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Chemical chaperones are low-molecular-weight compounds that suppress protein aggregation. They can influence different stages of the aggregation process—the stage of protein denaturation, the nucleation stage and the stage of aggregate growth—and this may lead to a change in the aggregation kinetic regime. Here, the possibility of changing the kinetic regime in the presence of a chemical chaperone 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD) was investigated for a test system based on the thermally induced aggregation of yeast alcohol dehydrogenase (yADH) at 56 °C. According to differential scanning calorimetry data, 2-HP-β-CD did not affect the stage of the protein molecule unfolding. Dynamic light scattering data indicated changes in the aggregation kinetics of yADH during the nucleation and aggregate growth stages in the presence of the chaperone. The analysis of kinetic curves showed that the order of aggregation with respect to protein (nc), calculated for the stage of aggregate growth, changed from nc = 1 to nc = 2 with the addition of 100 mM 2-HP-β-CD. The mechanism of 2-HP-β-CD action on the yADH thermal aggregation leading to a change in its kinetic regime of aggregation is discussed. [ABSTRACT FROM AUTHOR]

The importance of studying the structural stability of proteins is determined by the structure–function relationship. Protein stability is influenced by many factors among which are freeze–thaw and thermal stresses. The effect of trehalose, betaine, sorbitol and 2-hydroxypropyl-β-cyclodextrin (HPCD) on the stability and aggregation of bovine liver glutamate dehydrogenase (GDH) upon heating at 50 °C or freeze–thawing was studied by dynamic light scattering, differential scanning calorimetry, analytical ultracentrifugation and circular dichroism spectroscopy. A freeze–thaw cycle resulted in the complete loss of the secondary and tertiary structure, and aggregation of GDH. All the cosolutes suppressed freeze–thaw- and heat-induced aggregation of GDH and increased the protein thermal stability. The effective concentrations of the cosolutes during freeze–thawing were lower than during heating. Sorbitol exhibited the highest anti-aggregation activity under freeze–thaw stress, whereas the most effective agents stabilizing the tertiary structure of GDH were HPCD and betaine. HPCD and trehalose were the most effective agents suppressing GDH thermal aggregation. All the chemical chaperones stabilized various soluble oligomeric forms of GDH against both types of stress. The data on GDH were compared with the effects of the same cosolutes on glycogen phosphorylase b during thermal and freeze–thaw-induced aggregation. This research can find further application in biotechnology and pharmaceutics. [ABSTRACT FROM AUTHOR]

The Unfolded protein response (UPR), triggered by stress in the endoplasmic reticulum (ER), is a key driver of neurodegenerative diseases. GM2 gangliosidosis, which includes Tay-Sachs and Sandhoff disease, is caused by an accumulation of GM2, mainly in the brain, that leads to progressive neurodegeneration. Previously, we demonstrated in a cellular model of GM2 gangliosidosis that PERK, a UPR sensor, contributes to neuronal death. There is currently no approved treatment for these disorders. Chemical chaperones, such as ursodeoxycholic acid (UDCA), have been found to alleviate ER stress in cell and animal models. UDCA's ability to move across the blood-brain barrier makes it interesting as a therapeutic tool. Here, we found that UDCA significantly diminished the neurite atrophy induced by GM2 accumulation in primary neuron cultures. It also decreased the up-regulation of pro-apoptotic CHOP, a downstream PERK-signaling component. To explore its potential mechanisms of action, in vitro kinase assays and crosslinking experiments were performed with different variants of recombinant protein PERK, either in solution or in reconstituted liposomes. The results suggest a direct interaction between UDCA and the cytosolic domain of PERK, which promotes kinase phosphorylation and dimerization. [ABSTRACT FROM AUTHOR]

HMG-CoA reductase (HMGR) undergoes regulated degradation as part of feedback control of the sterol pathway. In yeast, the stability of the HMGR isozyme Hmg2 is controlled by the 20-carbon isoprenoid geranylgeranyl pyrophosphate (GGPP). Increasing GGPP levels cause more efficient degradation by the HMG-CoA reductase degradation (HRD) pathway, allowing for feedback regulation of HMGR. The HRD pathway is critical for the endoplasmic reticulum (ER)-associated degradation (ERAD) of misfolded ER proteins. Here, we have explored GGPP's role in HRD-dependent Hmg2 degradation. We found that GGPP potently regulates Hmg2 levels in vivo and causes reversible Hmg2 misfolding at nanomolar concentrations in vitro These GGPP-mediated effects were absent in several stabilized or nonregulated Hmg2 mutants. Consistent with its high potency, GGPP's effects were highly specific such that other structurally related molecules were ineffective in altering Hmg2 structure. For instance, two closely related GGPP analogues, 2F-GGPP and GGSPP, were completely inactive at all concentrations tested. Furthermore, GGSPP antagonized GGPP's effects in vivo and in vitro Chemical chaperones reversed GGPP's effects on Hmg2 structure and degradation, suggesting that GGPP causes selective Hmg2 misfolding. These results indicate that GGPP functions in a manner similar to an allosteric ligand, causing Hmg2 misfolding through interaction with a reversible, specific binding site. Consistent with this, the Hmg2 protein formed multimers, typical of allosteric proteins. We propose that this "allosteric misfolding," or mallostery, observed here for Hmg2 may be a widely used tactic of biological regulation with potential for development of therapeutic small molecules that induce selective misfolding.

