| 1. |
- Arike, Liisa, et al.
(författare)
-
The Densely O-Glycosylated MUC2 Mucin Protects the Intestine and Provides Food for the Commensal Bacteria.
- 2016
-
Ingår i: Journal of molecular biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 428:16, s. 3221-3229
-
Forskningsöversikt (refereegranskat)abstract
- All mucins are highly O-glycosylated by variable glycans depending on species, histoblood group and organ. This makes the intestinal main mucin MUC2 non-degradable by the host digestive system but well by both commensal and pathogenic bacteria. The MUC2 glycans are important for selection of the commensal bacteria and act as a nutritional source for the bacteria; this also helps the host to recover some of the energy spent on constantly renewing the protective mucus layer. Glycosylation is the most diverse and common posttranslational modification of cell surfaces and secreted proteins. N-Glycosylation is most well studied and predictable, whereas O-glycosylation is more diverse and less well understood. O-Glycosylation is also often called mucin-type glycosylation as it is typical for mucins that often have more than 80% of the mass as O-glycans. This review will discuss the mucin-type O-glycosylation and especially the O-glycosylation of human and mice intestinal mucin MUC2 in relation to bacteria and disease.
|
|
| 2. |
- Bauer, D W, et al.
(författare)
-
Influence of Internal DNA Pressure on Stability and Infectivity of Phage λ.
- 2015
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 427:20, s. 3189-3200
-
Tidskriftsartikel (refereegranskat)abstract
- Viruses must remain infectious while in harsh extracellular environments. An important aspect of viral particle stability for double-stranded DNA viruses is the energetically unfavorable state of the tightly confined DNA chain within the virus capsid creating pressures of tens of atmospheres. Here, we study the influence of internal genome pressure on the thermal stability of viral particles. Using differential scanning calorimetry to monitor genome loss upon heating, we find that internal pressure destabilizes the virion, resulting in a smaller activation energy barrier to trigger DNA release. These experiments are complemented by plaque assay and electron microscopy measurements to determine the influence of intra-capsid DNA pressure on the rates of viral infectivity loss. At higher temperatures (65-75°C), failure to retain the packaged genome is the dominant mechanism of viral inactivation. Conversely, at lower temperatures (40-55°C), a separate inactivation mechanism dominates, which results in non-infectious particles that still retain their packaged DNA. Most significantly, both mechanisms of infectivity loss are directly influenced by internal DNA pressure, with higher pressure resulting in a more rapid rate of inactivation at all temperatures.
|
|
| 3. |
- Ben-David, Moshe, et al.
(författare)
-
Catalytic Stimulation by Restrained Active-Site Floppiness-The Case of High Density Lipoprotein-Bound Serum Paraoxonase-1
- 2015
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 427:6, s. 1359-1374
-
Tidskriftsartikel (refereegranskat)abstract
- Despite the abundance of membrane-associated enzymes, the mechanism by which membrane binding stabilizes these enzymes and stimulates their catalysis remains largely unknown. Serum paraoxonase-1 (PON1) is a lipophilic lactonase whose stability and enzymatic activity are dramatically stimulated when associated with high-density lipoprotein (HDL) particles. Our mutational and structural analyses, combined with empirical valence bond simulations, reveal a network of hydrogen bonds that connect HDL binding residues with Asn168-a key catalytic residue residing >15 angstrom from the HDL contacting interface. This network ensures precise alignment of N168, which, in turn, ligates PON1's catalytic calcium and aligns the lactone substrate for catalysis. HDL binding restrains the overall motion of the active site and particularly of N168, thus reducing the catalytic activation energy barrier. We demonstrate herein that disturbance of this network, even at its most far-reaching periphery, undermines PON1's activity. Membrane binding thus immobilizes long-range interactions via second- and third-shell residues that reduce the active site's floppiness and pre-organize the catalytic residues. Although this network is critical for efficient catalysis, as demonstrated here, unraveling these long-rage interaction networks is challenging, let alone their implementation in artificial enzyme design.
|
|
| 4. |
- Borg, Anneli, et al.
