Real-world safety of Levetiracetam: Mining and analysis of its adverse drug reactions based on FAERS database.

INTRODUCTION: Levetiracetam is a relatively new and widely utilized anti-seizure medication; however, limited information is available regarding its adverse effects. This study aims to thoroughly investigate, evaluate, and present evidence on the safety profile of Levetiracetam, relying on data from the FDA Adverse Event Reporting System (FAERS) database to facilitate informed clinical decision-making. METHODS: We employed various statistical measures, including Reporting Odds Ratio (ROR), Proportionate Reporting Ratio (PRR), and analysis by the Medicines and Healthcare Products Regulatory Agency (MHRA), to identify signals of adverse reactions associated with Levetiracetam. Positive signals consistent with Designated Medical Event (DME) were singled out for focused comparison and discussion. RESULTS: The analysis of 26,182 adverse events linked to Levetiracetam as the primary suspected drug revealed 692 positive signals spanning 22 System Organ Classes (SOCs). Nervous system disorders were the most frequently reported, followed by psychiatric disorders, and general disorders and administration site conditions. 11 positive signals consistent with Preferred Terms (PTs) in DME were identified, predominantly concentrated in 6 SOCs. Among these, rhabdomyolysis, Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), drug reaction with eosinophilia and systemic symptoms (DRESS) exhibited relatively large values of A, ROR, and Chi-squared. Additionally, PTs related to spontaneous abortion, drug interaction, urethral atresia, ventricular septal defect, and atrial septal defect showed significant strength. CONCLUSIONS: The study indicates that Levetiracetam carries a potential risk of causing rhabdomyolysis, SJS, TEN, DRESS as well as spontaneous abortion. Signals related to drug interaction, urethral atresia, ventricular septal defect, and atrial septal defect warrant heightened attention in clinical use.

Structural insights into the functions of Raf1 and Bsd2 in hexadecameric Rubisco assembly.

Hexadecameric form I Rubisco, which consisting consists of eight large (RbcL) and eight small (RbcS) subunits, is the most abundant enzyme on earth. Extensive efforts to engineer an improved Rubisco to speed up its catalytic efficiency and ultimately increase agricultural productivity. However, difficulties with correct folding and assembly in foreign hosts or in vitro have hampered the genetic manipulation of hexadecameric Rubisco. In this study, we reconstituted Synechococcus sp. PCC6301 Rubisco in vitro using the chaperonin system and assembly factors from cyanobacteria and Arabidopsis thaliana (At). Rubisco holoenzyme was produced in the presence of cyanobacterial Rubisco accumulation factor 1 (Raf1) alone or both AtRaf1 and bundle-sheath defective-2 (AtBsd2) from Arabidopsis. RbcL released from GroEL is assembly capable in the presence of ATP, and AtBsd2 functions downstream of AtRaf1. Cryo-EM structures of RbcL8-AtRaf18, RbcL8-AtRaf14-AtBsd28, and RbcL8 revealed that the interactions between RbcL and AtRaf1 are looser than those between prokaryotic RbcL and Raf1, with AtRaf1 tilting 7° farther away from RbcL. AtBsd2 stabilizes the flexible regions of RbcL, including the N and C termini, the 60s loop, and loop 6. Using these data, combined with previous findings, we propose the possible biogenesis pathways of prokaryotic and eukaryotic Rubisco.

A structural basis for the functional differences between the cytosolic and plastid phosphoglucose isomerase isozymes.

