Prefoldins are novel regulators of the unfolded protein response in artemisinin resistant Plasmodium falciparum malaria.

Emerging Artemisinin (ART) resistance in Plasmodium falciparum (Pf) poses challenges for the discovery of novel drugs to tackle ART-resistant parasites. Concentrated efforts toward the ART resistance mechanism indicated a strong molecular link of ART resistance with upregulated expression of unfolded protein response pathways involving Prefoldins (PFDs). However, a complete characterization of PFDs as molecular players taking part in ART resistance mechanism, and discovery of small molecule inhibitors to block this process have not been identified to date. Here, we functionally characterized all Pf Prefoldin subunits (PFD1-6) and established a causative role played by PFDs in ART resistance by demonstrating their expression in intra-erythrocytic parasites along with their interactions with Kelch13 protein through immunoprecipitation coupled MS/MS analysis. Systematic biophysical interaction analysis between all subunits of PFDs revealed their potential to form a complex. The role of PFDs in ART resistance was confirmed in orthologous yeast PFD6 mutants, where PfPFD6 expression in yeast mutants reverted phenotype to ART resistance. We identified an FDA-approved drug "Biperiden" that restricts the formation of Prefoldin complex and inhibits its interaction with its key parasite protein substrates, MSP-1 and α-tubulin-I. Moreover, Biperiden treatment inhibits the parasite growth in ART-sensitive Pf3D7 and resistant Pf3D7k13R539T strains. Ring survival assays that are clinically relevant to analyze ART resistance in Pf3D7k13R539T parasites demonstrate the potency of BPD to inhibit the growth of survivor parasites. Overall, our study provides the first evidence of the role of PfPFDs in ART resistance mechanisms and opens new avenues for the management of resistant parasites.

STORM Super-Resolution Visualization of Self-Assembled γPFD Chaperone Ultrastructures in Methanocaldococcus jannaschii.

Gamma-prefoldin (γPFD), a unique chaperone found in the extremely thermophilic methanogen Methanocaldococcus jannaschii, self-assembles into filaments in vitro, which so far have been observed using transmission electron microscopy and cryo-electron microscopy. Utilizing three-dimensional stochastic optical reconstruction microscopy (3D-STORM), here we achieve ∼20 nm resolution by precisely locating individual fluorescent molecules, hence resolving γPFD ultrastructure both in vitro and in vivo. Through CF647 NHS ester labeling, we first demonstrate the accurate visualization of filaments and bundles with purified γPFD. Next, by implementing immunofluorescence labeling after creating a 3xFLAG-tagged γPFD strain, we successfully visualize γPFD in M. jannaschii cells. Through 3D-STORM and two-color STORM imaging with DNA, we show the widespread distribution of filamentous γPFD structures within the cell. These findings provide valuable insights into the structure and localization of γPFD, opening up possibilities for studying intriguing nanoscale components not only in archaea but also in other microorganisms.

Model of the external force field for the protein folding process-the role of prefoldin.

Introduction: The protein folding process is very sensitive to environmental conditions. Many possibilities in the form of numerous pathways for this process can-if an incorrect one is chosen-lead to the creation of forms described as misfolded. The aqueous environment is the natural one for the protein folding process. Nonetheless, other factors such as the cell membrane and the presence of specific molecules (chaperones) affect this process, ensuring the correct expected structural form to guarantee biological activity. All these factors can be considered components of the external force field for this process. Methods: The fuzzy oil drop-modified (FOD-M) model makes possible the quantitative evaluation of the modification of the external field, treating the aqueous environment as a reference. The FOD-M model (tested on membrane proteins) includes the component modifying the water environment, allowing the assessment of the external force field generated by prefoldin. Results: In this work, prefoldin was treated as the provider of a specific external force field for actin and tubulin. The discussed model can be applied to any folding process simulation, taking into account the changed external conditions. Hence, it can help simulate the in silico protein folding process under defined external conditions determined by the respective external force field. In this work, the structures of prefoldin and protein folded with the participation of prefoldin were analyzed. Discussion: Thus, the role of prefoldin can be treated as a provider of an external field comparable to other environmental factors affecting the protein folding process.

