UPS-dependent strategies of protein quality control degradation.

The degradation of damaged proteins is critical for tissue integrity and organismal health because damaged proteins have a high propensity to form aggregates. E3 ubiquitin ligases are key regulators of protein quality control (PQC) and mediate the selective degradation of damaged proteins, a process termed 'PQC degradation' (PQCD). The degradation signals (degrons) that trigger PQCD are based on hydrophobic sites that are normally buried within the native protein structure. However, an open question is how PQCD-specialized E3 ligases distinguish between transiently misfolded proteins, which can be efficiently refolded, and permanently damaged proteins, which must be degraded. While significant progress has been made in characterizing degradation determinants, understanding the key regulatory signals of cellular and organismal PQCD pathways remains a challenge.

Grp78 destabilization of infectious prions is strain-specific and modified by multiple factors including accessory chaperones and pH.

Lethal neurodegenerative prion diseases result from the continuous accumulation of infectious and variably protease-resistant prion protein aggregates (PrPD) which are misfolded forms of the normally detergent soluble and protease-sensitive cellular prion protein. Molecular chaperones like Grp78 have been found to reduce the accumulation of PrPD, but how different cellular environments and other chaperones influence the ability of Grp78 to modify PrPD is poorly understood. In this work, we investigated how pH and protease-mediated structural changes in PrPD from two mouse-adapted scrapie prion strains, 22L and 87V, influenced processing by Grp78 in the presence or absence of chaperones Hsp90, DnaJC1, and Stip1. We developed a cell-free in vitro system to monitor chaperone-mediated structural changes to, and disaggregation of, PrPD. For both strains, Grp78 was most effective at structurally altering PrPD at low pH, especially when additional chaperones were present. While Grp78, DnaJC1, Stip1, and Hsp90 were unable to disaggregate the majority of PrPD from either strain, pretreatment of PrPD with proteases increased disaggregation of 22L PrPD compared to 87V, indicating strain-specific differences in aggregate structure were impacting chaperone activity. Hsp90 also induced structural changes in 87V PrPD as indicated by an increase in the susceptibility of its n-terminus to proteases. Our data suggest that, while chaperones like Grp78, DnaJC1, Stip1, and Hsp90 disaggregate only a small fraction of PrPD, they may still facilitate its clearance by altering aggregate structure and sensitizing PrPD to proteases in a strain and pH-dependent manner.

Plastid HSP90C C-terminal extension region plays a regulatory role in chaperone activity and client binding.

HSP90Cs are essential molecular chaperones localized in the plastid stroma that maintain protein homeostasis and assist the import and thylakoid transport of chloroplast proteins. While HSP90C contains all conserved domains as an HSP90 family protein, it also possesses a unique feature in its variable C-terminal extension (CTE) region. This study elucidated the specific function of this HSP90C CTE region. Our phylogenetic analyses revealed that this intrinsically disordered region contains a highly conserved DPW motif in the green lineages. With biochemical assays, we showed that the CTE is required for the chaperone to effectively interact with client proteins PsbO1 and LHCB2 to regulate ATP-independent chaperone activity and to effectuate its ATP hydrolysis. The CTE truncation mutants could support plant growth and development reminiscing the wild type under normal conditions except for a minor phenotype in cotyledon when expressed at a level comparable to wild type. However, higher HSP90C expression was observed to correlate with a stronger response to specific photosystem II inhibitor DCMU, and CTE truncations dampened the response. Additionally, when treated with lincomycin to inhibit chloroplast protein translation, CTE truncation mutants showed a delayed response to PsbO1 expression repression, suggesting its role in chloroplast retrograde signaling. Our study therefore provides insights into the mechanism of HSP90C in client protein binding and the regulation of green chloroplast maturation and function, especially under stress conditions.

Invited review: Modeling milk stability.