Phosphoglucose isomerase (PGI) catalyzes the interconversion between glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P), thereby regulating sucrose synthesis in plant cells. In general, plants contain a pair of PGI isozymes located in two distinct compartments of the cell (cytosol and plastid) with differences in both the primary structure and the higher-order structure. Previously, we showed that the activity of cytosolic PGI (PGIc) is more robust (activity, thermal stability, substrate turnover rate, etc.) than that of the plastid counterpart (PGIp) in multiple organisms, including wheat, rice, and Arabidopsis. The crystal structures of apoTaPGIc (an isotype cytosol PGIc in Triticum aestivum), TaPGIc-G6P complex, and apoTaPGIp (an isotype plastid PGIp in Triticum aestivum) were first solved in higher plants, especially in crops. In this study, we detailed the structural characteristics related to the biochemical properties and functions of TaPGIs in different plant organelles. We found that the C-terminal domains (CTDs) of TaPGIc and TaPGIp are very different, which affects the stability of the dimerized enzyme, and that Lys213TaPGIc/Lys193TaPGIp and its surrounding residues at the binding pocket gateway may participate in the entrance and exit of substrates. Our findings provide a good example illuminating the evolution of proteins from primary to higher structures as a result of physical barriers and adaptation to the biochemical environment.

The cryo-EM structure of the chloroplast ClpP complex.

Protein homoeostasis in plastids is strategically regulated by the protein quality control system involving multiple chaperones and proteases, among them the Clp protease. Here, we determined the structure of the chloroplast ClpP complex from Chlamydomonas reinhardtii by cryo-electron microscopy. ClpP contains two heptameric catalytic rings without any symmetry. The top ring contains one ClpR6, three ClpP4 and three ClpP5 subunits while the bottom ring is composed of three ClpP1C subunits and one each of the ClpR1-4 subunits. ClpR3, ClpR4 and ClpT4 subunits connect the two rings and stabilize the complex. The chloroplast Cpn11/20/23 co-chaperonin, a co-factor of Cpn60, forms a cap on the top of ClpP by protruding mobile loops into hydrophobic clefts at the surface of the top ring. The co-chaperonin repressed ClpP proteolytic activity in vitro. By regulating Cpn60 chaperone and ClpP protease activity, the co-chaperonin may play a role in coordinating protein folding and degradation in the chloroplast.

Dynamical analysis of fractional-order Holling type-II food chain model.

This paper proposed a fractional-order Holling type-II food chain model. First, we verified the existence, uniqueness, nonnegativity and boundedness of the solution of the model, and some conditions for equilibrium existence and local stability were studied. Second, a controller was proposed, and the Lyapunov method was used to study the global stability of the positive equilibrium point. Finally, numerical simulations were performed to verify the theoretical results.

Engineering of the cytosolic form of phosphoglucose isomerase into chloroplasts improves plant photosynthesis and biomass.

Starch is the most abundant carbohydrate synthesized in plant chloroplast as the product of photosynthetic carbon assimilation, serving a crucial role in the carbon budget as storage energy. Phosphoglucose isomerase (PGI) catalyzes the interconversion between glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P), which are important metabolic molecules in starch synthesis within chloroplasts and sucrose synthesis in cytosol. Here, we found that the specific activity of recombinantly purified PGI localized in cytosolic PGI (PGIc) was much higher than its plastidic isoenzyme counterpart (PGIp) originated from wheat, rice and Arabidopsis, with wheat PGIc having by far the highest activity. Crystal structures of wheat TaPGIc and TaPGIp proteins were solved and the functional units were homodimers. The active sites of PGIc and PGIp, constituted by the same amino acids, formed different binding pockets. Moreover, PGIc showed slightly lower affinity to the substrate F6P but with much faster turnover rates. Engineering of TaPGIc into chloroplasts of a pgip mutant of Arabidopsis thaliana (atpgip) resulted in starch overaccumulation, increased CO2 assimilation, up to 19% more plant biomass and 27% seed yield productivity. These results show that manipulating starch metabolic pathways in chloroplasts can improve plant biomass and yield productivity.

Glymphatic Drainage Blocking Aggravates Brain Edema, Neuroinflammation via Modulating TNF-α, IL-10, and AQP4 After Intracerebral Hemorrhage in Rats.