Prefoldin Subunits and Its Associate Partners: Conservations and Specificities in Plants.

Prefoldins (PFDs) are ubiquitous co-chaperone proteins that originated in archaea during evolution and are present in all eukaryotes, including yeast, mammals, and plants. Typically, prefoldin subunits form hexameric PFD complex (PFDc) that, together with class II chaperonins, mediate the folding of nascent proteins, such as actin and tubulin. In addition to functioning as a co-chaperone in cytoplasm, prefoldin subunits are also localized in the nucleus, which is essential for transcription and post-transcription regulation. However, the specific and critical roles of prefoldins in plants have not been well summarized. In this review, we present an overview of plant prefoldin and its related proteins, summarize the structure of prefoldin/prefoldin-like complex (PFD/PFDLc), and analyze the versatile landscape by prefoldin subunits, from cytoplasm to nucleus regulation. We also focus the specific role of prefoldin-mediated phytohormone response and global plant development. Finally, we overview the emerging prefoldin-like (PFDL) subunits in plants and the novel roles in related processes, and discuss the next direction in further studies.

Prefoldin-5 Expression Is Elevated in Eutopic and Ectopic Endometriotic Epithelium and Modulates Endometriotic Epithelial Cell Proliferation and Migration In Vitro.

Endometriosis is a common disease among women of reproductive age in which endometrial tissue grows in ectopic localizations, primarily within the pelvic cavity. These ectopic "lesions" grow as well as migrate and invade underlying tissues. Despite the prevalence of the disease, an understanding of factors that contribute to these cellular attributes remains poorly understood. Prefoldin-5 (PFDN5) has been associated with both aberrant cell proliferation and migration, but a potential role in endometriosis is unknown. As such, the purpose of this study was to examine PFDN5 expression in endometriotic tissue. PFDN5 mRNA and protein were examined in ectopic (lesion) and eutopic endometrial tissue from women with endometriosis and in eutopic endometrium from those without endometriosis using qRT-PCR and immunohistochemistry, respectively, while function of PFDN5 in vitro was evaluated using cell count and migration assays. PFDN5 mRNA and protein were expressed in eutopic and ectopic endometrial tissue, predominantly in the glandular epithelium, but not in endometrium from control subjects. Expression of both mRNA and protein was variable among endometriotic eutopic and ectopic endometrial tissue but showed an overall net increase. Knockdown of PFDN5 by siRNA transfection of endometriotic epithelial 12Z cells was associated with reduced cell proliferation/survival and migration. PFDN5 is expressed in eutopic and ectopic glandular epithelium and may play a role in proliferation and migration of these cells contributing to disease pathophysiology.

Lysine demethylase 5C inhibits transcription of prefoldin subunit 5 to activate c-Myc signal transduction and colorectal cancer progression.

BACKGROUND: Lysine demethylase 5C (KDM5C) has been implicated in the development of several human cancers. This study aims to investigate the role of KDM5C in the progression of colorectal cancer (CRC) and explore the associated molecular mechanism. METHODS: Bioinformatics tools were employed to predict the target genes of KDM5C in CRC. The expression levels of KDM5C and prefoldin subunit 5 (PFDN5) in CRC cells were determined by RT-qPCR and western blot assays. The interaction between KDM5C, H3K4me3, and PFDN5 was validated by chromatin immunoprecipitation. Expression and prognostic values of KDM5C and PFDN5 in CRC were analyzed in a cohort of 72 patients. The function of KDM5C/PFDN5 in c-Myc signal transduction was analyzed by luciferase assay. Silencing of KDM5C and PFDN5 was induced in CRC cell lines to analyze the cell malignant phenotype in vitro and tumorigenic activity in nude mice. RESULTS: KDM5C exhibited high expression, while PFDN5 displayed low expression in CRC cells and clinical CRC samples. High KDM5C levels correlated with poor survival and unfavorable clinical presentation, whereas elevated PFDN5 correlated with improved patient outcomes. KDM5C mediated demethylation of H3K4me3 on the PFDN5 promoter, suppressing its transcription and thereby enhancing the transcriptional activity of c-Myc. KDM5C knockdown in CRC cells suppressed cell proliferation, migration and invasion, epithelial-mesenchymal transition, and tumorigenic activity while increasing autophagy and apoptosis rates. However, the malignant behavior of cells was restored by the further silencing of PFDN5. CONCLUSION: This study demonstrates that KDM5C inhibits PFDN5 transcription, thereby activating c-Myc signal transduction and promoting CRC progression.

