Academic literature on the topic 'Eukaryotic prefoldin'

Prefoldin is a hexameric molecular chaperone found in the cytosol of archaea and eukaryotes. Its hexameric complex is built from two related classes of subunits, and has the appearance of a jellyfish: Its body consists of a double β-barrel assembly with six long tentacle-like coiled coils protruding from it. Using the tentacles, prefoldin captures an unfolded protein substrate and transfers it to a group II chaperonin. Based on structural information from archaeal prefoldins, mechanisms of substrate recognition and prefoldin-chaperonin cooperation have been investigated. In contrast, the structure and mechanisms of eukaryotic prefoldins remain unknown. In this study, we succeeded in obtaining recombinant prefoldin from a thermophilic fungus, Chaetomium thermophilum (CtPFD). The recombinant CtPFD could not protect citrate synthase from thermal aggregation. However, CtPFD formed a complex with actin from chicken muscle and tubulin from porcine brain, suggesting substrate specificity. We succeeded in observing the complex formation of CtPFD and the group II chaperonin of C. thermophilum (CtCCT) by atomic force microscopy and electron microscopy. These interaction kinetics were analyzed by surface plasmon resonance using Biacore. Finally, we have shown the transfer of actin from CtPFD to CtCCT. The study of the folding pathway formed by CtPFD and CtCCT should provide important information on mechanisms of the eukaryotic prefoldin–chaperonin system.

Intracellular protein homeostasis is maintained by a network of chaperones that function to fold proteins into their native conformation. The eukaryotic TRiC chaperonin (TCP1-ring complex, also called CCT for cytosolic chaperonin containing TCP1) facilitates folding of a subset of proteins with folding constraints such as complex topologies. To better understand the mechanism of TRiC folding, we investigated the biogenesis of an obligate TRiC substrate, the reovirus σ3 capsid protein. We discovered that the σ3 protein interacts with a network of chaperones, including TRiC and prefoldin. Using a combination of cryoelectron microscopy, cross-linking mass spectrometry, and biochemical approaches, we establish functions for TRiC and prefoldin in folding σ3 and promoting its assembly into higher-order oligomers. These studies illuminate the molecular dynamics of σ3 folding and establish a biological function for TRiC in virus assembly. In addition, our findings provide structural and functional insight into the mechanism by which TRiC and prefoldin participate in the assembly of protein complexes.

Proper protein folding is an essential process for all organisms. Prefoldin (PFD) is a molecular chaperone that assists protein folding by delivering non-native proteins to group II chaperonin. A heterohexamer of eukaryotic PFD has been shown to specifically recognize and deliver non-native actin and tubulin to chaperonin-containing TCP-1 (CCT), but the mechanism of specific recognition is still unclear. To determine its crystal structure, recombinant human PFD was reconstituted, purified and crystallized. X-ray diffraction data were collected to 4.7 Å resolution. The crystals belonged to space groupP21212, with unit-cell parametersa= 123.2,b= 152.4,c= 105.9 Å.

A survey of archaeal genomes for the presence of homologues of bacterial and eukaryotic chaperones reveals several interesting features. All archaea contain chaperonins, also known as Hsp60s (where Hsp is heat-shock protein). These are more similar to the type II chaperonins found in the eukaryotic cytosol than to the type I chaperonins found in bacteria, mitochondria and chloroplasts, although some archaea also contain type I chaperonin homologues, presumably acquired by horizontal gene transfer. Most archaea contain several genes for these proteins. Our studies on the type II chaperonins of the genetically tractable archaeon Haloferax volcanii have shown that only one of the three genes has to be present for the organisms to grow, but that there is some evidence for functional specialization between the different chaperonin proteins. All archaea also possess genes for prefoldin proteins and for small heat-shock proteins, but they generally lack genes for Hsp90 and Hsp100 homologues. Genes for Hsp70 (DnaK) and Hsp40 (DnaJ) homologues are only found in a subset of archaea. Thus chaperone-assisted protein folding in archaea is likely to display some unique features when compared with that in eukaryotes and bacteria, and there may be important differences in the process between euryarchaea and crenarchaea.