Novel insights into the stability of milk and milk products during storage and processing result from describing caseins near neutral pH as hydrophilic, intrinsically disordered, proteins. Casein solubility is strongly influenced by pH and multivalent ion binding. Solubility is high at a neutral pH or above, but decreases as the casein net charge approaches zero, allowing a condensed casein phase or gel to form, then increases at lower pH. Of particular importance for casein micelle stability near neutral pH is the proportion of free caseins in the micelle (i.e., caseins not bound directly to nanoclusters of calcium phosphate). Free caseins are more soluble and better able to act as molecular chaperones (to prevent casein and whey protein aggregation) than bound caseins. Some free caseins are highly phosphorylated and can also act as mineral chaperones to inhibit the growth of calcium phosphate phases and prevent mineralized deposits from forming on membranes or heat exchangers. Thus, casein micelle stability is reduced when free caseins bind to amyloid fibrils, destabilized whey proteins or calcium phosphate. The multivalent-binding model of the casein micelle quantitatively describes these and other factors affecting the stability of milk and milk protein products during manufacture and storage.

Mizoribine Promotes Molecular Chaperone HSP60/HSP10 Complex Formation.

It has been reported that Mizoribine is an immunosuppressant used to suppress rejection in renal transplantation, nephrotic syndrome, lupus nephritis, and rheumatoid arthritis. The molecular chaperone HSP60 alone induces inflammatory cytokine IL-6 and the co-chaperone HSP10 alone inhibits IL-6 induction. HSP60 and HSP10 form a complex in the presence of ATP. We analyzed the effects of Mizoribine, which is structurally similar to ATP, on the structure and physiological functions of HSP60-HSP10 using Native/PAGE and transmission electron microscopy. At low concentrations of Mizoribine, no complex formation of HSP60-HSP10 was observed, nor was the expression of IL-6 affected. On the other hand, high concentrations of Mizoribine promoted HSP60-HSP10 complex formation and consequently suppressed IL-6 expression. Here, we propose a novel mechanism of immunosuppressive action of Mizoribine.

Molecular Chaperonin HSP60: Current Understanding and Future Prospects.

Molecular chaperones are highly conserved across evolution and play a crucial role in preserving protein homeostasis. The 60 kDa heat shock protein (HSP60), also referred to as chaperonin 60 (Cpn60), resides within mitochondria and is involved in maintaining the organelle's proteome integrity and homeostasis. The HSP60 family, encompassing Cpn60, plays diverse roles in cellular processes, including protein folding, cell signaling, and managing high-temperature stress. In prokaryotes, HSP60 is well understood as a GroEL/GroES complex, which forms a double-ring cavity and aids in protein folding. In eukaryotes, HSP60 is implicated in numerous biological functions, like facilitating the folding of native proteins and influencing disease and development processes. Notably, research highlights its critical involvement in sustaining oxidative stress and preserving mitochondrial integrity. HSP60 perturbation results in the loss of the mitochondria integrity and activates apoptosis. Currently, numerous clinical investigations are in progress to explore targeting HSP60 both in vivo and in vitro across various disease models. These studies aim to enhance our comprehension of disease mechanisms and potentially harness HSP60 as a therapeutic target for various conditions, including cancer, inflammatory disorders, and neurodegenerative diseases. This review delves into the diverse functions of HSP60 in regulating proteo-homeostasis, oxidative stress, ROS, apoptosis, and its implications in diseases like cancer and neurodegeneration.

Role of the Alpha-B-Crystallin Protein in Cardiomyopathic Disease.

Alpha-B-crystallin, a member of the small heat shock family of proteins, has been implicated in a variety of cardiomyopathies and in normal cardiac homeostasis. It is known to function as a molecular chaperone, particularly for desmin, but also interacts with a wide variety of additional proteins. The molecular chaperone function is also enhanced by signal-dependent phosphorylation at specific residues under stress conditions. Naturally occurring mutations in CRYAB, the gene that encodes alpha-B-crystallin, have been suggested to alter ionic intermolecular interactions that affect dimerization and chaperone function. These mutations have been associated with myofibrillar myopathy, restrictive cardiomyopathy, and hypertrophic cardiomyopathy and promote pathological hypertrophy through different mechanisms such as desmin aggregation, increased reductive stress, or activation of calcineurin-NFAT signaling. This review will discuss the known mechanisms by which alpha-B-crystallin functions in cardiac homeostasis and the pathogenesis of cardiomyopathies and provide insight into potential future areas of exploration.

