scholarly journals Cooperative Amyloid Fibre Binding and Disassembly by the Hsp70 disaggregase

2021 ◽  
Author(s):  
Joseph G Beton ◽  
Jim Monistrol ◽  
Anne Wentink ◽  
Erin C Johnston ◽  
Anthony J Roberts ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Fibre Surface ◽  
Nucleotide Exchange ◽  
Force Microscopy ◽  
Atomic Force ◽  
Nucleotide Exchange Factor ◽  
Cryo Electron Tomography ◽  
Active Zones ◽  
Human Hsp70

Although amyloid fibres are highly stable protein aggregates, a specific combination of human Hsp70 system chaperones can disassemble them, including fibres formed of α-synuclein, huntingtin or Tau. Disaggregation requires the ATPase activity of the constitutively expressed Hsp70, Hsc70, together with the J domain protein DNAJB1 and the nucleotide exchange factor Apg2. Recruitment and clustering of Hsc70 on the fibrils appear to be necessary for disassembly. Here we use atomic force microscopy (AFM) to show that segments of in vitro assembled α-synuclein fibrils are first coated with chaperones and then undergo bursts of rapid, unidirectional disassembly. Cryo-electron tomography reveals fibrils with regions of densely bound chaperones extending from the fibre surface, preferentially at one end of the fibre. Sub-stoichiometric amounts of Apg2 relative to Hsc70 dramatically increase recruitment of Hsc70 to the fibres, creating localised active zones that then undergo rapid disassembly at a rate of ~4 subunits per second.

scholarly journals Drebrin-like protein regulates body bending of C. elegans via suppression of NCA cation leak channels

2019 ◽  
Author(s):  
Eugenia Butkevich ◽  
Peter Weist ◽  
Daniel Härtter ◽  
Dieter R. Klopfenstein ◽  
Renata Garces ◽  
...  
Keyword(s):  
Muscle Tone ◽  
Adaptor Protein ◽  
Dominant Negative ◽  
Nucleotide Exchange ◽  
Force Microscopy ◽  
C Elegans ◽  
Guanine Nucleotide Exchange ◽  
Atomic Force ◽  
Nucleotide Exchange Factor ◽  
Genetic Epistasis

AbstractDrebrin-like protein (DBN-1) in C. elegans is an adaptor protein that connects different cellular pathways to the actin cytoskeleton. Using a CRISPR-Cas9 system, we generated a new dbn-1 allele, which lacks 80% of C-terminal part of DBN-1. The mutant displays a striking hyper-bending locomotion phenotype and body posture with two times stronger curvature than wild type. We show by atomic force microscopy that the muscle tone of the mutant remains unaffected. Aiming to track down the cause of hyper-bending, we performed genetic epistasis experiments. We found that mutations in the Rho-specific guanine-nucleotide exchange factor (GEF) domain of UNC-73 (Trio), pan-neuronal expression of dominant negative RHO-1 and mutations in NCA (NALCN) cation leak channels all suppressed hyper-bending in the dbn-1 mutant. These data indicate that DBN-1 negatively regulates the activity of both NCA-1 and NCA-2 channels, opposing RHO-1 in the non-canonical Gq pathway. We conclude that DBN-1 is an important component of the neuronal signaling cascade that controls the degree of C. elegans body bending during locomotion.

scholarly journals Disassembly of Tau fibrils by the human Hsp70 disaggregation machinery generates small seeding-competent species

2020 ◽  
Vol 295 (28) ◽  
pp. 9676-9690 ◽  
Author(s):  
Eliana Nachman ◽  
Anne S. Wentink ◽  
Karine Madiona ◽  
Luc Bousset ◽  
Taxiarchis Katsinelos ◽  
...  
Keyword(s):  
Protein Homeostasis ◽  
Nucleotide Exchange ◽  
Cell Culture Model ◽  
Nucleotide Exchange Factor ◽  
Culture Model ◽  
Class B ◽  
The Cost ◽  
Human Hsp70 ◽  
Tau Fibrils

The accumulation of amyloid Tau aggregates is implicated in Alzheimer's disease (AD) and other tauopathies. Molecular chaperones are known to maintain protein homeostasis. Here, we show that an ATP-dependent human chaperone system disassembles Tau fibrils in vitro. We found that this function is mediated by the core chaperone HSC70, assisted by specific cochaperones, in particular class B J-domain proteins and a heat shock protein 110 (Hsp110)-type nucleotide exchange factor (NEF). The Hsp70 disaggregation machinery processed recombinant fibrils assembled from all six Tau isoforms as well as Sarkosyl-resistant Tau aggregates extracted from cell cultures and human AD brain tissues, demonstrating the ability of the Hsp70 machinery to recognize a broad range of Tau aggregates. However, the chaperone activity released monomeric and small oligomeric Tau species, which induced the aggregation of self-propagating Tau conformers in a Tau cell culture model. We conclude that the activity of the Hsp70 disaggregation machinery is a double-edged sword, as it eliminates Tau amyloids at the cost of generating new seeds.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