(författare)
-
Determinants of the Rate of mRNA Translocation in Bacterial Protein Synthesis
- 2015
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 427:9, s. 1835-1847
-
Tidskriftsartikel (refereegranskat)abstract
- Studying the kinetics of translocation of mRNA and tRNAs on the translating ribosome is technically difficult since the rate-limiting steps involve large conformational changes without covalent bond formation or disruption. Here, we have developed a unique assay system for precise estimation of the full translocation cycle time at any position in any type of open reading frame (ORF). Using a buffer system optimized for high accuracy of tRNA selection together with high concentration of elongation factor G, we obtained in vivo compatible translocation rates. We found that translocation was comparatively slow early in the ORF and faster further downstream of the initiation codon. The maximal translocation rate decreased from the in vivo compatible value of 30 s(-1) at 1 mM free Mg2+ concentration to the detrimentally low value of 1 s(-1) at 6 mM free Mg2+ concentration. Thus, high and in vivo compatible accuracy of codon translation, as well as high and in vivo compatible translocation rate, required a remarkably low Mg2+ concentration. Finally, we found that the rate of translocation deep inside an ORF was not significantly affected upon variation of the standard free energy of interaction between a 6-nt upstream Shine-Dalgarno (SD)-like sequence and the anti-SD sequence of 16S rRNA in a range of 0-6 kcal/mol. Based on these experiments, we discuss the optimal choice of Mg2+ concentration for maximal fitness of the living cell by taking its effects on the accuracy of translation, the peptide bond formation rate and the translocation rate into account. (C) 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
|
|
| 5. |
- Brännström, Kristoffer, et al.
(författare)
-
The Properties of Amyloid-β Fibrils Are Determined by their Path of Formation
- 2018
-
Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 430:13, s. 1940-1949
-
Tidskriftsartikel (refereegranskat)abstract
- Fibril formation of the amyloid-β peptide (Aβ) follows a nucleation-dependent polymerization process and is associated with Alzheimer's disease. Several different lengths of Aβ are observed in vivo, but Aβ1-40 and Aβ1-42 are the dominant forms. The fibril architectures of Aβ1-40 and Aβ1-42 differ and Aβ1-42 assemblies are generally considered more pathogenic. We show here that monomeric Aβ1-42 can be cross-templated and incorporated into the ends of Aβ1-40 fibrils, while incorporation of Aβ1-40 monomers into Aβ1-42 fibrils is very poor. We also show that via cross-templating incorporated Aβ monomers acquire the properties of the parental fibrils. The suppressed ability of Aβ1-40 to incorporate into the ends of Aβ1-42 fibrils and the capacity of Aβ1-42 monomers to adopt the properties of Aβ1-40 fibrils may thus represent two mechanisms reducing the total load of fibrils having the intrinsic, and possibly pathogenic, features of Aβ1-42 fibrils in vivo. We also show that the transfer of fibrillar properties is restricted to fibril-end templating and does not apply to cross-nucleation via the recently described path of surface-catalyzed secondary nucleation, which instead generates similar structures to those acquired via de novo primary nucleation in the absence of catalyzing seeds. Taken together these results uncover an intrinsic barrier that prevents Aβ1-40 from adopting the fibrillar properties of Aβ1-42 and exposes that the transfer of properties between amyloid-β fibrils are determined by their path of formation.
|
|
| 6. |
- Cymer, Florian, et al.
(författare)
-
Mechanisms of Integral Membrane Protein Insertion and Folding
- 2015
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 427:5, s. 999-1022
-
Forskningsöversikt (refereegranskat)abstract
- The biogenesis, folding, and structure of alpha-helical membrane proteins (MPs) are important to understand because they underlie virtually all physiological processes in cells including key metabolic pathways, such as the respiratory chain and the photosystems, as well as the transport of solutes and signals across membranes. Nearly all MPs require translocons-often referred to as protein-conducting channels-for proper insertion into their target membrane. Remarkable progress toward understanding the structure and functioning of translocons has been made during the past decade. Here, we review and assess this progress critically. All available evidence indicates that MPs are equilibrium structures that achieve their final structural states by folding along thermodynamically controlled pathways. The main challenge for cells is the targeting and membrane insertion of highly hydrophobic amino acid sequences. Targeting and insertion are managed in cells principally by interactions between ribosomes and membrane-embedded translocons. Our review examines the biophysical and biological boundaries of MP insertion and the folding of polytopic MPs in vivo. A theme of the review is the under-appreciated role of basic thermodynamic principles in MP folding and assembly. Thermodynamics not only dictates the final folded structure but also is the driving force for the evolution of the ribosome-translocon system of assembly. We conclude the review with a perspective suggesting a new view of translocon-guided MP insertion. (C) 2014 Elsevier Ltd. All rights reserved.
|
|
| 7. |
- Engqvist, Martin, 1983, et al.
