scholarly journals Mechanistic insights into NAD synthase NMNAT chaperoning phosphorylated Tau from pathological aggregation

2019 ◽  
Author(s):  
Xiaojuan Ma ◽  
Jinxia Lu ◽  
Yi Zhu ◽  
Jingfei Xie ◽  
Chong Li ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Single Molecule ◽  
Neurofibrillary Tangles ◽  
Molecular Chaperones ◽  
Phosphorylated Tau ◽  
Amyloid Aggregation ◽  
Computational Approaches ◽  
Nad Metabolism ◽  
Nicotinamide Mononucleotide

AbstractTau hyper-phosphorylation and deposition into neurofibrillary tangles have been found in brains of patients with Alzheimer’s disease (AD) and other tauopathies. Molecular chaperones are involved in regulating the pathological aggregation of phosphorylated Tau (pTau) and modulating disease progression. Here, we report that nicotinamide mononucleotide adenylyltransferase (NMNAT), a well-known NAD synthase, serves as a chaperone of pTau to prevent its amyloid aggregation in vitro as well as mitigate its pathology in a fly tauopathy model. By combining NMR spectroscopy, crystallography, single-molecule and computational approaches, we revealed that NMNAT adopts its enzymatic pocket to specifically bind the phosphorylated sites of pTau, which can be competitively disrupted by the enzymatic substrates of NMNAT. Moreover, we found that NMNAT serves as a co-chaperone of Hsp90 for the specific recognition of pTau over Tau. Our work uncovers a dedicated chaperone of pTau and suggests NMNAT as a key node between NAD metabolism and Tau homeostasis in aging and neurodegeneration.

scholarly journals Nicotinamide mononucleotide adenylyltransferase uses its NAD+ substrate-binding site to chaperone phosphorylated Tau

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Xiaojuan Ma ◽  
Yi Zhu ◽  
Jinxia Lu ◽  
Jingfei Xie ◽  
Chong Li ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Single Molecule ◽  
Molecular Chaperones ◽  
Phosphorylated Tau ◽  
Amyloid Aggregation ◽  
Computational Approaches ◽  
Nad Metabolism ◽  
Nicotinamide Mononucleotide ◽  
Nicotinamide Mononucleotide Adenylyltransferase

Tau hyper-phosphorylation and deposition into neurofibrillary tangles have been found in brains of patients with Alzheimer’s disease (AD) and other tauopathies. Molecular chaperones are involved in regulating the pathological aggregation of phosphorylated Tau (pTau) and modulating disease progression. Here, we report that nicotinamide mononucleotide adenylyltransferase (NMNAT), a well-known NAD+ synthase, serves as a chaperone of pTau to prevent its amyloid aggregation in vitro as well as mitigate its pathology in a fly tauopathy model. By combining NMR spectroscopy, crystallography, single-molecule and computational approaches, we revealed that NMNAT adopts its enzymatic pocket to specifically bind the phosphorylated sites of pTau, which can be competitively disrupted by the enzymatic substrates of NMNAT. Moreover, we found that NMNAT serves as a co-chaperone of Hsp90 for the specific recognition of pTau over Tau. Our work uncovers a dedicated chaperone of pTau and suggests NMNAT as a key node between NAD+ metabolism and Tau homeostasis in aging and neurodegeneration.

scholarly journals The Amyloid Fibril-Forming β-Sheet Regions of Amyloid β and α-Synuclein Preferentially Interact with the Molecular Chaperone 14-3-3ζ

Molecules ◽  
2021 ◽  
Vol 26 (20) ◽  
pp. 6120
Author(s):  
Danielle M. Williams ◽  
David C. Thorn ◽  
Christopher M. Dobson ◽  
Sarah Meehan ◽  
Sophie E. Jackson ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Molecular Chaperones ◽  
Protein Unfolding ◽  
Amyloid Fibrils ◽  
Amyloid Β ◽  
Parkinson’S Diseases ◽  
Amino Acid Form ◽  
Shock Proteins

14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid β (Aβ) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer’s and Parkinson’s diseases, respectively, a process that is intimately linked to the diseases’ progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aβ (Aβ40) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of 15N-labeled Aβ40 and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aβ40 (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt β-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aβ40 and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy.

scholarly journals Dephosphorylation Passivates the Seeding Activity of Oligomeric Tau Derived From Alzheimer’s Brain