Molecular chaperones are a class of proteins responsible for proper folding of a large number of polypeptides in both prokaryotic and eukaryotic cells. Newly synthesized polypeptides are prone to nonspecific interactions, and many of them make toxic aggregates in absence of chaperones. The eukaryotic chaperonin CCT is a large, multisubunit, cylindrical structure having two identical rings stacked back to back. Each ring is composed of eight different but similar subunits and each subunit has three distinct domains. CCT assists folding of actin, tubulin, and numerous other cellular proteins in an ATP-dependent manner. The catalytic cooperativity of ATP binding/hydrolysis in CCT occurs in a sequential manner different from concerted cooperativity as shown for GroEL. Unlike GroEL, CCT does not have GroES-like cofactor, rather it has a built-in lid structure responsible for closing the central cavity. The CCT complex recognizes its substrates through diverse mechanisms involving hydrophobic or electrostatic interactions. Upstream factors like Hsp70 and Hsp90 also work in a concerted manner to transfer the substrate to CCT. Moreover, prefoldin, phosducin-like proteins, and Bag3 protein interact with CCT and modulate its function for the fine-tuning of protein folding process. Any misregulation of protein folding process leads to the formation of misfolded proteins or toxic aggregates which are linked to multiple pathological disorders.

Prefoldin is a hexameric protein complex ubiquitously expressed and found to influence the conformation of amyloidogenic peptides. Relatively high degrees of sequence identity and conservation across evolutionary lineages are observed, however differences in binding abilities have been noted between the homologs. This thesis describes work examining the structure of eukaryotic prefoldin and its biological activities with respect to interaction with amyloid β. The structure and biological activities of prefoldin’s individual subunits are also explored. Although many studies have investigated the structure of prokaryotic prefoldin, there is limited information available for eukaryotic prefoldin. Two-dimensional ¹H-¹H and ¹H-¹³C nuclear magnetic resonance (NMR) spectroscopy was utilised to probe the structure of both α and β human prefoldin subunits. The data revealed the highly alpha helical secondary structure of the subunits, which was further verified through far-UV circular dichroism. Further thermal aggregation assays utilising this technique have demonstrated the stability of the prefoldin subunits. The biological effect of prefoldin on the amyloid fibril formation of the Alzheimer’s disease related amyloid β peptide was investigated using a combination of dye-binding assays and cytotoxicity assays. The presence and absence of fibrils was confirmed by transmission electron microscopy. In terms of fibril formation, prefoldin and its subunits prevented in vitro conversion of the amyloid β peptide to amyloid fibrils. In some cases, total inhibition of fibril formation occurred and a 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted on the resultant products. The product was incubated with healthy PC-12 cells and induced cellular death, therefore establishing the cytotoxicity of the resultant oligomeric amyloid β form. Previous investigations into the binding capabilities of prokaryotic prefoldin identified the distal tips as an important structural aspect, interacting with the amyloidogenic peptide. The binding interface of prefoldin subunits 5 and 6 with amyloid β was probed using chemical cross-linking (CXL) experiments. Traditional methods to identify cross-linked peptides are challenging and the results are often ambiguous. In this study, CXL products were analysed by liquid chromatography-ion mobility-mass spectrometry (LC-IM-MS) to investigate the utility of IM in enhancing the CXL analytical workflow. The orthogonal separation of ion mobility enabled the identification of the cross-linked amino acids. The distal end of prefoldin subunit 5 was found to interact with the Nterminus of the amyloid peptide, whereas prefoldin subunit 6 was identified to interact with the peptide in the middle of its sequence. Ion mobility-mass spectrometry (IM-MS) analysis of the eukaryotic prefoldin complex identified the collisional cross section of the intact hexamer. Solution disruption experiments of the intact complex revealed the disengaging sub-complexes, and information on the intersubunit contacts and relative interfacial strengths were obtained. A capillary temperature controller (CTC) was developed to observe the thermal dissociation of the complex using nano-electrospray IM-MS. The combination of these results confirmed a structural aspect common to both mammalian prefoldin and prokaryotic prefoldin, despite the primary sequence differences. The biological assays revealed the ability of prefoldin to prevent the aggregation and amyloid fibril formation of amyloid β, and low resolution MS techniques were able to postulate the arrangement of the subunits and the possible interface interactions of the hexameric complex with the amyloidogenic peptide. This thesis has therefore provided an in-depth investigation of the structural characteristics of eukaryotic prefoldin and its chaperoning capability, therefore implicating a potential role for prefoldin in modulating protein misfolding and aggregation.
Thesis (Ph.D.) -- University of Adelaide, School of Physical Sciences, 2018