The association of the RSC remodeler complex with chromatin is influenced by the prefoldin-like Bud27 and determines nucleosome positioning and polyadenylation sites usage in Saccharomyces cerevisiae.

The tripartite interaction between the chromatin remodeler complex RSC, RNA polymerase subunit Rpb5 and prefoldin-like Bud27 is necessary for proper RNA pol II elongation. Indeed lack of Bud27 alters this association and affects transcription elongation. This work investigates the consequences of lack of Bud27 on the chromatin association of RSC and RNA pol II, and on nucleosome positioning. Our results demonstrate that RSC binds chromatin in gene bodies and lack of Bud27 alters this association, mainly around polyA sites. This alteration impacts chromatin organization and leads to the accumulation of RNA pol II molecules around polyA sites, likely due to pausing or arrest. Our data suggest that RSC is necessary to maintain chromatin organization around those sites, and any alteration of this organization results in the widespread use of alternative polyA sites. Finally, we also find a similar molecular phenotype that occurs upon TOR inhibition with rapamycin, which suggests that alternative polyadenylation observed upon TOR inhibition is likely Bud27-dependent.

Putative prefoldin complex subunit 5 of Plasmodium berghei is crucial for microtubule formation and parasite development in the mosquito.

Plasmodium sporozoite development in and egress from oocysts in the Anopheles mosquito remains largely enigmatic. In a previously performed high-throughput knockout screen, the putative subunit 5 of the prefoldin complex (PbPCS5, PBANKA_0920100) was identified as essential for parasite development during mosquito and liver stage development. Here we generated and analyzed a PbPCS5 knockout parasite line during its development in the mosquito. Interestingly, PbPCS5 deletion does not significantly affect oocyst formation but leads to a growth defect resulting in aberrantly shaped sporozoites. Sporozoites produced in the absence of PbPCS5 were thinner, markedly elongated, and did, in most cases, not contain a nucleus. Sporozoites contained fewer subpellicular microtubules, which reached deep into the sporoblast during sporogony where they contacted and indented nuclei. These aberrantly shaped sporozoites did not reach the salivary glands, and we, therefore, conclude that PbPCS5 is essential for sporogony and the life cycle progression of the parasite during its mosquito stage.

Prefoldin 2 contributes to mitochondrial morphology and function.

BACKGROUND: Prefoldin is an evolutionarily conserved co-chaperone of the tailless complex polypeptide 1 ring complex (TRiC)/chaperonin containing tailless complex 1 (CCT). The prefoldin complex consists of six subunits that are known to transfer newly produced cytoskeletal proteins to TRiC/CCT for folding polypeptides. Prefoldin function was recently linked to the maintenance of protein homeostasis, suggesting a more general function of the co-chaperone during cellular stress conditions. Prefoldin acts in an adenosine triphosphate (ATP)-independent manner, making it a suitable candidate to operate during stress conditions, such as mitochondrial dysfunction. Mitochondrial function depends on the production of mitochondrial proteins in the cytosol. Mechanisms that sustain cytosolic protein homeostasis are vital for the quality control of proteins destined for the organelle and such mechanisms among others include chaperones. RESULTS: We analyzed consequences of the loss of prefoldin subunits on the cell proliferation and survival of Saccharomyces cerevisiae upon exposure to various cellular stress conditions. We found that prefoldin subunits support cell growth under heat stress. Moreover, prefoldin facilitates the growth of cells under respiratory growth conditions. We showed that mitochondrial morphology and abundance of some respiratory chain complexes was supported by the prefoldin 2 (Pfd2/Gim4) subunit. We also found that Pfd2 interacts with Tom70, a receptor of mitochondrial precursor proteins that are targeted into mitochondria. CONCLUSIONS: Our findings link the cytosolic prefoldin complex to mitochondrial function. Loss of the prefoldin complex subunit Pfd2 results in adaptive cellular responses on the proteome level under physiological conditions suggesting a continuous need of Pfd2 for maintenance of cellular homeostasis. Within this framework, Pfd2 might support mitochondrial function directly as part of the cytosolic quality control system of mitochondrial proteins or indirectly as a component of the protein homeostasis network.