Highly efficient expression of Rasamsonia emersonii lipase in Pichia pastoris: characterization and gastrointestinal simulated digestion in vitro.

BACKGROUND: Acidic lipases with high catalytic activities under acidic conditions have important application values in the food, feed and pharmaceutical industries. However, the availability of acidic lipases is still the main obstacle to their industrial applications. Although a novel acidic lipase Rasamsonia emersonii (LIPR) was heterologously expressed in Escherichia coli, the expression level was unsatisfactory. RESULTS: To achieve the high-efficiency expression and secretion of LIPR in Pichia pastoris GS115, the combinatorial optimization strategy was adopted including gene codon preference, signal peptide, molecular chaperone co-expression and disruption of vacuolar sorting receptor VPS10. The activity of the combinatorial optimization engineered strain in a shake flask reached 1480 U mL-1, which was 8.13 times greater than the P. pastoris GS115 parental strain. After high-density fermentation in a 5-L bioreactor, the highest enzyme activity reached as high as 11 820 U mL-1. LIPR showed the highest activity at 40 °C and pH 4.0 in the presence of Ca2+ ion. LIPR exhibited strong tolerance to methanol, indicating its potential application in biodiesel biosynthesis. Moreover, the gastrointestinal digestion simulation results demonstrated that LIPR was tolerant to pepsin and trypsin, but its activity was inhibited by sodium taurodeoxycholate. CONCLUSION: This study provided an effective approach for the high expression of acidic lipase LIPR. LIPR was more appropriate for lipid digestion in the stomach than in intestine according to the gastrointestinal digestion simulation results. © 2024 Society of Chemical Industry.

The role of molecular chaperone CCT/TRiC in translation elongation: A literature review.

Protein synthesis from mRNA is an energy-intensive and strictly controlled biological process. Translation elongation is a well-coordinated and multifactorial step in translation that ensures the accurate and efficient addition of amino acids to a growing nascent-peptide chain encoded in the sequence of messenger RNA (mRNA). Which undergoes dynamic regulation due to cellular state and environmental determinants. An expanding body of research points to translational elongation as a crucial process that controls the translation of an mRNA through multiple feedback mechanisms. Molecular chaperones are key players in protein homeostasis to keep the balance between protein synthesis, folding, assembly, and degradation. Chaperonin-containing tailless complex polypeptide 1 (CCT) or tailless complex polypeptide 1 ring complex (TRiC) is an essential eukaryotic molecular chaperone that plays an essential role in assisting cellular protein folding and suppressing protein aggregation. In this review, we give an overview of the factors that influence translation elongation, focusing on different functions of molecular chaperones in translation elongation, including how they affect translation rates and post-translational modifications. We also provide an understanding of the mechanisms by which the molecular chaperone CCT plays multiple roles in the elongation phase of eukaryotic protein synthesis.

ArHsp90 is important in stress tolerance and embryo development of the brine shrimp, Artemia franciscana.

Females of the extremophile crustacean, Artemia franciscana, either release motile nauplii via the ovoviviparous pathway or encysted embryos (cysts) via the oviparous pathway. Cysts contain an abundant amount of the ATP-independent small heat shock protein that contributes to stress tolerance and embryo development, however, little is known of the role of ATP-dependent molecular chaperone, heat shock protein 90 (Hsp90) in the two processes. In this study, a hsp90 was cloned from A. franciscana. Characteristic domains of ArHsp90 were simulated from the deduced amino acid sequence, and 3D structures of ArHsp90 and Hsp90s of organisms from different groups were aligned. RNA interference was then employed to characterize ArHsp90 in A. franciscana nauplii and cysts. The partial knockdown of ArHsp90 slowed the development of nauplius-destined, but not cyst-destined embryos. ArHsp90 knockdown also reduced the survival and stress tolerance of nauplii newly released from A. franciscana females. Although the reduction of ArHsp90 had no effect on the development of diapause-destined embryos, the resulting cysts displayed reduced tolerance to desiccation and low temperature, two stresses normally encountered by A. franciscana in its natural environment. The results reveal that Hsp90 contributes to the development, growth, and stress tolerance of A. franciscana, an organism of practical importance as a feed source in aquaculture.

