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Autophagy May 2019Studies regarding macroautophagic/autophagic regulation in endothelial cells (ECs) under diabetic conditions are very limited. Clinical evidence establishes an...
Studies regarding macroautophagic/autophagic regulation in endothelial cells (ECs) under diabetic conditions are very limited. Clinical evidence establishes an endothelial protective effect of metformin, but the underlying mechanisms remain unclear. We aimed to investigate whether metformin exerts its protective role against hyperglycemia-induced endothelial impairment through the autophagy machinery. db/db mice were treated with intravitreal metformin injections. Human umbilical vein endothelial cells (HUVECs) were cultured either in normal glucose (NG, 5.5 mM) or high glucose (HG, 33 mM) medium in the presence or absence of metformin for 72 h. We observed an obvious inhibition of hyperglycemia-triggered autophagosome synthesis in both the diabetic retinal vasculature and cultured HUVECs by metformin, along with restoration of hyperglycemia-impaired Hedgehog (Hh) pathway activity. Specifically, deletion of ATG7 in retinal vascular ECs of db/db mice and cultured HUVECs indicated a detrimental role of autophagy in hyperglycemia-induced endothelial dysfunction. Pretreatment with GANT61, a Hh pathway inhibitor, abolished the metformin-mediated downregulation of autophagy and endothelial protective action. Furthermore, GLI-family (transcription factors of the Hh pathway) knockdown in HUVECs and retinal vasculature revealed that downregulation of hyperglycemia-activated autophagy by the metformin-mediated Hh pathway activation was GLI1 dependent. Mechanistically, GLI1 knockdown-triggered autophagy was related to upregulation of BNIP3, which subsequently disrupted the association of BECN1/Beclin 1 and BCL2. The role of BNIP3 in BECN1 dissociation from BCL2 was further confirmed by BNIP3 overexpression or BNIP3 RNAi. Taken together, the endothelial protective effect of metformin under hyperglycemia conditions could be partly attributed to its role in downregulating autophagy via Hh pathway activation. Abbreviations: 3-MA = 3-methyladenine; 8×GLI BS-FL = 8×GLI-binding site firefly luciferase; AAV = adeno-associated virus; AAV-Cdh5-sh-Atg7 = AAV vectors carrying shRNA against murine Atg7 under control of murine Cdh5 promoter; AAV-Cdh5-sh-Gli1 = AAV vectors carrying shRNA against murine Gli1 under control of murine Cdh5 promoter; AAV-Cdh5-Gli1 = AAV vectors carrying murine Gli1 cDNA under the control of murine Cdh5 core promoter; ACAC = acetyl-CoA carboxylase; Ad-BNIP3 = adenoviruses harboring human BNIP3`; Ad-GLI1 = adenoviruses harboring human GLI1; Ad-sh-ATG7 = adenoviruses harboring shRNA against human ATG7; Ad-sh-BNIP3 = adenoviruses harboring shRNA against human BNIP3; Ad-sh-GLI = adenoviruses harboring shRNA against human GLI; AGEs = advanced glycation end products; ATG = autophagy-related; atg7 mice = mice bearing an Atg7 allele, in which exon 14 of the Atg7 gene is flanked by 2 loxP sites; BafA1 = bafilomycin A; BECN1 = beclin 1; CDH5/VE-cadherin = cadherin 5; CASP3 = caspase 3; CASP8 = caspase 8; CASP9 = caspase 9; ECs = endothelial cells; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; GCL = ganglion cell layer; GFP-LC3B = green fluorescent protein labelled LC3B; HG = high glucose; Hh = Hedgehog; HHIP = hedgehog interacting protein; HUVECs = human umbilical vein endothelial cells; IB4 = isolectin B4; INL = inner nuclear layer; i.p. = intraperitoneal; MAP1LC3/LC3 = microtubule-associated protein 1 light chain 3; MAN = mannitol; MET = metformin; NG = normal glucose; ONL = outer nuclear layer; p-ACAC = phosphorylated acetyl-CoA carboxylase; PECAM1/CD31= platelet/endothelial cell adhesion molecule 1; PRKAA1/2 = protein kinase AMP-activated catalytic subunits alpha 1/2; p-PRKAA1/2 = phosphorylated PRKAA1/2; PTCH1 = patched 1; RAPA = rapamycin; RL = Renilla luciferase; SHH = sonic hedgehog; shRNA = short hairpin RNA; sh-PRKAA1/2 = short hairpin RNA against human PRKAA1/2; scrambled shRNA = the scrambled short hairpin RNA serves as a negative control for the target-specific short hairpin RNA, which has the same nucleotide composition as the input sequence and has no match with any mRNA of the selected organism database; SMO = smoothened, frizzled class receptor; sqRT-PCR = semi-quantitative RT-PCR; TEK/Tie2 = TEK receptor tyrosine kinase; Tek-Cre (+) mice = a mouse strain expressing Cre recombinase under the control of the promoter/enhancer of Tek, in a pan-endothelial fashion; TUNEL = terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling.
