| 1. |
- Andersson, Fredrik, 1977, et al.
(författare)
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Structure and function of a novel type of ATP-dependent Clp protease.
- 2009
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Ingår i: The Journal of biological chemistry. - 0021-9258 .- 1083-351X. ; 284:20, s. 13519-32
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Tidskriftsartikel (refereegranskat)abstract
- The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. The main constitutive Clp protease in photosynthetic organisms has evolved into a functionally essential and structurally intricate enzyme. The model Clp protease from the cyanobacterium Synechococcus consists of the HSP100 molecular chaperone ClpC and a mixed proteolytic core comprised of two distinct subunits, ClpP3 and ClpR. We have purified the ClpP3/R complex, the first for a Clp proteolytic core comprised of heterologous subunits. The ClpP3/R complex has unique functional and structural features, consisting of twin heptameric rings each with an identical ClpP3(3)ClpR(4) configuration. As predicted by its lack of an obvious catalytic triad, the ClpR subunit is shown to be proteolytically inactive. Interestingly, extensive modification to ClpR to restore proteolytic activity to this subunit showed that its presence in the core complex is not rate-limiting for the overall proteolytic activity of the ClpCP3/R protease. Altogether, the ClpP3/R complex shows remarkable similarities to the 20 S core of the proteasome, revealing a far greater degree of convergent evolution than previously thought between the development of the Clp protease in photosynthetic organisms and that of the eukaryotic 26 S proteasome.
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| 2. |
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| 3. |
- Tryggvesson, Anders, 1975
(författare)
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Biochemical studies of the essential Clp protease in cyanobacteria and its associated adaptor proteins
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are an essential part of all organisms and are involved in many cellular processes. To regulate the function of proteins and facilitate their removal when damaged or otherwise compromised, sophisticated control systems have evolved that include molecular chaperones and proteases. These regulatory proteins attempt to repair damaged polypeptides and if necessary degrade them before they can interfere with cellular activities. Clp/Hsp100 proteins are a family of chaperones that belong to the broader family of AAA+ proteins (ATPases associated with diverse cellular activities) that are present in a wide range of organisms. Many of these AAA+ Clp proteins function as the chaperone partner within Clp proteases, conferring substrate specificity and transferring the unfolded protein substrate to the proteolytic component for degradation. The Clp chaperones form single hexameric rings that associate to a proteolytic complex consisting of two opposing heptameric rings comprised typically of a single type of subunit, ClpP. The catalytic sites of these ClpP subunits are sequestered within the tetradecamer to avoid inadvertent protein degradation.The Clp protease in E. coli is the best studied Clp proteases to date, with two distinct types depending on if the chaperone partner is ClpA or ClpX. Also present are adaptor proteins that modify the substrate specificity of the chaperone component, such as ClpS that redirects the ClpAP protease to degrade N-end rule substrates. Although Clp proteins are found in a wide range of organisms, those in photosynthetic organisms such as cyanobacteria and vascular plants are by far the most numerous and diverse. In the cyanobacterium Synechococcus elongatus (Synechococcus) two types of mixed Clp proteolytic cores exist; ClpP3/R and ClpP1/P2. The ClpP3/R core associates to the chaperone ClpC to produce a protease that is essential for phototrophic growth, whereas ClpP1/P2 binds to ClpX to form a second Clp protease whose activity is non-essential. This thesis work has examined the structure and function of the mixed proteolytic core within the essential ClpCP3/R protease and its associated adapter proteins ClpS1 and ClpS2. This has been done using molecular and biochemical methods to purify recombinant versions of each Clp protein or complex and analyzing them in vitro. In Paper I, the ClpP3/R complex was over-expressed in E. coli and purified by column chromatography. The proteolytic core was shown to consist of two identical heptameric rings, each with three ClpP3 and four ClpR subunits in an alternating configuration. The ClpR subunit is catalytically inactive but its inclusion within the ClpP3/R core did not appear rate-limiting for the activity of the ClpCP3/R protease. The general architecture of ClpP3/R mirrored that of the proteolytic core within the eukaryotic 26S proteasome, with three active and four inactive subunits in the central heptameric rings. A model of ClpP3/R was also presented in this paper, along with the finding that the ClpS1 adaptor protein binds to ClpC and modifies its substrate specificity. In Paper II, two N-terminal regions in ClpR (the Tyr- and Pro motifs) and one in ClpP3 (the MPIG motif) were shown to be important for the interaction with ClpC and correct assembly of the ClpP3/R tetradecamer. We also identified a motif in the C-terminal region of ClpC (the R-motif) that confers the specific association to the ClpP3/R core. In Paper III, we investigated the essential adaptor protein ClpS2 that is so far unique to cyanobacteria. A recombinant version of ClpS2 was purified and its activity compared to that of ClpS1. Like ClpS1, ClpS2 binds to ClpC and alters its substrate specificity. However, ClpS1 and ClpS2 recognize different destabilizing residues and thus target different N-end rule substrates for degradation by the ClpCP3/R protease. Overall, this thesis provides new insights into the structure and function of the essential ClpCP3/R protease in cyanobacteria and how its substrate specificity is modified by the ClpS adaptor proteins.
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| 4. |
- Tryggvesson, Anders, 1975, et al.
(författare)
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Characterization of ClpS2, an essential adaptor protein for the cyanobacterium Synechococcus elongatus
- 2015
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Ingår i: Febs Letters. - : Wiley. - 0014-5793. ; 589:24, s. 4039-4046
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Tidskriftsartikel (refereegranskat)abstract
- The adaptor protein ClpS associates to the Clp protease and promotes degradation of N-end rule substrates in eubacteria and in algal/plant chloroplasts. Cyanobacteria are unusual in having two distinct ClpS paralogs. Although ClpS1 is typical of bacterial ClpS, ClpS2 differs in crucial ways. ClpS2 in Synechococcus elongatus is a relatively low- abundant, soluble protein essential for phototrophic growth. Like ClpS1, ClpS2 binds to the ClpCP3/R protease to block alpha-casein degradation and promote that of N-end rule substrates in vitro. However, their substrate specificity differs, with ClpS1 recognizing destabilizing Phe and Tyr residues at the substrate N-terminus whereas ClpS2 recognizes Leu. Overall, ClpS2 appears to have independently evolved in cyanobacteria to degrade a particular group of proteins, whose turnover is vital for cell viability. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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| 5. |
- Tryggvesson, Anders, 1975, et al.
(författare)
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Interaction specificity between the chaperone and proteolytic components of the cyanobacterial Clp protease
- 2012
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 446, s. 311-320
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Tidskriftsartikel (refereegranskat)abstract
- The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. In plant chloroplasts and cyanobacteria, the essential constitutive Clp protease consists of the Hsp100/ClpC chaperone partnering a proteolytic core of catalytic ClpP and noncatalytic ClpR subunits. In the present study, we have examined putative determinants conferring the highly specific association between ClpC and the ClpP3/R core from the model cyanobacterium Synechococcus elongatus. Two conserved sequences in the N-terminus of ClpR (tyrosine and proline motifs) and one in the N-terminus of ClpP3 (MPIG motif) were identified as being crucial for the ClpC-ClpP3/R association. These N-terminal domains also influence the stability of the ClpP3/R core complex itself. A unique C-terminal sequence was also found in plant and cyanobacterial ClpC orthologues just downstream of the P-loop region previously shown in Escherichia coli to be important for Hsp100 association to ClpP. This R motif in Synechococcus ClpC confers specificity for the ClpP3/R core and prevents association with E. coli ClpP; its removal from ClpC reverses this core specificity.
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