Objective: The "Glymphatic" system, a network of perivascular tunnels wrapped by astrocyte endfeet, was reported to be closely associated with the diseases of the central nervous system. Here, we investigated the role of the glymphatic system in intracerebral hemorrhage (ICH) and its protective mechanism. Method: Experimental ICH model was induced by type IV collagenase in rats. Cerebral lymphatic blockage was induced by ligation and removal of cervical lymph nodes. The experimental rats were divided into sham-operated (SO) group, ICH group, and cerebral lymphatic blocking and ICH (ICH + CLB) group. Neurological scores were measured using the Garcia scoring system on the third and seventh day after ICH. Active caspase-3 was immunostained to evaluate neuronal apoptosis. Brain water content was calculated using the dry-wet specific gravity method. The expression of inflammatory factors TNF-α, IL-1ß, and IL-10 were detected using ELISA. Aquaporins-4 (AQP-4) and glial fibrillary acidic protein (GFAP) were detected using western blot analysis. Results: The neurological scores of rats in the CLB + ICH group were significantly lower than those in the in ICH group. The number of active caspase-3 neurons was significantly higher in the CLB + ICH group compared to the ICH group. CLB significantly aggravated ICH-induced brain edema 3 d after ICH. There was an increase in the expression of TNF-α, IL-1ß, IL-10, AQP-4, GFAP after ICH. The expression of TNF-α was significantly higher in the CLB + ICH group compared to ICH group 3 d after ICH while there was no difference 7 d after ICH. There was no statistical difference in the expression of IL-1ß between the ICH group and CLB + ICH group. However, the expression of IL-10 in the CLB + ICH group was significantly lower than that in the ICH group. Lastly, AQP-4 expression was significantly lower in the CLB + ICH group compared to the ICH group while the expression of GFAP was higher in the CLB + ICH group compared to the ICH group. Conclusion: CLB exacerbated cerebral edema, neuroinflammation, neuronal apoptosis and caused neurological deficits in rats with ICH via down-regulating AQP-4, up-regulating inflammatory TNF-α and inhibiting IL-10 expression. The glymphatic drainage system protects against neurologic injury after ICH induction in rats under normal physiological conditions.

A rare gain of function mutation in a wheat tandem kinase confers resistance to powdery mildew.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most destructive diseases that pose a great threat to wheat production. Wheat landraces represent a rich source of powdery mildew resistance. Here, we report the map-based cloning of powdery mildew resistance gene Pm24 from Chinese wheat landrace Hulutou. It encodes a tandem kinase protein (TKP) with putative kinase-pseudokinase domains, designated WHEAT TANDEM KINASE 3 (WTK3). The resistance function of Pm24 was validated by transgenic assay, independent mutants, and allelic association analyses. Haplotype analysis revealed that a rare 6-bp natural deletion of lysine-glycine codons, endemic to wheat landraces of Shaanxi Province, China, in the kinase I domain (Kin I) of WTK3 is critical for the resistance function. Transgenic assay of WTK3 chimeric variants revealed that only the specific two amino acid deletion, rather than any of the single or more amino acid deletions, in the Kin I of WTK3 is responsible for gaining the resistance function of WTK3 against the Bgt fungus.

Hetero-oligomeric CPN60 resembles highly symmetric group-I chaperonin structure revealed by Cryo-EM.

