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Chemical chaperones are small molecules that improve protein folding, alleviating aberrant pathological phenotypes due to protein misfolding. Recent reports suggest that, in parallel with their role in relieving endoplasmic reticulum (ER) stress, chemical chaperones rescue mitochondrial function and insulin signaling. These effects may underlie their pharmacological action on metabolically demanding tissues. [ABSTRACT FROM AUTHOR]

The concentration of intracellular and extracellular potassium is tightly regulated due to the action of various ion transporters, channels, and pumps, which reside primarily in the kidney. Yet, potassium transporters and cotransporters play vital roles in all organs and cell types. Perhaps not surprisingly, defects in the biogenesis, function, and/or regulation of these proteins are linked to range of catastrophic human diseases, but to date, few drugs have been approved to treat these maladies. In this review, we discuss the structure, function, and activity of a group of potassium-chloride cotransporters, the KCCs, as well as the related sodium-potassium-chloride cotransporters, the NKCCs. Diseases associated with each of the four KCCs and two NKCCs are also discussed. Particular emphasis is placed on how these complex membrane proteins fold and mature in the endoplasmic reticulum, how non-native forms of the cotransporters are destroyed in the cell, and which cellular factors oversee their maturation and transport to the cell surface. When known, we also outline how the levels and activities of each cotransporter are regulated. Open questions in the field and avenues for future investigations are further outlined. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is an extremely devastating neurodegenerative disease, and there is no cure for it. AD is specified as the misfolding and aggregation of amyloid-β protein (Aβ) and abnormalities in hyperphosphorylated tau protein. Current approaches to treat Alzheimer's disease have had some success in slowing down the disease's progression. However, attempts to find a cure have been largely unsuccessful, most likely due to the complexity associated with AD pathogenesis. Hence, a shift in focus to better understand the molecular mechanism of Aβ processing and to consider alternative options such as chaperone proteins seems promising. Chaperone proteins act as molecular caretakers to facilitate cellular homeostasis under standard conditions. Chaperone proteins like heat shock proteins (Hsps) serve a pivotal role in correctly folding amyloid peptides, inhibiting mitochondrial dysfunction, and peptide aggregation. For instance, Hsp90 plays a significant role in maintaining cellular homeostasis through its protein folding mechanisms. In this review, we analyze the most recent studies from 2020 to 2023 and provide updates on Aβ regulation by Hsp90, BRICHOS domain chaperone, and distinctive newly reported chaperones. [ABSTRACT FROM AUTHOR]

Airway inflammation and pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα) underlie the pathophysiology of respiratory diseases, including asthma. Previously, we showed that TNFα activates the inositol-requiring enzyme 1α (IRE1α)/X-box binding protein 1 spliced (XBP1s) endoplasmic reticulum (ER) stress pathway in human airway smooth muscle (hASM) cells. The ER stress pathway is activated by the accumulation of unfolded proteins in the ER. Accordingly, chemical chaperones such as 4-phenylbutyric acid (4-PBA) may reduce ER stress activation. In the present study, we hypothesized that chemical chaperone 4-PBA mitigates TNFα-induced ER stress in hASM cells. hASM cells were isolated from bronchiolar tissue obtained from five patients with no history of smoking or respiratory diseases. The hASM cells' phenotype was confirmed via the expression of alpha-smooth muscle actin and elongated morphology. hASM cells from the same patient sample were then separated into three 12 h treatment groups: (1) TNFα (20 ng/mL), (2) TNFα + 4-PBA (1 μM, 30 min pretreatment), and (3) untreated control. The expressions of total IRE1α and phosphorylated IRE1α (pIRE1αS724) were determined through Western blotting. The splicing of XBP1 mRNA was analyzed using RT-PCR. We found that TNFα induced an increase in pIRE1αS724 phosphorylation, which was mitigated by treatment with chemical chaperone 4-PBA. We also found that TNFα induced an increase in XBP1s mRNA, which was also mitigated by treatment with chemical chaperone 4-PBA. These results support our hypothesis and indicate that chemical chaperone 4-PBA treatment mitigates TNFα-induced ER stress in hASM cells. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a heterogeneous neurodegenerative disorder with complex etiology that eventually leads to dementia. The main culprit of AD is the extracellular deposition of β-amyloid (Aβ) and intracellular neurofibrillary tangles. The protein conformational change and protein misfolding are the key events of AD pathophysiology; therefore, endoplasmic reticulum (ER) stress is an apparent consequence. ER, stress-induced unfolded protein response (UPR) mediators (viz. PERK, IRE1, and ATF6) have been reported widely in the AD brain. Considering these factors, preventing protein misfolding or aggregation of tau or amyloidogenic proteins appears to be the best approach to halt its pathogenesis. Therefore, therapies through chemical and pharmacological chaperones came to light as an alternative for the treatment of AD. Diverse studies have demonstrated 4-phenylbutyric acid (4-PBA) as a potential therapeutic agent in AD. The current review outlined the mechanism of protein misfolding, different etiological features behind the progression of AD, the significance of ER stress in AD, and the potential therapeutic role of different chaperones to counter AD. The study also highlights the gaps in current knowledge of the chaperones-based therapeutic approach and the possibility of developing chaperones as a potential therapeutic agent for AD treatment. [ABSTRACT FROM AUTHOR]

