of all possible CXXX sequences as FTase targets using the reporter Ydj1, an Hsp40 chaperone that only requires farnesylation for its activity. Our genetic and high-throughput sequencing approach reveals an unprecedented profile of sequences that yeast FTase can recognize in vivo, which effectively expands the potential target space of FTase within the yeast proteome. We also document that yeast FTase specificity is majorly influenced by restrictive amino acids at a2 and X positions as opposed to the resemblance of CaaX motif as previously regarded. This first complete evaluation of CXXX space expands the complexity of protein isoprenylation and marks a key step forward in understanding the potential scope of targets for this isoprenylation pathway.


Asunto(s)
Transferasas Alquil y Aril , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Farnesiltransferasa/genética , Farnesiltransferasa/metabolismo , Secuencia de Aminoácidos , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Prenilación de Proteína , Proteínas/genética , Especificidad por Sustrato
11.
Int J Mol Sci ; 24(8)2023 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-37108334

RESUMEN

Protein prenylation is an important protein modification that is responsible for diverse physiological activities in eukaryotic cells. This modification is generally catalyzed by three types of prenyl transferases, which include farnesyl transferase (FT), geranylgeranyl transferase (GGT-1) and Rab geranylgeranyl transferase (GGT-2). Studies in malaria parasites showed that these parasites contain prenylated proteins, which are proposed to play multiple functions in parasites. However, the prenyl transferases have not been functionally characterized in parasites of subphylum Apicomplexa. Here, we functionally dissected functions of three of the prenyl transferases in the Apicomplexa model organism Toxoplasma gondii (T. gondii) using a plant auxin-inducible degron system. The homologous genes of the beta subunit of FT, GGT-1 and GGT-2 were endogenously tagged with AID at the C-terminus in the TIR1 parental line using a CRISPR-Cas9 approach. Upon depletion of these prenyl transferases, GGT-1 and GGT-2 had a strong defect on parasite replication. Fluorescent assay using diverse protein markers showed that the protein markers ROP5 and GRA7 were diffused in the parasites depleted with GGT-1 and GGT-2, while the mitochondrion was strongly affected in parasites depleted with GGT-1. Importantly, depletion of GGT-2 caused the stronger defect to the sorting of rhoptry protein and the parasite morphology. Furthermore, parasite motility was observed to be affected in parasites depleted with GGT-2. Taken together, this study functionally characterized the prenyl transferases, which contributed to an overall understanding of protein prenylation in T. gondii and potentially in other related parasites.


Asunto(s)
Parásitos , Toxoplasma , Animales , Transferasas/metabolismo , Parásitos/metabolismo , Toxoplasma/metabolismo , Farnesiltransferasa/metabolismo , Prenilación de Proteína , Transporte de Proteínas , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo
12.
Plant Cell ; 35(6): 2293-2315, 2023 05 29.
Artículo en Inglés | MEDLINE | ID: mdl-36929908

RESUMEN

Terpenoids constitute the largest class of plant primary and secondary metabolites with a broad range of biological and ecological functions. They are synthesized from isopentenyl diphosphate and dimethylallyl diphosphate, which in plastids are condensed by geranylgeranyl diphosphate synthases (GGPPSs) to produce GGPP (C20) for diterpene biosynthesis and by geranyl diphosphate synthases (GPPSs) to form GPP (C10) for monoterpene production. Depending on the plant species, unlike homomeric GGPPSs, GPPSs exist as homo- and heteromers, the latter of which contain catalytically inactive GGPPS-homologous small subunits (SSUs) that can interact with GGPPSs. By combining phylogenetic analysis with functional characterization of GGPPS homologs from a wide range of photosynthetic organisms, we investigated how different GPPS architectures have evolved within the GGPPS protein family. Our results reveal that GGPPS gene family expansion and functional divergence began early in nonvascular plants, and that independent parallel evolutionary processes gave rise to homomeric and heteromeric GPPSs. By site-directed mutagenesis and molecular dynamics simulations, we also discovered that Leu-Val/Val-Ala pairs of amino acid residues were pivotal in the functional divergence of homomeric GPPSs and GGPPSs. Overall, our study elucidated an evolutionary path for the formation of GPPSs with different architectures from GGPPSs and uncovered the molecular mechanisms involved in this differentiation.