Targeting Breast Cancer Cells with G4 PAMAM Dendrimers and Valproic Acid Derivative Complexes

2020 ◽  
Vol 20 (15) ◽  
pp. 1857-1872
Author(s):  
Alberto M. Muñoz ◽  
Manuel J. Fragoso-Vázquez ◽  
Berenice P. Martel ◽  
Alma Chávez-Blanco ◽  
Alfonso Dueñas-González ◽  
...  
Keyword(s):  
Valproic Acid ◽  
Cell Lines ◽  
Water Solubility ◽  
Md Simulations ◽  
Pamam Dendrimer ◽  
Pamam Dendrimers ◽  
Force Microscopy ◽  
Micromolar Concentration ◽  
Atomic Force

Background: Our research group has developed some Valproic Acid (VPA) derivatives employed as anti-proliferative compounds targeting the HDAC8 enzyme. However, some of these compounds are poorly soluble in water. Objective: Employed the four generations of Polyamidoamine (G4 PAMAM) dendrimers as drug carriers of these compounds to increase their water solubility for further in vitro evaluation. Methods: VPA derivatives were subjected to Docking and Molecular Dynamics (MD) simulations to evaluate their affinity on G4 PAMAM. Then, HPLC-UV/VIS, 1H NMR, MALDI-TOF and atomic force microscopy were employed to establish the formation of the drug-G4 PAMAM complexes. Results: The docking results showed that the amide groups of VPA derivatives make polar interactions with G4 PAMAM, whereas MD simulations corroborated the stability of the complexes. HPLC UV/VIS experiments showed an increase in the drug water solubility which was found to be directly proportional to the amount of G4 PAMAM. 1H NMR showed a disappearance of the proton amine group signals, correlating with docking results. MALDI-TOF and atomic force microscopy suggested the drug-G4 PAMAM dendrimer complexes formation. Discussion: In vitro studies showed that G4 PAMAM has toxicity in the micromolar concentration in MDAMB- 231, MCF7, and 3T3-L1 cell lines. VPA CF-G4 PAMAM dendrimer complex showed anti-proliferative properties in the micromolar concentration in MCF-7 and 3T3-L1, and in the milimolar concentration in MDAMB- 231, whereas VPA MF-G4 PAMAM dendrimer complex didn’t show effects on the three cell lines employed. Conclusion: These results demonstrate that G4 PAMAM dendrimers are capableof transporting poorly watersoluble aryl-VPA derivate compounds to increase its cytotoxic activity against neoplastic cell lines.

scholarly journals Polymorphic Protective Dps–DNA Co-Crystals by Cryo Electron Tomography and Small Angle X-Ray Scattering

Biomolecules ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 39 ◽  
Author(s):  
Roman Kamyshinsky ◽  
Yury Chesnokov ◽  
Liubov Dadinova ◽  
Andrey Mozhaev ◽  
Ivan Orlov ◽  
...  
Keyword(s):  
Small Angle ◽  
Electron Tomography ◽  
Actual Structure ◽  
X Ray ◽  
Cell Parameters ◽  
X Ray Scattering ◽  
Cryo Electron Tomography ◽  
Dps Protein ◽  
Ray Scattering

Rapid increase of intracellular synthesis of specific histone-like Dps protein that binds DNA to protect the genome against deleterious factors leads to in cellulo crystallization—one of the most curious processes in the area of life science at the moment. However, the actual structure of the Dps–DNA co-crystals remained uncertain in the details for more than two decades. Cryo-electron tomography and small-angle X-ray scattering revealed polymorphous modifications of the co-crystals depending on the buffer parameters. Two different types of the Dps–DNA co-crystals are formed in vitro: triclinic and cubic. Three-dimensional reconstruction revealed DNA and Dps molecules in cubic co-crystals, and the unit cell parameters of cubic lattice were determined consistently by both methods.

scholarly journals Protective Dps–DNA co‐crystallization in stressed cells: an in vitro structural study by small‐angle X‐ray scattering and cryo‐electron tomography