Quantitative proteomic analysis reveals unique Hsp90 cycle-dependent client interactions.

Hsp90 is an abundant and essential molecular chaperone that mediates the folding and activation of client proteins in a nucleotide-dependent cycle. Hsp90 inhibition directly or indirectly impacts the function of 10-15% of all proteins due to degradation of client proteins or indirect downstream effects. Due to its role in chaperoning oncogenic proteins, Hsp90 is an important drug target. However, compounds that occupy the ATP-binding pocket and broadly inhibit function have not achieved widespread use due to negative effects. More selective inhibitors are needed; however, it is unclear how to achieve selective inhibition. We conducted a quantitative proteomic analysis of soluble proteins in yeast strains expressing wild-type Hsp90 or mutants that disrupt different steps in the client folding pathway. Out of 2,482 proteins in our sample set (approximately 38% of yeast proteins), we observed statistically significant changes in abundance of 350 (14%) of those proteins (log2 fold change ≥ 1.5). Of these, 257/350 (∼73%) with the strongest differences in abundance were previously connected to Hsp90 function. Principal component analysis of the entire dataset revealed that the effects of the mutants could be separated into 3 primary clusters. As evidence that Hsp90 mutants affect different pools of clients, simultaneous co-expression of 2 mutants in different clusters restored wild-type growth. Our data suggest that the ability of Hsp90 to sample a wide range of conformations allows the chaperone to mediate folding of a broad array of clients and that disruption of conformational flexibility results in client defects dependent on those states.

Type I Hsp40s/DnaJs aggregates exhibit features reminiscent of amyloidogenic structures.

A rise in temperature triggers a structural change in the human Type I 40 kDa heat shock protein (Hsp40/DnaJ), known as DNAJA1. This change leads to a less compact structure, characterized by an increased presence of solvent-exposed hydrophobic patches and ß-sheet-rich regions. This transformation is validated by circular dichroism, thioflavin T binding, and Bis-ANS assays. The formation of this ß-sheet-rich conformation, which is amplified in the absence of zinc, leads to protein aggregation. This aggregation is induced not only by high temperatures but also by low ionic strength and high protein concentration. The aggregated conformation exhibits characteristics of an amyloidogenic structure, including a distinctive X-ray diffraction pattern, seeding competence (which stimulates the formation of amyloid-like aggregates), cytotoxicity, resistance to SDS, and fibril formation. Interestingly, the yeast Type I Ydj1 also tends to adopt a similar ß-sheet-rich structure under comparable conditions, whereas Type II Hsp40s, whether human or from yeast, do not. Moreover, Ydj1 aggregates were found to be cytotoxic. Studies using DNAJA1- and Ydj1-deleted mutants suggest that the zinc-finger region plays a crucial role in amyloid formation. Our discovery of amyloid aggregation in a C-terminal deletion mutant of DNAJA1, which resembles a spliced homolog expressed in the testis, implies that Type I Hsp40 co-chaperones may generate amyloidogenic species in vivo.

Dataset of wheat HSP90.2 chaperome.

Wheat (Triticum aestivum L.) is one of the world's most important staple crops, whose production is critical to feed the expanding population worldwide. The 90-kDa Heat Shock Protein 90 (HSP90) is a highly abundant chaperone protein involved in multiple cellular processes. It facilitates the folding of nascent preproteins for their maturation and functioning. This data described HSP90.2 clients identified from the whole genome of wheat. The HSP90.2 chaperome contains over 1500 proteins, most detected by the C terminus and full-length of HSP90.2. Over 60 % of the clients reside in the cytosol, nucleus, and chloroplasts. Cytoskeleton-related proteins are enriched in the chaperome of the N terminus of HSP90.2. The clients of the middle part of HSP90.2 contains several factors involved in ethylene biosynthesis and extracellular vesicle or organelle-related activities. Some clients related to plant hypersensitive response are induced by stripe rust. The presented dataset could isolate proteins regulated by HSP90.2 at the post-translational level.

Molecular basis for different substrate-binding sites and chaperone functions of the BRICHOS domain.