The chloroplast chaperonin system is indispensable for the biogenesis of Rubisco, the key enzyme in photosynthesis. Using Chlamydomonas reinhardtii as a model system, we found that in vivo the chloroplast chaperonin consists of CPN60α, CPN60ß1 and CPN60ß2 and the co-chaperonin of the three subunits CPN20, CPN11 and CPN23. In Escherichia coli, CPN20 homo-oligomers and all possible other chloroplast co-chaperonin hetero-oligomers are functional, but only that consisting of CPN11/20/23-CPN60αß1ß2 can fully replace GroES/GroEL under stringent stress conditions. Endogenous CPN60 was purified and its stoichiometry was determined to be 6:2:6 for CPN60α:CPN60ß1:CPN60ß2. The cryo-EM structures of endogenous CPN60αß1ß2/ADP and CPN60αß1ß2/co-chaperonin/ADP were solved at resolutions of 4.06 and 3.82 Å, respectively. In both hetero-oligomeric complexes the chaperonin subunits within each ring are highly symmetric. Through hetero-oligomerization, the chloroplast co-chaperonin CPN11/20/23 forms seven GroES-like domains, which symmetrically interact with CPN60αß1ß2. Our structure also reveals an uneven distribution of roof-forming domains in the dome-shaped CPN11/20/23 co-chaperonin and potentially diversified surface properties in the folding cavity of the CPN60αß1ß2 chaperonin that might enable the chloroplast chaperonin system to assist in the folding of specific substrates.

A Novel N-Methyltransferase in Arabidopsis Appears to Feed a Conserved Pathway for Nicotinate Detoxification among Land Plants and Is Associated with Lignin Biosynthesis.

The Preiss-Handler pathway, which salvages nicotinate (NA) for NAD synthesis, is an indispensable biochemical pathway in land plants. Various NA conjugations (mainly methylation and glycosylation) have been detected and have long been proposed for NA detoxification in plants. Previously, we demonstrated that NA O-glucosylation functions as a mobilizable storage form for NAD biosynthesis in the Brassicaceae. However, little is known about the functions of other NA conjugations in plants. In this study, we first found that N-methylnicotinate is a ubiquitous NA conjugation in land plants. Furthermore, we functionally identified a novel methyltransferase (At3g53140; NANMT), which is mainly responsible for N-methylnicotinate formation, from Arabidopsis (Arabidopsis thaliana). We also established that trigonelline is a detoxification form of endogenous NA in plants. Combined phylogenetic analysis and enzymatic assays revealed that NA N-methylation activity was likely derived from the duplication and subfunctionalization of an ancestral caffeic acid O-methyltransferase (COMT) gene in the course of land plant evolution. COMT enzymes, which function in S-lignin biosynthesis, also have weak NANMT activity. Our data suggest that NA detoxification conferred by NANMT and COMT might have facilitated the retention of the Preiss-Handler pathway in land plants.

Two Novel Vesicle-Inducing Proteins in Plastids 1 Genes Cloned and Characterized in Triticum urartu.

Vesicle-inducing protein in plastids 1 (Vipp1) is thought to play an important role both in thylakoid biogenesis and chloroplast envelope maintenance during stress. Vipp1 is conserved in photosynthetic organisms and forms a high homo-oligomer complex structure that may help sustain the membrane integrity of chloroplasts. This study cloned two novel VIPP1 genes from Triticum urartu and named them TuVipp1 and TuVipp2. Both proteins shared high identity with the homologous proteins AtVipp1 and CrVipp1. TuVipp1 and TuVipp2 were expressed in various organs of common wheat, and both genes were induced by light and various stress treatments. Purified TuVipp1 and TuVipp2 proteins showed secondary and advanced structures similar to those of the homologous proteins. Similar to AtVipp1, TuVipp1 is a chloroplast target protein. Additionally, TuVipp1 was able to rescue the phenotypes of pale leaves, lethality, and disordered chloroplast structures of AtVipp1 (-/-) mutant lines. Collectively, our data demonstrate that TuVipp1 and TuVipp2 are functional proteins in chloroplasts in wheat and may be critical for maintaining the chloroplast envelope under stress and membrane biogenesis upon photosynthesis.

Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes.