Introduction: During tooth development, proper protein folding and trafficking are significant processes as newly synthesized proteins proceed to form designated tissues. Endoplasmic reticulum (ER) stress occurs inevitably in tooth development as unfolded and misfolded proteins accumulate in ER. 4- Phenylbutyric acid (4PBA) is a FDA approved drug and known as a chemical chaperone which alleviates the ER stress. Recently, several studies showed that 4PBA performs therapeutic effects in some genetic diseases due to misfolding of proteins, metabolic related-diseases and apoptosis due to ER stress. However, the roles of 4PBA during odontogenesis are not elucidated. This study revealed the effects of 4PBA during molar development in mice. Methods: We employed in vitro organ cultivation and renal transplantation methods which would mimic the permanent tooth development in an infant period of human. The in vitro cultivated tooth germs and renal calcified teeth were examined by histology and immunohistochemical analysis. Results and Discussion: Our results revealed that treatment of 4PBA altered expression patterns of enamel knot related signaling molecules, and consequently affected cellular secretion and patterned formation of dental hard tissues including dentin and enamel during tooth morphogenesis. The alteration of ER stress by 4PBA treatment during organogenesis would suggest that proper ER stress is important for pattern formation during tooth development and morphogenesis, and 4PBA as a chemical chaperone would be one of the candidate molecules for dental and hard tissue regeneration. [ABSTRACT FROM AUTHOR]

Protein aggregation and subsequent accumulation of insoluble amyloid fibrils with cross-β structure is an intrinsic characteristic of amyloid diseases, i.e., amyloidoses. Amyloid formation involves a series of on-pathway and off-pathway protein aggregation events, leading to mature insoluble fibrils that eventually accumulate in multiple tissues. In this cascade of events, soluble oligomeric species are formed, which are among the most cytotoxic molecular entities along the amyloid cascade. The direct or indirect action of these amyloid soluble oligomers and amyloid protofibrils and fibrils in several tissues and organs lead to cell death in some cases and organ disfunction in general. There are dozens of different proteins and peptides causing multiple amyloid pathologies, chief among them Alzheimer's, Parkinson's, Huntington's, and several other neurodegenerative diseases. Amyloid fibril disassembly is among the disease-modifying therapeutic strategies being pursued to overcome amyloid pathologies. The clearance of preformed amyloids and consequently the arresting of the progression of organ deterioration may increase patient survival and quality of life. In this review, we compiled from the literature many examples of chemical and biochemical agents able to disaggregate preformed amyloids, which have been classified as molecular chaperones, chemical chaperones, and pharmacological chaperones. We focused on their mode of action, chemical structure, interactions with the fibrillar structures, morphology and toxicity of the disaggregation products, and the potential use of disaggregation agents as a treatment option in amyloidosis. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the degeneration of motor neurons. Mutations in the superoxide dismutase (SOD1) gene, causing protein misfolding and aggregation, were suggested as the pathogenic mechanisms involved in familial ALS cases. In the present study, we investigated the potential therapeutic effect of C4 and C5, two derivatives of the chemical chaperone 4-phenylbutyric acid (4-PBA). By combining in vivo and in vitro techniques, we show that, although C4 and C5 successfully inhibited amyloid aggregation of recombinant mutant SOD1 in a dose-dependent manner, they failed to suppress the accumulation of misfolded SOD1. Moreover, C4 or C5 daily injections to SOD1G93A mice following onset had no effect on either the accumulation of misfolded SOD1 or the neuroinflammatory response in the spinal cord and, consequently, failed to extend the survival of SOD1G93A mice or to improve their motor symptoms. Finally, pharmacokinetic (PK) studies demonstrated that high concentrations of C4 and C5 reached the brain and spinal cord but only for a short period of time. Thus, our findings suggest that use of such chemical chaperones for ALS drug development may need to be optimized for more effective results. [ABSTRACT FROM AUTHOR]

Osmolytes are naturally occurring small molecular weight organic molecules, which are accumulated in large amounts in all life forms to maintain the stability of cellular proteins and hence preserve their functions during adverse environmental conditions. Trimethylamine N-oxide (TMAO) and N,N,N-trimethylglycine (betaine) are methylamine osmolytes that have been extensively studied for their diverse roles in humans and have demonstrated opposing relations with human health. These osmolytes are obtained from food and synthesized endogenously using dietary constituents like choline and carnitine. Especially, gut microbiota plays a vital role in TMAO synthesis and contributes significantly to plasma TMAO levels. The elevated plasma TMAO has been reported to be correlated with the pathogenesis of numerous human diseases, including cardiovascular disease, heart failure, kidney diseases, metabolic syndrome, etc.; Hence, TMAO has been recognized as a novel biomarker for the detection/prediction of several human diseases. In contrast, betaine acts as a methyl donor in one-carbon metabolism, maintains cellular S-adenosylmethionine levels, and protects the cells from the harmful effects of increased plasma homocysteine. Betaine also demonstrates antioxidant and anti-inflammatory activities and has a promising therapeutic value in several human diseases, including homocystinuria and fatty liver disease. The present review examines the multifarious functions of TMAO and betaine with possible molecular mechanisms towards a better understanding of their emerging and diverging functions with probable implications in the prevention, diagnosis, and treatment of human diseases.