Proteins can misfold into fibrillar or amorphous aggregates and molecular chaperones act as crucial guardians against these undesirable processes. The BRICHOS chaperone domain, found in several otherwise unrelated proproteins that contain amyloidogenic regions, effectively inhibits amyloid formation and toxicity but can in some cases also prevent non-fibrillar, amorphous protein aggregation. Here, we elucidate the molecular basis behind the multifaceted chaperone activities of the BRICHOS domain from the Bri2 proprotein. High-confidence AlphaFold2 and RoseTTAFold predictions suggest that the intramolecular amyloidogenic region (Bri23) is part of the hydrophobic core of the proprotein, where it occupies the proposed amyloid binding site, explaining the markedly reduced ability of the proprotein to prevent an exogenous amyloidogenic peptide from aggregating. However, the BRICHOS-Bri23 complex maintains its ability to form large polydisperse oligomers that prevent amorphous protein aggregation. A cryo-EM-derived model of the Bri2 BRICHOS oligomer is compatible with surface-exposed hydrophobic motifs that get exposed and come together during oligomerization, explaining its effects against amorphous aggregation. These findings provide a molecular basis for the BRICHOS chaperone domain function, where distinct surfaces are employed against different forms of protein aggregation.

Investigation of heat stress responses and adaptation mechanisms by integrative metabolome and transcriptome analysis in tea plants (Camellia sinensis).

Extreme high temperature has deleterious impact on the yield and quality of tea production, which has aroused the attention of growers and breeders. However, the mechanisms by which tea plant varieties respond to extreme environmental heat is not clear. In this study, we analyzed physiological indices, metabolites and transcriptome differences in three different heat-tolerant tea plant F1 hybrid progenies. Results showed that the antioxidant enzyme activity, proline, and malondialdehyde were significantly decreased in heat-sensitive 'FWS' variety, and the accumulation of reactive oxygen molecules such as H2O2 and O2- was remarkably increased during heat stress. Metabolomic analysis was used to investigate the metabolite accumulation pattern of different varieties in response to heat stress. The result showed that a total of 810 metabolites were identified and more than 300 metabolites were differentially accumulated. Transcriptional profiling of three tea varieties found that such genes encoding proteins with chaperon domains were preferentially expressed in heat-tolerant varieties under heat stress, including universal stress protein (USP32, USP-like), chaperonin-like protein 2 (CLP2), small heat shock protein (HSP18.1), and late embryogenesis abundant protein (LEA5). Combining metabolomic with transcriptomic analyses discovered that the flavonoids biosynthesis pathway was affected by heat stress and most flavonols were up-regulated in heat-tolerant varieties, which owe to the preferential expression of key FLS genes controlling flavonol biosynthesis. Take together, molecular chaperons, or chaperon-like proteins, flavonols accumulation collaboratively contributed to the heat stress adaptation in tea plant. The present study elucidated the differences in metabolite accumulation and gene expression patterns among three different heat-tolerant tea varieties under extreme ambient high temperatures, which helps to reveal the regulatory mechanisms of tea plant adaptation to heat stress, and provides a reference for the breeding of heat-tolerant tea plant varieties.

Insights into Hsp90 mechanism and in vivo functions learned from studies in the yeast, Saccharomyces cerevisiae.

The molecular chaperone Hsp90 (Heat shock protein, 90 kDa) is an abundant and essential cytosolic protein required for the stability and/or folding of hundreds of client proteins. Hsp90, along with helper cochaperone proteins, assists client protein folding in an ATP-dependent pathway. The laboratory of Susan Lindquist, in collaboration with other researchers, was the first to establish the yeast Saccharomyces cerevisiae as a model organism to study the functional interaction between Hsp90 and clients. Important insights from studies in her lab were that Hsp90 is essential, and that Hsp90 functions and cochaperone interactions are highly conserved between yeast and mammalian cells. Here, we describe key mechanistic insights into the Hsp90 folding cycle that were obtained using the yeast system. We highlight the early contributions of the laboratory of Susan Lindquist and extend our analysis into the broader use of the yeast system to analyze the understanding of the conformational cycle of Hsp90 and the impact of altered Hsp90 function on the proteome.

Specific inhibition of α-synuclein oligomer generation and toxicity by the chaperone domain Bri2 BRICHOS.

