scholarly journals Tau amyloidogenesis begins with a loss of its conformational polymorphism

2020 ◽  
Author(s):  
María del Carmen Fernández-Ramírez ◽  
Rubén Hervás ◽  
Margarita Menéndez ◽  
Douglas V. Laurents ◽  
Mariano Carrión-Vázquez
Keyword(s):  
Single Molecule ◽  
Molecular Mechanisms ◽  
Conformational Space ◽  
Dependent Manner ◽  
Single Molecule Force Spectroscopy ◽  
Conformational Polymorphism ◽  
Specific Chemical ◽  
Chemical Conditions ◽  
Tau Aggregation ◽  
Amyloid Assembly

AbstractKnowledge on the molecular bases of early amyloid assembly is fundamental to understand its structure-dysfunction relationship during disease progression. Tauopathies, a well-defined set of neurodegenerative disorders that includes Alzheimer’s disease, are characterized by the pathological amyloid aggregation of tau. However, the underlying molecular mechanisms that trigger tau aggregation and toxicity are poorly understood. Here, using a single-molecule approach, AFM-based single molecule-force spectroscopy (AFM-SMFS), combined with a protein-engineering mechanical protection strategy, we have analyzed the fluctuations of the conformational space of tau during the start of its pathological amyloid assembly. Specifically, we have analyzed the region that includes the four tau microtubule-binding repeats, known to play a key role on tau aggregation. We find that, unlike other amyloid-forming proteins, tau aggregation is accompanied by a decrease of conformational polymorphism, which is driven by amyloid-promoting factors, such as the Δ280K and P301L mutations, linked to Frontotemporal Dementia-17, or by specific chemical conditions. Such perturbations have distinct effects and lead to different tau (aggregate) structures. In addition to providing insight into how tau aggregates in a context dependent manner, these findings may help delve into how protein aggregation-based diseases, like Alzheimer’s, might be treated using monomer fluctuations as a pharmacological target.Abstract Figure

scholarly journals Pausing controls branching between productive and non-productive pathways during initial transcription

2017 ◽  
Author(s):  
David Dulin ◽  
David L. V. Bauer ◽  
Anssi M. Malinen ◽  
Jacob J. W. Bakermans ◽  
Martin Kaller ◽  
...  
Keyword(s):  
Single Molecule ◽  
Transcription Initiation ◽  
Rna Synthesis ◽  
Molecular Mechanisms ◽  
Dependent Manner ◽  
Single Molecule Fret ◽  
Bacterial Rna ◽  
Tightly Coupled ◽  
Relationship Of ◽  
The Relationship

AbstractTranscription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. Recently, initial RNA synthesis by the bacterial RNA polymerase (RNAP) has been shown to be interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Here, we employed single-molecule FRET and biochemical analysis to disentangle the pausing-related pathways of bacterial initial transcription. We present further evidence that region σ3.2 constitutes a barrier after the initial transcribing complex synthesizes a 6-nt RNA (ITC6), halting transcription. We also show that the paused ITC6 state acts as a checkpoint that directs RNAP, in an NTP-dependent manner, to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway that blocks transcription initiation. Our results show that abortive RNA release and DNA unscrunching are not as tightly coupled as previously thought.

scholarly journals DDK regulates replication initiation by controlling the multiplicity of Cdc45-GINS binding to Mcm2-7

2020 ◽  
Author(s):  
Lorraine De Jesus Kim ◽  
Larry J Friedman ◽  
Christian Ramsoomair ◽  
Jeff Gelles ◽  
Stephen P Bell
Keyword(s):  
Single Molecule ◽  
Replication Origin ◽  
Molecular Mechanisms ◽  
Replication Initiation ◽  
Phosphorylation Sites ◽  
Dependent Manner ◽  
Multiplicity Results ◽  
Biochemical Assays ◽  
Eukaryotic Dna ◽  
Two Stages

