Structures,Functions, and Interactions of ClpT1 and ClpT2 in the Clp Protease System of Arabidopsis Chloroplasts

Plastid ClpT1 and ClpT2 are plant-specific proteins that associate with the ClpPR protease. However, their physiological significance and structures are not understood. Arabidopsis thaliana loss-of-function single clpt1 and clpt2 mutants showed no visible phenotypes, whereas clpt1 clpt2 double mutants showed delayed development, reduced plant growth, and virescent, serrated leaves but were viable and produced seed. The clpt1 and clpt1 clpt2 mutants showed partial destabilization of the ClpPR complex, whereas clpt2 null mutants showed only marginal destabilization. Comparative proteomics of clpt1 clpt2 plants showed a proteostasis phenotype similar to viable mutants in ClpPR core subunits, indicating reduced Clp protease capacity. In vivo and in vitro assays showed that ClpT1 and ClpT2 can independently interact with the single ClpP ring and ClpPR core, but not with the single ClpR ring. We determined ClpT1 and ClpT2 structures (2.4- and 2.0-Å resolution) and detailed the similarities to the N-domains of bacterial ClpA/C chaperones. The ClpT structures suggested a conserved MYFF motif for interaction with the ClpPR core near the interface between the P- and R-rings. In vivo complementation showed that ClpT function and ClpPR core stabilization require the MYFF motif. Several models are presented that may explain how ClpT1,2 contribute to ClpPR protease activity.     

The Crystal Structure of D-Threonine Aldolase from Alcaligenes xylosoxidans Provides Insight into a Metal Ion Assisted PLP-Dependent Mechanism

Threonine aldolases catalyze the pyridoxal phosphate (PLP) dependent cleavage of threonine into glycine and acetaldehyde and play a major role in the degradation of this amino acid. In nature, L- as well as D-specific enzymes have been identified, but the exact physiological function of D-threonine aldolases (DTAs) is still largely unknown. Both types of enantio-complementary enzymes have a considerable potential in biocatalysis for the stereospecific synthesis of various β-hydroxy amino acids, which are valuable building blocks for the production of pharmaceuticals. While several structures of L-threonine aldolases (LTAs) have already been determined, no structure of a DTA is available to date. Here, we report on the determination of the crystal structure of the DTA from Alcaligenes xylosoxidans (AxDTA) at 1.5 Å resolution. Our results underline the close relationship of DTAs and alanine racemases and allow the identification of a metal binding site close to the PLP-cofactor in the active site of the enzyme which is consistent with the previous observation that divalent cations are essential for DTA activity. Modeling of AxDTA substrate complexes provides a rationale for this metal dependence and indicates that binding of the β-hydroxy group of the substrate to the metal ion very likely activates this group and facilitates its deprotonation by His193. An equivalent involvement of a metal ion has been implicated in the mechanism of a serine dehydratase, which harbors a metal ion binding site in the vicinity of the PLP cofactor at the same position as in DTA. The structure of AxDTA is completely different to available structures of LTAs. The enantio-complementarity of DTAs and LTAs can be explained by an approximate mirror symmetry of crucial active site residues relative to the PLP-cofactor.     

Proteomics,phylogenetics, and coexpression analyses indicate novel interactions in the plastid CLP chaperone-protease system

The chloroplast chaperone CLPC1 unfolds and delivers substrates to the stromal CLPPRT protease complex for degradation. We previously used an in vivo trapping approach to identify interactors with CLPC1 in Arabidopsis thaliana by expressing a STREPII-tagged copy of CLPC1 mutated in its Walker B domains (CLPC1-TRAP) followed by affinity purification and mass spectrometry. To create a larger pool of candidate substrates, adaptors, or regulators, we carried out a far more sensitive and comprehensive in vivo protein trapping analysis. We identified 59 highly enriched CLPC1 protein interactors, in particular proteins belonging to families of unknown functions (DUF760, DUF179, DUF3143, UVR-DUF151, HugZ/DUF2470), as well as the UVR domain proteins EXE1 and EXE2 implicated in singlet oxygen damage and signaling. Phylogenetic and functional domain analyses identified other members of these families that appear to localize (nearly) exclusively to plastids. In addition, several of these DUF proteins are of very low abundance as determined through the Arabidopsis PeptideAtlas http://www.peptideatlas.org/builds/arabidopsis/ showing that enrichment in the CLPC1-TRAP was extremely selective. Evolutionary rate covariation indicated that the HugZ/DUF2470 family coevolved with the plastid CLP machinery suggesting functional and/or physical interactions. Finally, mRNA-based coexpression networks showed that all 12 CLP protease subunits tightly coexpressed as a single cluster with deep connections to DUF760-3. Coexpression modules for other trapped proteins suggested specific functions in biological processes, e.g., UVR2 and UVR3 were associated with extraplastidic degradation, whereas DUF760-6 is likely involved in senescence. This study provides a strong foundation for discovery of substrate selection by the chloroplast CLP protease system.     

