| 1. |
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| 2. |
- Trillo-Muyo, Sergio, 1983, et al.
(författare)
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Granule-stored MUC5B mucins are packed by the non-covalent formation of N-terminal head-to-head tetramers
- 2018
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 293:15, s. 5746-5754
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Tidskriftsartikel (refereegranskat)abstract
- Most MUC5B mucin polymers in the upper airways of humans and pigs are produced by submucosal glands. MUC5B forms N-terminal covalent dimers that are further packed into larger assemblies because of low pH and high Ca2+ in the secretory granule of the mucin-producing cell. We purified the recombinant MUC5B N-terminal covalent dimer and used single-particle electron microscopy to study its structure under intracellular conditions. We found that, at intragranular pH, the dimeric MUC5B organized into head-to-head noncovalent tetramers where the von Willebrand D1-D2 domains hooked into each other. These N-terminal tetramers further formed long linear complexes from which, we suggest, the mucin domains and their C termini project radially outwards. Using conventional and video microscopy, we observed that, upon secretion into the submucosal gland ducts, a flow of bicarbonate-rich fluid passes the mucin-secreting cells. We suggest that this unfolds and pulls out the MUC5B assemblies into long linear threads. These further assemble into thicker mucin bundles in the glandular ducts before emerging at the gland duct opening. We conclude that the combination of intracellular packing of the MUC5B mucin and the submucosal gland morphology creates an efficient machine for producing linear mucin bundles.
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| 3. |
- Chen, G., et al.
(författare)
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Abilities of the BRICHOS domain to prevent neurotoxicity and fibril formation are dependent on a highly conserved Asp residue
- 2022
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Ingår i: RSC Chemical Biology. - : Royal Society of Chemistry (RSC). - 2633-0679. ; 3:11, s. 1342-1358
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Tidskriftsartikel (refereegranskat)abstract
- Proteins can self-assemble into amyloid fibrils or amorphous aggregates and thereby cause disease. Molecular chaperones can prevent both these types of protein aggregation, but to what extent the respective mechanisms are overlapping is not fully understood. The BRICHOS domain constitutes a disease-associated chaperone family, with activities against amyloid neurotoxicity, fibril formation, and amorphous protein aggregation. Here, we show that the activities of BRICHOS against amyloid-induced neurotoxicity and fibril formation, respectively, are oppositely dependent on a conserved aspartate residue, while the ability to suppress amorphous protein aggregation is unchanged by Asp to Asn mutations. The Asp is evolutionarily highly conserved in >3000 analysed BRICHOS domains but is replaced by Asn in some BRICHOS families. The conserved Asp in its ionized state promotes structural flexibility and has a pKa value between pH 6.0 and 7.0, suggesting that chaperone effects can be differently affected by physiological pH variations.
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| 4. |
- Kaldmäe, Margit, et al.
(författare)
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A “spindle and thread” mechanism unblocks p53 translation by modulating N-terminal disorder
- 2022
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 30:5, s. 733-742, e1-e7
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Tidskriftsartikel (refereegranskat)abstract
- Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of “life on the edge of solubility.” Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT∗). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT∗ domain. We conclude that interactions with NT∗ help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT∗. In summary, we demonstrate that inducing co-translational folding via a molecular “spindle and thread” mechanism unblocks protein translation in vitro.
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| 5. |
- Koeck, Philip J. B., et al.
(författare)
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3D-correlation-averaging for membrane-protein-crystals
- 2008
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Ingår i: EMC 2008 14th European Microscopy Congress. - Berlin, Heidelberg : Springer Berlin Heidelberg. ; , s. 55-56
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Konferensbidrag (refereegranskat)abstract
- Few 2-dimensional protein crystals can be used to determine high-resolution structures, whereas most electron crystallography projects remain at a resolution around 10 Ångström. This might be partly due to lack of flatness of many two-dimensional crystals [1]. We have investigated this problem and suggest single particle projection matching (3D-correlation averaging) of locally averaged unit cells to improve the quality of three-dimensional maps. Theoretical considerations and tests on simulated data demonstrate the feasibility of this refinement method [2].
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| 6. |
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| 7. |
- Wu, S.R., et al.