A structural vista of phosducin-like PhLP2A-chaperonin TRiC cooperation during the ATP-driven folding cycle.

Proper cellular proteostasis, essential for viability, requires a network of chaperones and cochaperones. ATP-dependent chaperonin TRiC/CCT partners with cochaperones prefoldin (PFD) and phosducin-like proteins (PhLPs) to facilitate the folding of essential eukaryotic proteins. Using cryoEM and biochemical analyses, we determine the ATP-driven cycle of TRiC-PFD-PhLP2A interaction. In the open TRiC state, PhLP2A binds to the chamber's equator while its N-terminal H3-domain binds to the apical domains of CCT3/4, thereby displacing PFD from TRiC. ATP-induced TRiC closure rearranges the contacts of PhLP2A domains within the closed chamber. In the presence of substrate, actin and PhLP2A segregate into opposing chambers, each binding to the positively charged inner surfaces formed by CCT1/3/6/8. Notably, actin induces a conformational change in PhLP2A, causing its N-terminal helices to extend across the inter-ring interface to directly contact a hydrophobic groove in actin. Our findings reveal an ATP-driven PhLP2A structural rearrangement cycle within the TRiC chamber to facilitate folding.

Horseradish Peroxidase Catalyzed Silk-Prefoldin Composite Hydrogel Networks.

Protein-based hydrogel biomaterials provide a platform for different biological applications, including the encapsulation and stabilization of different biomolecules. These hydrogel properties can be modulated by controlling the design parameters to match specific needs; thus, multicomponent hydrogels have distinct advantages over single-component hydrogels due to their enhanced versatility. Here, silk fibroin and γ-prefoldin chaperone protein based composite hydrogels were prepared and studied. Different ratios of the proteins were chosen, and the hydrogels were prepared by enzyme-assisted cross-linking. The secondary structure of the two proteins, dityrosine bond formation, and mechanical properties were assessed. The results obtained can be used as a platform for the rational design of composite thermostable hydrogel biomaterials to facilitate protection (due to hydrogel mechanics) and retention of bioactivity (e.g., of enzymes and other biomolecules) due to chaperone-like properties of γ-prefoldin.

Structural visualization of the tubulin folding pathway directed by human chaperonin TRiC/CCT.

The ATP-dependent ring-shaped chaperonin TRiC/CCT is essential for cellular proteostasis. To uncover why some eukaryotic proteins can only fold with TRiC assistance, we reconstituted the folding of ß-tubulin using human prefoldin and TRiC. We find unstructured ß-tubulin is delivered by prefoldin to the open TRiC chamber followed by ATP-dependent chamber closure. Cryo-EM resolves four near-atomic-resolution structures containing progressively folded ß-tubulin intermediates within the closed TRiC chamber, culminating in native tubulin. This substrate folding pathway appears closely guided by site-specific interactions with conserved regions in the TRiC chamber. Initial electrostatic interactions between the TRiC interior wall and both the folded tubulin N domain and its C-terminal E-hook tail establish the native substrate topology, thus enabling C-domain folding. Intrinsically disordered CCT C termini within the chamber promote subsequent folding of tubulin's core and middle domains and GTP-binding. Thus, TRiC's chamber provides chemical and topological directives that shape the folding landscape of its obligate substrates.

The unconventional prefoldin RPB5 interactor mediates the gravitropic response by modulating cytoskeleton organization and auxin transport in Arabidopsis.