Topics: Animals; Autophagy; Capillary Permeability; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Down-Regulation; Endothelium, Vascular; Gene Expression Regulation; HEK293 Cells; Hedgehog Proteins; Human Umbilical Vein Endothelial Cells; Humans; Hyperglycemia; Metformin; Mice; Mice, Inbred C57BL; Mice, Transgenic; Retina; Signal Transduction
PubMed: 30653446
DOI: 10.1080/15548627.2019.1569913 -
Autophagy Jan 2020Autophagy is a conserved catabolic process in eukaryotes that contributes to cell survival in response to multiple stresses and is important for organism fitness. In ,...
Autophagy is a conserved catabolic process in eukaryotes that contributes to cell survival in response to multiple stresses and is important for organism fitness. In , the core machinery of autophagy is well defined, but its transcriptional regulation is largely unknown. The ATG8 (autophagy-related 8) protein plays central roles in decorating autophagosomes and binding to specific cargo receptors to recruit cargo to autophagosomes. We propose that the transcriptional control of genes is important during the formation of autophagosomes and therefore contributes to survival during stress. Here, we describe a yeast one-hybrid (Y1H) screen for transcription factors (TFs) that regulate gene expression in , using the promoters of 4 genes. We identified a total of 225 TFs from 35 families that bind these promoters. The TF- promoter interactions revealed a wide array of diverse TF families for different promoters, as well as enrichment for families of TFs that bound to specific fragments. These TFs are not only involved in plant developmental processes but also in the response to environmental stresses. TGA9 (TGACG (TGA) motif-binding protein 9)/AT1G08320 was confirmed as a positive regulator of autophagy. TGA9 overexpression activated autophagy under both control and stress conditions and transcriptionally up-regulated expression of and additional genes via binding to their promoters. Our results provide a comprehensive resource of TFs that regulate gene expression and lay a foundation for understanding the transcriptional regulation of plant autophagy. ABRC: biological resource center; AP2-EREBP: APETALA2/Ethylene-responsive element binding protein; ARF: auxin response factor; ATF4: activating transcription factor 4; ATG: autophagy-related; ChIP: chromatin immunoprecipitation; DAP-seq: DNA affinity purification sequencing; FOXO: forkhead box O; GFP: green fluorescent protein; GO: gene ontologies; HB: homeobox; LD: long-day; LUC: firefly luciferase; MAP1LC3: microtubule associated protein 1 light chain 3; MDC: monodansylcadaverine; 3-MA: 3-methyladenine; OE: overexpressing; PCD: programmed cell death; qPCR: quantitative polymerase chain reaction; REN: renilla luciferase; RT: room temperature; SD: standard deviation; TF: transcription factor; TFEB: transcription factor EB; TGA: TGACG motif; TOR: target of rapamycin; TSS: transcription start site; WT: wild-type; Y1H: yeast one-hybrid.
Topics: Arabidopsis; Arabidopsis Proteins; Autophagosomes; Autophagy; Autophagy-Related Protein 8 Family; Carrier Proteins; DNA-Binding Proteins; Plant Proteins; Promoter Regions, Genetic; Stress, Physiological; Transcription Factors
PubMed: 30909785
DOI: 10.1080/15548627.2019.1598753 -
Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.ACS Chemical Biology Nov 2012Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have...
Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ~2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ~150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.