The committed step of eukaryotic DNA replication occurs when the replicative Mcm2-7 helicase pairs that license each replication origin are activated. Helicase activation requires the recruitment of Cdc45 and GINS to Mcm2-7, forming Cdc45-Mcm2-7-GINS complexes (CMGs). Using single-molecule biochemical assays to monitor CMG formation, we found that Cdc45 and GINS are recruited to loaded Mcm2-7 in two stages. Initially, Cdc45 and GINS are individually recruited to unstructured Mcm2-7 N-terminal tails in a Dbf4-dependent kinase (DDK)-dependent manner, forming Cdc45-tail-GINS intermediates (CtGs). The multiple phosphorylation sites on the Mcm2-7 tails promote DDK-dependent modulation of the number of CtGs formed per Mcm2-7. In a second, inefficient event, a subset of CtGs transfer their Cdc45 and GINS components to form CMGs. Importantly, higher CtG multiplicity results in increased frequency of CMG formation. Our findings reveal molecular mechanisms sensitizing helicase activation to DDK levels with implications for the control of replication origin efficiency and timing.

scholarly journals Life in extreme environments: single molecule force spectroscopy as a tool to explore proteins from extremophilic organisms

2015 ◽  
Vol 43 (2) ◽  
pp. 179-185 ◽  
Author(s):  
Katarzyna M. Tych ◽  
Toni Hoffmann ◽  
Matthew Batchelor ◽  
Megan L. Hughes ◽  
Katherine E. Kendrick ◽  
...  
Keyword(s):  
Single Molecule ◽  
Extreme Environments ◽  
Force Spectroscopy ◽  
Industrial Applications ◽  
Short Article ◽  
Single Molecule Force Spectroscopy ◽  
Recent Developments ◽  
Chemical Conditions ◽  
Physical And Chemical ◽  
Structural Adaptations

Extremophiles are organisms which survive and thrive in extreme environments. The proteins from extremophilic single-celled organisms have received considerable attention as they are structurally stable and functionally active under extreme physical and chemical conditions. In this short article, we provide an introduction to extremophiles, the structural adaptations of proteins from extremophilic organisms and the exploitation of these proteins in industrial applications. We provide a review of recent developments which have utilized single molecule force spectroscopy to mechanically manipulate proteins from extremophilic organisms and the information which has been gained about their stability, flexibility and underlying energy landscapes.

scholarly journals Characterization of G-Quadruplexes Folding/Unfolding Dynamics and Interactions with Proteins from Single-Molecule Force Spectroscopy

Biomolecules ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1579
Author(s):  
Yuanlei Cheng ◽  
Yashuo Zhang ◽  
Huijuan You
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Molecular Mechanisms ◽  
Force Spectroscopy ◽  
Magnetic Tweezers ◽  
Single Molecule Force Spectroscopy ◽  
Force Microscopy ◽  
Multiple Structures ◽  
Regulation Functions ◽  
Nucleic Acid Structures

G-quadruplexes (G4s) are stable secondary nucleic acid structures that play crucial roles in many fundamental biological processes. The folding/unfolding dynamics of G4 structures are associated with the replication and transcription regulation functions of G4s. However, many DNA G4 sequences can adopt a variety of topologies and have complex folding/unfolding dynamics. Determining the dynamics of G4s and their regulation by proteins remains challenging due to the coexistence of multiple structures in a heterogeneous sample. Here, in this mini-review, we introduce the application of single-molecule force–spectroscopy methods, such as magnetic tweezers, optical tweezers, and atomic force microscopy, to characterize the polymorphism and folding/unfolding dynamics of G4s. We also briefly introduce recent studies using single-molecule force spectroscopy to study the molecular mechanisms of G4-interacting proteins.

scholarly journals Stoichiometry and Mobility Switching of a Morphogenetic Protein in Live Differentiating Cells

2018 ◽  
Author(s):  
Adam J. M. Wollman ◽  
Katarína Muchová ◽  
Zuzana Chromiková ◽  
Anthony J. Wilkinson ◽  
Imrich Barák ◽  
...  
Keyword(s):  
Cell Division ◽  
Single Molecule ◽  
High Speed ◽  
Molecular Mechanisms ◽  
Asymmetric Cell Division ◽  
Fluorescent Protein ◽  
Dependent Manner ◽  
Cell Stage ◽  
Additional Information ◽  
Morphological Asymmetry

