scholarly journals WldS protein requires Nmnat activity and a short N-terminal sequence to protect axons in mice

2009 ◽  
Vol 184 (4) ◽  
pp. 491-500 ◽  
Author(s):  
Laura Conforti ◽  
Anna Wilbrey ◽  
Giacomo Morreale ◽  
Lucie Janeckova ◽  
Bogdan Beirowski ◽  
...  
Keyword(s):  
Nicotinamide Adenine Dinucleotide ◽  
Wallerian Degeneration ◽  
Adenosine Triphosphatase ◽  
Chimeric Protein ◽  
Biochemical Property ◽  
Terminal Sequence ◽  
Protective Function ◽  
Nicotinamide Mononucleotide ◽  
Binding Sequence

The slow Wallerian degeneration (WldS) protein protects injured axons from degeneration. This unusual chimeric protein fuses a 70–amino acid N-terminal sequence from the Ube4b multiubiquitination factor with the nicotinamide adenine dinucleotide–synthesizing enzyme nicotinamide mononucleotide adenylyl transferase 1. The requirement for these components and the mechanism of WldS-mediated neuroprotection remain highly controversial. The Ube4b domain is necessary for the protective phenotype in mice, but precisely which sequence is essential and why are unclear. Binding to the AAA adenosine triphosphatase valosin-containing protein (VCP)/p97 is the only known biochemical property of the Ube4b domain. Using an in vivo approach, we show that removing the VCP-binding sequence abolishes axon protection. Replacing the WldS VCP-binding domain with an alternative ataxin-3–derived VCP-binding sequence restores its protective function. Enzyme-dead WldS is unable to delay Wallerian degeneration in mice. Thus, neither domain is effective without the function of the other. WldS requires both of its components to protect axons from degeneration.

scholarly journals WldS requires Nmnat1 enzymatic activity and N16–VCP interactions to suppress Wallerian degeneration

2009 ◽  
Vol 184 (4) ◽  
pp. 501-513 ◽  
Author(s):  
Michelle A. Avery ◽  
Amy E. Sheehan ◽  
Kimberly S. Kerr ◽  
Jing Wang ◽  
Marc R. Freeman
Keyword(s):  
Drosophila Melanogaster ◽  
Amino Acid ◽  
Enzymatic Activity ◽  
Wallerian Degeneration ◽  
Protective Activity ◽  
Protective Function ◽  
Nicotinamide Mononucleotide ◽  
Severed Axons ◽  
Drosophila Cells

Slow Wallerian degeneration (WldS) encodes a chimeric Ube4b/nicotinamide mononucleotide adenylyl transferase 1 (Nmnat1) fusion protein that potently suppresses Wallerian degeneration, but the mechanistic action of WldS remains controversial. In this study, we characterize WldS-mediated axon protection in vivo using Drosophila melanogaster. We show that Nmnat1 can protect severed axons from autodestruction but at levels significantly lower than WldS, and enzyme-dead versions of Nmnat1 and WldS exhibit severely reduced axon-protective function. Interestingly, a 16–amino acid N-terminal domain of WldS (termed N16) accounts for the differences in axon-sparing activity between WldS and Nmnat1, and N16-dependent enhancement of Nmnat1-protective activity in WldS requires the N16-binding protein valosin-containing protein (VCP)/TER94. Thus, WldS-mediated suppression of Wallerian degeneration results from VCP–N16 interactions and Nmnat1 activity converging in vivo. Surprisingly, mouse Nmnat3, a mitochondrial Nmnat enzyme that localizes to the cytoplasm in Drosophila cells, protects severed axons at levels indistinguishable from WldS. Thus, nuclear Nmnat activity does not appear to be essential for WldS-like axon protection.

scholarly journals Oral Administration of Nicotinamide Mononucleotide Increases Nicotinamide Adenine Dinucleotide Level in an Animal Brain

Nutrients ◽  
2022 ◽  
Vol 14 (2) ◽  
pp. 300
Author(s):  
Chidambaram Ramanathan ◽  
Thomas Lackie ◽  
Drake H. Williams ◽  
Paul S. Simone ◽  
Yufeng Zhang ◽  
...  
Keyword(s):  
Oral Administration ◽  
Neurodegenerative Diseases ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine ◽  
Disease States ◽  
Nicotinamide Mononucleotide ◽  
Age Related ◽  
Mice Brain ◽  
The Brain