Gravity-induced root curvature involves the asymmetric distribution of the phytohormone auxin. This response depends on the concerted activities of the auxin transporters such as PIN-FORMED (PIN) proteins for auxin efflux and AUXIN RESISTANT 1 (AUX1) for auxin influx. However, how the auxin gradient is established remains elusive. Here we identified a new mutant with a short root, strong auxin distribution in the lateral root cap and an impaired gravitropic response. The causal gene encoded an Arabidopsis homolog of the human unconventional prefoldin RPB5 interactor (URI). AtURI interacted with prefoldin 2 (PFD2) and PFD6, two ß-type PFD members that modulate actin and tubulin patterning in roots. The auxin reporter DR5rev :GFP showed that asymmetric auxin redistribution after gravistimulation is disordered in aturi-1 root tips. Treatment with the endomembrane protein trafficking inhibitor brefeldin A indicated that recycling of the auxin transporter PIN2 is disrupted in aturi-1 roots as well as in pfd mutants. We propose that AtURI cooperates with PFDs to recycle PIN2 and modulate auxin distribution.

A distinct P-body-like granule is induced in response to the disruption of microtubule integrity in Saccharomyces cerevisiae.

The Processing-body is a conserved membraneless organelle that has been implicated in the storage and/or decay of mRNAs. Although Processing-bodies have been shown to be induced by a variety of conditions, the mechanisms controlling their assembly and their precise physiological roles in eukaryotic cells are still being worked out. In this study, we find that a distinct subtype of Processing-body is induced in response to conditions that disrupt microtubule integrity in the budding yeast, Saccharomyces cerevisiae. For example, treatment with the microtubule-destabilizing agent, benomyl, led to the induction of these novel ribonucleoprotein granules. A link to microtubules had been noted previously and the observations here extend our understanding by demonstrating that the induced foci differ from traditional P-bodies in a number of significant ways. These include differences in overall granule morphology, protein composition, and the manner in which their induction is regulated. Of particular note, several key Processing-body constituents are absent from these benomyl-induced granules, including the Pat1 protein that is normally required for efficient Processing-body assembly. However, these novel ribonucleoprotein structures still contain many known Processing-body proteins and exhibit similar hallmarks of a liquid-like compartment. In all, the data suggest that the disruption of microtubule integrity leads to the formation of a novel type of Processing-body granule that may have distinct biological activities in the cell. Future work will aim to identify the biological activities of these benomyl-induced granules and to determine, in turn, whether these Processing-body-like granules have any role in the regulation of microtubule dynamics.

A High-Copy Suppressor Screen Reveals a Broad Role of Prefoldin-like Bud27 in the TOR Signaling Pathway in Saccharomyces cerevisiae.

Bud27 is a prefoldin-like, a member of the family of ATP-independent molecular chaperones that associates with RNA polymerases I, II, and III in Saccharomyces cerevisiae. Bud27 and its human ortholog URI perform several functions in the cytoplasm and the nucleus. Both proteins participate in the TOR signaling cascade by coordinating nutrient availability with gene expression, and lack of Bud27 partially mimics TOR pathway inactivation. Bud27 regulates the transcription of the three RNA polymerases to mediate the synthesis of ribosomal components for ribosome biogenesis through the TOR cascade. This work presents a high-copy suppression screening of the temperature sensitivity of the bud27Δ mutant. It shows that Bud27 influences different TOR-dependent processes. Our data also suggest that Bud27 can impact some of these TOR-dependent processes: cell wall integrity and autophagy induction.

Prefoldin and prefoldin-like complex subunits as predictive biomarkers for hepatocellular carcinoma immunotherapy.

