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Cell Death and Differentiation Jul 2023SREBP2 is a master regulator of the mevalonate pathway (MVP), a biosynthetic process that drives the synthesis of dolichol, heme A, ubiquinone and cholesterol and also...
SREBP2 is a master regulator of the mevalonate pathway (MVP), a biosynthetic process that drives the synthesis of dolichol, heme A, ubiquinone and cholesterol and also provides substrates for protein prenylation. Here, we identify SREBP2 as a novel substrate for USP28, a deubiquitinating enzyme that is frequently upregulated in squamous cancers. Our results show that silencing of USP28 reduces expression of MVP enzymes and lowers metabolic flux into this pathway. We also show that USP28 binds to mature SREBP2, leading to its deubiquitination and stabilisation. USP28 depletion rendered cancer cells highly sensitive to MVP inhibition by statins, which was rescued by the addition of geranyl-geranyl pyrophosphate. Analysis of human tissue microarrays revealed elevated expression of USP28, SREBP2 and MVP enzymes in lung squamous cell carcinoma (LSCC) compared to lung adenocarcinoma (LADC). Moreover, CRISPR/Cas-mediated deletion of SREBP2 selectively attenuated tumour growth in a KRas/p53/LKB1 mutant mouse model of lung cancer. Finally, we demonstrate that statins synergise with a dual USP28/25 inhibitor to reduce viability of SCC cells. Our findings suggest that combinatorial targeting of MVP and USP28 could be a therapeutic strategy for the treatment of squamous cell carcinomas.
Topics: Mice; Animals; Humans; Mevalonic Acid; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Carcinoma, Non-Small-Cell Lung; Lung Neoplasms; Carcinoma, Squamous Cell; Ubiquitin Thiolesterase
PubMed: 37202505
DOI: 10.1038/s41418-023-01173-6 -
Cell Death & Disease Jan 2024Chronic metabolic stress paradoxically elicits pro-tumorigenic signals that facilitate cancer stem cell (CSC) development. Therefore, elucidating the metabolic sensing...
Chronic metabolic stress paradoxically elicits pro-tumorigenic signals that facilitate cancer stem cell (CSC) development. Therefore, elucidating the metabolic sensing and signaling mechanisms governing cancer cell stemness can provide insights into ameliorating cancer relapse and therapeutic resistance. Here, we provide convincing evidence that chronic metabolic stress triggered by hyaluronan production augments CSC-like traits and chemoresistance by partially impairing nucleotide sugar metabolism, dolichol lipid-linked oligosaccharide (LLO) biosynthesis and N-glycan assembly. Notably, preconditioning with either low-dose tunicamycin or 2-deoxy-D-glucose, which partially interferes with LLO biosynthesis, reproduced the promoting effects of hyaluronan production on CSCs. Multi-omics revealed characteristic changes in N-glycan profiles and Notch signaling activation in cancer cells exposed to mild glycometabolic stress. Restoration of N-glycan assembly with glucosamine and mannose supplementation and Notch signaling blockade attenuated CSC-like properties and further enhanced the therapeutic efficacy of cisplatin. Therefore, our findings uncover a novel mechanism by which tolerable glycometabolic stress boosts cancer cell resilience through altered N-glycosylation and Notch signaling activation.
Topics: Humans; Glycosylation; Hyaluronic Acid; Resilience, Psychological; Neoplasm Recurrence, Local; Polysaccharides; Dietary Supplements; Neoplastic Stem Cells
PubMed: 38225221
DOI: 10.1038/s41419-024-06432-z -
Molecules (Basel, Switzerland) Dec 2023Dolichols are isoprenoid end-products of the mevalonate and 2-methyl-D-erythritol-4-phosphate pathways. The synthesis of dolichols is initiated with the addition of...
Dolichols are isoprenoid end-products of the mevalonate and 2-methyl-D-erythritol-4-phosphate pathways. The synthesis of dolichols is initiated with the addition of several molecules of isopentenyl diphosphate to farnesyl diphosphate. This reaction is catalyzed by a -prenyltransferase and leads to the formation of polyprenyl diphosphate. Subsequent steps involve the dephosphorylation and reduction of the α-isoprene unit by a polyprenol reductase, resulting in the generation of dolichol. The size of the dolichol varies, depending on the number of isoprene units incorporated. In eukaryotes, dolichols are synthesized as a mixture of four or more different lengths. Their biosynthesis is predicted to occur in the endoplasmic reticulum, where dolichols play an essential role in protein glycosylation. In this study, we have developed a selection of aptamers targeting dolichols and enhanced their specificity by incorporating fatty acids for negative selection. One aptamer showed high enrichment and specificity for linear polyisoprenoids containing at least one oxygen atom, such as an alcohol or aldehyde, in the α-isoprene unit. The selected aptamer proved to be a valuable tool for the subcellular localization of polyisoprenoids in the malaria parasite. To the best of our knowledge, this is the first time that polyisoprenoids have been localized within a cell using aptamer-based imaging techniques.