Protein–protein interactions (PPIs) play an important role in many biological processes in a living cell. Among them chaperone–client interactions are the most important. In this work PPIs of αB-crystallin and glycogen phosphorylase b (Phb) in the presence of betaine (Bet) and arginine (Arg) at 48 °C and ionic strength of 0.15 M were studied using methods of dynamic light scattering, differential scanning calorimetry, and analytical ultracentrifugation. It was shown that Bet enhanced, while Arg reduced both the stability of αB-crystallin and its adsorption capacity (AC0) to the target protein at the stage of aggregate growth. Thus, the anti-aggregation activity of αB-crystallin increased in the presence of Bet and decreased under the influence of Arg, which resulted in inhibition or acceleration of Phb aggregation, respectively. Our data show that chemical chaperones can influence the tertiary and quaternary structure of both the target protein and the protein chaperone. The presence of the substrate protein also affects the quaternary structure of αB-crystallin, causing its disassembly. This is inextricably linked to the anti-aggregation activity of αB-crystallin, which in turn affects its PPI with the target protein. Thus, our studies contribute to understanding the mechanism of interaction between chaperones and proteins. [ABSTRACT FROM AUTHOR]

Abstract Background Congenital coagulation factor (F) VII deficiency is a rare bleeding disorder caused by mutations in the F7 gene. The missense factor FVII variant p.Q160R is the disease-causing mutation in all Norwegian FVII deficient patients and results in reduced biological activity and antigen levels of FVII in patient plasma. Previous in vitro studies on this variant demonstrated impaired intracellular trafficking and reduced secretion, possibly due to protein misfolding. The aim of the study was therefore to assess the impact of chemical chaperones on cellular processing and secretion of this variant using a cell model based on overexpression of the recombinant protein. Results Through screening of compounds, we identified 4-phenylbutyrate (4-PBA) to increase the secretion of recombinant (r) FVII-160R by ~ 2.5-fold. Additionally, treatment with 4-PBA resulted in a modest increase in specific biological activity. Intracellular localization studies revealed that upon treatment with 4-PBA, rFVII-160R was secreted through Golgi and Golgi reassembly-stacking protein (GRASP)-structures. Conclusions The present study demonstrates that the chemical chaperone 4-PBA, restores intracellular trafficking and increases the secretion of a missense FVII variant with functional properties in the extrinsic coagulation pathway.

(1) Background: Congenital factor (F) VII deficiency is caused by mutations in the F7 gene. Patients with modest differences in FVII levels may display large differences in clinical severity. The variant p.A354V-p.P464Hfs is associated with reduced FVII antigen and activity. The aim of the study was to investigate the clinical manifestation of this variant and the underlying molecular mechanisms. (2) Methods: Analyses were conducted in 37 homozygous patients. The recombinant variant was produced in mammalian cells. (3) Results: We report a large variation in clinical phenotypes, which points out genetic and acquired components beyond F7 mutations as a source of variability. In contrast, patients displayed similarly reduced FVII plasma levels with antigen higher than its activity. Comparative analysis of the recombinant variant and of plasma samples from a subset of patients indicated the presence of an elongated variant with indistinguishable migration. Treatment of cells with the chemical chaperone 4-phenylbutyrate (4-PBA) improved the intracellular trafficking of the variant and increased its secretion to the conditioned medium up to 2-fold. However, the effect of 4-PBA on biological activity was marginal. (4) Conclusions: Chemical chaperones can be used as biochemical tools to study the intracellular fate of a trafficking-defective FVII variant. [ABSTRACT FROM AUTHOR]

Abstract Background Cyclodextrin glycosyltransferases (CGTases) catalyze the synthesis of cyclodextrins, which are circular α-(1,4)-linked glucans used in many applications in the industries related to food, pharmaceuticals, cosmetics, chemicals, and agriculture, among others. Economic use of these CGTases, particularly γ-CGTase, requires their efficient production. In this study, the effects of chemical chaperones, temperature and inducers on cell growth and the production of soluble γ-CGTase by Escherichia coli were investigated. Results The yield of soluble γ-CGTase in shake-flask culture approximately doubled when β-cyclodextrin was added to the culture medium as a chemical chaperone. When a modified two-stage feeding strategy incorporating 7.5 mM β-cyclodextrin was used in a 3-L fermenter, a dry cell weight of 70.3 g·L− 1 was achieved. Using this cultivation approach, the total yield of γ-CGTase activity (50.29 U·mL− 1) was 1.71-fold greater than that observed in the absence of β-cyclodextrin (29.33 U·mL− 1). Conclusions Since β-cyclodextrin is inexpensive and nontoxic to microbes, these results suggest its universal application during recombinant protein production. The higher expression of soluble γ-CGTase in a semi-synthetic medium showed the potential of the proposed process for the economical production of many enzymes on an industrial scale.

Abstract Background Classic galactosemia is a rare genetic metabolic disease with an unmet treatment need. Current standard of care fails to prevent chronically-debilitating brain and gonadal complications. Many mutations in the GALT gene responsible for classic galactosemia have been described to give rise to variants with conformational abnormalities. This pathogenic mechanism is highly amenable to a therapeutic strategy based on chemical/pharmacological chaperones. Arginine, a chemical chaperone, has shown beneficial effect in other inherited metabolic disorders, as well as in a prokaryotic model of classic galactosemia. The p.Q188R mutation presents a high prevalence in the Caucasian population, making it a very clinically relevant mutation. This mutation gives rise to a protein with lower conformational stability and lower catalytic activity. The aim of this study is to assess the potential therapeutic role of arginine for this mutation. Methods Arginine aspartate administration to four patients with the p.Q188R/p.Q188R mutation, in vitro studies with three fibroblast cell lines derived from classic galactosemia patients as well as recombinant protein experiments were used to evaluate the effect of arginine in galactose metabolism. This study has been registered at https://clinicaltrials.gov (NCT03580122) on 09 July 2018. Retrospectively registered. Results Following a month of arginine administration, patients did not show a significant improvement of whole-body galactose oxidative capacity (p = 0.22), erythrocyte GALT activity (p = 0.87), urinary galactose (p = 0.52) and urinary galactitol levels (p = 0.41). Patients’ fibroblasts exposed to arginine did not show changes in GALT activity. Thermal shift analysis of recombinant p.Q188R GALT protein in the presence of arginine did not exhibit a positive effect. Conclusions This short pilot study in four patients homozygous for the p.Q188R/p.Q188R mutation reveals that arginine has no potential therapeutic role for galactosemia patients homozygous for the p.Q188R mutation.

