A Distinct Mechanism to Achieve Efficient Signal Recognition Particle (SRP)-SRP Receptor Interaction by the Chloroplast SRP Pathway

Cotranslational protein targeting by the signal recognition particle (SRP) requires the SRP RNA, which accelerates the interaction between the SRP and SRP receptor 200-fold. This otherwise universally conserved SRP RNA is missing in the chloroplast SRP (cpSRP) pathway. Instead, the cpSRP and cpSRP receptor (cpFtsY) by themselves can interact 200-fold faster than their bacterial homologues. Here, cross-complementation analyses revealed the molecular origin underlying their efficient interaction. We found that cpFtsY is 5- to 10-fold more efficient than Escherichia coli FtsY at interacting with the GTPase domain of SRP from both chloroplast and bacteria, suggesting that cpFtsY is preorganized into a conformation more conducive to complex formation. Furthermore, the cargo-binding M-domain of cpSRP provides an additional 100-fold acceleration for the interaction between the chloroplast GTPases, functionally mimicking the effect of the SRP RNA in the cotranslational targeting pathway. The stimulatory effect of the SRP RNA or the M-domain of cpSRP is specific to the homologous SRP receptor in each pathway. These results strongly suggest that the M-domain of SRP actively communicates with the SRP and SR GTPases and that the cytosolic and chloroplast SRP pathways have evolved distinct molecular mechanisms (RNA vs. protein) to mediate this communication

Mechanism of an ATP-independent Protein Disaggregase - I. Structure of a Membrane Protein Aggregate Reveals a Mechanism of Recognition by its Chaperone

Protein aggregation is detrimental to the maintenance of proper protein homeostasis in all cells. To overcome this problem, cells have evolved a network of molecular chaperones to prevent protein aggregation and even reverse existing protein aggregates. The most extensively studied disaggregase systems are ATP-driven macromolecular machines. Recently, we reported an alternative disaggregase system in which the 38-kDa subunit of chloroplast signal recognition particle (cpSRP43) efficiently reverses the aggregation of its substrates, the light-harvesting chlorophyll a/b-binding (LHC) proteins, in the absence of external energy input. To understand the molecular mechanism of this novel activity, here we used biophysical and biochemical methods to characterize the structure and nature of LHC protein aggregates. We show that LHC proteins form micellar, disc-shaped aggregates that are kinetically stable and detergent-resistant. Despite the nonamyloidal nature, the LHC aggregates have a defined global organization, displaying the chaperone recognition motif on its solvent-accessible surface. These findings suggest an attractive mechanism for recognition of the LHC aggregate by cpSRP43 and provide important constraints to define the capability of this chaperone

ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP subunit

Membrane proteins impose enormous challenges to cellular protein homeostasis during their post-translational targeting, and they require chaperones to keep them soluble and translocation competent. Here we show that a novel targeting factor in the chloroplast signal recognition particle (cpSRP), cpSRP43, is a highly specific molecular chaperone that efficiently reverses the aggregation of its substrate proteins. In contrast to 'ATPases associated with various cellular activities' (AAA+) chaperones, cpSRP43 uses specific binding interactions with its substrate to mediate its 'disaggregase' activity. This disaggregase capability can allow targeting machineries to more effectively capture their protein substrates and emphasizes a close connection between protein folding and trafficking processes. Moreover, cpSRP43 provides the first example to our knowledge of an ATP-independent disaggregase and shows that efficient reversal of protein aggregation can be attained by specific binding interactions between a chaperone and its substrate

Biochemical and functional characterization of Plasmodium falciparum GTP cyclohydrolase I

BACKGROUND: Antifolates are currently in clinical use for malaria preventive therapy and treatment. The drugs kill the parasites by targeting the enzymes in the de novo folate pathway. The use of antifolates has now been limited by the spread of drug-resistant mutations. GTP cyclohydrolase I (GCH1) is the first and the rate-limiting enzyme in the folate pathway. The amplification of the gch1 gene found in certain Plasmodium falciparum isolates can cause antifolate resistance and influence the course of antifolate resistance evolution. These findings showed the importance of P. falciparum GCH1 in drug resistance intervention. However, little is known about P. falciparum GCH1 in terms of kinetic parameters and functional assays, precluding the opportunity to obtain the key information on its catalytic reaction and to eventually develop this enzyme as a drug target. METHODS: Plasmodium falciparum GCH1 was cloned and expressed in bacteria. Enzymatic activity was determined by the measurement of fluorescent converted neopterin with assay validation by using mutant and GTP analogue. The genetic complementation study was performed in ∆folE bacteria to functionally identify the residues and domains of P. falciparum GCH1 required for its enzymatic activity. Plasmodial GCH1 sequences were aligned and structurally modeled to reveal conserved catalytic residues. RESULTS: Kinetic parameters and optimal conditions for enzymatic reactions were determined by the fluorescence-based assay. The inhibitor test against P. falciparum GCH1 is now possible as indicated by the inhibitory effect by 8-oxo-GTP. Genetic complementation was proven to be a convenient method to study the function of P. falciparum GCH1. A series of domain truncations revealed that the conserved core domain of GCH1 is responsible for its enzymatic activity. Homology modelling fits P. falciparum GCH1 into the classic Tunnelling-fold structure with well-conserved catalytic residues at the active site. CONCLUSIONS: Functional assays for P. falciparum GCH1 based on enzymatic activity and genetic complementation were successfully developed. The assays in combination with a homology model characterized the enzymatic activity of P. falciparum GCH1 and the importance of its key amino acid residues. The potential to use the assay for inhibitor screening was validated by 8-oxo-GTP, a known GTP analogue inhibitor