Substantial efforts are being made to optimize the CRISPR/Cas9 system for precision crop breeding. The avoidance of transgene integration and reduction of off-target mutations are the most important targets for optimization. Here, we describe an efficient genome editing method for bread wheat using CRISPR/Cas9 ribonucleoproteins (RNPs). Starting from RNP preparation, the whole protocol takes only seven to nine weeks, with four to five independent mutants produced from 100 immature wheat embryos. Deep sequencing reveals that the chance of off-target mutations in wheat cells is much lower in RNP mediated genome editing than in editing with CRISPR/Cas9 DNA. Consistent with this finding, no off-target mutations are detected in the mutant plants. Because no foreign DNA is used in CRISPR/Cas9 RNP mediated genome editing, the mutants obtained are completely transgene free. This method may be widely applicable for producing genome edited crop plants and has a good prospect of being commercialized.

Chloroplast Chaperonin: An Intricate Protein Folding Machine for Photosynthesis.

Group I chaperonins are large cylindrical-shaped nano-machines that function as a central hub in the protein quality control system in the bacterial cytosol, mitochondria and chloroplasts. In chloroplasts, proteins newly synthesized by chloroplast ribosomes, unfolded by diverse stresses, or translocated from the cytosol run the risk of aberrant folding and aggregation. The chloroplast chaperonin system assists these proteins in folding into their native states. A widely known protein folded by chloroplast chaperonin is the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), an enzyme responsible for the fixation of inorganic CO2 into organic carbohydrates during photosynthesis. Chloroplast chaperonin was initially identified as a Rubisco-binding protein. All photosynthetic eucaryotes genomes encode multiple chaperonin genes which can be divided into α and ß subtypes. Unlike the homo-oligomeric chaperonins from bacteria and mitochondria, chloroplast chaperonins are more complex and exists as intricate hetero-oligomers containing both subtypes. The Group I chaperonin requires proper interaction with a detachable lid-like co-chaperonin in the presence of ATP and Mg2+ for substrate encapsulation and conformational transition. Besides the typical Cpn10-like co-chaperonin, a unique co-chaperonin consisting of two tandem Cpn10-like domains joined head-to-tail exists in chloroplasts. Since chloroplasts were proposed as sensors to various environmental stresses, this diversified chloroplast chaperonin system has the potential to adapt to complex conditions by accommodating specific substrates or through regulation at both the transcriptional and post-translational levels. In this review, we discuss recent progress on the unique structure and function of the chloroplast chaperonin system based on model organisms Chlamydomonas reinhardtii and Arabidopsis thaliana. Knowledge of the chloroplast chaperonin system may ultimately lead to successful reconstitution of eukaryotic Rubisco in vitro.

Structural insight into the cooperation of chloroplast chaperonin subunits.

BACKGROUND: Chloroplast chaperonin, consisting of multiple subunits, mediates folding of the highly abundant protein Rubisco with the assistance of co-chaperonins. ATP hydrolysis drives the chaperonin allosteric cycle to assist substrate folding and promotes disassembly of chloroplast chaperonin. The ways in which the subunits cooperate during this cycle remain unclear. RESULTS: Here, we report the first crystal structure of Chlamydomonas chloroplast chaperonin homo-oligomer (CPN60ß1) at 3.8 Å, which shares structural topology with typical type I chaperonins but with looser compaction, and possesses a larger central cavity, less contact sites and an enlarged ATP binding pocket compared to GroEL. The overall structure of Cpn60 resembles the GroEL allosteric intermediate state. Moreover, two amino acid (aa) residues (G153, G154) conserved among Cpn60s are involved in ATPase activity regulated by co-chaperonins. Domain swapping analysis revealed that the monomeric state of CPN60α is controlled by its equatorial domain. Furthermore, the C-terminal segment (aa 484-547) of CPN60ß influenced oligomer disassembly and allosteric rearrangement driven by ATP hydrolysis. The entire equatorial domain and at least one part of the intermediate domain from CPN60α are indispensable for functional cooperation with CPN60ß1, and this functional cooperation is strictly dependent on a conserved aa residue (E461) in the CPN60α subunit. CONCLUSIONS: The first crystal structure of Chlamydomonas chloroplast chaperonin homo-oligomer (CPN60ß1) is reported. The equatorial domain maintained the monomeric state of CPN60α and the C-terminus of CPN60ß affected oligomer disassembly driven by ATP. The cooperative roles of CPN60 subunits were also established.