Genome-wide association studies (GWASs) have identified many disease-associated variant alleles, but understanding whether and how different genes/loci interact requires a platform for probing how the variant alleles act mechanistically. Isogenic mutant human embryonic stem cells (hESCs) provide an unlimited resource to derive and study human disease-relevant cells. Here, we focused on CDKAL1, linked by GWASs to diabetes. Through transcript profiling, we find that expression of the metallothionein (MT) gene family, also linked by GWASs to diabetes, is significantly downregulated in CDKAL1−/− cells that have been differentiated to insulin-expressing pancreatic beta-like cells. Forced MT1E expression rescues both hypersensitivity of CDKAL1 mutant cells to glycolipotoxicity and pancreatic beta-cell dysfunction in vitro and in vivo. MT1E functions at least in part through relief of ER stress. This study establishes an isogenic hESC-based platform to study the interaction of GWAS-identified diabetes gene variants and illuminate the molecular network impacting disease progression.

The transmembrane protein CD19 is exclusively expressed on normal and malignant B cells and therefore constitutes the target of approved CAR-T cell-based cancer immunotherapies. Current efforts to assess CAR-T cell functionality in a quantitative fashion both in vitro and in vivo are hampered by the limited availability of the properly folded recombinant extracellular domain of CD19 (CD19-ECD) considered as “difficult-to-express” (DTE) protein. Here, we successfully expressed a novel fusion construct consisting of the full-length extracellular domain of CD19 and domain 2 of human serum albumin (CD19-AD2), which was integrated into the Rosa26 bacterial artificial chromosome vector backbone for generation of a recombinant CHO-K1 production cell line. Product titers could be further boosted using valproic acid as a chemical chaperone. Purified monomeric CD19-AD2 proved stable as shown by non-reduced SDS-PAGE and SEC-MALS measurements. Moreover, flow cytometric analysis revealed specific binding of CD19-AD2 to CD19-CAR-T cells. Finally, we demonstrate biological activity of our CD19-AD2 fusion construct as we succeeded in stimulating CD19-CAR-T cells effectively with the use of CD19-AD2-decorated planar supported lipid bilayers.

(1) Background: Congenital factor (F) VII deficiency is caused by mutations in the F7 gene. Patients with modest differences in FVII levels may display large differences in clinical severity. The variant p.A354V-p.P464Hfs is associated with reduced FVII antigen and activity. The aim of the study was to investigate the clinical manifestation of this variant and the underlying molecular mechanisms. (2) Methods: Analyses were conducted in 37 homozygous patients. The recombinant variant was produced in mammalian cells. (3) Results: We report a large variation in clinical phenotypes, which points out genetic and acquired components beyond F7 mutations as a source of variability. In contrast, patients displayed similarly reduced FVII plasma levels with antigen higher than its activity. Comparative analysis of the recombinant variant and of plasma samples from a subset of patients indicated the presence of an elongated variant with indistinguishable migration. Treatment of cells with the chemical chaperone 4-phenylbutyrate (4-PBA) improved the intracellular trafficking of the variant and increased its secretion to the conditioned medium up to 2-fold. However, the effect of 4-PBA on biological activity was marginal. (4) Conclusions: Chemical chaperones can be used as biochemical tools to study the intracellular fate of a trafficking-defective FVII variant.

Background: From their initial, accidental discovery 50 years ago, the highly conserved Heat Shock Proteins (HSPs) continue to exhibit fundamental roles in the protection of cell integrity. Meanwhile, in the midst of an obesity epidemic, research demonstrates a key involvement of low grade inflammation, and mitochondrial dysfunction amongst other mechanisms, in the pathology of insulin resistance and type 2 diabetes mellitus (T2DM). In particular, tumor necrosis factor alpha (TNFα), endoplasmic reticulum (ER) and oxidative stress all appear to be associated with obesity and stimulate inflammatory kinases such as c jun amino terminal kinase (JNK), inhibitor of NF-κβ kinase (IKK) and protein kinase C (PKC) which in turn, inhibit insulin signaling. Mitochondrial dysfunction in skeletal muscle has also been proposed to be prominent in the pathogenesis of T2DM either by reducing the ability to oxidize fatty acids, leading to the accumulation of deleterious lipid species in peripheral tissues such as skeletal muscle and liver, or by altering the cellular redox state. Since HSPs act as molecular chaperones and demonstrate crucial protective functions in stressed cells, we and others have postulated that the manipulation of HSP expression in metabolically relevant tissues represents a therapeutic avenue for obesity-induced insulin resistance. Scope of Review: This review summarizes the literature from both animal and human studies, that has examined how HSPs, particularly the inducible HSP, Heat Shock Protein 72 (Hsp72) alters glucose homeostasis and the possible approaches to modulating Hsp72 expression. A summation of the role of chemical chaperones in metabolic disorders is also included. Major Conclusions: Targeted manipulation of Hsp72 or use of chemical chaperiones may have clinical utility in treating metabolic disorders such as insulin resistance and T2DM.