Topics: Animals; Parasites; Diagnostic Imaging; Dolichols; Malaria; Butadienes; Hemiterpenes
PubMed: 38202761
DOI: 10.3390/molecules29010178 -
Cell Death & Disease Jul 2023Retinitis pigmentosa (RP) defines a group of hereditary progressive rod-cone degenerations that exhibit a common phenotype caused by variants in over 70 genes. While...
Retinitis pigmentosa (RP) defines a group of hereditary progressive rod-cone degenerations that exhibit a common phenotype caused by variants in over 70 genes. While most variants in the dehydrodolichyl diphosphate synthase (DHDDS) gene result in syndromic abnormalities, some variants cause non-syndromic RP (RP59). DHDDS encodes one subunit of the enzyme cis-prenyltransferase (CPT), which is required for the synthesis of dolichol (Dol), that is a necessary protein glycosylation cofactor. We previously reported the creation and initial characterization of a knock-in (KI) mouse model harboring the most prevalent RP59-associated DHDDS variant (K42E) to understand how defects in DHDDS lead to retina-specific pathology. This model exhibited no profound retinal degeneration, nor protein N-glycosylation defects. Here, we report that the Dol isoprenylogue species in retina, liver, and brain of the K42E mouse model are statistically shorter than in the corresponding tissues of age-matched controls, as reported in blood and urine of RP59 patients. Retinal transcriptome analysis demonstrated elevation of many genes encoding proteins involved in synaptogenesis and synaptic function. Quantitative retinal cell layer thickness measurements demonstrated a significant reduction in the inner nuclear layer (INL) and total retinal thickness (TRT) beginning at postnatal (PN) ∼2 months, progressively increasing to PN 18-mo. Histological analysis revealed cell loss in the INL, outer plexiform layer (OPL) disruption, and ectopic localization of outer nuclear layer (ONL) nuclei into the OPL of K42E mutant retinas, relative to controls. Electroretinograms (ERGs) of mutant mice exhibited reduced b-wave amplitudes beginning at PN 1-mo, progressively declining through PN 18-mo, without appreciable a-wave attenuation, relative to controls. Our results suggest that the underlying cause of DHDDS K42E variant driven RP59 retinal pathology is defective synaptic transmission from outer to inner retina.
Topics: Animals; Mice; Retina; Retinal Degeneration; Retinitis Pigmentosa; Electroretinography; Synaptic Transmission
PubMed: 37443173
DOI: 10.1038/s41419-023-05936-4 -
JIMD Reports May 2024ALG1-CDG is a rare, clinically variable metabolic disease, caused by the defect of adding the first mannose (Man) to N-acetylglucosamine (GlcNAc)-pyrophosphate...
ALG1-CDG is a rare, clinically variable metabolic disease, caused by the defect of adding the first mannose (Man) to N-acetylglucosamine (GlcNAc)-pyrophosphate (PP)-dolichol to the growing oligosaccharide chain, resulting in impaired N-glycosylation of proteins. N-glycosylation has a key role in functionality, stability, and half-life of most proteins. Therefore, congenital defects of glycosylation typically are multisystem disorders. Here we report a 3-year-old patient with severe neurological, cardiovascular, respiratory, musculoskeletal and gastrointestinal symptoms. ALG1-CDG was suggested based on exome sequencing and Western blot analysis. Despite her severe clinical manifestations and genetic diagnosis, serum transferrin glycoform analysis was normal. Western blot analysis of highly glycosylated proteins in fibroblasts revealed decreased intercellular adhesion molecule 1 (ICAM1), but normal lysosomal associated membrane protein 1 and 2 (LAMP1 and LAMP2) expression levels. Glycoproteomics in fibroblasts showed the presence of the abnormal tetrasacharide. Reviewing the literature, we found 86 reported ALG1-CDG patients, but only one with normal transferrin analysis. Based on our results we would like to highlight the importance of multiple approaches in diagnosing ALG1-CDG, as normal serum transferrin glycosylation or other biomarkers with normal expression levels can occur.
PubMed: 38736633
DOI: 10.1002/jmd2.12415 -
PLoS Pathogens Jan 2024A proposed treatment for malaria is a combination of fosmidomycin and clindamycin. Both compounds inhibit the methylerythritol 4-phosphate (MEP) pathway, the parasitic...