Asunto(s)
Dimetilaliltranstransferasa , Diterpenos , Farnesiltransferasa/genética , Farnesiltransferasa/metabolismo , Filogenia , Dimetilaliltranstransferasa/genética , Dimetilaliltranstransferasa/metabolismo , Diterpenos/metabolismo
13.
J Neurosci ; 43(14): 2615-2629, 2023 04 05.
Artículo en Inglés | MEDLINE | ID: mdl-36788031

RESUMEN

Macroautophagy is a catabolic process that coordinates with lysosomes to degrade aggregation-prone proteins and damaged organelles. Loss of macroautophagy preferentially affects neuron viability and is associated with age-related neurodegeneration. We previously found that α-synuclein (α-syn) inhibits lysosomal function by blocking ykt6, a farnesyl-regulated soluble NSF attachment protein receptor (SNARE) protein that is essential for hydrolase trafficking in midbrain neurons. Using Parkinson's disease (PD) patient iPSC-derived midbrain cultures, we find that chronic, endogenous accumulation of α-syn directly inhibits autophagosome-lysosome fusion by impairing ykt6-SNAP-29 complexes. In wild-type (WT) cultures, ykt6 depletion caused a near-complete block of autophagic flux, highlighting its critical role for autophagy in human iPSC-derived neurons. In PD, macroautophagy impairment was associated with increased farnesyltransferase (FTase) activity, and FTase inhibitors restored macroautophagic flux through promoting active forms of ykt6 in human cultures, and male and female mice. Our findings indicate that ykt6 mediates cellular clearance by coordinating autophagic-lysosomal fusion and hydrolase trafficking, and that macroautophagy impairment in PD can be rescued by FTase inhibitors.SIGNIFICANCE STATEMENT The pathogenic mechanisms that lead to the death of neurons in Parkinson's disease (PD) and Dementia with Lewy bodies (LBD) are currently unknown. Furthermore, disease modifying treatments for these diseases do not exist. Our study indicates that a cellular clearance pathway termed autophagy is impaired in patient-derived culture models of PD and in vivo We identified a novel druggable target, a soluble NSF attachment protein receptor (SNARE) protein called ykt6, that rescues autophagy in vitro and in vivo upon blocking its farnesylation. Our work suggests that farnesyltransferase (FTase) inhibitors may be useful therapies for PD and DLB through enhancing autophagic-lysosomal clearance of aggregated proteins.


Asunto(s)
Enfermedad de Parkinson , Humanos , Masculino , Ratones , Animales , Femenino , Enfermedad de Parkinson/metabolismo , Farnesiltransferasa/metabolismo , alfa-Sinucleína/metabolismo , Autofagia/fisiología , Mesencéfalo/metabolismo , Neuronas/metabolismo , Lisosomas/metabolismo , Proteínas SNARE/metabolismo , Hidrolasas/metabolismo , Proteínas R-SNARE/metabolismo
14.
Adv Biol (Weinh) ; 7(9): e2200150, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-36599632

RESUMEN

There are no known approved pharmacotherapies for non-alcoholic fatty liver disease (NAFLD) in the clinical setting. Although studies have provided substantial evidence that geranylgeranyl diphosphate synthase (GGPPS) is a potential therapeutic target for the treatment of NAFLD corresponding drug screening is rare. A GGPPS-targeted inhibitor is identified using a structure-based virtual small molecule screening method. The interaction of 4-AZ and GGPPS is detected by microscale thermophoresis. 4-AZ degradation of GGPPS by the ubiquitin-proteasome pathway is detected by western blotting. The anti-steatotic effect of 4-AZ in vivo is detected by CT. Lipid-related gene detection is detected by real-time PCR both in primary hepatocytes and mice. The compound inhibits the accumulation of lipids in primary hepatocytes and decreases lipogenic gene expression through GGPPS. Pharmacological studies show that 4-AZ can attenuate hepatic steatosis and improve liver injury in high-fat diet-induced mice. This data provides a novel application of 4-AZ NAFLD therapy, proving that the inhibition of GGPPS is a novel strategy for the treatment of NAFLD.


Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Ratones , Animales , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/genética , Farnesiltransferasa/genética , Farnesiltransferasa/metabolismo , Complejo de la Endopetidasa Proteasomal , Ubiquitinas
15.
Clin Transl Med ; 13(1): e1167, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36650113

RESUMEN

Geranylgeranyl diphosphate synthase (GGDPS), an enzyme in the isoprenoid biosynthesis pathway, is responsible for the production of geranylgeranyl pyrophosphate (GGPP). GGPP serves as a substrate for the post-translational modification (geranylgeranylation) of proteins, including those belonging to the Ras superfamily of small GTPases. These proteins play key roles in signalling pathways, cytoskeletal regulation and intracellular transport, and in the absence of the prenylation modification, cannot properly localise and function. Aberrant expression of GGDPS has been implicated in various human pathologies, including liver disease, type 2 diabetes, pulmonary disease and malignancy. Thus, this enzyme is of particular interest from a therapeutic perspective. Here, we review the physiological function of GGDPS as well as its role in pathophysiological processes. We discuss the current GGDPS inhibitors under development and the therapeutic implications of targeting this enzyme.


Asunto(s)
Diabetes Mellitus Tipo 2 , Inhibidores Enzimáticos , Humanos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Farnesiltransferasa/metabolismo , Fosfatos de Poliisoprenilo/metabolismo
16.
Microb Cell Fact ; 22(1): 17, 2023 Jan 24.
Artículo en Inglés | MEDLINE | ID: mdl-36694175

RESUMEN

BACKGROUND: The tobacco leaf-derived cembratriene-ol exhibits anti-insect effects, but its content in plants is scarce. Cembratriene-ol is difficult and inefficiently chemically synthesised due to its complex structure. Moreover, the titer of reported recombinant hosts producing cembratriene-ol was low and cannot be applied to industrial production. RESULTS: In this study, Pantoea ananatis geranylgeranyl diphosphate synthase (CrtE) and Nicotiana tabacum cembratriene-ol synthase (CBTS) were heterologously expressed to synthsize the cembratriene-ol in Escherichia coli. Overexpression of cbts*, the 1-deoxy-D-xylulose 5-phosphate synthase gene dxs, and isopentenyl diphosphate isomerase gene idi promoted the production of cembratriene-ol. The cembratriene-ol titer was 1.53-folds higher than that of E. coli Z17 due to the systematic regulation of ggpps, cbts*, dxs, and idi expression. The production of cembratriene-ol was boosted via the overexpression of genes ispA, ispD, and ispF. The production level of cembratriene-ol in the optimal medium at 72 h was 8.55-folds higher than that before fermentation optimisation. The cembratriene-ol titer in the 15-L fermenter reached 371.2 mg L- 1, which was the highest titer reported. CONCLUSION: In this study, the production of cembratriene-ol in E. coli was significantly enhanced via systematic optimization. It was suggested that the recombinant E. coli producing cembratriene-ol constructed in this study has potential for industrial production and applications.


Asunto(s)
Diterpenos , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Diterpenos/metabolismo , Farnesiltransferasa/metabolismo
17.
Int J Mol Sci ; 24(2)2023 Jan 04.
Artículo en Inglés | MEDLINE | ID: mdl-36674507

RESUMEN

As one of the most imperative antioxidants in higher plants, carotenoids serve as accessory pigments to harvest light for photosynthesis and photoprotectors for plants to adapt to high light stress. Here, we report a small subunit (SSU) of geranylgeranyl diphosphate synthase (GGPPS) in Nicotiana tabacum, NtSSU II, which takes part in the regulation carotenoid biosynthesis by forming multiple enzymatic components with NtGGPPS1 and downstream phytoene synthase (NtPSY1). NtSSU II transcript is widely distributed in various tissues and stimulated by low light and high light treatments. The confocal image revealed that NtSSU II was localized in the chloroplast. Bimolecular fluorescence complementation (BiFC) indicated that NtSSU II and NtGGPPS1 formed heterodimers, which were able to interact with phytoene synthase (NtPSY1) to channel GGPP into the carotenoid production. CRISPR/Cas9-induced ntssu II mutant exhibited decreased leaf area and biomass, along with a decline in carotenoid and chlorophyll accumulation. Moreover, the genes involved in carotenoid biosynthesis were also downregulated in transgenic plants of ntssu II mutant. Taken together, the newly identified NtSSU II could form multiple enzymatic components with NtGGPPS1 and NtPSY1 to regulate carotenoid biosynthesis in N. tabacum, in addition to the co-expression of genes in carotenoids biosynthetic pathways.