Ectoine and hydroxyectoine are widely synthesized by members of the Bacteria and a few members of the Archaea as potent osmostress protectants. We have studied the salient features of the osmostress-responsive promoter directing the transcription of the ectoine/hydroxyectoine biosynthetic gene cluster from the plant-root-associated bacterium Pseudomonas stutzeri by transferring it into Escherichia coli, an enterobacterium that does not produce ectoines naturally. Using ectlacZ reporter fusions, we found that the heterologous ect promoter reacted with exquisite sensitivity in its transcriptional profile to graded increases in sustained high salinity, responded to a true osmotic signal, and required the buildup of an osmotically effective gradient across the cytoplasmic membrane for its induction. The involvement of the -10, -35, and spacer regions of the sigma-70-type ect promoter in setting promoter strength and response to osmotic stress was assessed through site-directed mutagenesis. Moderate changes in the ect promoter sequence that increase its resemblance to housekeeping sigma-70-type promoters of E. coli afforded substantially enhanced expression, both in the absence and in the presence of osmotic stress. Building on this set of ect promoter mutants, we engineered an E. coli chassis strain for the heterologous production of ectoines. This synthetic cell factory lacks the genes for the osmostress-responsive synthesis of trehalose and the compatible solute importers ProP and ProU, and it continuously excretes ectoines into the growth medium. By combining appropriate host strains and different plasmid variants, excretion of ectoine, hydroxyectoine, or a mixture of both compounds was achieved under mild osmotic stress conditions. [ABSTRACT FROM AUTHOR]

Background & Aims: Today, diabetic nephropathy is considered to be one of the most common causes of end stage renal disease. Uncontrolled hyperglycemia, and consequently, production of advanced glycation end products activate pathways which play key roles in diabetic nephropathy. Among these pathways, high expression of receptor for advanced glycation end products (RAGE) and transforming growth factor beta (TGFβ) are notable. In this study, in order to find compounds which can prevent the incidence or progression of diabetic nephropathy, we examined the effects of glycine and lysine amino acids on expression of RAGE and TGFβ in kidney tissue of diabetic rats. Methods: After rendering rats with diabetes with streptozotocin (STZ), they were divided into different groups and were treated with oral 1% glycine and 0.1% lysine in drinking water for 12 weeks. Blood glucose and serum AGEs were measured during this time. Changes in RAGE and TGFβ expression were assessed by semi quantitative reverse transcription polymerase chain reaction (RT-PCR) method. Results: Results show that both glycine and lysine administration for 12 weeks not only caused a significant reduction in blood glucose and AGEs in diabetic rats, but also led to a significant reduction in RAGE and TGFβ expression in comparison to non-treated diabetic rats. Conclusion: These results show that oral glycine and lysine, as chemical chaperones, have the ability to prevent diabetic nephropathy by decreasing RAGE and TGFβ expression. This may be due to the effect of these chemical chaperones in the reduction of hyperglycemia and serum AGEs in diabetic rats. Since the positive effects of these amino acids in diabetic nephropathy have been observed in previous studies, the determination of their dose in future studies seems necessary

In many cases of familial amyotrophic lateral sclerosis (ALS), mutant forms of the Cu,Zn superoxide dismutase protein (SOD1) misfold and aggregate in motor neurons. Monomers of the normally homodimeric SOD1 have been found in patient tissue, presymptomatic mouse models of ALS, and in vitro misfolding assays which suggests that monomerization might be an early step in the pathological SOD1 misfolding pathway. In this study, we targeted the dimer interface with small molecules that might act as chemical chaperones to stabilize the native dimer and prevent downstream misfolding and aggregation. We performed a computational screen with a library of ~4400 drugs and natural compounds that were docked to two pockets around the SOD1 dimer interface. Of the resultant hits, seven were tested for misfolding and aggregation inhibition activity with A4V mutant SOD1. Quercitrin, quercetin-3-β-D-glucoside (Q3BDG), and, to a markedly lesser extent, epigallocatechin gallate (EGCG) were found to combat misfolding and aggregation induced by hydrogen peroxide, a physiologically relevant stress, as assessed by a gel-based assay and 8-anilinonaphthalene-1-suflonic acid (ANS) fluorescence. Isothermal titration calorimetry (ITC) and a colourimetric assay determined that these molecules directly bind A4V SOD1. Based on these findings, we speculate that quercitrin and Q3BDG may be potential therapeutic inhibitors of misfolding and aggregation in SOD1-associated ALS. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) is an intracellular compartment dedicated to the synthesis and maturation of secretory and membrane proteins, totalling about 30% of the total eukaryotic cells proteome. The capacity to produce correctly folded polypeptides and to transport them to their correct intra- or extracellular destinations relies on proteostasis networks that regulate and balance the activity of protein folding, quality control, transport and degradation machineries. Nutrient and environmental changes, pathogen infection aging and, more relevant for the topics discussed in this review, mutations that impair attainment of the correct 3D structure of nascent polypeptide chains may compromise the activity of the proteostasis networks with devastating consequences on cells, organs and organisms’ homeostasis. Here we present a review of mechanisms regulating folding and quality control of proteins expressed in the ER, and we describe the protein degradation and the ER stress pathways activated by the expression of misfolded proteins in the ER lumen. Finally, we highlight select examples of proteopathies (also known as conformational disorders or protein misfolding diseases) caused by protein misfolding in the ER and/or affecting cellular proteostasis and therapeutic interventions that might alleviate or cure the disease symptoms.

