| 1. |
- Kampinga, Harm H., et al.
(författare)
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Function, evolution, and structure of J-domain proteins
- 2019
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Ingår i: Cell stress & chaperones (Print). - : Springer Science and Business Media LLC. - 1355-8145 .- 1466-1268. ; 24:1, s. 7-15
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Forskningsöversikt (refereegranskat)abstract
- Hsp70 chaperone systems are very versatile machines present in nearly all living organisms and in nearly all intracellular compartments. They function in many fundamental processes through their facilitation of protein (re)folding, trafficking, remodeling, disaggregation, and degradation. Hsp70 machines are regulated by co-chaperones. J-domain containing proteins (JDPs) are the largest family of Hsp70 co-chaperones and play a determining role functionally specifying and directing Hsp70 functions. Many features of JDPs are not understood; however, a number of JDP experts gathered at a recent CSSI-sponsored workshop in Gdansk (Poland) to discuss various aspects of J-domain protein function, evolution, and structure. In this report, we present the main findings and the consensus reached to help direct future developments in the field of Hsp70 research.
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| 2. |
- Marszalek, Jaroslaw, et al.
(författare)
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J-domain proteins : From molecular mechanisms to diseases
- 2024
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Ingår i: Cell stress & chaperones (Print). - 1355-8145 .- 1466-1268. ; 29:1, s. 21-33
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Tidskriftsartikel (refereegranskat)abstract
- J-domain proteins (JDPs) are the largest family of chaperones in most organisms, but much of how they function within the network of other chaperones and protein quality control machineries is still an enigma. Here, we report on the latest findings related to JDP functions presented at a dedicated JDP workshop in Gdansk, Poland. The report does not include all (details) of what was shared and discussed at the meeting, because some of these original data have not yet been accepted for publication elsewhere or represented still preliminary observations at the time.
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| 3. |
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| 4. |
- Götzke, Hansjörg, et al.
(författare)
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YfgM Is an Ancillary Subunit of the SecYEG Translocon in Escherichia coli
- 2014
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 289:27, s. 19089-19097
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Tidskriftsartikel (refereegranskat)abstract
- Protein secretion in Gram-negative bacteria is essential for both cell viability and pathogenesis. The vast majority of secreted proteins exit the cytoplasm through a transmembrane conduit called the Sec translocon in a process that is facilitated by ancillary modules, such as SecA, SecDF-YajC, YidC, and PpiD. In this study we have characterized YfgM, a protein with no annotated function. We found it to be a novel ancillary subunit of the Sec translocon as it co-purifies with both PpiD and the SecYEG translocon after immunoprecipitation and blue native/SDS-PAGE. Phenotypic analyses of strains lacking yfgM suggest that its physiological role in the cell overlaps with the periplasmic chaperones SurA and Skp. We, therefore, propose a role for YfgM in mediating the trafficking of proteins from the Sec translocon to the periplasmic chaperone network that contains SurA, Skp, DegP, PpiD, and FkpA.
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| 5. |
- Maddalo, Gianluca, et al.
(författare)
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Systematic Analysis of Native Membrane Protein Complexes in Escherichia coli
- 2011
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 10:4, s. 1848-1859
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Tidskriftsartikel (refereegranskat)abstract
- The cell envelope of Escherichia coli is an essential structure that modulates exchanges between the cell and the extra-cellular milieu. Previous proteomic analyses have suggested that it contains a significant number of proteins with no annotated function. To gain insight into these proteins and the general organization of the cell envelope proteome, we have carried out a systematic analysis of native membrane protein complexes. We have identified 30 membrane protein complexes (6 of which are novel) and present reference maps that can be used for cell envelope profiling. In one instance, we identified a protein with no annotated function (YfgM) in a complex with a well-characterized periplasmic chaperone (PpiD). Using the guilt by association principle, we suggest that YfgM is also part of the periplasmic chaperone network. The approach we present circumvents the need for engineering of tags and protein overexpression. It is applicable for the analysis of membrane protein complexes in any organism and will be particularly useful for less-characterized organisms where conventional strategies that require protein engineering (i.e., 2-hybrid based approaches and TAP-tagging) are not feasible.
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| 6. |
- Marani, Paula, et al.
(författare)
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New Escherichia coli outer membrane proteins identified through prediction and experimental verification
- 2006
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 15:4, s. 884-889
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Tidskriftsartikel (refereegranskat)abstract
- Many new Escherichia coli outer membrane proteins have recently been identified by proteomics techniques. However, poorly expressed proteins and proteins expressed only under certain conditions may escape detection when wild-type cells are grown under standard conditions. Here, we have taken a complementary approach where candidate outer membrane proteins have been identified by bioinformatics prediction, cloned and overexpressed, and finally localized by cell fractionation experiments. Out of eight predicted outer membrane proteins, we have confirmed the outer membrane localization for five-YftM, YaiO, YfaZ, CsgF, and YliI - and also provide preliminary data indicating that a sixth -YfaL- may be an outer membrane autotransporter.
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| 7. |
- Schlegel, Susan, et al.