AbstractSpore formation following asymmetric cell division in Bacillus subtilis offers a model system to study development, morphogenesis and signal transduction in more complex organisms. Extensive biochemical and genetic details of its sporulation factors are known, however, the molecular mechanisms by which asymmetry is generated remain unclear. A crucial membrane phosphatase, SpoIIE, couples gene regulation to morphology changes, but how it performs different functions dependent on cell stage is unknown. We addressed this puzzle using high-speed single-molecule fluorescence microscopy on live B. subtilis expressing genomically encoded SpoIIE fluorescent protein fusions during sporulation. Copy number analysis indicated a few tens of SpoIIE at sporulation onset increasing to 400-600 molecules per cell following asymmetric cell division with up to 30% greater proportion in the forespore, corresponding to a concentration enhancement in the smaller forespore sufficient for differential dephosphorylation of an anti-sigma factor antagonist and activation of the forespore specific transcription factor, σF. Step-wise photobleach analysis indicates that SpoIIE forms tetramers capable of reversible oligomerisation to form clusters correlated with stage-specific functions. Specifically, low mobility SpoIIE clusters which initially localize to the asymmetric septum are released as mobile SpoIIE clusters around the forespore when phosphatase activity is manifested. SpoIIE is subsequently recaptured at the septum in a SpoIIQ-dependent manner. After mother cell engulfment of the forespore, SpoIIE is released as a mix of higher mobility clusters and tetramers. Our findings suggest that additional information captured in the changing state of multimerization and mobility enable one protein to perform different roles at different cell stages.Significance/impactCertain bacteria undergo sporulation involving cells dividing asymmetrically. A crucial protein SpoIIE facilitates this morphological asymmetry and directly links it to asymmetry in gene expression. Here, we used specialized light microscopy, capable of observing single molecules, plus biophysics, genetics and biochemical tools, to monitor SpoIIE in single living bacteria in real time, allowing us to count how many molecules are present in different cell regions, and how mobile they are. We find that SpoIIE clusters and moves depending on development stages, indicating that it has different roles depending on other binding proteins and their cellular locations. Our results suggest that changes in molecular stoichiometry and mobility may be used as switches in more complex cell processes.

scholarly journals TFE and Spt4/5 open and close the RNA polymerase clamp during the transcription cycle

2016 ◽  
Vol 113 (13) ◽  
pp. E1816-E1825 ◽  
Author(s):  
Sarah Schulz ◽  
Andreas Gietl ◽  
Katherine Smollett ◽  
Philip Tinnefeld ◽  
Finn Werner ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Nucleic Acids ◽  
Single Molecule ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Conformational Space ◽  
Single Molecule Fret ◽  
Transcription Complexes ◽  
Basal Transcription Factors ◽  
Transcription Cycle

Transcription is an intrinsically dynamic process and requires the coordinated interplay of RNA polymerases (RNAPs) with nucleic acids and transcription factors. Classical structural biology techniques have revealed detailed snapshots of a subset of conformational states of the RNAP as they exist in crystals. A detailed view of the conformational space sampled by the RNAP and the molecular mechanisms of the basal transcription factors E (TFE) and Spt4/5 through conformational constraints has remained elusive. We monitored the conformational changes of the flexible clamp of the RNAP by combining a fluorescently labeled recombinant 12-subunit RNAP system with single-molecule FRET measurements. We measured and compared the distances across the DNA binding channel of the archaeal RNAP. Our results show that the transition of the closed to the open initiation complex, which occurs concomitant with DNA melting, is coordinated with an opening of the RNAP clamp that is stimulated by TFE. We show that the clamp in elongation complexes is modulated by the nontemplate strand and by the processivity factor Spt4/5, both of which stimulate transcription processivity. Taken together, our results reveal an intricate network of interactions within transcription complexes between RNAP, transcription factors, and nucleic acids that allosterically modulate the RNAP during the transcription cycle.

scholarly journals Amyloid β-protein oligomers promote the uptake of tau fibril seeds potentiating intracellular tau aggregation