(författare)
-
Directed evolution of gloeobacter violaceus rhodopsin spectral properties
- 2015
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 427:1, s. 205-220
-
Tidskriftsartikel (refereegranskat)abstract
- Proton-pumping rhodopsins (PPRs) are photoactive retinal-binding proteins that transport ions across biological membranes in response to light. These proteins are interesting for light-harvesting applications in bioenergy production, in optogenetics applications in neuroscience, and as fluorescent sensors of membrane potential. Little is known, however, about how the protein sequence determines the considerable variation in spectral properties of PPRs from different biological niches or how to engineer these properties in a given PPR. Here we report a comprehensive study of amino acid substitutions in the retinal-binding pocket of Gloeobacter violaceus rhodopsin (GR) that tune its spectral properties. Directed evolution generated 70 GR variants with absorption maxima shifted by up to ± 80 nm, extending the protein's light absorption significantly beyond the range of known natural PPRs. While proton-pumping activity was disrupted in many of the spectrally shifted variants, we identified single tuning mutations that incurred blue and red shifts of 42 nm and 22 nm, respectively, that did not disrupt proton pumping. Blue-shifting mutations were distributed evenly along the retinal molecule while red-shifting mutations were clustered near the residue K257, which forms a covalent bond with retinal through a Schiff base linkage. Thirty eight of the identified tuning mutations are not found in known microbial rhodopsins. We discovered a subset of red-shifted GRs that exhibit high levels of fluorescence relative to the WT (wild-type) protein.
|
|
| 8. |
- Ganesan, Ashok, et al.
(författare)
-
Selectivity of Aggregation-Determining Interactions
- 2015
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 427:2, s. 236-247
-
Tidskriftsartikel (refereegranskat)abstract
- Protein aggregation is sequence specific, favoring self-assembly over cross-seeding with non-homologous sequences. Still, as the majority of proteins in a proteome are aggregation prone, the high level of homogeneity of protein inclusions in vivo both during recombinant overexpression and in disease remains surprising. To investigate the selectivity of protein aggregation in a proteomic context, we here compared the selectivity of aggregation-determined interactions with antibody binding. To that purpose, we synthesized biotin-labeled peptides, corresponding to aggregation-determining sequences of the bacterial protein β-galactosidase and two human disease biomarkers: C-reactive protein and prostate-specific antigen. We analyzed the selectivity of their interactions in Escherichiacoli lysate, human serum and human seminal plasma, respectively, using a Western blot-like approach in which the aggregating peptides replace the conventional antibody. We observed specific peptide accumulation in the same bands detected by antibody staining. Combined spectroscopic and mutagenic studies confirmed accumulation resulted from binding of the peptide on the identical sequence of the immobilized target protein. Further, we analyzed the sequence redundancy of aggregating sequences and found that about 90% of them are unique within their proteome. As a result, the combined specificity and low sequence redundancy of aggregating sequences therefore contribute to the observed homogeneity of protein aggregation in vivo. This suggests that these intrinsic proteomic properties naturally compartmentalize aggregation events in sequence space. In the event of physiological stress, this might benefit the ability of cells to respond to proteostatic stress by allowing chaperones to focus on specific aggregation events rather than having to face systemic proteostatic failure.
|
|
| 9. |
- Grannas, Karin, et al.
(författare)
-
Crosstalk between Hippo and TGF beta : Subcellular Localization of YAP/TAZ/Smad Complexes
- 2015
-
Ingår i: Journal of Molecular Biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 427:21, s. 3407-3415
-
Tidskriftsartikel (refereegranskat)abstract
- The Hippo pathway plays a crucial role in growth control, proliferation and tumor suppression. Activity of the signaling pathway is associated with cell density sensing and tissue organization. Furthermore, the Hippo pathway helps to coordinate cellular processes through crosstalk with growth-factor-mediated signaling pathways such as TGF beta. Here we have examined the localization of interactions between proteins of the Hippo pathway (YAP/TAZ) and TGF beta (Smad2/3) signaling pathway by using in situ proximity ligation assays. We investigated the formation of protein complexes between YAP/TAZ and Smad2/3 and examined how these interactions were affected by TGF beta stimulation and cell density in HaCaT keratinocytes and in Smad4-deficient HT29 colon cancer cells. We demonstrate that TGF beta induces formation of YAP/TAZ-Smad2/3 complexes in HaCaT cells. Under sparse cell conditions, the complexes were detected to a higher degree and were predominantly located in the nucleus, while under dense culture conditions, the complexes were fewer and mainly located in the cytoplasm. Surprisingly, we could not detect any YAP/TAZ Smad2/3 complexes in HT29 cells. To examine if Smad4 deficiency was responsible for the absence of interactions, we treated HaCaT cells with siRNA targeting Smad4. However, we could still observe complex formation in the siRNA-treated cells, suggesting that Smad4 is not essential for the YAP Smad2/3 interaction. In conclusion, this study shows localized, density-dependent formation of YAP/TAZ Smad2/3 complexes in HaCaT cells and provides evidence supporting a crosstalk between the Hippo and the TGF beta signaling pathways.
|
|
| 10. |
|
|