A proposed treatment for malaria is a combination of fosmidomycin and clindamycin. Both compounds inhibit the methylerythritol 4-phosphate (MEP) pathway, the parasitic source of farnesyl and geranylgeranyl pyrophosphate (FPP and GGPP, respectively). Both FPP and GGPP are crucial for the biosynthesis of several essential metabolites such as ubiquinone and dolichol, as well as for protein prenylation. Dietary prenols, such as farnesol (FOH) and geranylgeraniol (GGOH), can rescue parasites from MEP inhibitors, suggesting the existence of a missing pathway for prenol salvage via phosphorylation. In this study, we identified a gene in the genome of P. falciparum, encoding a transmembrane prenol kinase (PolK) involved in the salvage of FOH and GGOH. The enzyme was expressed in Saccharomyces cerevisiae, and its FOH/GGOH kinase activities were experimentally validated. Furthermore, conditional knockout parasites (Δ-PolK) were created to investigate the biological importance of the FOH/GGOH salvage pathway. Δ-PolK parasites were viable but displayed increased susceptibility to fosmidomycin. Their sensitivity to MEP inhibitors could not be rescued by adding prenols. Additionally, Δ-PolK parasites lost their capability to utilize prenols for protein prenylation. Experiments using culture medium supplemented with whole/delipidated human plasma in transgenic parasites revealed that human plasma has components that can diminish the effectiveness of fosmidomycin. Mass spectrometry tests indicated that both bovine supplements used in culture and human plasma contain GGOH. These findings suggest that the FOH/GGOH salvage pathway might offer an alternate source of isoprenoids for malaria parasites when de novo biosynthesis is inhibited. This study also identifies a novel kind of enzyme related to isoprenoid metabolism.
Topics: Humans; Animals; Cattle; Parasites; Phosphates; Terpenes; Pentanols; Diterpenes; Fosfomycin; Hemiterpenes
PubMed: 38277417
DOI: 10.1371/journal.ppat.1011557 -
Molecular Syndromology Aug 2023Congenital glycosylation disorders are multisystem diseases with heterogeneous clinical manifestations caused by defects in the synthesis of the glycan moiety of...
INTRODUCTION
Congenital glycosylation disorders are multisystem diseases with heterogeneous clinical manifestations caused by defects in the synthesis of the glycan moiety of glycoproteins or glycolipids or the binding of glycans to proteins and lipids. DPAGT1 (UDP-GlcNAc: dolichol phosphate N-acetylglucosamine-1-phosphotransferase) is an initiating protein in the biosynthetic pathway of dolichol-linked oligosaccharides required for protein N-glycosylation. Pathogenic variants in (UDP-GlcNAc: dolichol phosphate N-acetylglucosamine-1-phosphotransferase) gene cause a rare type of congenital glycosylation disorder called DPAGT1-CDG (formerly CDG-Ij) (OMIM #608093). It is a rare autosomal recessive disease or a milder version with congenital myasthenic syndrome known as DPAGT1-CMS. A severe disease course with hypotonia, cataracts, skeletal deformities, resistant epilepsy, intellectual disability, global developmental delay, premature death has been described in most patients with DPAGT1-CDG.
PATIENT PRESENTATION
We describe two patients with variants in the gene: an 8-month-old boy with a homozygous, missense :c.339T>G (p.Phe113Leu) novel variant and a 13-year-old female patient with compound heterozygous variants, :c.466C>T (p.Arg156Cys, R156C) and :c.161+5G>A. While the 8-month-old patient was diagnosed with congenital cataract at the age of 1 month, had dysmorphic findings, and epilepsy, clinical symptoms in the other patient appeared later but with more prominent muscle weakness, behavioral disorder, dysmorphic findings, and no epilepsy.
DISCUSSION
Cholinesterase inhibitor therapy was found to be effective in patients against muscle weakness, supporting deficiency as the underlying etiology. We started pyridostigmine treatment in our patient with more pronounced muscle weakness, and we saw its benefit. We aimed to present our patients diagnosed with DPAGT1-CDG due to different variants in the same gene and different clinical presentations, treatment and to compare them with other patients in the literature.
PubMed: 37766827
DOI: 10.1159/000529494 -
Poultry Science Jan 2024Heterosis is the major benefit of crossbreeding and has been exploited in laying hens breeding for a long time. This genetic phenomenon has been linked to various modes...
Transcriptome analysis of ovarian tissues highlights genes controlling energy homeostasis and oxidative stress as potential drivers of heterosis for egg number and clutch size in crossbred laying hens.