2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Woo Shik Shin ◽  
Jing Di ◽  
Qin Cao ◽  
Binsen Li ◽  
Paul M. Seidler ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Amyloid Β ◽  
Tau Proteins ◽  
Amyloid Β Protein ◽  
Dependent Manner ◽  
Aβ Oligomers ◽  
Human Neuroblastoma ◽  
Primary Mouse ◽  
Tau Aggregation ◽  
Aβ Oligomer

Abstract Background Repeated failure of drug candidates targeting Alzheimer’s disease (AD) in clinical trials likely stems from a lack of understanding of the molecular mechanisms underlying AD pathogenesis. Recent research has highlighted synergistic interactions between aggregated amyloid-β (Aβ) and tau proteins in AD, but the molecular details of how these interactions drive AD pathology remain elusive and speculative. Methods Here, we test the hypothesis that Aβ potentiates intracellular tau aggregation, and show that oligomeric Aβ specifically exacerbates proteopathic seeding by tau. Using tau-biosensor cells, we show that treatment with sub-toxic concentrations of Aβ oligomers, but not monomers or fibrils, “primes” cells, making them more susceptible to tau seeding. The treatment with Aβ oligomers enhances intracellular tau aggregation in a dose-dependent manner when the cells are seeded with either recombinant or brain-derived tau fibrils, whereas little or no aggregation is observed in the absence of Aβ-oligomer priming. Results Priming by Aβ oligomers appears to be specific to tau, as α-synuclein seeding is unaffected by this treatment. Aβ oligomer-enhanced tau seeding also occurs in primary mouse neurons and human neuroblastoma cells. Using fluorescently labeled tau seeds, we find that treatment with Aβ oligomers significantly enhances the cellular uptake of tau seeds, whereas a known tau-uptake inhibitor blocks the effect of Aβ on tau uptake. Conclusion The ability of Aβ to promote tau seeding suggests a specific and plausible mechanism by which extracellular Aβ initiates a deleterious cascade that is unique to AD. These data suggest that the Aβ-mediated potentiation of tau uptake into cells should also be taken into account when designing Aβ-targeted therapeutics.

scholarly journals DDK regulates replication initiation by controlling the multiplicity of Cdc45-GINS binding to Mcm2-7

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Lorraine De Jesus Kim ◽  
Larry J Friedman ◽  
Marko Looke ◽  
Christian K Ramsoomair ◽  
Jeff Gelles ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Replication Origin ◽  
Molecular Mechanisms ◽  
Replication Initiation ◽  
Phosphorylation Sites ◽  
Dependent Manner ◽  
Biochemical Assays ◽  
Eukaryotic Dna ◽  
Two Stages

The committed step of eukaryotic DNA replication occurs when the pairs of Mcm2-7 replicative helicases that license each replication origin are activated. Helicase activation requires the recruitment of Cdc45 and GINS to Mcm2-7, forming Cdc45-Mcm2-7-GINS complexes (CMGs). Using single-molecule biochemical assays to monitor CMG formation, we found that Cdc45 and GINS are recruited to loaded Mcm2-7 in two stages. Initially, Cdc45, GINS, and likely additional proteins are recruited to unstructured Mcm2-7 N-terminal tails in a Dbf4-dependent kinase (DDK)-dependent manner, forming Cdc45-tail-GINS intermediates (CtGs). DDK phosphorylation of multiple phosphorylation sites on the Mcm2‑7 tails modulates the number of CtGs formed per Mcm2-7. In a second, inefficient event, a subset of CtGs transfer their Cdc45 and GINS components to form CMGs. Importantly, higher CtG multiplicity increases the frequency of CMG formation. Our findings reveal molecular mechanisms sensitizing helicase activation to DDK levels with implications for control of replication origin efficiency and timing.