(författare)
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Single-particle cryoelectron microscopy analysis reveals the HIV-1 spike as a tripod structure
- 2010
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 107:44, s. 18844-18849
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Tidskriftsartikel (refereegranskat)abstract
- The HIV-1 spike is a trimer of the transmembrane gp41 and the peripheral gp120 subunit pair. It is activated for virus-cell membrane fusion by binding sequentially to CD4 and to a chemokine receptor. Here we have studied the structural transition of the trimeric spike during the activation process. We solubilized and isolated unliganded and CD4-bound spikes from virus-like particles and used cryoelectron microscopy to reconstruct their 3D structures. In order to increase the yield and stability of the spike, we used an endodomain deleted and gp120-gp41 disulfide-linked variant. The unliganded spike displayed a hollow cage-like structure where the gp120-gp41 protomeric units formed a roof and bottom, and separated lobes and legs on the sides. The tripod structure was verified by fitting the recent atomic core structure of gp120 with intact N- and C-terminal ends into the spike density map. This defined the lobe as gp120 core, showed that the legs contained the polypeptide termini, and suggested the deleted variable loops V1/V2 and V3 to occupy the roof and gp41 the bottom. CD4 binding shifted the roof density peripherally and condensed the bottom density centrally. Fitting with a V3 containing gp120 core suggested that the V1/V2 loops in the roof were displaced laterally and the V3 lifted up, while the core and leg were kept in place. The loop displacements probably prepared the spike for coreceptor interaction and roof opening so that a new fusion-active gp41 structure, assembled at the center of the cage bottom, could reach the target membrane.
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| 8. |
- Wu, S.R., et al.
(författare)
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Turning of the receptor binding domains opens up the murine leukaemia virus Env for membrane fusion
- 2008
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 27:20, s. 2799-2808
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Tidskriftsartikel (refereegranskat)abstract
- The activity of the membrane fusion protein Env of Moloney mouse leukaemia virus is controlled by isomerization of the disulphide that couples its transmembrane (TM) and surface (SU) subunits. We have arrested Env activation at a stage prior to isomerization by alkylating the active thiol in SU and compared the structure of isomerization-arrested Env with that of native Env. Env trimers of respective form were isolated from solubilized particles by sedimentation and their structures were reconstructed from electron microscopic images of both vitrified and negatively stained samples. We found that the protomeric unit of both trimers formed three protrusions, a top, middle and a lower one. The atomic structure of the receptor-binding domain of SU fitted into the upper protrusion. This was formed similar to a bent finger. Significantly, in native Env the tips of the fingers were directed against each other enclosing a cavity below, whereas they had turned outward in isomerization-arrested Env transforming the cavity into an open well. This might subsequently guide the fusion peptides in extended TM subunits into the target membrane.
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| 9. |
- Zhong, Xueying, et al.
(författare)
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Amyloid Fibril Formation of Arctic Amyloid-β 1-42 Peptide is Efficiently Inhibited by the BRICHOS Domain
- 2022
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Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 17:8, s. 2201-2211
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Tidskriftsartikel (refereegranskat)abstract
- Amyloid-β peptide (Aβ) aggregation is one of the hallmarks of Alzheimer's disease (AD). Mutations in Aβ are associated with early onset familial AD, and the Arctic mutant E22G (Aβarc) is an extremely aggregation-prone variant. Here, we show that BRICHOS, a natural anti-amyloid chaperone domain, from Bri2 efficiently inhibits aggregation of Aβarcby mainly interfering with secondary nucleation. This is qualitatively different from the microscopic inhibition mechanism for the wild-type Aβ, against which Bri2 BRICHOS has a major effect on both secondary nucleation and fibril end elongation. The monomeric Aβ42arcpeptide aggregates into amyloid fibrils significantly faster than wild-type Aβ (Aβ42wt), as monitored by thioflavin T (ThT) binding, but the final ThT intensity was strikingly lower for Aβ42arccompared to Aβ42wtfibrils. The Aβ42arcpeptide formed large aggregates, single-filament fibrils, and multiple-filament fibrils without obvious twists, while Aβ42wtfibrils displayed a polymorphic pattern with typical twisted fibril architecture. Recombinant human Bri2 BRICHOS binds to the Aβ42arcfibril surface and interferes with the macroscopic fibril arrangement by promoting single-filament fibril formation. This study provides mechanistic insights on how BRICHOS efficiently affects the aggressive Aβ42arcaggregation, resulting in both delayed fibril formation kinetics and altered fibril structure.
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| 10. |
- Ambort, Daniel, 1978, et al.
(författare)
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Calcium and pH-dependent packing and release of the gel-forming MUC2 mucin.