2021 ◽  
Vol 14 ◽  
Author(s):  
Ruozhen Wu ◽  
Longfei Li ◽  
Ruirui Shi ◽  
Yan Zhou ◽  
Nana Jin ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Neurofibrillary Tangles ◽  
Cultured Cells ◽  
Phosphorylated Tau ◽  
Western Blots ◽  
Tau Aggregation

Accumulation of intracellular neurofibrillary tangles (NFTs), which are constituted of abnormally phosphorylated tau, is one of the neuropathological hallmarks of Alzheimer’s disease (AD). The oligomeric aggregates of tau in AD brain (AD O-tau) are believed to trigger NFT spreading by seeding normal tau aggregation as toxic seeds, in a prion-like fashion. Here, we revealed the features of AD O-tau by Western blots using antibodies against various epitopes and determined the effect of dephosphorylation on the seeding activity of AD O-tau by capture and seeded aggregation assays. We found that N-terminal truncated and C-terminalhyperphosphorylated tau species were enriched in AD O-tau. Dephosphorylation of AD O-tau by alkaline phosphatasediminished its activity in capturing tau in vitro and ininducing insoluble aggregates in cultured cells. Our resultssuggested that dephosphorylation passivated the seeding activity ofAD O-tau. Inhibition of phosphorylation may be a potentstrategy to prevent the spreading of tau patho3logy.

scholarly journals Nicotinamide Mononucleotide Supplementation

2021 ◽  
Author(s):  
Tamás Kiss
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Molecular Mechanisms ◽  
Bioinformatic Analysis ◽  
Vascular Cognitive Impairment ◽  
Vascular Aging ◽  
Nad Metabolism ◽  
Nicotinamide Mononucleotide

Vascular cognitive impairment (VCI) is a major cause of dementia among elderly individuals. Understanding molecular mechanisms behind vascular aging is essential to develop novel interventional strategies for the treatment and prevention of VCI. Recent studies have provided critical evidence that vascular aging is characterized by cellular NAD+ depletion. In our studies we systematically investigated the effects of boosting cellular NAD+ levels by the use of nicotinamide mononucleotide (NMN), an intermediate of NAD+ metabolism. First, we conducted in vitro examination of cultured cerebromicrovascular endothelial cells (CMVES) isolated from young and aged F344xBN rats. We have shown that NMN treatment attenuates oxidative stress and rescues angiogenic capacity in aged CMVES. Next, in vivo aged C57BL/6 mice were treated daily with NMN for 14 days. NMN treatment rescued cognitive performance, motor function and neurovascular coupling in aged animals. To understand the fundamental gene regulation underlying the beneficial effects of NMN treatment, we generated a miRNA profile from the aorta and a gene expression profile from isolated brain endothelial cells. Bioinformatic analysis revealed that the effects of NMN treatment were mediated by the sirtuin pathway and induced gene expression changes associated with mitochondrial rejuvenation, anti-inflammatory and anti- apoptotic pathways.

NMR spectroscopy of the neuronal tau protein: normal function and implication in Alzheimer's disease

2010 ◽  
Vol 38 (4) ◽  
pp. 1006-1011 ◽  
Author(s):  
Isabelle Landrieu ◽  
Arnaud Leroy ◽  
Caroline Smet-Nocca ◽  
Isabelle Huvent ◽  
Laziza Amniai ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Current Knowledge ◽  
Normal Function ◽  
Phosphorylated Tau ◽  
Functional Assays ◽  
Protein Partners ◽  
Proteomics Tool ◽  
Functional Consequences ◽  
Dependent Protein

NMR spectroscopy was used to explore the different aspects of the normal and pathological functions of tau, but proved challenging because the protein contains 441 amino acids and has poor signal dispersion. We have set out to dissect the phosphorylation patterns of tau in order to understand better its role in the aggregation process and microtubule-binding regulation. Our current knowledge on the functional consequences of specific phosphorylations is still limited, mainly because producing and assessing quantitatively phosphorylated tau samples is far from straightforward, even in vitro. We use NMR spectroscopy as a proteomics tool to characterize the phosphorylation patterns of tau, after in vitro phosphorylation by recombinant kinases. The phosphorylated tau can next be use for functional assays or interaction assays with phospho-dependent protein partners, such as the prolyl cis–trans isomerase Pin1.