Topics: Animals; Cell Line; Crustacea; Enzyme Stability; Fireflies; Gene Expression; Genes, Reporter; Humans; Luciferases; Luminescent Agents; Models, Molecular; Protein Engineering; Pyrazines; Recombinant Fusion Proteins; Renilla; Temperature
PubMed: 22894855
DOI: 10.1021/cb3002478 -
The FEBS Journal Apr 2020Bioluminescence occurs when an enzyme, known as a luciferase, oxidizes a small-molecule substrate, known as a luciferin. Nature has evolved multiple distinct luciferases... (Review)
Review
Bioluminescence occurs when an enzyme, known as a luciferase, oxidizes a small-molecule substrate, known as a luciferin. Nature has evolved multiple distinct luciferases and luciferins independently, all of which accomplish the impressive feat of light emission. One of the best-known examples of bioluminescence is exhibited by fireflies, a class of beetles that use d-luciferin as their substrate. The evolution of bioluminescence in beetles is thought to have emerged from ancestral fatty acyl-CoA synthetase (ACS) enzymes present in all insects. This theory is supported by multiple lines of evidence: Beetle luciferases share high sequence identity with these enzymes, often retain ACS activity, and some ACS enzymes from nonluminous insects can catalyze bioluminescence from synthetic d-luciferin analogues. Recent sequencing of firefly genomes and transcriptomes further illuminates how the duplication of ACS enzymes and subsequent diversification drove the evolution of bioluminescence. These genetic analyses have also uncovered candidate enzymes that may participate in luciferin metabolism. With the publication of the genomes and transcriptomes of fireflies and related insects, we are now better positioned to dissect and learn from the evolution of bioluminescence in beetles.
Topics: Animals; Benzothiazoles; Biocatalysis; Coenzyme A Ligases; Evolution, Molecular; Luciferases, Firefly; Luminescence; Luminescent Measurements; Substrate Specificity
PubMed: 31828943
DOI: 10.1111/febs.15176 -
The Indian Medical Gazette Oct 1875
PubMed: 28999481
DOI: No ID Found -
Biosensors Jun 2022Firefly luciferases catalyze the efficient production of yellow-green light under normal physiological conditions, having been extensively used for bioanalytical... (Review)
Review
Firefly luciferases catalyze the efficient production of yellow-green light under normal physiological conditions, having been extensively used for bioanalytical purposes for over 5 decades. Under acidic conditions, high temperatures and the presence of heavy metals, they produce red light, a property that is called pH-sensitivity or pH-dependency. Despite the demand for physiological intracellular biosensors for pH and heavy metals, firefly luciferase pH and metal sensitivities were considered drawbacks in analytical assays. We first demonstrated that firefly luciferases and their pH and metal sensitivities can be harnessed to estimate intracellular pH variations and toxic metal concentrations through ratiometric analysis. Using sp2 firefly luciferase, the intracellular pH could be ratiometrically estimated in bacteria and then in mammalian cells. The luciferases of sp2 and fireflies were also harnessed to ratiometrically estimate zinc, mercury and other toxic metal concentrations in the micromolar range. The temperature was also ratiometrically estimated using firefly luciferases. The identification and engineering of metal-binding sites have allowed the development of novel luciferases that are more specific to certain metals. The luciferase of the firefly was selected for its special sensitivity to cadmium and mercury, and for its stability at higher temperatures. These color-tuning luciferases can potentially be used with smartphones for hands-on field analysis of water contamination and biochemistry teaching assays. Thus, firefly luciferases are novel color-tuning sensors for intracellular pH and toxic metals. Furthermore, a single luciferase gene is potentially useful as a dual bioluminescent reporter to simultaneously report intracellular ATP and/or luciferase concentrations luminometrically, and pH or metal concentrations ratiometrically, providing a useful tool for real-time imaging of intracellular dynamics and stress.
Topics: Animals; Fireflies; Hydrogen-Ion Concentration; Luciferases; Luciferases, Firefly; Luminescent Measurements; Mammals; Mercury; Metals, Heavy
PubMed: 35735548
DOI: 10.3390/bios12060400 -
Scientific Reports Jul 2022Luciferases are often used as a sensitive, versatile reporter in cell-free transcription-translation (TXTL) systems, for research and practical applications such as...
Luciferases are often used as a sensitive, versatile reporter in cell-free transcription-translation (TXTL) systems, for research and practical applications such as engineering genetic parts, validating genetic circuits, and biosensor outputs. Currently, only two luciferases (Firefly and Renilla) are commonly used without substrate cross-talk. Here we demonstrate the expansion of the cell-free luciferase reporter system, with two orthogonal luciferase reporters: N. nambi luciferase (Luz) and LuxAB. These luciferases do not have cross-reactivity with the Firefly and Renilla substrates. We also demonstrate a substrate regeneration pathway for one of the new luciferases, enabling long-term time courses of protein expression monitoring in the cell-free system. Furthermore, we reduced the number of genes required in TXTL expression, by engineering a cell extract containing part of the luciferase enzymes. Our findings lead to an expanded platform with multiple orthogonal luminescence translation readouts for in vitro protein expression.
Topics: Animals; Fireflies; Genes, Reporter; Indicators and Reagents; Luciferases; Luciferases, Firefly; Luminescence
PubMed: 35798760
DOI: 10.1038/s41598-022-15624-6 -
Proceedings. Biological Sciences Jul 2022We understand very little about the timing and origins of bioluminescence, particularly as a predator avoidance strategy. Understanding the timing of its origins,...