- 2012
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 109:15, s. 5645-50
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Tidskriftsartikel (refereegranskat)abstract
- MUC2, the major colonic mucin, forms large polymers by N-terminal trimerization and C-terminal dimerization. Although the assembly process for MUC2 is established, it is not known how MUC2 is packed in the regulated secretory granulae of the goblet cell. When the N-terminal VWD1-D2-D'D3 domains (MUC2-N) were expressed in a goblet-like cell line, the protein was stored together with full-length MUC2. By mimicking the pH and calcium conditions of the secretory pathway we analyzed purified MUC2-N by gel filtration, density gradient centrifugation, and transmission electron microscopy. At pH 7.4 the MUC2-N trimer eluted as a single peak by gel filtration. At pH 6.2 with Ca(2+) it formed large aggregates that did not enter the gel filtration column but were made visible after density gradient centrifugation. Electron microscopy studies revealed that the aggregates were composed of rings also observed in secretory granulae of colon tissue sections. The MUC2-N aggregates were dissolved by removing Ca(2+) and raising pH. After release from goblet cells, the unfolded full-length MUC2 formed stratified layers. These findings suggest a model for mucin packing in the granulae and the mechanism for mucin release, unfolding, and expansion.
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| 11. |
- Chen, Gefei, et al.
(författare)
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Bri2 BRICHOS client specificity and chaperone activity are governed by assembly state
- 2017
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- . Protein misfolding and aggregation is increasingly being recognized as a cause of disease. In Alzheimer's disease the amyloid-beta peptide (A beta) misfolds into neurotoxic oligomers and assembles into amyloid fibrils. The Bri2 protein associated with Familial British and Danish dementias contains a BRICHOS domain, which reduces A beta fibrillization as well as neurotoxicity in vitro and in a Drosophila model, but also rescues proteins from irreversible nonfibrillar aggregation. How these different activities are mediated is not known. Here we show that Bri2 BRICHOS monomers potently prevent neuronal network toxicity of A beta, while dimers strongly suppress A beta fibril formation. The dimers assemble into high-molecular-weight oligomers with an apparent two-fold symmetry, which are efficient inhibitors of non-fibrillar protein aggregation. These results indicate that Bri2 BRICHOS affects qualitatively different aspects of protein misfolding and toxicity via different quaternary structures, suggesting a means to generate molecular chaperone diversity.
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| 12. |
- Chen, Gefei, et al.
(författare)
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Molecular basis for different substrate-binding sites and chaperone functions of the BRICHOS domain
- 2024
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 33:7
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Tidskriftsartikel (refereegranskat)abstract
- Proteins can misfold into fibrillar or amorphous aggregates and molecular chaperones act as crucial guardians against these undesirable processes. The BRICHOS chaperone domain, found in several otherwise unrelated proproteins that contain amyloidogenic regions, effectively inhibits amyloid formation and toxicity but can in some cases also prevent non-fibrillar, amorphous protein aggregation. Here, we elucidate the molecular basis behind the multifaceted chaperone activities of the BRICHOS domain from the Bri2 proprotein. High-confidence AlphaFold2 and RoseTTAFold predictions suggest that the intramolecular amyloidogenic region (Bri23) is part of the hydrophobic core of the proprotein, where it occupies the proposed amyloid binding site, explaining the markedly reduced ability of the proprotein to prevent an exogenous amyloidogenic peptide from aggregating. However, the BRICHOS-Bri23 complex maintains its ability to form large polydisperse oligomers that prevent amorphous protein aggregation. A cryo-EM-derived model of the Bri2 BRICHOS oligomer is compatible with surface-exposed hydrophobic motifs that get exposed and come together during oligomerization, explaining its effects against amorphous aggregation. These findings provide a molecular basis for the BRICHOS chaperone domain function, where distinct surfaces are employed against different forms of protein aggregation.
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| 13. |
- Chen, Gefei, et al.
(författare)
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Short hydrophobic loop motifs in BRICHOS domains determine chaperone activity against amorphous protein aggregation but not against amyloid formation
- 2023
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Ingår i: Communications Biology. - : Springer Nature. - 2399-3642. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- BRICHOS domain oligomerization exposes three short hydrophobic motifs that are necessary for efficient chaperone activity against amorphous protein aggregation. ATP-independent molecular chaperones are important for maintaining cellular fitness but the molecular determinants for preventing aggregation of partly unfolded protein substrates remain unclear, particularly regarding assembly state and basis for substrate recognition. The BRICHOS domain can perform small heat shock (sHSP)-like chaperone functions to widely different degrees depending on its assembly state and sequence. Here, we observed three hydrophobic sequence motifs in chaperone-active domains, and found that they get surface-exposed when the BRICHOS domain assembles into larger oligomers. Studies of loop-swap variants and site-specific mutants further revealed that the biological hydrophobicities of the three short motifs linearly correlate with the efficiency to prevent amorphous protein aggregation. At the same time, they do not at all correlate with the ability to prevent ordered amyloid fibril formation. The linear correlations also accurately predict activities of chimeras containing short hydrophobic sequence motifs from a sHSP that is unrelated to BRICHOS. Our data indicate that short, exposed hydrophobic motifs brought together by oligomerisation are sufficient and necessary for efficient chaperone activity against amorphous protein aggregation.