Protein misfolding and aggregation are involved in several neurodegenerative disorders, such as α-synuclein (αSyn) implicated in Parkinson's disease, where new therapeutic approaches remain essential to combat these devastating diseases. Elucidating the microscopic nucleation mechanisms has opened new opportunities to develop therapeutics against toxic mechanisms and species. Here, we show that naturally occurring molecular chaperones, represented by the anti-amyloid Bri2 BRICHOS domain, can be used to target αSyn-associated nucleation processes and structural species related to neurotoxicity. Our findings revealed that BRICHOS predominantly suppresses the formation of new nucleation units on the fibrils surface (secondary nucleation), decreasing the oligomer generation rate. Further, BRICHOS directly binds to oligomeric αSyn species and effectively diminishes αSyn fibril-related toxicity. Hence, our studies show that molecular chaperones can be utilized as tools to target molecular processes and structural species related to αSyn neurotoxicity and have the potential as protein-based treatments against neurodegenerative disorders.

Recent developments of HSP90 inhibitors: an updated patent review (2020-present).

INTRODUCTION: The 90-kDa heat shock protein (HSP90) functions as a molecular chaperone, it assumes a significant role in diseases such as cancer, inflammation, neurodegeneration, and infection. Therefore, the research and development of HSP90 inhibitors have garnered considerable attention. AREAS COVERED: The primary references source for this review is patents obtained from SciFinder, encompassing patents on HSP90 inhibitors from the period of 2020 to 2023.This review includes a thorough analysis of their structural attributes, pharmacological properties, and potential clinical utilities. EXPERT OPINION: In the past few years, HSP90 inhibitors targeting ATP binding pocket are still predominate and one of them has been launched, besides, novel drug design strategies like C-terminal targeting, isoform selective inhibiting and bifunctional molecules are booming, aiming to improve the efficacy and safety. With expanded drug types and applications, HSP90 inhibitors may gradually becoming a sagacious option for treating various diseases.

Improvement of Carotenoids' Production by Increasing the Activity of Beta-Carotene Ketolase with Different Strategies.

Canthaxanthin is an important antioxidant with wide application prospects, and ß-carotene ketolase is the key enzyme involved in the biosynthesis of canthaxanthin. However, the challenge for the soluble expression of ß-carotene ketolase is that it hinders the large-scale production of carotenoids such as canthaxanthin and astaxanthin. Hence, this study employed several strategies aiming to improve the soluble expression of ß-carotene ketolase and its activity, including selecting optimal expression vectors, screening induction temperatures, adding soluble expression tags, and adding a molecular chaperone. Results showed that all these strategies can improve the soluble expression and activity of ß-carotene ketolase in Escherichia coli. In particular, the production of soluble ß-carotene ketolase was increased 8 times, with a commercial molecular chaperon of pG-KJE8, leading to a 1.16-fold enhancement in the canthaxanthin production from ß-carotene. Interestingly, pG-KJE8 could also enhance the soluble expression of ß-carotene ketolase derived from eukaryotic microalgae. Further research showed that the production of canthaxanthin and echinenone was significantly improved by as many as 30.77 times when the pG-KJE8 was added, indicating the molecular chaperone performed differently among different ß-carotene ketolase. This study not only laid a foundation for further research on the improvement of ß-carotene ketolase activity but also provided new ideas for the improvement of carotenoid production.

Comparative structural and functional analysis of the glycine-rich regions of Class A and B J-domain protein cochaperones of Hsp70.

J-domain proteins are critical Hsp70 co-chaperones. A and B types have a poorly understood glycine-rich region (Grich) adjacent to their N-terminal J-domain (Jdom). We analyzed the ability of Jdom/Grich segments of yeast Class B Sis1 and a suppressor variant of Class A, Ydj1, to rescue the inviability of sis1-∆. In each, we identified a cluster of Grich residues required for rescue. Both contain conserved hydrophobic and acidic residues and are predicted to form helices. While, as expected, the Sis1 segment docks on its J-domain, that of Ydj1 does not. However, data suggest both interact with Hsp70. We speculate that the Grich-Hsp70 interaction of Classes A and B J-domain proteins can fine tune the activity of Hsp70, thus being particularly important for the function of Class B.