Asunto(s)
Carotenoides , Nicotiana , Farnesiltransferasa/genética , Farnesiltransferasa/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Carotenoides/metabolismo , Fotosíntesis , Geranilgeranil-Difosfato Geranilgeraniltransferasa/genética , Geranilgeranil-Difosfato Geranilgeraniltransferasa/metabolismo
18.
Drug Dev Res ; 84(1): 62-74, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36433690

RESUMEN

Rab GTPases are critical regulators of protein trafficking in the cell. To ensure proper cellular localization and function, Rab proteins must undergo a posttranslational modification, termed geranylgeranylation. In the isoprenoid biosynthesis pathway, the enzyme geranylgeranyl diphosphate synthase (GGDPS) generates the 20-carbon isoprenoid donor (geranylgeranyl pyrophosphate [GGPP]), which is utilized in the prenylation of Rab proteins. We have pursued the development of GGDPS inhibitors (GGSI) as a novel means to target Rab activity in cancer cells. Osteosarcoma (OS) and Ewing sarcoma (ES) are aggressive childhood bone cancers with stagnant survival statistics and limited treatment options. Here we show that GGSI treatment induces markers of the unfolded protein response (UPR) and triggers apoptotic cell death in a variety of OS and ES cell lines. Confirmation that these effects were secondary to cellular depletion of GGPP and disruption of Rab geranylgeranylation was confirmed via experiments using exogenous GGPP or specific geranylgeranyl transferase inhibitors. Furthermore, GGSI treatment disrupts cellular migration and invasion in vitro. Metabolomic profiles of OS and ES cell lines identify distinct changes in purine metabolism in GGSI-treated cells. Lastly, we demonstrate that GGSI treatment slows tumor growth in a mouse model of ES. Collectively, these studies support further development of GGSIs as a novel treatment for OS and ES.


Asunto(s)
Neoplasias Óseas , Osteosarcoma , Sarcoma de Ewing , Animales , Ratones , Neoplasias Óseas/tratamiento farmacológico , Farnesiltransferasa/metabolismo , Osteosarcoma/tratamiento farmacológico , Sarcoma de Ewing/tratamiento farmacológico , Terpenos
19.
Magy Onkol ; 67(3): 223-235, 2023 Sep 28.
Artículo en Húngaro | MEDLINE | ID: mdl-38484318

RESUMEN

In silico studies raised the possibility that farnesyltransferase inhibitors (FTIs) may have antitumoral effects on KRAS mutant cancer cells. Accordingly, we have tested FTIs (tipifarnib and lonafarnib) in G12C mutant human cancer cell lines in vitro and in vivo. We have discovered that the combination of the two drugs has a synergistic antitumoral effect. Next, we have tested FTIs on G12D mutant human cancer cell lines and found that the combination has antitumoral effect in various preclinical cancer models. At last, we have also tested FTIs on G12V mutant human cancer cells and again we have detected antitumoral effects. We suggest that FTIs may have clinical relevance outside the HRAS mutant cancers.


Asunto(s)
Neoplasias , Proteínas Proto-Oncogénicas p21(ras) , Humanos , Farnesiltransferasa/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/genética
20.
ACS Chem Biol ; 17(10): 2945-2953, 2022 10 21.
Artículo en Inglés | MEDLINE | ID: mdl-36194691

RESUMEN

Photoswitchable lipids have emerged as attractive tools for the optical control of lipid bioactivity, metabolism, and biophysical properties. Their design is typically based on the incorporation of an azobenzene photoswitch into the hydrophobic lipid tail, which can be switched between its trans- and cis-form using two different wavelengths of light. While glycero- and sphingolipids have been successfully designed to be photoswitchable, isoprenoid lipids have not yet been investigated. Herein, we describe the development of photoswitchable analogs of an isoprenoid lipid and systematically assess their potential for the optical control of various steps in the isoprenylation processing pathway of CaaX proteins in Saccharomyces cerevisiae. One photoswitchable analog of farnesyl diphosphate (AzoFPP-1) allowed effective optical control of substrate prenylation by farnesyltransferase. The subsequent steps of isoprenylation processing (proteolysis by either Ste24 or Rce1 and carboxyl methylation by Ste14) were less affected by photoisomerization of the group introduced into the lipid moiety of the substrate a-factor, a mating pheromone from yeast. We assessed both proteolysis and methylation of the a-factor analogs in vitro and the bioactivity of a fully processed a-factor analog containing the photoswitch, exogenously added to cognate yeast cells. Combined, these data describe the first successful conversion of an isoprenoid lipid into a photolipid and suggest the utility of this approach for the optical control of protein prenylation.


Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Terpenos/metabolismo , Farnesiltransferasa/metabolismo , Péptidos/química , Prenilación de Proteína , Feromonas , Lípidos , Esfingolípidos/metabolismo , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
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