BACKGROUND: Prefoldin complex subunits (PFDNs) and prefoldin-like complex subunits (PFDLNs) collaborate in protein folding, modulate endoplasmic reticulum stress. The association between PFDN/PFDLN and the immune microenvironment of HCC remains unclear. We investigated the biological significance of PFDNs and PFDLNs in HCC using bioinformatics. METHODS: The relationship between PFDNs/PFDLNs and HCC was analysed using TCGA, and Human Protein Atlas. The protein-protein interaction (PPI) network was performed through String and Cytoscape. In addition, mutations in PFDNs and PFDLNs were analysed using cBioPortal. Clinical correlation analysis, survival analysis was conducted by using UALCAN and Kaplan-Meier analysis. The protein-protein interaction (PPI) network was performed through String and Cytoscape. The GO and KEGG enrichment analyses were also carried out. CCK-8 and Flow cytometry analysis were used to detect the proliferation and apoptosis of PFDN1 and UXT knockdown HCC cells. Immune infiltrates analyses was were conducted using the TIMER and TISIDB to determine whether PFDNs/PFDLNs are predictive biomarkers of immune cell infiltration. RESULTS: We observed that PFDNs and PFDLNs were significantly overexpressed in HCC tissues compared to normal liver tissues. This abnormal expression was associated with worse clinicopathological features and negatively affected patient survival. PFDNs and PFDLNs have varying degrees of mutations in HCC, which may be related to their abnormal expression. In addition, up-regulated PFDN1 and UXT were found to promote HCC proliferation and inhibit apoptosis in vitro. Finally, the expression of certain PFDNs and PFDLNs in the tumour microenvironment was positively correlated with the level of tumour-infiltrating immune cells and significantly enhanced the infiltration of immune cells in the microenvironment. CONCLUSIONS: PFDNs and PFDLNs are valuable predictive biomarkers for immune infiltration in HCC and may assist in tumour immunotherapy research and prognosis prediction in the future.

Comparative structural insight into prefoldin subunints of archaea and eukaryotes with special emphasis on unexplored prefoldin of Plasmodium falciparum.

Prefoldin (PFD) is a heterohexameric molecular chaperone which bind unfolded proteins and subsequently deliver them to a group II chaperonin for correct folding. Although there is structural and functional information available for humans and archaea PFDs, their existence and functions in malaria parasite remains uncharacterized. In the present review, we have collected the available information on prefoldin family members of archaea and humans and attempted to analyze unexplored PFD subunits of Plasmodium falciparum (Pf). Our review enhances the understanding of probable functions, structure and mechanism of substrate binding of Pf prefoldin by comparing with the available information of its homologs in archaea and H. sapiens. Three PfPFD out of six and a Pf prefoldin-like protein are reported to be essential for parasite survival that signifies their importance in malaria parasite biology. Transcriptome analyses suggest that PfPFD subunits are up-regulated at the mRNA level during asexual and sexual stages of parasite life cycle. Our in silico analysis suggested several pivotal proteins like myosin E, cytoskeletal protein (tubulin), merozoite surface protein and ring exported protein 3 as their interacting partners. Based on structural information of archaeal and H. sapiens PFDs, P. falciparum counterparts have been modelled and key interface residues were identified that are critical for oligomerization of PfPFD subunits. We collated information on PFD-substrate binding and PFD-chaperonin interaction in detail to understand the mechanism of substrate delivery in archaea and humans. Overall, our review enables readers to view the PFD family comprehensively. Communicated by Ramaswamy H. SarmaAbbreviations: HSP: Heat shock proteins; CCT: Chaperonin containing TCP-1; PFD: Prefoldin; PFLP: Prefoldin like protein; PfPFD: Plasmodium falciparum prefoldin; Pf: Plasmodium falciparum; H. sapiens: Homo sapiens; M. thermoautotrophicus: Methanobacterium thermoautotrophicus; P. horikoshii: Pyrococcus horikoshii.

Prefoldin subunit 6 of Plasmodium falciparum binds merozoite surface protein-1.