As a redox-sensitive coenzyme, nicotinamide adenine dinucleotide (NAD+) plays a central role in cellular energy metabolism and homeostasis. Low NAD+ levels are linked to multiple disease states, including age-related diseases, such as metabolic and neurodegenerative diseases. Consequently, restoring/increasing NAD+ levels in vivo has emerged as an important intervention targeting age-related neurodegenerative diseases. One of the widely studied approaches to increase NAD+ levels in vivo is accomplished by using NAD+ precursors, such as nicotinamide mononucleotide (NMN). Oral administration of NMN has been shown to successfully increase NAD+ levels in a variety of tissues; however, it remains unclear whether NMN can cross the blood–brain barrier to increase brain NAD+ levels. This study evaluated the effects of oral NMN administration on NAD+ levels in C57/B6J mice brain tissues. Our results demonstrate that oral gavage of 400 mg/kg NMN successfully increases brain NAD+ levels in mice after 45 min. These findings provide evidence that NMN may be used as an intervention to increase NAD+ levels in the brain.

scholarly journals Creating enzymes and self-sufficient cells for biosynthesis of the non-natural cofactor nicotinamide cytosine dinucleotide

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xueying Wang ◽  
Yanbin Feng ◽  
Xiaojia Guo ◽  
Qian Wang ◽  
Siyang Ning ◽  
...  
Keyword(s):  
Biological Sciences ◽  
Escherichia Coli ◽  
Nicotinamide Adenine Dinucleotide ◽  
Chemical Biology ◽  
Reduced Form ◽  
Redox Transformations ◽  
Nicotinamide Mononucleotide ◽  
Self Sufficiency ◽  
Binding Pockets ◽  
Pathway Regulation

AbstractNicotinamide adenine dinucleotide (NAD) and its reduced form are indispensable cofactors in life. Diverse NAD mimics have been developed for applications in chemical and biological sciences. Nicotinamide cytosine dinucleotide (NCD) has emerged as a non-natural cofactor to mediate redox transformations, while cells are fed with chemically synthesized NCD. Here, we create NCD synthetase (NcdS) by reprograming the substrate binding pockets of nicotinic acid mononucleotide (NaMN) adenylyltransferase to favor cytidine triphosphate and nicotinamide mononucleotide over their regular substrates ATP and NaMN, respectively. Overexpression of NcdS alone in the model host Escherichia coli facilitated intracellular production of NCD, and higher NCD levels up to 5.0 mM were achieved upon further pathway regulation. Finally, the non-natural cofactor self-sufficiency was confirmed by mediating an NCD-linked metabolic circuit to convert L-malate into D-lactate. NcdS together with NCD-linked enzymes offer unique tools and opportunities for intriguing studies in chemical biology and synthetic biology.

scholarly journals Hyperglycaemia up-regulates placental growth factor (PlGF) expression and secretion in endothelial cells via suppression of PI3 kinase-Akt signalling and activation of FOXO1

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Samir Sissaoui ◽  
Stuart Egginton ◽  
Ling Ting ◽  
Asif Ahmed ◽  
Peter W. Hewett
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Endothelial Dysfunction ◽  
Akt Activation ◽  
Forkhead Box ◽  
Pi3k Activity ◽  
Binding Sequence

AbstractPlacenta growth factor (PlGF) is a pro-inflammatory angiogenic mediator that promotes many pathologies including diabetic complications and atherosclerosis. Widespread endothelial dysfunction precedes the onset of these conditions. As very little is known of the mechanism(s) controlling PlGF expression in pathology we investigated the role of hyperglycaemia in the regulation of PlGF production in endothelial cells. Hyperglycaemia stimulated PlGF secretion in cultured primary endothelial cells, which was suppressed by IGF-1-mediated PI3K/Akt activation. Inhibition of PI3K activity resulted in significant PlGF mRNA up-regulation and protein secretion. Similarly, loss or inhibition of Akt activity significantly increased basal PlGF expression and prevented any further PlGF secretion in hyperglycaemia. Conversely, constitutive Akt activation blocked PlGF secretion irrespective of upstream PI3K activity demonstrating that Akt is a central regulator of PlGF expression. Knock-down of the Forkhead box O-1 (FOXO1) transcription factor, which is negatively regulated by Akt, suppressed both basal and hyperglycaemia-induced PlGF secretion, whilst FOXO1 gain-of-function up-regulated PlGF in vitro and in vivo. FOXO1 association to a FOXO binding sequence identified in the PlGF promoter also increased in hyperglycaemia. This study identifies the PI3K/Akt/FOXO1 signalling axis as a key regulator of PlGF expression and unifying pathway by which PlGF may contribute to common disorders characterised by endothelial dysfunction, providing a target for therapy.