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| 14. |
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| 15. |
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| 16. |
- Cheng, Kimberley, et al.
(författare)
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Rapana thomasiana hemocyanin (RtH): Comparison of the two isoforms, RtH1 and RtH2, at 19 angstrom 16 angstrom resolution
- 2006
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Ingår i: Micron. - : Elsevier BV. - 0968-4328 .- 1878-4291. ; 37:6, s. 566-576
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Forskningsöversikt (refereegranskat)abstract
- Three-dimensional (3D) reconstructions of the two 8.4 MDa Rapana thomasiana hemocyanin isoforms, RtH1 and RtH2, have been obtained by cryoelectron microscopy of molecules embedded in vitreous ice and single particle image processing. The final 3D structures of the RtH1 and RtH2 didecamers at 19 angstrom and 16 angstrom resolution, respectively, are very similar to earlier reconstructions of gastropodan hemocyanins, revealing structural features such as the obliquely oriented subunits, the five- and two-fold symmetrical axes. Three new interactions are defined; two of them connecting the arch and the wall while the third is formed between the collar and the wall. The collar-wall connection and one of the arch-wall connections are positioned between two individual subunit dimers, while the second arch-wall connection is located between two subunits within the subunit dimer. All three interactions establish connections to the first tier of the wall. Furthermore, for each interaction we have allocated two first tier functional units most likely involved in forming the connections. (
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| 17. |
- Elmlund, Hans, et al.
(författare)
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The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II
- 2006
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 103:43, s. 15788-15793
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Tidskriftsartikel (refereegranskat)abstract
- CDK8 (cyclin-dependent kinase 8), along with CycC, Med12, and Med13, form a repressive module (the Cdk8 module) that prevents RNA polymerase II (pol II) interactions with Mediator. Here, we report that the ability of the Cdk8 module to prevent pol II interactions is independent of the Cdk8-dependent kinase activity. We use electron microscopy and single-particle reconstruction to demonstrate that the Cdk8 module forms a distinct structural entity that binds to the head and middle region of Mediator, thereby sterically blocking interactions with pol II.
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| 18. |
- Härmark, Johan, et al.
(författare)
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Shell thickness determination of polymer-shelled microbubbles using transmission electron microscopy
- 2016
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Ingår i: Micron. - : Elsevier. - 0968-4328 .- 1878-4291. ; 85, s. 39-43
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Tidskriftsartikel (refereegranskat)abstract
- Intravenously injected microbubbles (MBs) can be utilized as ultrasound contrast agent (CA) resulting in enhanced image quality. A novel CA, consisting of air filled MBs stabilized with a shell of polyvinyl alcohol (PVA) has been developed. These spherical MBs have been decorated with superparamagnetic iron oxide nanoparticles (SPIONs) in order to serve as both ultrasound and magnetic resonance imaging (MRI) CA. In this study, a mathematical model was introduced that determined the shell thickness of two types of SPIONs decorated MBs (Type A and Type B). The shell thickness of MBs is important to determine, as it affects the acoustical properties. In order to investigate the shell thickness, thin sections of plastic embedded MBs were prepared and imaged using transmission electron microscopy (TEM). However, the sections were cut at random distances from the MB center, which affected the observed shell thickness. Hence, the model determined the average shell thickness of the MBs from corrected mean values of the outer and inner radii observed in the TEM sections. The model was validated using simulated slices of MBs with known shell thickness and radius. The average shell thickness of Type A and Type B MBs were 651nm and 637nm, respectively.
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| 19. |
- Koeck, Philip J. B.
(författare)
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Alternative Forward Models for Imaging Thick Specimens in Transmission Electron Microscopy
- 2023
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Ingår i: Microscopy and Microanalysis. - : Oxford University Press (OUP). - 1431-9276 .- 1435-8115. ; 29:3, s. 1071-1076
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Tidskriftsartikel (refereegranskat)abstract
- I have investigated two different forward models for image formation in transmission electron microscopy of thick specimens, the 3DCtf model, which introduces a defocus gradient in the linear approximation, and the multislice model. An important result is that the 3DCtf model does not seem to be compatible with the multislice image formation model. A second very useful finding is that the exit wave in the multislice model has an imaginary part, which, in first-order approximation, is a pure projection of the specimen and is not affected by the defocus gradient. The defocus gradient only comes into play in real valued and higher-order imaginary terms. If the multislice model is closer to reality than the 3DCtf-model, then the best way to retrieve the specimen projection for thicker specimens should be a procedure for retrieving the exit wave's imaginary term, for example using images recorded at different defocus values.