Malaria is a human disease caused by eukaryotic protozoan parasites of the Plasmodium genus. Plasmodium falciparum (Pf) causes the most lethal form of human malaria and is responsible for widespread mortality worldwide. Prefoldin is a heterohexameric molecular complex that binds and delivers unfolded proteins to chaperonin for correct folding. The prefoldin PFD6 is predicted to interact with merozoite surface protein-1 (MSP-1), a protein well known to play a pivotal role in erythrocyte binding and invasion by Plasmodium merozoites. We previously found that the P. falciparum (Pf) genome contains six prefoldin genes and a prefoldin-like gene whose molecular functions are unidentified. Here, we analyzed the expression of PfPFD-6 during the asexual blood stages of the parasite and investigated its interacting partners. PfPFD-6 was found to be significantly expressed at the trophozoite and schizont stages. Pull-down assays suggest PfPFD-6 interacts with MSP-1. In silico analysis suggested critical residues involved in the PfPFD-6-MSP-1 interaction. Our data suggest PfPFD-6 may play a role in stabilizing or trafficking MSP-1.

Influence of prefoldin subunit 4 on the tolerance of Kluyveromyces marxianus to lignocellulosic biomass-derived inhibitors.

BACKGROUND: Kluyveromyces marxianus is a potentially excellent host for microbial cell factories using lignocellulosic biomass, due to its thermotolerance, high growth rate, and wide substrate spectrum. However, its tolerance to inhibitors derived from lignocellulosic biomass pretreatment needs to be improved. The prefoldin complex assists the folding of cytoskeleton which relates to the stress tolerance, moreover, several subunits of prefoldin have been verified to be involved in gene expression regulation. With the presence of inhibitors, the expression of a gene coding the subunit 4 of prefoldin (KmPFD4), a possible transcription factor, was significantly changed. Therefore, KmPFD4 was selected to evaluate its functions in inhibitors tolerance. RESULTS: In this study, the disruption of the prefoldin subunit 4 gene (KmPFD4) led to increased concentration of intracellular reactive oxygen species (ROS) and disturbed the assembly of actin and tubulin in the presence of inhibitors, resulting in reduced inhibitor tolerance. Nuclear localization of KmPFD4 indicated that it could regulate gene expression. Transcriptomic analysis showed that upregulated gene expression related to ROS elimination, ATP production, and NAD+ synthesis, which is a response to the presence of inhibitors, disappeared in KmPFD4-disrupted cells. Thus, KmPFD4 impacts inhibitor tolerance by maintaining integration of the cytoskeleton and directly or indirectly affecting the expression of genes in response to inhibitors. Finally, overexpression of KmPFD4 enhanced ethanol fermentation with a 46.27% improvement in productivity in presence of the inhibitors. CONCLUSION: This study demonstrated that KmPFD4 plays a positive role in the inhibitor tolerance and can be applied for the development of inhibitor-tolerant platform strains.

Improved folding of recombinant protein via co-expression of exogenous chaperones.

Expression of heterologous genes in Escherichia coli is a routine technology for recombinant protein production, but the predictable recovery of properly folded and uniformly bioactive material remains a challenge. Misfolded proteins typically accumulate as insoluble inclusion bodies, and a variety of strategies have been employed in efforts to increase the yield of soluble product. One technique is the overexpression of E. coli protein chaperones during recombinant protein induction, in an effort to increase the folding capacity of the bacterial host. We have developed an alternative approach, by supplementing the host protein folding machinery with chaperones from other species. Extremophiles have evolved under conditions (extremes of temperature, salinity, pressure, and/or pH) that make them attractive candidates for possessing chaperones with novel folding activities. The green fluorescent protein (GFP) of Aequorea victoria, which is predominantly insoluble under typical recombinant expression culture conditions, was employed as an in vivo indicator of protein folding activity for chaperone homologs from a variety of extremophiles. For a subset of the chaperones tested, co-expression with GFP promoted an increase in both fluorescence signal intensity as well as the amount of GFP recovered in the soluble protein fraction. Several archaeal chaperones were also found to be able to refold soluble Lyt_Orn C40 peptidase from inclusion bodies in vitro. In particular, Pf Cpn(MA), a mutant chaperonin which exhibited significant refolding activity, is also shown to deconstruct the morphology and structure of inclusion bodies (Kurouski et al., 2012). Hence, the simple and rapid GFP assay provides a tool to screen for extremophilic chaperones that exhibit folding activity under E. coli growth conditions, and suggests that increasing the repertoire of heterologous chaperones might provide a partial but general solution to the problem of recombinant protein insolubility.