| 1. |
- Byström, Roberth, et al.
(författare)
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SOD1 Mutations Targeting Surface Hydrogen Bonds Promote Amyotrophic Lateral Sclerosis without Reducing Apo-state Stability
- 2010
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 285:25, s. 19544-19552
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Tidskriftsartikel (refereegranskat)abstract
- In good accord with the protein aggregation hypothesis for neurodegenerative diseases, ALS-associated SOD1 mutations are found to reduce structural stability or net repulsive charge. Moreover there are weak indications that the ALS disease progression rate is correlated with the degree of mutational impact on the apoSOD1 structure. A bottleneck for obtaining more conclusive information about these structure-disease relationships, however, is the large intrinsic variability in patient survival times and insufficient disease statistics for the majority of ALS- provoking mutations. As an alternative test of the structure- disease relationship we focus here on the SOD1 mutations that appear to be outliers in the data set. The results identify several ALS- provoking mutations whose only effect on apoSOD1 is the elimination or introduction of a single charge, i.e. D76V/Y, D101N, and N139D/K. The thermodynamic stability and folding behavior of these mutants are indistinguishable from the wild-type control. Moreover, D101N is an outlier in the plot of stability loss versus patient survival time by having rapid disease progression. Commonto the identified mutations is that they truncate conserved salt-links and/or H-bond networks in the functional loops IV or VII. The results show that the local impact of ALS- associated mutations on the SOD1 molecule can sometimes overrun their global effects on apo-state stability and net repulsive charge, and point at the analysis of property outliers as an efficient strategy for mapping out new ALS- provoking features.
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| 2. |
- Jong, Wouter SP, et al.
(författare)
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A structurally informed autotransporter platform for efficient heterologous protein secretion and display
- 2012
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859 .- 1475-2859. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The self-sufficient autotransporter (AT) pathway, ubiquitous in Gram-negative bacteria, combines a relatively simple protein secretion mechanism with a high transport capacity. ATs consist of a secreted passenger domain and a β-domain that facilitates transfer of the passenger across the cell-envelope. They have a great potential for the extracellular expression of recombinant proteins but their exploitation has suffered from the limited structural knowledge of carrier ATs. Capitalizing on its crystal structure, we have engineered the Escherichia coli AT Hemoglobin protease (Hbp) into a platform for the secretion and surface display of heterologous proteins, using the Mycobacterium tuberculosis vaccine target ESAT6 as a model protein.RESULTS: Based on the Hbp crystal structure, five passenger side domains were selected and one by one replaced by ESAT6, whereas a β-helical core structure (β-stem) was left intact. The resulting Hbp-ESAT6 chimeras were efficiently and stably secreted into the culture medium of E. coli. On the other hand, Hbp-ESAT6 fusions containing a truncated β-stem appeared unstable after translocation, demonstrating the importance of an intact β-stem. By interrupting the cleavage site between passenger and β-domain, Hbp-ESAT6 display variants were constructed that remain cell associated and facilitate efficient surface exposure of ESAT6 as judged by proteinase K accessibility and whole cell immuno-EM analysis. Upon replacement of the passenger side domain of an alternative AT, EspC, ESAT6 was also efficiently secreted, showing the approach is more generally applicable to ATs. Furthermore, Hbp-ESAT6 was efficiently displayed in an attenuated Salmonella typhimurium strain upon chromosomal integration of a single encoding gene copy, demonstrating the potential of the Hbp platform for live vaccine development.CONCLUSIONS: We developed the first structurally informed AT platform for efficient secretion and surface display of heterologous proteins. The platform has potential with regard to the development of recombinant live vaccines and may be useful for other biotechnological applications that require high-level secretion or display of recombinant proteins by bacteria.
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| 3. |
- Jong, Wouter S. P., et al.
(författare)
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An autotransporter display platform for the development of multivalent recombinant bacterial vector vaccines
- 2014
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Ingår i: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859 .- 1475-2859. ; 13, s. -162
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Tidskriftsartikel (refereegranskat)abstract
- Background: The Autotransporter pathway, ubiquitous in Gram-negative bacteria, allows the efficient secretion of large passenger proteins via a relatively simple mechanism. Capitalizing on its crystal structure, we have engineered the Escherichia coli autotransporter Hemoglobin protease (Hbp) into a versatile platform for secretion and surface display of multiple heterologous proteins in one carrier molecule. Results: As proof-of-concept, we demonstrate efficient secretion and high-density display of the sizeable Mycobacterium tuberculosis antigens ESAT6, Ag85B and Rv2660c in E. coli simultaneously. Furthermore, we show stable multivalent display of these antigens in an attenuated Salmonella Typhimurium strain upon chromosomal integration. To emphasize the versatility of the Hbp platform, we also demonstrate efficient expression of multiple sizeable antigenic fragments from Chlamydia trachomatis and the influenza A virus at the Salmonella cell surface. Conclusions: The successful efficient cell surface display of multiple antigens from various pathogenic organisms highlights the potential of Hbp as a universal platform for the development of multivalent recombinant bacterial vector vaccines.
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| 4. |
- Meikle, W. P. S., et al.
(författare)
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Dust and the type II-Plateau supernova 2004dj
- 2011
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Ingår i: Astrophysical Journal. - 0004-637X .- 1538-4357. ; 732:2, s. 109-
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Tidskriftsartikel (refereegranskat)abstract
- We present mid-infrared (MIR) spectroscopy of a Type II-plateau supernova, SN 2004dj, obtained with the Spitzer Space Telescope, spanning 106-1393 days after explosion. MIR photometry plus optical/near-IR observations are also reported. An early-time MIR excess is attributed to emission from non-silicate dust formed within a cool dense shell (CDS). Most of the CDS dust condensed between 50 days and 165 days, reaching a mass of 0.3 x 10(-5) M(circle dot). Throughout the observations, much of the longer wavelength (> 10 mu m) part of the continuum is explained as an IR echo from interstellar dust. The MIR excess strengthened at later times. We show that this was due to thermal emission from warm, non-silicate dust formed in the ejecta. Using optical/near-IR line profiles and the MIR continua, we show that the dust was distributed as a disk whose radius appeared to be shrinking slowly. The disk radius may correspond to a grain destruction zone caused by a reverse shock which also heated the dust. The dust-disk lay nearly face-on, had high opacities in the optical/near-IR regions, but remained optically thin in the MIR over much of the period studied. Assuming a uniform dust density, the ejecta dust mass by 996 days was (0.5 +/- 0.1) x 10(-4) M(circle dot) and exceeded 10(-4) M(circle dot) by 1393 days. For a dust density rising toward the center the limit is higher. Nevertheless, this study suggests that the amount of freshly synthesized dust in the SN 2004dj ejecta is consistent with that found from previous studies and adds further weight to the claim that such events could not have been major contributors to the cosmic dust budget.
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