scholarly journals In VivoVisualization of Heterogeneous Intratumoral Distribution of Hypoxia-Inducible Factor-1αActivity by the Fusion of High-Resolution SPECT and Morphological Imaging Tests

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Hirofumi Fujii ◽  
Masayuki Yamaguchi ◽  
Kazumasa Inoue ◽  
Yasuko Mutou ◽  
Masashi Ueda ◽  
...  
Keyword(s):  
High Resolution ◽  
Ex Vivo ◽  
Hypoxia Inducible Factor ◽  
Chimeric Protein ◽  
Small Animal ◽  
Ct Scanner ◽  
Heterogeneous Distribution ◽  
Fusion Images ◽  
Intratumoral Distribution

Purpose. We aimed to clearly visualize heterogeneous distribution of hypoxia-inducible factor 1α(HIF) activity in tumor tissuesin vivo.Methods. We synthesized of125I-IPOS, a125I labeled chimeric protein probe, that would visualize HIF activity. The biodistribution of125I-IPOS in FM3A tumor-bearing mice was evaluated. Then, the intratumoral localization of this probe was observed by autoradiography, and it was compared with histopathological findings. The distribution of125I-IPOS in tumors was imaged by a small animal SPECT/CT scanner. The obtainedin vivoSPECT-CT fusion images were compared withex vivoimages of excised tumors. Fusion imaging with MRI was also examined.Results.125I-IPOS well accumulated in FM3A tumors. The intratumoral distribution of125I-IPOS by autoradiography was quite heterogeneous, and it partially overlapped with that of pimonidazole. High-resolution SPECT-CT fusion images successfully demonstrated the heterogeneity of125I-IPOS distribution inside tumors. SPECT-MRI fusion images could give more detailed information about the intratumoral distribution of125I-IPOS.Conclusion. High-resolution SPECT images successfully demonstrated heterogeneous intratumoral distribution of125I-IPOS. SPECT-CT fusion images, more favorably SPECT-MRI fusion images, would be useful to understand the features of heterogeneous intratumoral expression of HIF activityin vivo.

scholarly journals Detection and pharmacological modulation of nicotinamide mononucleotide (NMN) in vitro and in vivo

2009 ◽  
Vol 77 (10) ◽  
pp. 1612-1620 ◽  
Author(s):  
Laura Formentini ◽  
Flavio Moroni ◽  
Alberto Chiarugi
Keyword(s):  
Pharmacological Modulation ◽  
Nicotinamide Mononucleotide

scholarly journals A nucleosome-positioning sequence is required for GCN4 to activate transcription in the absence of a TATA element.

1990 ◽  
Vol 10 (8) ◽  
pp. 4256-4265 ◽  
Author(s):  
C J Brandl ◽  
K Struhl
Keyword(s):  
Binding Site ◽  
Transcriptional Activation ◽  
Nucleosome Positioning ◽  
Alternative Mechanism ◽  
Tata Element ◽  
Binding Factor ◽  
Positioning Analysis ◽  
Binding Sequence