Some inherited coagulation factor deficiencies are caused by intracellular retention or degradation of misfolded proteins, and chemical chaperones have been shown to reverse protein misfolding. The purpose of the present study was to investigate whether chemical chaperones may improve secretion of the protein CA267T (PCA267T) mutant in a cellular model. Using stably transfected Chinese hamster ovary cells (CHO-K1) expressing PCA267T we demonstrate that sodium 4-phenylbutyrate (PBA) increased the secretion of PCA267T by approximately 4-fold in comparison with untreated cells, and that this secretion seemed to follow an unconventional pathway via the Golgi reassembly stacking protein (GRASP55). [ABSTRACT FROM AUTHOR]

Background From their initial, accidental discovery 50 years ago, the highly conserved Heat Shock Proteins (HSPs) continue to exhibit fundamental roles in the protection of cell integrity. Meanwhile, in the midst of an obesity epidemic, research demonstrates a key involvement of low grade inflammation, and mitochondrial dysfunction amongst other mechanisms, in the pathology of insulin resistance and type 2 diabetes mellitus (T2DM). In particular, tumor necrosis factor alpha (TNFα), endoplasmic reticulum (ER) and oxidative stress all appear to be associated with obesity and stimulate inflammatory kinases such as c jun amino terminal kinase (JNK), inhibitor of NF-κβ kinase (IKK) and protein kinase C (PKC) which in turn, inhibit insulin signaling. Mitochondrial dysfunction in skeletal muscle has also been proposed to be prominent in the pathogenesis of T2DM either by reducing the ability to oxidize fatty acids, leading to the accumulation of deleterious lipid species in peripheral tissues such as skeletal muscle and liver, or by altering the cellular redox state. Since HSPs act as molecular chaperones and demonstrate crucial protective functions in stressed cells, we and others have postulated that the manipulation of HSP expression in metabolically relevant tissues represents a therapeutic avenue for obesity-induced insulin resistance. Scope of Review This review summarizes the literature from both animal and human studies, that has examined how HSPs, particularly the inducible HSP, Heat Shock Protein 72 (Hsp72) alters glucose homeostasis and the possible approaches to modulating Hsp72 expression. A summation of the role of chemical chaperones in metabolic disorders is also included. Major Conclusions Targeted manipulation of Hsp72 or use of chemical chaperiones may have clinical utility in treating metabolic disorders such as insulin resistance and T2DM. [ABSTRACT FROM AUTHOR]

Abstract: Multidrug resistance protein 1 (MRP1) extrudes drugs as well as pharmacologically and physiologically important organic anions across the plasma membrane in an ATP-dependent manner. We previously showed that Ala substitutions of Lys513 and Lys516 in the cytoplasmic loop (CL5) connecting transmembrane helix 9 (TM9) to TM10 cause misfolding of MRP1, abrogating its expression at the plasma membrane in transfected human embryonic kidney (HEK) cells. Exposure of HEK cells to the chemical chaperones glycerol, DMSO, polyethylene glycol (PEG) and 4-aminobutyric acid (4-PBA) improved levels of K513A to wild-type MRP1 levels but transport activity was only fully restored by 4-PBA or DMSO treatments. Tryptic fragmentation patterns and conformation-dependent antibody immunoreactivity of the transport-deficient PEG- and glycerol-rescued K513A proteins indicated that the second nucleotide binding domain (NBD2) had adopted a more open conformation than in wild-type MRP1. This structural change was accompanied by differences in ATP binding and hydrolysis but no changes in substrate Km . In contrast to K513A, K516A levels in HEK cells were not significantly enhanced by chemical chaperones. In more permissive insect cells, however, K516A levels were comparable to wild-type MRP1. Nevertheless, organic anion transport by K516A in insect cell membranes was reduced by >80% due to reduced substrate Km . Tryptic fragmentation patterns indicated a more open conformation of the third membrane spanning domain of MRP1. Thus, despite their close proximity to one another in CL5, Lys513 and Lys516 participate in different interdomain interactions crucial for the proper folding and assembly of MRP1. [Copyright &y& Elsevier]

Background The stabilizing and function-preserving effects of ectoines have attracted considerable biotechnological interest up to industrial scale processes for their production. These rely on the release of ectoines from high-salinity-cultivated microbial producer cells upon an osmotic down-shock in rather complex processor configurations. There is growing interest in uncoupling the production of ectoines from the typical conditions required for their synthesis, and instead design strains that naturally release ectoines into the medium without the need for osmotic changes, since the use of high-salinity media in the fermentation process imposes notable constraints on the costs, design, and durability of fermenter systems. Results Here, we used a Corynebacterium glutamicum strain as a cellular chassis to establish a microbial cell factory for the biotechnological production of ectoines. The implementation of a mutant aspartokinase enzyme ensured efficient supply of L-aspartate-beta-semialdehyde, the precursor for ectoine biosynthesis. We further engineered the genome of the basic C. glutamicum strain by integrating a codon-optimized synthetic ectABCD gene cluster under expressional control of the strong and constitutive C. glutamicum tuf promoter. The resulting recombinant strain produced ectoine and excreted it into the medium; however, lysine was still found as a by-product. Subsequent inactivation of the L-lysine exporter prevented the undesired excretion of lysine while ectoine was still exported. Using the streamlined cell factory, a fed-batch process was established that allowed the production of ectoine with an overall productivity of 6.7 g L-1 day-1 under growth conditions that did not rely on the use of high-salinity media. Conclusions The present study describes the construction of a stable microbial cell factory for recombinant production of ectoine. We successfully applied metabolic engineering strategies to optimize its synthetic production in the industrial workhorse C. glutamicum and thereby paved the way for further improvements in ectoine yield and biotechnological process optimization. [ABSTRACT FROM AUTHOR]