(författare)
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De-convoluting the Genetic Adaptations of E-coli C41(DE3) in Real Time Reveals How Alleviating Protein Production Stress Improves Yields
- 2015
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 10:10, s. 1758-1766
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Tidskriftsartikel (refereegranskat)abstract
- The well-established E. coli protein production strain C41(DE3) was isolated from the T7 RNA polymerase-based BL21(DE3) strain for its ability to produce difficult recombinant proteins, and it acquired multiple mutations during its isolation. Standard allelic replacement and competition experiments were insufficient to de-convolute these mutations. By reconstructing the evolution of C41(DE3) in real time, we identified the time frames when the different mutations occurred, enabling us to link them to particular stress events. Starvation stress imposed by the isolation procedure selected for mutations enhancing nutrient uptake, and protein production stress for mutations weakening the lacUV5 promoter, which governs t7rnap expression. Moreover, recapitulating protein production stress in BL21(DE3) showed that mutations weakening the lacUV5 promoter occur through RecA-dependent recombination with the wild-type lac-promoter and are selected for upon the production of any protein. Thus, the instability of the lacUV5 promoter in BL21(DE3) alleviates protein production stress and can be harnessed to enhance production.
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| 8. |
- Schlegel, Susan, et al.
(författare)
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Isolating Escherichia coli strains for recombinant protein production
- 2017
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Ingår i: Cellular and Molecular Life Sciences (CMLS). - : Springer Science and Business Media LLC. - 1420-682X .- 1420-9071. ; 74:5, s. 891-908
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Forskningsöversikt (refereegranskat)abstract
- Escherichia coli has been widely used for the production of recombinant proteins. To improve protein production yields in E. coli, directed engineering approaches have been commonly used. However, there are only few reported examples of the isolation of E. coli protein production strains using evolutionary approaches. Here, we first give an introduction to bacterial evolution and mutagenesis to set the stage for discussing how so far selection- and screening-based approaches have been used to isolate E. coli protein production strains. Finally, we discuss how evolutionary approaches may be used in the future to isolate E. coli strains with improved protein production characteristics.
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| 9. |
- Schroeder, Kristen, 1986-
(författare)
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Caulobacter crescentus folding machines at the interface of inheritance, cell division, and energy metabolism
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- All living cells must perform essential biological processes while monitoring and responding to environmental cues. Bacteria are accessible experimental systems in which to study the function of conserved central processes. One of the most highly conserved systems between different organisms is the proteostasis network; a group of chaperones and proteases that work collectively to repair and remove damaged proteins that accumulate in living systems. In the work of this thesis, we investigate how folding machines of the proteostasis network are integrated with central biological processes in the model organism Caulobacter crescentus, and examine how these relationships change during stress.Asymmetrically-dividing C. crescentus has previously been described to undergo aging, with the accumulation of protein aggregates in the larger stalked cell proposed to drive replicative decline in this organism. In study I, we establish C. crescentus as a model for monitoring the dynamic cellular response to protein aggregation. Using this system, we demonstrate that protein aggregates are shared during division, and do not preferentially collect in one cell type.The ubiquitous GroESL folding machine, which provides a specialized environment for folding specific proteins, has been previously linked to the C. crescentus cell cycle through an unknown mechanism. In study II, we discover that GroESL folding is required to support division both in optimal conditions and during mild stress. Specifically, we find that GroESL supports the function of proteins that interact with the highly conserved bacterial division scaffold FtsZ, as well as proteins that direct synthesis of the peptidoglycan cell envelope layer.In study III we investigate the functional link between GroESL folding and energy metabolism, and find that the chaperonin has a conserved role in folding respiratory and metabolic proteins, thereby supporting the central pathways these proteins function in. Furthermore, we find that GroESL protects several of these proteins from aggregation during stress.Taken together, the work of this thesis addresses current models of prokaryotic damage segregation and aging, expands on how chaperonin folding is integrated into the essential process of division, and demonstrates a functional role for protein folding in protecting energy metabolism during stress. The findings of this research thereby provide novel insight into how fundamental biological processes interface with protein folding machines.
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| 10. |
- Stårsta, Magnus
(författare)
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The effects of internally expressed Contact-Dependent growth Inhibition (CDI) toxins in bacteria
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bacteria, both pathogenic and non-pathogenic, have developed multiple forms of competition mechanisms to combat each other including, but not limited to, Contact-Dependent growth Inhibition (CDI) systems, Type VI Secretion Systems and the associated Rearrangement hotspot (Rhs) toxin system. These systems usually confers a great fitness advantage as they allow for precise delivery of toxic molecules into competing bacteria whilst sister cells are protected from auto-inhibition by producing a cognate immunity protein. Delivery between sister cells may serve as a form of “self-recognition” whilst maintaining selection pressure for these genes within the population. How these genes are maintained in conditions where delivery does not occur has until now not been fully understood.This thesis describes secondary functions, maintained selection pressure and regulation of Rhs and CDI systems in three parts. In Paper I, we made a novel discovery that rhs toxin and immunity genes from Salmonella enterica serovar Typhimurium are transcribed from internal transcriptional start sites independent of the full length delivery gene. This results in functional cytosolic proteins capable of regulating proliferation and growth rate of S. Typhimurium during infection of RAW264.7 cells. In Paper II, we continued our work from Paper I and studied growth effects in vitro as well as regulation of the internal expression. Our findings show that Rhs causes a small fitness cost also in rich laboratory medium and is regulated by alternate sigma factor RpoS, two-component system PhoP/Q and DNA binding protein H-NS. In Paper III, we made a discovery similar to our findings in Paper I and II by observing internally transcribed toxin and immunity genes of multiple CDI systems from E. coli regulated by RpoS. We propose that CDI toxin-immunity pairs function as selfish genetic elements that maintain gene selection whilst simultaneously retaining the ability to protect the cell from externally delivered toxins.
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