| 1. |
- De Oliveira, Danilo Hirabae, et al.
(författare)
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Silk Assembly against Hydrophobic Surfaces?Modeling and Imaging of Formation of Nanofibrils
- 2023
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Ingår i: ACS Applied Bio Materials. - : AMER CHEMICAL SOC. - 2576-6422. ; 6:3, s. 1011-1018
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Tidskriftsartikel (refereegranskat)abstract
- A detailed insight about the molecular organization behind spider silk assembly is valuable for the decoding of the unique properties of silk. The recombinant partial spider silk protein 4RepCT contains four poly-alanine/glycine-rich repeats followed by an amphiphilic C-terminal domain and has shown the capacity to self-assemble into fibrils on hydrophobic surfaces. We herein use molecular dynamic simulations to address the structure of 4RepCT and its different parts on hydrophobic versus hydrophilic surfaces. When 4RepCT is placed in a wing arrangement model and periodically repeated on a hydrophobic surface, fi-sheet structures of the poly-alanine repeats are preserved, while the CT part is settled on top, presenting a fibril with a height of similar to 7 nm and a width of similar to 11 nm. Both atomic force microscopy and cryo-electron microscopy imaging support this model as a possible fibril formation on hydrophobic surfaces. These results contribute to the understanding of silk assembly and alignment mechanism onto hydrophobic surfaces.
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| 2. |
- Farsi, Zohreh, et al.
(författare)
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Clathrin coat controls synaptic vesicle acidification by blocking vacuolar ATPase activity
- 2018
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Ingår i: eLIFE. - : ELIFE SCIENCES PUBLICATIONS LTD. - 2050-084X. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Newly-formed synaptic vesicles (SVs) are rapidly acidified by vacuolar adenosine triphosphatases (vATPases), generating a proton electrochemical gradient that drives neurotransmitter loading. Clathrin-mediated endocytosis is needed for the formation of new SVs, yet it is unclear when endocytosed vesicles acidify and refill at the synapse. Here, we isolated clathrin-coated vesicles (CCVs) from mouse brain to measure their acidification directly at the single vesicle level. We observed that the ATP-induced acidification of CCVs was strikingly reduced in comparison to SVs. Remarkably, when the coat was removed from CCVs, uncoated vesicles regained ATP-dependent acidification, demonstrating that CCVs contain the functional vATPase, yet its function is inhibited by the clathrin coat. Considering the known structures of the vATPase and clathrin coat, we propose a model in which the formation of the coat surrounds the vATPase and blocks its activity. Such inhibition is likely fundamental for the proper timing of SV refilling.
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| 3. |
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| 4. |
- Kerr, A. G., et al.
(författare)
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The long noncoding RNA ADIPINT regulates human adipocyte metabolism via pyruvate carboxylase
- 2022
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- The pleiotropic function of long noncoding RNAs is well recognized, but their direct role in governing metabolic homeostasis is less understood. Here, we describe a human adipocyte-specific lncRNA, ADIPINT, that regulates pyruvate carboxylase, a pivotal enzyme in energy metabolism. We developed an approach, Targeted RNA-protein identification using Orthogonal Organic Phase Separation, which identifies that ADIPINT binds to pyruvate carboxylase and validated the interaction with electron microscopy. ADIPINT knockdown alters the interactome and decreases the abundance and enzymatic activity of pyruvate carboxylase in the mitochondria. Reduced ADIPINT or pyruvate carboxylase expression lowers adipocyte lipid synthesis, breakdown, and lipid content. In human white adipose tissue, ADIPINT expression is increased in obesity and linked to fat cell size, adipose insulin resistance, and pyruvate carboxylase activity. Thus, we identify ADIPINT as a regulator of lipid metabolism in human white adipocytes, which at least in part is mediated through its interaction with pyruvate carboxylase.
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| 5. |
- Sporny, Michael, et al.
(författare)
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Structural basis for SARM1 inhibition and activation under energetic stress
- 2020
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Ingår i: eLIFE. - : eLife Sciences Publications, Ltd. - 2050-084X. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- SARM1, an executor of axonal degeneration, displays NADase activity that depletes the key cellular metabolite, NAD+, in response to nerve injury. The basis of SARM1 inhibition and its activation under stress conditions are still unknown. Here, we present cryo-EM maps of SARM1 at 2.9 and 2.7 angstrom resolutions. These indicate that SARM1 homo-octamer avoids premature activation by assuming a packed conformation, with ordered inner and peripheral rings, that prevents dimerization and activation of the catalytic domains. This inactive conformation is stabilized by binding of SARM1's own substrate NAD+ in an allosteric location, away from the catalytic sites. This model was validated by mutagenesis of the allosteric site, which led to constitutively active SARM1. We propose that the reduction of cellular NAD+ concentration contributes to the disassembly of SARM1's peripheral ring, which allows formation of active NADase domain dimers, thereby further depleting NAD+ to cause an energetic catastrophe and cell death.
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| 6. |
- Wang, Zuoneng, 1991-, et al.
(författare)
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Optimizing purification of the peripheral membrane protein FAM92A1 fused to a modified spidroin tag
- 2022
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Ingår i: Protein Expression and Purification. - : Elsevier. - 1046-5928 .- 1096-0279. ; 189, s. 105992-105992
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Cryo-electron microscopy has revolutionized structural biology. In particular structures of proteins at themembrane interface have been a major contribution of cryoEM. Yet, visualization and characterization of peripheralmembrane proteins remains challenging; mostly because there is no unified purification strategy forthese proteins. FAM92A1 is a novel peripheral membrane protein that binds to the mitochondrial inner membrane.There, FAM92A1 dimers bind to the membrane and play an essential role in regulating the mitochondrialultrastructure. Curiously, FAM92A1 has also an important function in ciliogenesis. FAM92A1 is part of themembrane bending Bin1/Amphiphsyin/RVS (BAR) domain protein family. Currently, there is no structure ofFAM92A1, mostly because FAM92A1 is unstable and insoluble at high concentrations, like many BAR domainproteins. Yet, pure and concentrated protein is a necessity for screening to generate samples suitable for structuredetermination. Here, we present an optimized purification and expression strategy for dimeric FAM92A1. To ourknowledge, we are the first to use the spidroin tag NT* to successfully purify a peripheral membrane protein. Ourresults show that NT* not only increases solubility but stabilizes FAM92A1 as a dimer. FAM92A1 fused to NT* isactive because it is able to efficiently bend membranes. Taken together, our strategy indicates that this is apossible avenue to express and purify other challenging BAR domain proteins.
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