MHC class I molecules bind only those peptides with high affinity that conform to stringent length and sequence requirements. We have now investigated which peptides can aid the in vitro folding of class I molecules, and we find that the dipeptide glycyl-leucine efficiently supports the folding of HLA-A*02:01 and H-2Kb into a peptide-receptive conformation that rapidly binds high-affinity peptides. Treatment of cells with glycyl-leucine induces accumulation of peptide-receptive H-Kb and HLA-A*02:01 at the surface of cells. Other dipeptides with a hydrophobic second amino acid show similar enhancement effects. Our data suggest that the dipeptides bind into the F pocket like the C-terminal amino acids of a high-affinity peptide. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of nonalcoholic steatohepatitis. The ER stress response is activated in the livers of mice fed a methionine- and choline-deficient (MCD) diet, yet the role of ER stress in the pathogenesis of MCD diet-induced steatohepatitis is unknown. Using chemical chaperones on hepatic steatosis and markers of inflammation and fibrosis in mice fed a MCD diet, we aim to determine the effects of reducing ER stress. C57BL/6J mice were fed a MCD diet with or without the ER chemical chaperones 4-phenylbutyric acid (PBA) and tauroursodeoxycholic acid (TUDCA) for 2 wk. TUDCA and PBA effectively attenuated the ER stress response in MCD diet-fed mice, as evidenced by reduced protein levels of phosphorylated eukaryotic initiation factor 2α and phosphorylated JNK and suppression of mRNA levels of CCAAT/enhancer binding protein homologous protein, glucose-regulated protein 78 kDa, and X-box binding protein 1. However, PBA and TUDCA did not decrease MCD diet-induced hepatic steatosis. MCD diet-induced hepatic inflammation, as evidenced by increased plasma alanine aminotransferase and induction of hepatic TNFα expression, was also not reduced by PBA or TUDCA. PBA and TUDCA did not attenuate MCD diet-induced upregulation of the fibrosis-associated genes tissue inhibitor of metalloproteinase-1 and matrix metalloproteinase-9. ER chemical chaperones reduce MCD diet-induced ER stress, yet they do not improve MCD diet-induced hepatic steatosis, inflammation, or activation of genes associated with fibrosis. These data suggest that although the ER stress response is activated by the MCD diet, it does not have a primary role in the pathogenesis of MCD diet-induced steatohepatitis. [ABSTRACT FROM AUTHOR]

Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizing homology models of CFTR. The intracellular segment of CFTR was modeled and three cavities were identified at inter-domain interfaces: (1) Interface between the two Nucleotide Binding Domains (NBDs); (2) Interface between NBD1 and Intracellular Loop (ICL) 4, in the region of the F508 deletion; (3) multi-domain interface between NBD1:2:ICL1:2:4. We hypothesized that compounds binding at these interfaces may improve the stability of the protein, potentially affecting the folding yield or surface stability. In silico structure-based screening was performed at the putative binding-sites and a total of 496 candidate compounds from all three sites were tested in functional assays. A total of 15 compounds, representing diverse chemotypes, were identified as F508del folding correctors. This corresponds to a 3% hit rate, ~tenfold higher than hit rates obtained in corresponding high-throughput screening campaigns. The same binding sites also yielded potentiators and, most notably, compounds with a dual corrector-potentiator activity (dual-acting). Compounds harboring both activity types may prove to be better leads for the development of CF therapeutics than either pure correctors or pure potentiators. To the best of our knowledge this is the first report of structure-based discovery of CFTR modulators. [ABSTRACT FROM AUTHOR]

To cope with the accumulation of unfolded or misfolded proteins the endoplasmic reticulum (ER) has evolved specific signalling pathways collectively called the unfolded protein response (UPR). Elucidation of the mechanisms governing ER stress signalling has linked this response to the regulation of diverse physiologic processes as well as to the progression of a number of diseases. Interest in hereditary haemochromatosis (HH) has focused on the study of proteins implicated in iron homeostasis and on the identification of new alleles related with the disease. HFE has been amongst the preferred targets of interest, since the discovery that its C282Y mutation was associated with HH. However, the discrepancies between the disease penetrance and the frequency of this mutation have raised the possibility that its contribution to disease progression might go beyond the mere involvement in regulation of cellular iron uptake. Recent findings revealed that activation of the UPR is a feature of HH and that this stress response may be involved in the genesis of immunological anomalies associated with the disease. This review addresses the connection of the UPR with HH, including its role in MHC-I antigen presentation pathway and possible implications for new clinical approaches to HH. [ABSTRACT FROM AUTHOR]

N-Alkylation at the ring nitrogen of the D-galactosidase inhibitor 1-deoxygalactonojirimycin with a functionalised C6 alkyl chain followed by modification with different aromatic substituents provided lipophilic 1-deoxygalactonojirimycin derivatives which exhibit inhibitory properties against β-glycosidases from E. coli and Agrobacterium sp. as well as green coffee bean α-galactosidase. In preliminary studies, these compounds also showed potential as chemical chaperones for GM1-gangliosidosis related β-galactosidase mutants.