The Anti-Atherogenic Properties of Sesamin are Mediated via Improved Macrophage Cholesterol Efflux Through PPARγ1-LXRα and MAPK Signaling

2014 ◽  
Vol 84 (1-2) ◽  
pp. 79-91 ◽  
Author(s):  
Amin F. Majdalawieh ◽  
Hyo-Sung Ro
Keyword(s):  
Cholesterol Efflux ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Foam Cell ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Foam Cell Formation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Macrophage Cholesterol Efflux

Background: Foam cell formation resulting from disrupted macrophage cholesterol efflux, which is triggered by PPARγ1 and LXRα, is a hallmark of atherosclerosis. Sesamin and sesame oil exert anti-atherogenic effects in vivo. However, the exact molecular mechanisms underlying such effects are not fully understood. Aim: This study examines the potential effects of sesamin (0, 25, 50, 75, 100 μM) on PPARγ1 and LXRα expression and transcriptional activity as well as macrophage cholesterol efflux. Methods: PPARγ1 and LXRα expression and transcriptional activity are assessed by luciferase reporter assays. Macrophage cholesterol efflux is evaluated by ApoAI-specific cholesterol efflux assays. Results: The 50 μM, 75 μM, and 100 μM concentrations of sesamin up-regulated the expression of PPARγ1 (p< 0.001, p < 0.001, p < 0.001, respectively) and LXRα (p = 0.002, p < 0.001, p < 0.001, respectively) in a concentration-dependent manner. Moreover, 75 μM and 100 μM concentrations of sesamin led to 5.2-fold (p < 0.001) and 6.0-fold (p<0.001) increases in PPAR transcriptional activity and 3.9-fold (p< 0.001) and 4.2-fold (p < 0.001) increases in LXR transcriptional activity, respectively, in a concentration- and time-dependent manner via MAPK signaling. Consistently, 50 μM, 75 μM, and 100 μM concentrations of sesamin improved macrophage cholesterol efflux by 2.7-fold (p < 0.001), 4.2-fold (p < 0.001), and 4.2-fold (p < 0.001), respectively, via MAPK signaling. Conclusion: Our findings shed light on the molecular mechanism(s) underlying sesamin’s anti-atherogenic effects, which seem to be due, at least in part, to its ability to up-regulate PPARγ1 and LXRα expression and transcriptional activity, improving macrophage cholesterol efflux. We anticipate that sesamin may be used as a therapeutic agent for treating atherosclerosis.

Neuroblastoma: An Updated Review on Biology and Treatment

2020 ◽  
Vol 20 (13) ◽  
pp. 1014-1022 ◽  
Author(s):  
Suresh Mallepalli ◽  
Manoj Kumar Gupta ◽  
Ramakrishna Vadde
Keyword(s):  
Survival Rate ◽  
High Risk ◽  
Molecular Mechanisms ◽  
Definitive Treatment ◽  
Therapeutic Drug ◽  
Mycn Amplification ◽  
Dependent Manner ◽  
High Risk Patients ◽  
Treatment And Prevention ◽  
Risk Patients

Background: Neuroblastoma (NB) is the second leading extracranial solid tumors of early childhood and clinically characterized by the presence of round, small, monomorphic cells with excess nuclear pigmentation (hyperchromasia).Owing to a lack of definitive treatment against NB and less survival rate in high-risk patients, there is an urgent requirement to understand molecular mechanisms associated with NB in a better way, which in turn can be utilized for developing drugs towards the treatment of NB in human. Objectives: In this review, an approach was adopted to understand major risk factors, pathophysiology, the molecular mechanism associated with NB, and various therapeutic agents that can serve as drugs towards the treatment of NB in humans. Conclusions: Numerous genetic (e.g., MYCN amplification), perinatal, and gestational factors are responsible for developing NB. However, no definite environmental or parental exposures responsible for causing NB have been confirmed to date. Though intensive multimodal treatment approaches, namely, chemotherapy, surgery &radiation, may help in improving the survival rate in children, these approaches have several side effects and do not work efficiently in high-risk patients. However, recent studies suggested that numerous phytochemicals, namely, vincristine, and matrine have a minimal side effect in the human body and may serve as a therapeutic drug during the treatment of NB. Most of these phytochemicals work in a dose-dependent manner and hence must be prescribed very cautiously. The information discussed in the present review will be useful in the drug discovery process as well as treatment and prevention on NB in humans.

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