UBIQUITIN-SPECIFIC PROTEASE14 Interacts with ULTRAVIOLET-B INSENSITIVE4 to Regulate Endoreduplication and Cell and Organ Growth in Arabidopsis.

Organ growth is determined by a coordinated combination of cell proliferation and cell growth and differentiation. Endoreduplication is often coupled with cell growth and differentiation, but the genetic and molecular mechanisms that link endoreduplication with cell and organ growth are largely unknown. Here, we describe UBIQUITIN-SPECIFIC PROTEASE14 (UBP14), encoded by the DA3 gene, which functions as a negative regulator of endoreduplication. The Arabidopsis thaliana da3-1 mutant shows large cotyledons, leaves, and flowers with higher ploidy levels. UBP14 acts along with UV-B-INSENSITIVE4 (UVI4), an inhibitor of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase, to repress endoreduplication. Also, UBP14 functions antagonistically with CELL CYCLE SWITCH52 A1 (CCS52A1), an activator of APC/C, to regulate endoreduplication. UBP14 physically associates with UVI4 both in vitro and in vivo but does not directly interact with CCS52A1. Further results reveal that UBP14 influences the stability of cyclin A2;3 (CYCA2;3) and cyclin-dependent kinase B1;1 (CDKB1;1), two downstream components of the APC/C Thus, our findings show how endoreduplication is linked with cell and organ growth by revealing important genetic and molecular functions for the ubiquitin-specific protease UBP14 and for the key cell cycle regulators UVI4, CCS52A1, CYCA2;3, and CDKB1;1.

Asymmetric functional interaction between chaperonin and its plastidic cofactors.

The specific cochaperonin, chloroplast chaperonin (Cpn)20, consisting of two tandem GroES-like domains, is present abundantly in plant and algal chloroplasts, in addition to Cpn10, which is similar in size to GroES. How Cpn20 oligomers, containing six or eight 10-kDa domains, cooperate with the heptameric ring of chaperonin at the same time as encountering symmetry mismatch is unclear. In the present study, we characterized the functional cooperation of cochaperonins, including two plastidic Cpn20 homo-oligomers from Arabidopsis (AtCpn20) and Chlamydomonas (CrCPN20), and one algal CrCPNs hetero-oligomer, consisting of three cochaperonins, CrCPN11, CrCPN20 and CrCPN23, with two chaperonins, Escherichia coli GroEL and Chlamydomonas CrCPN60. AtCpn20 and CrCPNs were functional for assisting both chaperonins in folding model substrates ribulose bisphosphate carboxylase oxygenase from Rhodospirillum rubrum (RrRubisco) in vitro and complementing GroES function in E. coli. CrCPN20 cooperated only with CrCPN60 (and not GroEL) to refold RrRubisco in vitro and showed differential complementation with the two chaperonins in E. coli. Cochaperonin concatamers, consisting of six to eight covalently linked 10-kDa domains, were functionally similar to their respective native forms. Our results indicate that symmetrical match between chaperonin and cochaperonin is not an absolute requisite for functional cooperation.

Structural Analysis of the Rubisco-Assembly Chaperone RbcX-II from Chlamydomonas reinhardtii.