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| 20. |
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| 21. |
- Koeck, Philip J. B.
(författare)
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Annular dark field transmission electron microscopy for protein structure determination
- 2016
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Ingår i: Ultramicroscopy. - : Elsevier. - 0304-3991 .- 1879-2723. ; 161, s. 98-104
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Tidskriftsartikel (refereegranskat)abstract
- Recently annular dark field (ADF) transmission electron microscopy (TEM) has been advocated as a means of recording images of biological specimens with better signal to noise ratio (SNR) than regular bright field images. I investigate whether and how such images could be used to determine the three-dimensional structure of proteins given that an ADF aperture with a suitable pass-band can be manufactured and used in practice. I develop an approximate theory of ADF-TEM image formation for weak amplitude and phase objects and test this theory using computer simulations. I also test whether these simulated images can be used to calculate a three-dimensional model of the protein using standard software and discuss problems and possible ways to overcome these.
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| 22. |
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| 23. |
- Koeck, Philip J. B.
(författare)
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Design of an Electrostatic Phase Shifting Device for Biological Transmission Electron Microscopy
- 2018
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Ingår i: Ultramicroscopy. - : Elsevier. - 0304-3991 .- 1879-2723. ; 187, s. 107-112
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Tidskriftsartikel (refereegranskat)abstract
- I suggest an electrostatic phase plate designed to broaden the contrast transfer function of a transmission electron microscope operated close to Scherzer defocus primarily in the low resolution direction. At higher defocus the low frequency behavior is equal to that close to Scherzer defocus, but CTF-correction becomes necessary to extend image interpretation to higher resolution. One simple realization of the phase plate consists of two ring shaped electrodes symmetrically surrounding the central beam. Since no physical components come into contact with the central beam and charge on the electrodes is controlled by an external voltage supply, problems with uncontrolled charging are expected to be reduced.
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| 24. |
- Koeck, Philip J. B.
(författare)
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Improved Hilbert phase contrast for transmission electron microscopy
- 2015
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Ingår i: Ultramicroscopy. - : Elsevier BV. - 0304-3991 .- 1879-2723. ; 154, s. 37-41
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Tidskriftsartikel (refereegranskat)abstract
- Hilbert phase contrast has been recognized as a means of recording high resolution images with high contrast using a transmission electron microscope. This imaging mode could be used to image typical phase objects such as unstained biological molecules or cryo sections of biological tissue. According to the original proposal by (Danev et al., 2002) the Hilbert phase plate applies a phase shift of π to approximately half the focal plane (for example the right half excluding the central beam) and an image is recorded at Gaussian focus. After correction for the inbuilt asymmetry of differential phase contrast this image will have an almost perfect contrast transfer function (close to 1) from the lowest spatial frequency up to a maximum resolution determined by the wave length and spherical aberration of the microscope. In this paper I present theory and simulations showing that this maximum spatial frequency can be increased considerably almost without loss of contrast by using a Hilbert phase plate of half the thickness, leading to a phase shift of π/2, and recording images at Scherzer defocus. The maximum resolution can be improved even more by imaging at extended Scherzer defocus, though at the cost of contrast loss at lower spatial frequencies.
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| 25. |
- Koeck, Philip J. B.
(författare)
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Missing data in image and signal processing : The case of binary objects
- 2004
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Ingår i: Optik (Stuttgart). - : Elsevier BV. - 0030-4026 .- 1618-1336. ; 115:10, s. 459-472
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Tidskriftsartikel (refereegranskat)abstract
- I investigated which portions of the Fourier transform of binary signals, images and three-dimensional objects are necessary to correctly identify an object in the presence of noise. This is practically possible for very small binary data sets since the total number of possible objects is then very limited. There are for example 512 different binary images with 9 pixels. It is easy to see that this number soon becomes impractically large for bigger images or if one allows more than two possible pixel values. It turns out that even in the presence of large amounts of noise a relatively small portion of the Fourier transform is essential for deciding which of all possible binary objects the Fourier transform belongs to. These 'decision experiments' can be used as a standard for how well algorithms for retrieval of missing Fourier components perform. In another set of computer experiments I investigate the possibility of retrieving various missing Fourier components algorithmically. The main finding of this second set of computer experiments is that the simple retrieval algorithm (a limited form of 'projection onto convex sets') used falls very much short of what one might expect from the 'decision experiments'. I conclude with a discussion what this discrepancy might be due to and some suggestions how to improve the performance of retrieval algorithms for binary objects.
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