Heterosis is the major benefit of crossbreeding and has been exploited in laying hens breeding for a long time. This genetic phenomenon has been linked to various modes of nonadditive gene action. However, the molecular mechanism of heterosis for egg production in laying hens has not been fully elucidated. To fill this research gap, we sequenced mRNAs and lncRNAs of the ovary stroma containing prehierarchical follicles in White Leghorn, Rhode Island Red chickens as well as their reciprocal crossbreds that demonstrated heterosis for egg number and clutch size. We further delineated the modes of mRNAs and lncRNAs expression to identify their potential functions in the observed heterosis. Results showed that dominance was the principal mode of nonadditive expression exhibited by mRNAs and lncRNAs in the prehierarchical follicles of crossbred hens. Specifically, low-parent dominance was the main mode of mRNA expression, while high-parent dominance was the predominant mode of lncRNA expression. Important pathways enriched by genes that showed higher expression in crossbreds compared to either one or both parental lines were cell adhesion molecules, tyrosine and purine metabolism. In contrast, ECM-receptor interaction, focal adhesion, PPAR signaling, and ferroptosis were enriched in genes with lower expression in the crossbred. Protein network interaction identified nonadditively expressed genes including apolipoprotein B (APOB), transferrin, acyl-CoA synthetase medium-chain family member (APOBEC) 3, APOBEC1 complementation factor, and cathepsin S as hub genes. Among these potential hub genes, APOB was the only gene with underdominance expression common to the 2 reciprocal crossbred lines, and has been linked to oxidative stress. LncRNAs with nonadditive expression in the crossbred hens targeted natriuretic peptide receptor 1, epidermal differentiation protein beta, spermatogenesis-associated gene 22, sperm-associated antigen 16, melanocortin 2 receptor, dolichol kinase, glycine amiinotransferase, and prolactin releasing hormone receptor. In conclusion, genes with nonadditive expression in the crossbred may play crucial roles in follicle growth and atresia by improving follicle competence and increasing oxidative stress, respectively. These 2 phenomena could underpin heterosis for egg production in crossbred laying hens.
Topics: Male; Animals; Female; Chickens; Clutch Size; Hybrid Vigor; RNA, Long Noncoding; Plant Breeding; Gene Expression Profiling; Homeostasis; Oxidative Stress; Apolipoproteins B
PubMed: 37980751
DOI: 10.1016/j.psj.2023.103163 -
BioRxiv : the Preprint Server For... Apr 2024The oligosaccharide needed for protein -glycosylation is assembled on a lipid carrier via a multi-step pathway. Synthesis is initiated on the cytoplasmic face of the...
The oligosaccharide needed for protein -glycosylation is assembled on a lipid carrier via a multi-step pathway. Synthesis is initiated on the cytoplasmic face of the endoplasmic reticulum (ER) and completed on the luminal side after transbilayer translocation of a heptasaccharide lipid intermediate. More than 30 Congenital Disorders of Glycosylation (CDGs) are associated with this pathway, including CDG 1N which results from defects in the membrane protein Rft1. Rft1 is essential for the viability of yeast and mammalian cells and was proposed as the transporter needed to flip the heptasaccharide lipid intermediate across the ER membrane. However, other studies indicated that Rft1 is not required for heptasaccharide lipid flipping in microsomes or unilamellar vesicles reconstituted with ER membrane proteins, nor is it required for the viability of at least one eukaryote. It is therefore not known what role Rft1 plays in -glycosylation. Here, we present a molecular characterization of human Rft1, using yeast cells as a reporter system. We show that it is a multi-spanning membrane protein located in the ER, with its N and C-termini facing the cytoplasm. It is not -glycosylated. The majority of CDG 1N mutations map to highly conserved regions of the protein. We identify key residues that are important for Rft1's ability to support glycosylation and cell viability. Our results provide a necessary platform for future work on this enigmatic protein.
PubMed: 38617304
DOI: 10.1101/2024.04.03.587922 -
African Journal of Infectious Diseases 2024Global mycosis is still a problem. One of these is the cryptococcal disease. A systemic mycosis brought on by Cryptococcus is called cryptococcosis. Host immunological...
Global mycosis is still a problem. One of these is the cryptococcal disease. A systemic mycosis brought on by Cryptococcus is called cryptococcosis. Host immunological conditions influence infection with Cryptococcosis. When environmental spores are inhaled by the host, the spores get to the lungs, an infection is created. Alveolar macrophages and other immune cells recognize in the lung. The initial line of defense against pathogens in the phagolysosome is provided by alveolar macrophages found in the lungs. When the immune system is weak, Cryptococcus uses the evasion system as a molecular interaction with the immune system and persists in the lungs without causing any symptoms such as Factor Transcription, Cell masking, N-glycan structure, Extracellular molecule, and Antioxidant system. The evasion mechanism protects and makes Cryptococcus disseminate throughout the other organs, especially CNS. If Cryptococcus escapes against the host immune system, it will disseminate to other organs, especially Cerebrospinal System by Three mechanisms. There are Trojan Horse, Paracellular, and Transcellular interactions with Blood-Brain Barrier. Disease severity is determined by the Interaction between the host's immune system and the fungus.
PubMed: 38058414
DOI: 10.21010/Ajidv18i1.3