Immobilization of the first dimension in 2D blue native/SDS-PAGE allows the relative quantification of membrane proteomes

In biological membranes many proteins are organized in complexes. The method of choice for the global analysis of the subunits of these complexes is two-dimensional blue native (2D BN)/SDS-PAGE. In the 1st dimension complexes are separated by BN-PAGE, and in the 2nd dimension their subunits are resolved by SDS-PAGE. In the currently available protocols the 1st dimension BN gel lanes get distorted during their transfer to the 2nd dimension separation gels. This leads to low reproducibility and high variation of 2D BN/SDS-gels, rendering them unsuitable for comparative analysis. We have developed a 2D BN/SDS-PAGE protocol where the 1st dimension BN gel is cast on a GelBond PAG film. Immobilization prevents distortion of BN gel lanes, which lowers variation and greatly improves reproducibility of 2D BN/SDS-gels. 2D BN/SDS-PAGE with an immobilized 1st dimension was used for the comparative analysis of the cytoplasmic membrane proteomes of Escherichia coli cells overexpressing a membrane protein and to create a 2D BN/SDS-PAGE reference map of the E. coli cytoplasmic membrane proteome with 143 identified proteins from 165 different protein spots.     

Quantitative proteomics of a chloroplast SRP54 sorting mutant and its genetic interactions with CLPC1 in Arabidopsis

cpSRP54 (for chloroplast SIGNAL RECOGNITION PARTICLE54) is involved in cotranslational and posttranslational sorting of thylakoid proteins. The Arabidopsis (Arabidopsis thaliana) cpSRP54 null mutant, ffc1-2, is pale green with delayed development. Western-blot analysis of individual leaves showed that the SRP sorting pathway, but not the SecY/E translocon, was strongly down-regulated with progressive leaf development in both wild-type and ffc1-2 plants. To further understand the impact of cpSRP54 deletion, a quantitative comparison of ffc2-1 was carried out for total leaf proteomes of young seedlings and for chloroplast proteomes of fully developed leaves using stable isotope labeling (isobaric stable isotope labeling and isotope-coded affinity tags) and two-dimensional gels. This showed that cpSRP54 deletion led to a change in light-harvesting complex composition, an increase of PsbS, and a decreased photosystem I/II ratio. Moreover, the cpSRP54 deletion led in young leaves to up-regulation of thylakoid proteases and stromal chaperones, including ClpC. In contrast, the stromal protein homeostasis machinery returned to wild-type levels in mature leaves, consistent with the developmental down-regulation of the SRP pathway. A differential response between young and mature leaves was also found in carbon metabolism, with an up-regulation of the Calvin cycle and the photorespiratory pathway in peroxisomes and mitochondria in young leaves but not in old leaves. The Calvin cycle was down-regulated in mature leaves to adjust to the reduced capacity of the light reaction, while reactive oxygen species defense proteins were up-regulated. The significance of ClpC up-regulation was confirmed through the generation of an ffc2-1 clpc1 double mutant. This mutant was seedling lethal under autotrophic conditions but could be partially rescued under heterotrophic conditions.     