FEBS Letters ◽  
2019 ◽  
Vol 593 (12) ◽  
pp. 1360-1371 ◽  
Author(s):  
Liubov A. Dadinova ◽  
Yurii M. Chesnokov ◽  
Roman A. Kamyshinsky ◽  
Ivan A. Orlov ◽  
Maxim V. Petoukhov ◽  
...  
Keyword(s):  
Structural Study ◽  
Small Angle ◽  
Electron Tomography ◽  
X Ray ◽  
X Ray Scattering ◽  
Cryo Electron Tomography ◽  
Stressed Cells ◽  
Ray Scattering

scholarly journals The nucleoporin RanBP2 tethers the cAMP effector Epac1 and inhibits its catalytic activity

2011 ◽  
Vol 193 (6) ◽  
pp. 1009-1020 ◽  
Author(s):  
Martijn Gloerich ◽  
Marjolein J. Vliem ◽  
Esther Prummel ◽  
Lars A.T. Meijer ◽  
Marije G.A. Rensen ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Negative Regulator ◽  
Camp Signaling ◽  
Nuclear Pore ◽  
Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Wide Range

Cyclic adenosine monophosphate (cAMP) is a second messenger that relays a wide range of hormone responses. In this paper, we demonstrate that the nuclear pore component RanBP2 acts as a negative regulator of cAMP signaling through Epac1, a cAMP-regulated guanine nucleotide exchange factor for Rap. We show that Epac1 directly interacts with the zinc fingers (ZNFs) of RanBP2, tethering Epac1 to the nuclear pore complex (NPC). RanBP2 inhibits the catalytic activity of Epac1 in vitro by binding to its catalytic CDC25 homology domain. Accordingly, cellular depletion of RanBP2 releases Epac1 from the NPC and enhances cAMP-induced Rap activation and cell adhesion. Epac1 also is released upon phosphorylation of the ZNFs of RanBP2, demonstrating that the interaction can be regulated by posttranslational modification. These results reveal a novel mechanism of Epac1 regulation and elucidate an unexpected link between the NPC and cAMP signaling.

scholarly journals Osteoblast-Like Cell Behavior on Porous Scaffolds Based on Poly(styrene) Fibers

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Andrada Serafim ◽  
Romain Mallet ◽  
Florence Pascaretti-Grizon ◽  
Izabela-Cristina Stancu ◽  
Daniel Chappard
Keyword(s):  
Interference Microscopy ◽  
Porous Scaffolds ◽  
Allogenic Bone ◽  
Force Microscopy ◽  
Atomic Force ◽  
Dry Spinning ◽  
Bone Trabeculae ◽  
Large Bone

Scaffolds of nonresorbable biomaterials can represent an interesting alternative for replacing large bone defects in some particular clinical cases with massive bone loss. Poly(styrene) microfibers were prepared by a dry spinning method. They were partially melted to provide 3D porous scaffolds. The quality of the material was assessed by Raman spectroscopy. Surface roughness was determined by atomic force microscopy and vertical interference microscopy. Saos-2 osteoblast-like cells were seeded on the surface of the fibers and left to proliferate. Cell morphology, evaluated by scanning electron microscopy, revealed that they can spread and elongate on the rough microfiber surface. Porous 3D scaffolds made of nonresorbable poly(styrene) fibers are cytocompatible biomaterials mimicking allogenic bone trabeculae and allowing the growth and development of osteoblast-like cellsin vitro.

scholarly journals Nanostructural and mechanical changes in the sclera following proteoglycan depletion

2018 ◽  
Vol 2 (2) ◽  
pp. 14-17
Author(s):  
Zhuola Zhuola ◽  
Steve Barrett ◽  
Yalda Ashraf Kharaz ◽  
Riaz Akhtar
Keyword(s):  
Mechanical Properties ◽  
Collagen Fibril ◽  
Enzymatic Degradation ◽  
Ocular Tissues ◽  
Force Microscopy ◽  
Nanomechanical Properties ◽  
Atomic Force ◽  
Fibril Morphology

The mechanical properties of ocular tissues, such as the sclera, have a major impact on healthy eye function, and are governed by the properties and composition of the microstructural components. For example, biomechanical degradation associated with myopia occurs alongside a reduction of proteoglycans (PGs). In this study, the role of PG degradation in the nanomechanical properties of the porcine sclera is explored. In-vitro enzymatic degradation of PGs was conducted with α-amylase and chondroitinase ABC enzymes. Collagen fibril morphology and nanomechanical stiffness were measured with atomic force microscopy (AFM). The elastic modulus of the tissue was reduced in all enzyme-treated samples relative to controls. In addition, collagen fibril organization was disrupted by PG depletion. Our data demonstrate that PGs play an important role in determining not only the mechanical properties at these length scales, but also collagen fibril arrangement.

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