Mechanism of an ATP-independent Protein Disaggregase. II. Distinct Molecular Interactions Drive Multiple Steps During Aggregate Disassembly

The ability of molecular chaperones to overcome the misfolding and aggregation of proteins is essential for the maintenance of proper protein homeostasis in all cells. Thus far, the best studied disaggregase systems are the Clp/Hsp100 family of “ATPases associated with various cellular activities” (AAA^+) ATPases, which use mechanical forces powered by ATP hydrolysis to remodel protein aggregates. An alternative system to disassemble large protein aggregates is provided by the 38-kDa subunit of the chloroplast signal recognition particle (cpSRP43), which uses binding energy with its substrate proteins to drive disaggregation. The mechanism of this novel chaperone remains unclear. Here, molecular genetics and structure-activity analyses show that the action of cpSRP43 can be dissected into two steps with distinct molecular requirements: (i) initial recognition, during which cpSRP43 binds specifically to a recognition motif displayed on the surface of the aggregate; and (ii) aggregate remodeling, during which highly adaptable binding interactions of cpSRP43 with hydrophobic transmembrane domains of the substrate protein compete with the packing interactions within the aggregate. This establishes a useful framework to understand the molecular mechanism by which binding interactions from a molecular chaperone can be used to overcome protein aggregates in the absence of external energy input from ATP

Evaluation of Methylotrophic Yeast Ogataea thermomethanolica TBRC 656 as a Heterologous Host for Production of an Animal Vaccine Candidate

Multiple yeast strains have been developed into versatile heterologous protein expression platforms. Earlier works showed that Ogataea thermomethanolica TBRC 656 (OT), a thermotolerant methylotrophic yeast, can efficiently produce several industrial enzymes. In this work, we demonstrated the potential of this platform for biopharmaceutical manufacturing. Using a swine vaccine candidate as a model, we showed that OT can be optimized to express and secrete the antigen based on porcine circovirus type 2d capsid protein at a respectable yield. Crucial steps for yield improvement include codon optimization and reduction of OT protease activities. The antigen produced in this system could be purified efficiently and induce robust antibody response in test animals. Improvements in this platform, especially more efficient secretion and reduced extracellular proteases, would extend its potential as a competitive platform for biopharmaceutical industries

Novel constructs and 1-step chromatography protocols for the production of Porcine Circovirus 2d (PCV2d) and Circovirus 3 (PCV3) subunit vaccine candidates

Porcine circovirus type 2 (PCV2) has been a major problem for the pig production industry worldwide for decades. While the majority of commercially available vaccines are based on the original PCV2a genotype, the current dominant genotype is PCV2d. The notable differences between genotypes could lead to incomplete cross-protection. Moreover, most current subunit PCV2 vaccines are generated from expensive insect cell culture technology. In this work, we present a new workflow for production of an updated and relatively inexpensive PCV2d vaccine candidate. After expression in fed-batch Escherichia coli fermentation systems with a simple one-step ion-exchange chromatography purification protocol, the yield of purified PCV2d-based antigen reached over 1 g per litre bacterial culture. Using similar procedures, we also demonstrated even higher PCV2d-based antigen yields from a chimeric PCV2d-PCV3 capsid construct, which is cleaved during fermentation to release PCV2d- and PCV3-related polypeptides. Although the PCV2d-based recombinant protein from this protocol did not form viral-like particles as analysed by size-exclusion chromatography, it could effectively induce capsid-specific and PCV2d-neutralising antibodies in immunised animals, indicating significant potential as a new vaccine candidate that can be easily manufactured at commercial scale

Post-Translational Membrane Protein Targeting by the Chloroplast Signal Recognition Particle

Post-translational transport of membrane proteins poses enormous challenges to the cells. The transport factors must accurately select and deliver the cargos to the appropriate target membranes. In addition, they have to provide chaperone for their hydrophobic cargos. To understand capacity and limitation of a post-translational transport factor, we studied one of the most efficient membrane protein transport pathways, the delivery of light-harvesting chlorophyll-binding (LHC) proteins to the thylakoid membrane. This targeting reaction is mediated by the chloroplast Signal Recognition Particle (cpSRP) and its receptor. Although the core SRP GTPases are close homologues of those in cytosolic SRP pathways, the unique features of cpSRP that might reflect its adaptation to the challenges in post-translational targeting include (i) the lack of the otherwise universally conserved SRP RNA, and (ii) the exclusive presence of a novel protein, cpSRP43. In the first part of this thesis, we define the thermodynamic and kinetic framework for the GTPase cycles of cpSRP and its receptor and uncover the molecular bases that enable their intrinsically fast interactions, such that they can bypass an SRP RNA, an essential accelerator for the cytosolic SRP–receptor interaction. The second part of the thesis is devoted to characterization of the chaperone function of cpSRP43. We show that cpSRP43 specifically and effectively prevents and reverses the aggregation of its cargo, LHC proteins. We further investigate the molecular mechanism of this novel disaggregase activity, using a combination of biochemical and structural approaches. In summary, this dissertation aims to understand how cpSRP and its receptor adapt to their unique requirements in efficiently transporting a family of highly abundant membrane proteins