The most prevalent form of the Rubisco enzyme is a complex of eight catalytic large subunits (RbcL) and eight regulatory small subunits (RbcS). Rubisco biogenesis depends on the assistance by specific molecular chaperones. The assembly chaperone RbcX stabilizes the RbcL subunits after folding by chaperonin and mediates their assembly to the RbcL8 core complex, from which RbcX is displaced by RbcS to form active holoenzyme. Two isoforms of RbcX are found in eukaryotes, RbcX-I, which is more closely related to cyanobacterial RbcX, and the more distant RbcX-II. The green algae Chlamydomonas reinhardtii contains only RbcX-II isoforms, CrRbcX-IIa and CrRbcX-IIb. Here we solved the crystal structure of CrRbcX-IIa and show that it forms an arc-shaped dimer with a central hydrophobic cleft for binding the C-terminal sequence of RbcL. Like other RbcX proteins, CrRbcX-IIa supports the assembly of cyanobacterial Rubisco in vitro, albeit with reduced activity relative to cyanobacterial RbcX-I. Structural analysis of a fusion protein of CrRbcX-IIa and the C-terminal peptide of RbcL suggests that the peptide binding mode of RbcX-II may differ from that of cyanobacterial RbcX. RbcX homologs appear to have adapted to their cognate Rubisco clients as a result of co-evolution.

α-Helical Domains Affecting the Oligomerization of Vipp1 and Its Interaction with Hsp70/DnaK in Chlamydomonas.

Vesicle-inducing protein in plastids 1 (Vipp1) forms >1 MDa ordered homo-oligomeric complexes in chloroplasts and is involved in the biogenesis of photosynthetic machinery. The Hsp70 chaperone system has been shown to interact with Vipp1, influencing its higher-order structure. In this study, a series of deletion mutants in Chlamydomonas reinhardii Vipp1 (CrVipp1) is used to investigate the role of the α-helical domains (H1-H7) in mediating its structure and interaction with DnaK, an Hsp70 orthologue. Results from these analyses demonstrate that α-helical domains H1-H6 of CrVipp1 are required for its efficient accumulation in protease-resistant large complexes, termed superoligomers. Deletions of these α-helical domains, either individually or in combination, cause CrVipp1 to assemble into a heterogeneous mixture of smaller, protease-sensitive oligomers. Furthermore, domains H2 and H3 are required to form a stable structural core in mutant oligomers, whereas domains H1 and H4-H6 likely function downstream in assembly of the superoligomer. DnaK binds only weakly to any form of CrVipp1 that efficiently assembles into superoligomers. In contrast, the interaction with DnaK is much more robust with certain misfolded CrVipp1 oligomers in a process mediated by their H4 and H7 domains. DnaK also interacts with full-length CrVipp1 at an early stage of CrVipp1 biosynthesis, perhaps during initial steps in the oligomerization pathway. Taken together, these data suggest that not only α-helical domains but also the oligomeric states of CrVipp1 influence its interaction with DnaK. It is therefore plausible that the Hsp70/DnaK system may be involved in the assembly of Vipp1 superoligomers.

Protomer Roles in Chloroplast Chaperonin Assembly and Function.

The individual roles of three chloroplast CPN60 protomers (CPN60α, CPN60ß1, and CPN60ß2) and whether and how they are assembled into functional chaperonin complexes are investigated in Chlamydomonas reinhardtii. Protein complexes containing all three potential subunits were identified in Chlamydomonas, and their co-expression in Escherichia coli yielded a homogeneous population of oligomers containing all three subunits (CPN60αß1ß2), with a molecular weight consistent with a tetradecameric structure. While homo-oligomers of CPN60ß could form, they were dramatically reduced when CPN60α was present and homo-oligomers of CPN60ß2 were readily changed into hetero-oligomers in the presence of ATP and other protomers. ATP hydrolysis caused CPN60 oligomers to disassemble and drove the purified protomers to reconstitute oligomers in vitro, suggesting that the dynamic nature of CPN60 oligomers is dependent on ATP. Only hetero-oligomeric CPN60αß1ß2, containing CPN60α, CPN60ß1, and CPN60ß2 subunits in a 5:6:3 ratio, cooperated functionally with GroES. The combination of CPN60α and CPN60ß subunits, but not the individual subunits alone, complemented GroEL function in E. coli with subunit recognition specificity. Down-regulation of the CPN60α subunit in Chlamydomonas resulted in a slow growth defect and an inability to grow autotrophically, indicating the essential role of CPN60α in vivo.