We understand very little about the timing and origins of bioluminescence, particularly as a predator avoidance strategy. Understanding the timing of its origins, however, can help elucidate the evolution of this ecologically important signal. Using fireflies, a prevalent bioluminescent group where bioluminescence primarily functions as aposematic and sexual signals, we explore the origins of this signal in the context of their potential predators. Divergence time estimations were performed using genomic-scale datasets providing a robust estimate for the origin of firefly bioluminescence as both a terrestrial and as an aerial signal. Our results recover the origin of terrestrial beetle bioluminescence at 141.17 (122.63-161.17) Ma and firefly aerial bioluminescence at 133.18 (117.86-152.47) Ma using a large dataset focused on Lampyridae; and terrestrial bioluminescence at 148.03 (130.12-166.80) Ma, with the age of aerial bioluminescence at 104.97 (99.00-120.90) Ma using a complementary Elateroidea dataset. These ages pre-date the origins of all known extant aerial predators (i.e. bats and birds) and support much older terrestrial predators (assassin bugs, frogs, ground beetles, lizards, snakes, hunting spiders and harvestmen) as the drivers of terrestrial bioluminescence in beetles. These ages also support the hypothesis that sexual signalling was probably the original function of this signal in aerial fireflies.
Topics: Animals; Chiroptera; Coleoptera; Fireflies; Genomics; Phylogeny
PubMed: 35855602
DOI: 10.1098/rspb.2022.0821 -
PeerJ 2021Fireflies (Coleoptera: Lampyridae) are commonly recognized by adult traits, such as a soft exoskeleton, lanterns and associated glow and flash patterns, but their larval...
BACKGROUND
Fireflies (Coleoptera: Lampyridae) are commonly recognized by adult traits, such as a soft exoskeleton, lanterns and associated glow and flash patterns, but their larval stage is far less appreciated. However, fireflies spend most of their lives as larvae, and adults of most species rely solely on resources previously obtained. Therefore, studying the immature stages is imperative towards a comprehensive understanding of fireflies. This paper reviews and indicates key gaps in the biology of firefly larvae based on available literature.
METHODOLOGY
We reviewed the literature on firefly larvae to identify key issues and important taxonomic, geographic, and subject biases and gaps.
RESULTS
We found 376 papers that included information on firefly larvae. Only 139 species in 47 genera across eight of eleven lampyrid subfamilies have been studied during larval stages. These numbers reveal a staggering gap, since 94% of species and over half of the genera of fireflies were never studied in a crucial stage of their life cycle. Most studies on firefly larvae focus on two subfamilies (Luciolinae and Lampyrinae) in four zoogeographic regions (Sino-Japanese, Oriental, Nearctic, and Palearctic), whereas the other subfamilies and regions remain largely unstudied. These studies mainly dealt with morphology and behavior, other subjects remaining greatly understudied by comparison, including habitats, life cycle, physiology and interactions.
CONCLUSIONS
Together, these literature biases and gaps highlight how little is known about firefly larvae, and warmly invite basic and applied research, in the field and in the lab, to overcome these limitations and improve our understanding of firefly biology to better preserve them.
PubMed: 34616609
DOI: 10.7717/peerj.12121 -
Current Opinion in Insect Science Apr 2022Fireflies are one of the best-known bioluminescent organisms, and the reaction mechanism and ecological utility of bioluminescence have been well-studied. Genome... (Review)
Review
Fireflies are one of the best-known bioluminescent organisms, and the reaction mechanism and ecological utility of bioluminescence have been well-studied. Genome assemblies of six species of bioluminescent beetles have recently been published. These studies have focused on the evolution of novelties; luciferase, and the biosynthesis of luciferin and defensive chemicals. For example, clustering of the luciferase gene with acyl-CoA synthetase genes on a chromosome in luminous beetle genomes suggests the involvement of tandem gene duplications and neofunctionalization during the evolution of beetle bioluminescence. Several candidate genes for critical roles in beetle bioluminescence have been identified, but their functional analyses are still ongoing. The establishment of a long-term mass-rearing system and strain will be the key for the post-genome research on bioluminescent beetles. Lastly, the application of contemporary chromosome-scale genome assembly techniques to luminous beetles will help resolve outstanding evolutionary questions, such as how many times bioluminescence evolved in this clade.
Topics: Animals; Coleoptera; Fireflies; Luciferases
PubMed: 35091104
DOI: 10.1016/j.cois.2022.100879