Gene therapy of Hunter syndrome: evaluation of the efficiency of muscle electro gene transfer for the production and release of recombinant iduronate-2-sulfatase (IDS)

Mucopolysaccharidosis type II (MPSII) is an inherited disorder due to a deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). The disease is characterized by a considerable deposition of heparan- and dermatan-sulfate, causing a general impairment of physiological functions. Most of the therapeutic protocols proposed so far are mainly based upon enzyme replacement therapy which is very expensive. There is a requirement for an alternative approach, and to this aim, we evaluated the feasibility of muscle electro gene transfer (EGT) performed in the IDS-knockout (IDS-ko) mouse model. EGT is a highly efficient method of delivering exogenous molecules into different tissues. More recently, pre-treatment with bovine hyaluronidase has shown to further improve transfection efficiency of muscle EGT. We here show that, by applying such procedure, IDS was very efficiently produced inside the muscle. However, no induced IDS activity was measured in the IDS-ko mice plasma, in contrast to matched healthy controls. In the same samples, an anticipated and rapidly increasing immune response against the recombinant protein was observed in the IDS-ko vs control mice, although reaching the same levels at 5 weeks post-injection. Additional experiments performed on healthy mice showed a significant contribution of hyaluronidase pre-treatment in increasing the immune response.     

Reduction of GAG storage in MPS II mouse model following implantation of encapsulated recombinant myoblasts

BACKGROUND: Hunter syndrome, mucopolysaccharidosis type II (MPS II), is a X-linked inherited disorder caused by the deficiency of the enzyme iduronate-2-sulfatase (IDS), involved in the lysosomal catabolism of the glycosaminoglycans (GAG) dermatan and heparan sulfate. Such a deficiency leads to the intracellular accumulation of undegraded GAG and eventually to a progressive severe clinical pattern. Many attempts have been made in the last two to three decades to identify possible therapeutic strategies for the disorder, including gene therapy and somatic cell therapy. METHODS: In this study we evaluated the intraperitoneal implantation of allogeneic myoblasts over-expressing IDS, enclosed in alginate microcapsules, in the MPS II mouse model. Animals were monitored for 8 weeks post-implantation, during which plasma and tissue IDS levels, as well as tissue and urinary GAG contents, were measured. RESULTS AND CONCLUSIONS: Induced enzyme activity occurred both in the plasma and in the different tissues analyzed. A significant decrease in urinary undegraded GAG between the fourth and the sixth week of treatment was observed. Moreover, a biochemical reduction of GAG deposits was measured 8 weeks after treatment in the liver and kidney, on average 30 and 38%, respectively, while in the spleen GAG levels were almost normalized. Finally, the therapeutic effect was confirmed by histolochemical examination of the same tissues. Such effects were obtained following implantation of about 1.5 x 10(6) recombinant cells/animal. Taken together, these results represent a clear evidence of the therapeutic efficacy of this strategy in the MPS II mouse model, and encourage further evaluation of this approach for potential treatment of human beings.     

Constitutive expression of pea Lhcb 1–2 in tobacco affects plant development, morphology and photosynthetic capacity

Lhcb1–2 from pea was constitutively expressed in transgenic tobacco plants and assessed for functional impact. The successful assembly of the encoded proteins into LHCII trimers was confirmed by electrospray tandem mass spectrometry. Constitutive production of LHCb1–2 led to increased number of thylakoid membranes per chloroplast, increased grana stacking, higher chloroplast numbers per palisade cell and increased photosynthetic capacity at low irradiance, both on a chlorophyll and leaf area basis. The transgenic plants also displayed increased cell volume, larger leaves, higher leaf number per plant at flowering, increased biomass and increased seed weight, when grown under low irradiance levels. Under high irradiance, both transgenic and wild type plants displayed similar photosynthetic rates when tested at 25°C; however, the non-photochemical quenching (NPQ) and qE values increased in the transgenic plants. The exposure of transgenic plants to a photoinhibitory treatment (4°C for 4h, under continuous illumination) resulted in more detrimental impairment of photosynthesis, since recovery was slower than the non-transgenic plants. These data indicate that constitutive expression of additional Lhcb1–2 transgenes led to a series of changes at all levels of the plant (cellular, leaf and whole organism), and a delay in flowering and senescence. The additional production of the pea protein appears to be accommodated by increasing cellular structures such as the number of thylakoids per chloroplast, organelle volume, organelles per cell, and leaf expansion. The presence of the trimeric pea protein in the tobacco LHCII, however, caused a possible change in the organization of the associated super-complex, that in turn limited photosynthesis at low temperature.