The formation of amyloid-like structures by metabolites is associated with several inborn errors of metabolism (IEMs). These structures display most of the biological, chemical and physical properties of protein amyloids. However, the molecular interactions underlying the assembly remain elusive, and so far, no modulating therapeutic agents are available for clinical use. Chemical chaperones are known to inhibit protein and peptide amyloid formation and stabilize misfolded enzymes. Here, we provide an in-depth characterization of the inhibitory effect of osmolytes and hydrophobic chemical chaperones on metabolite assemblies, thus extending their functional repertoire. We applied a combined in vivo-in vitro-in silico approach and show their ability to inhibit metabolite amyloid-induced toxicity and reduce cellular amyloid content in yeast. We further used various biophysical techniques demonstrating direct inhibition of adenine self-assembly and alteration of fibril morphology by chemical chaperones. Using a scaffold-based approach, we analyzed the physiochemical properties of various dimethyl sulfoxide derivatives and their role in inhibiting metabolite self-assembly. Lastly, we employed whole-atom molecular dynamics simulations to elucidate the role of hydrogen bonds in osmolyte inhibition. Our results imply a dual mode of action of chemical chaperones as IEMs therapeutics, that could be implemented in the rational design of novel lead-like molecules. [ABSTRACT FROM AUTHOR]

Cell surface and secreted proteins provide essential functions for multicellular life. They enter the endoplasmic reticulum (ER) lumen co-translationally, where they mature and fold into their complex three-dimensional structures. The ER is populated with a host of molecular chaperones, associated co-factors, and enzymes that assist and stabilize folded states. Together, they ensure that nascent proteins mature properly or, if this process fails, target them for degradation. BiP, the ER HSP70 chaperone, interacts with unfolded client proteins in a nucleotide-dependent manner, which is tightly regulated by eight DnaJ-type proteins and two nucleotide exchange factors (NEFs), SIL1 and GRP170. Loss of SIL1′s function is the leading cause of Marinesco-Sjögren syndrome (MSS), an autosomal recessive, multisystem disorder. The development of animal models has provided insights into SIL1′s functions and MSS-associated pathologies. This review provides an in-depth update on the current understanding of the molecular mechanisms underlying SIL1′s NEF activity and its role in maintaining ER homeostasis and normal physiology. A precise understanding of the underlying molecular mechanisms associated with the loss of SIL1 may allow for the development of new pharmacological approaches to treat MSS. [ABSTRACT FROM AUTHOR]

Intravenous infusion of alpha-1 antitrypsin (AAT) was approved by the United States Food and Drug Administration (FDA) to treat emphysema associated with AAT deficiency (AATD) in 1987 and there are now several FDA-approved therapy products on the market, all of which are derived from pooled human plasma. Intravenous AAT therapy has proven clinical efficacy in slowing the decline of lung function associated with AATD progression; however, it is only recommended for individuals with the most severe forms of AATD as there is a lack of evidence that this treatment is effective in treating wild-type heterozygotes (e.g., PI*MS and PI*MZ genotypes), for which the prevalence may be much higher than previously thought. There are large numbers of individuals that are currently left untreated despite displaying symptoms of AATD. Furthermore, not all countries offer AAT augmentation therapy due to its expense and inconvenience for patients. More cost-effective treatments are now being sought that show efficacy for less severe forms of AATD and many new therapeutic technologies are being investigated, such as gene repair and other interference strategies, as well as the use of chemical chaperones. New sources of AAT are also being investigated to ensure there are enough supplies to meet future demand, and new methods of assessing response to treatment are being evaluated. There is currently extensive research into AATD and its treatment, and this chapter aims to highlight important emerging treatment strategies that aim to improve the lives of patients with AATD., Competing Interests: Conflict of interest statement: FFR is a consultant, speaker, and researcher for Takeda and Grifols, and a consultant for CSL Behring., (© The Author(s), 2021.)

Introduction: The monocarboxylate transporter 8 (MCT8; SLC16A2) is a specific transporter for thyroid hormones. MCT8 deficiency, formerly known as the Allan-Herndon-Dudley syndrome, is a rare genetic disease that leads to neurological impairments and muscle weakness. Current experimental treatment options rely on thyromimetic agonists that do not depend on MCT8 for cellular uptake. Another approach comes from studies with the chemical chaperone sodium phenylbutyrate (NaPB), which was able to stabilize MCT8 mutants having protein folding defects in vitro. In addition, NaPB is known as a compound that assists with plasma membrane translocation., Objective: The pathogenic MCT8 L291R leads to the same severe neurological impairments found for other MCT8-deficient patients but, unexpectedly, lacks alterations in plasma 3,3',5-triiodothyronine (T 3 ) levels. Here we tried to unravel the underlying mechanism of MCT8 deficiency and tested whether the pathogenic MCT8 L291R mutant responds to NaPB treatment. Therefore, we overexpressed the mutant in Madin-Darby canine kidney cells in the human choriocarcinoma cell line JEG1 and in COS7 cells of African green monkey origin., Results: In our recent study we describe that the MCT8 L291R mutation most likely leads to a translocation defect. The pathogenic mutant is not located at the plasma membrane, but shows overlapping expression with a marker protein of the lysosome. Mutation of the corresponding amino acid in murine Mct8 (Mct8 L223R ) displays a similar effect on cell surface expression and transport function as seen before for MCT8 L291R . NaPB was able to correct the translocation defect of MCT8 L291R /Mct8 L223R and restored protein function by increasing T 3 transport activity. Furthermore, we detected enhanced mRNA levels of wild-type and mutant MCT8/Mct8 after NaPB treatment. The increase in mRNA levels could be an explanation for the positive effect on protein expression and function detected for wild-type MCT8., Conclusion: NaPB is not only suitable for the treatment of mutations leading to misfolding and protein degradation, but also for a mutant wrongly sorted inside a cell which is otherwise functional., Competing Interests: No competing financial interests exist., (Copyright © 2020 by S. Karger AG, Basel.)