scholarly journals The Design and Preclinical Evaluation of a Single-Label Bimodal Nanobody Tracer for Image-Guided Surgery

Biomolecules ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 360
Author(s):  
Pieterjan Debie ◽  
Noemi B. Declerck ◽  
Danny van Willigen ◽  
Celine M. Huygen ◽  
Bieke De Sloovere ◽  
...  
Keyword(s):  
Single Molecule ◽  
Gamma Ray ◽  
Nuclear Imaging ◽  
Primary Amines ◽  
Resection Margins ◽  
Fluorescent Light ◽  
Single Structure ◽  
Fluorescence Images

Intraoperative guidance using targeted fluorescent tracers can potentially provide surgeons with real-time feedback on the presence of tumor tissue in resection margins. To overcome the limited depth penetration of fluorescent light, combining fluorescence with SPECT/CT imaging and/or gamma-ray tracing has been proposed. Here, we describe the design and preclinical validation of a novel bimodal nanobody-tracer, labeled using a “multifunctional single attachment point” (MSAP) label, integrating a Cy5 fluorophore and a diethylenetriaminepentaacetic acid (DTPA) chelator into a single structure. After conjugation of the bimodal MSAP to primary amines of the anti-HER2 nanobody 2Rs15d and 111In-labeling of DTPA, the tracer’s characteristics were evaluated in vitro. Subsequently, its biodistribution and tumor targeting were assessed by SPECT/CT and fluorescence imaging over 24 h. Finally, the tracer’s ability to identify small, disseminated tumor lesions was investigated in mice bearing HER2-overexpressing SKOV3.IP1 peritoneal lesions. [111In]In-MSAP.2Rs15d retained its affinity following conjugation and remained stable for 24 h. In vivo SPECT/CT and fluorescence images showed specific uptake in HER2-overexpressing tumors with low background. High tumor-to-muscle ratios were obtained at 1h p.i. and remained 19-fold on SPECT/CT and 3-fold on fluorescence images over 24 h. In the intraperitoneally disseminated model, the tracer allowed detection of larger lesions via nuclear imaging, while fluorescence enabled accurate removal of submillimeter lesions. Bimodal nuclear/fluorescent nanobody-tracers can thus be conveniently designed by conjugation of a single-molecule MSAP-reagent carrying a fluorophore and chelator for radioactive labeling. Such tracers hold promise for clinical applications.

scholarly journals DNA replication machinery: Insights from in vitro single-molecule approaches

2021 ◽  
Vol 19 ◽  
pp. 2057-2069
Author(s):  
Rebeca Bocanegra ◽  
G.A. Ismael Plaza ◽  
Carlos R. Pulido ◽  
Borja Ibarra
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Replication Machinery

scholarly journals Infrared nanospectroscopy reveals the molecular interaction fingerprint of an aggregation inhibitor with single Aβ42 oligomers

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Francesco Simone Ruggeri ◽  
Johnny Habchi ◽  
Sean Chia ◽  
Robert I. Horne ◽  
Michele Vendruscolo ◽  
...  
Keyword(s):  
Small Molecules ◽  
Single Molecule ◽  
Protein Misfolding ◽  
Molecular Mechanisms ◽  
Chemical Sensitivity ◽  
Incomplete Knowledge ◽  
Parkinson’S Diseases ◽  
Aggregation Inhibitor ◽  
Misfolding Diseases

AbstractSignificant efforts have been devoted in the last twenty years to developing compounds that can interfere with the aggregation pathways of proteins related to misfolding disorders, including Alzheimer’s and Parkinson’s diseases. However, no disease-modifying drug has become available for clinical use to date for these conditions. One of the main reasons for this failure is the incomplete knowledge of the molecular mechanisms underlying the process by which small molecules interact with protein aggregates and interfere with their aggregation pathways. Here, we leverage the single molecule morphological and chemical sensitivity of infrared nanospectroscopy to provide the first direct measurement of the structure and interaction between single Aβ42 oligomeric and fibrillar species and an aggregation inhibitor, bexarotene, which is able to prevent Aβ42 aggregation in vitro and reverses its neurotoxicity in cell and animal models of Alzheimer’s disease. Our results demonstrate that the carboxyl group of this compound interacts with Aβ42 aggregates through a single hydrogen bond. These results establish infrared nanospectroscopy as a powerful tool in structure-based drug discovery for protein misfolding diseases.

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