In the gal-his3 hybrid promoter his3-GG1, the yeast upstream activator protein GCN4 stimulates transcription when bound at the position normally occupied by the TATA element. This TATA-independent activation by GCN4 requires two additional elements in the gal enhancer region that are distinct from those involved in normal galactose induction. Both additional elements appear to be functionally distinct from a classical TATA element because they cannot be replaced by the TFIID-binding sequence TATAAA. One of these elements, termed Q, is essential for GCN4-activated transcription and contains the sequence GTCAC CCG, which overlaps (but is distinct from) a GAL4 binding site. Surprisingly, relatively small increases in the distance between Q and the GCN4 binding site significantly reduce the level of transcription. The Q element specifically interacts with a yeast protein (Q-binding protein [QBP]) that may be equivalent to Y, a protein that binds at a sequence that forms a constraint to nucleosome positioning. Analysis of various deletion mutants indicates that the sequence requirements for binding by QBP in vitro are indistinguishable from those necessary for Q activity in vivo, strongly suggesting that QBP is required for the function of this TATA-independent promoter. These results support the view that transcriptional activation can occur by an alternative mechanism in which the TATA-binding factor TFIID either is not required or is not directly bound to DNA. In addition, they suggest a potential role of nucleosome positioning for the activity of a promoter.

scholarly journals Dimeric heat shock protein 40 binds radial spokes for generating coupled power strokes and recovery strokes of 9 + 2 flagella

2008 ◽  
Vol 180 (2) ◽  
pp. 403-415 ◽  
Author(s):  
Chun Yang ◽  
Heather A. Owen ◽  
Pinfen Yang
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Adenosine Triphosphatase ◽  
Stable Conformation ◽  
Initiation And Propagation ◽  
Evolutionarily Conserved ◽  
Radial Spokes ◽  
Cilia And Flagella ◽  
Hsp 40

T-shape radial spokes regulate flagellar beating. However, the precise function and molecular mechanism of these spokes remain unclear. Interestingly, Chlamydomonas reinhardtii flagella lacking a dimeric heat shock protein (HSP) 40 at the spokehead–spokestalk juncture appear normal in length and composition but twitch actively while cells jiggle without procession, resembling a central pair (CP) mutant. HSP40− cells begin swimming upon electroporation with recombinant HSP40. Surprisingly, the rescue doesn't require the signature DnaJ domain. Furthermore, the His-Pro-Asp tripeptide that is essential for stimulating HSP70 adenosine triphosphatase diverges in candidate orthologues, including human DnaJB13. Video microscopy reveals hesitance in bend initiation and propagation as well as irregular stalling and stroke switching despite fairly normal waveform. The in vivo evidence suggests that the evolutionarily conserved HSP40 specifically transforms multiple spoke proteins into stable conformation capable of mechanically coupling the CP with dynein motors. This enables 9 + 2 cilia and flagella to bend and switch to generate alternate power strokes and recovery strokes.

Chronic nicotinamide mononucleotide supplementation elevates blood nicotinamide adenine dinucleotide levels and alters muscle motility in healthy old men

2021 ◽  
Author(s):  
Masaki Igarashi ◽  
Masaomi Miura ◽  
Yoshiko Nakagawa-Nagahama ◽  
Keisuke Yaku ◽  
Kosuke Kashiwabara ◽  
...  
Keyword(s):  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine ◽  
Double Blind ◽  
Oral Supplementation ◽  
Healthy Elderly ◽  
Nicotinamide Mononucleotide ◽  
Parallel Group ◽  
And Performance ◽  
Old Men ◽  
Parallel Group Trial

Abstract Preclinical studies have revealed that the elevation of nicotinamide adenine dinucleotide (NAD+) levels on administration of an NAD+ precursor, nicotinamide mononucleotide (NMN), can mitigate aging-related disorders; however, human data are sparse. Therefore, we aimed to investigate whether the chronic oral supplementation of NMN can elevate blood NAD+ levels and alter physiological dysfunctions, including muscle weakness, in healthy elderly participants. We administered 250 mg NMN per day to aged men for 6 or 12 weeks (n=21 for 6 weeks, n=10 for 12 weeks) in a placebo-controlled, randomized, double blind, parallel-group trial. Chronic supplementation with NMN was well tolerated and did not cause any significant deleterious effect. Metabolomic analysis of whole blood demonstrated that the oral supplementation of NMN significantly increased the concentrations of NAD+ and NAD+ metabolites. Moreover, NMN significantly improved muscle strength and performance, which were evaluated using the 30-second chair stand test, walking speed, and grip strength, and it showed no significant effect on body composition. Thus, our evidence indicates that chronic oral NMN supplementation can be an efficient NAD+ booster for preventing aging-related muscle dysfunctions in humans.

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