Jae-Ho Jeon,1,* Somyoung Im,1,* Hyo Shin Kim,1 Dongyun Lee,1 Kwiwan Jeong,2 Jin-Mo Ku,2 Tae-Gyu Nam1 1Department of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University ERICA campus, Ansan, Gyeonggi-do, 15588, Republic of Korea; 2Gyeonggi Bio-Center, Gyeonggido Business and Science Accelerator, Suwon, Gyeonggi-do, 16229, Republic of Korea*These authors contributed equally to this workCorrespondence: Tae-Gyu Nam, Tel +82-31-400-5807, Fax +82-31-400-5958, Email tnam@hanyang.ac.krAbstract: The endoplasmic reticulum (ER) is responsible for structural transformation or folding of de novo proteins for transport to the Golgi. When the folding capacity of the ER is exceeded or excessive accumulation of misfolded proteins occurs, the ER enters a stressed condition (ER stress) and unfolded protein responses (UPR) are triggered in order to rescue cells from the stress. Recovery of ER proceeds toward either survival or cell apoptosis. ER stress is implicated in many pathologies, such as diabetes, cardiovascular diseases, inflammatory diseases, neurodegeneration, and lysosomal storage diseases. As a survival or adaptation mechanism, chaperone molecules are upregulated to manage ER stress. Chemical versions of chaperone have been developed in search of drug candidates for ER stress-related diseases. In this review, synthetic or semi-synthetic chemical chaperones are categorized according to potential therapeutic area and listed along with their chemical structure and activity. Although only a few chemical chaperones have been approved as pharmaceutical drugs, a dramatic increase in literatures over the recent decades indicates enormous amount of efforts paid by many researchers. The efforts warrant clearer understanding of ER stress and the related diseases and consequently will offer a promising drug discovery platform with chaperone activity.Keywords: endoplasmic reticulum stress, unfolded protein response, chemical chaperone, drug discovery, diabetes, cardiovascular disease, neurodegeneration, lysosomal storage disease

Formation and accumulation of protein aggregates adversely affect intracellular processes in living cells and are negative factors in the production and storage of protein preparations. Chemical chaperones can prevent protein aggregation, but this effect is not universal and depends on the target protein structure and kinetics of its aggregation. We studied the effect of betaine (Bet) and lysine (Lys) on thermal aggregation of muscle glycogen phosphorylase b (Phb) at 48°C (aggregation order, n = 0.5), UV-irradiated Phb (UV-Phb) at 37°C (n = 1), and apo-form of Phb (apo-Phb) at 37°C (n = 2). Using dynamic light scattering, differential scanning calorimetry, and analytical ultracentrifugation, we have shown that Bet protected Phb and apo-Phb from aggregation, but accelerated the aggregation of UV-Phb. At the same time, Lys prevented UV-Phb and apo-Phb aggregation, but increased the rate of Phb aggregation. The mechanisms of chemical chaperone action on the tertiary and quaternary structures and kinetics of thermal aggregation of the target proteins are discussed. Comparison of the effects of chemical chaperones on the proteins with different aggregation kinetics provides more complete information on the mechanism of their action. [ABSTRACT FROM AUTHOR]

Organic cation transporters (OCTs) are membrane proteins that take up monoamines, cationic drugs and xenobiotics. We previously reported novel missense mutations of organic cation transporter 3 (OCT3, SLC22A3), some with drastically impacted transport capabilities compared to wildtype. For some variants, this was due to ER retention and subsequent degradation of the misfolded transporter. For other transporter families, it was previously shown that treatment of misfolded variants with pharmacological and chemical chaperones could restore transport function to a certain degree. To investigate two potentially ER-bound, misfolded variants (D340G and R348W), we employed confocal and biochemical analyses. In addition, radiotracer uptake assays were conducted to assess whether pre-treatment with chaperones could restore transporter function. We show that pre-treatment of cells with the chemical chaperone 4-PBA (4-phenyl butyric acid) leads to increased membrane expression of misfolded variants and is associated with increased transport capacity of D340G (8-fold) and R348W (1.5 times) compared to untreated variants. We herein present proof of principle that folding-deficient SLC22 transporter variants, in particular those of OCT3, are amenable to rescue by chaperones. These findings need to be extended to other SLC22 members with corroborated disease associations. [ABSTRACT FROM AUTHOR]

Alzheimer’s disease (AD) is an extremely devastating neurodegenerative disease, and there is no cure for it. AD is specified as the misfolding and aggregation of amyloid-β protein (Aβ) and abnormalities in hyperphosphorylated tau protein. Current approaches to treat Alzheimer’s disease have had some success in slowing down the disease’s progression. However, attempts to find a cure have been largely unsuccessful, most likely due to the complexity associated with AD pathogenesis. Hence, a shift in focus to better understand the molecular mechanism of Aβ processing and to consider alternative options such as chaperone proteins seems promising. Chaperone proteins act as molecular caretakers to facilitate cellular homeostasis under standard conditions. Chaperone proteins like heat shock proteins (Hsps) serve a pivotal role in correctly folding amyloid peptides, inhibiting mitochondrial dysfunction, and peptide aggregation. For instance, Hsp90 plays a significant role in maintaining cellular homeostasis through its protein folding mechanisms. In this review, we analyze the most recent studies from 2020 to 2023 and provide updates on Aβ regulation by Hsp90, BRICHOS domain chaperone, and distinctive newly reported chaperones.

Flurbiprofen, a nonsteroidal anti-inflammatory drug, reportedly exhibits chemical chaperone activity. Herein, we investigated the role of flurbiprofen in regulating serotonin transporter (SERT) function via membrane trafficking. We used COS-7 cells transiently expressing wild-type (WT) SERT or a C-terminus-deleted mutant of SERT (SERTΔCT), a misfolded protein. Flurbiprofen treatment reduced the expression of immaturely glycosylated SERT and enhanced the expression of maturely glycosylated SERT. In addition, we observed increased serotonin uptake in SERT-expressing cells. These results suggest that flurbiprofen modulates SERT function by promoting membrane trafficking. In SERTΔCT-expressing cells, flurbiprofen reduced the protein expression and uptake activity of SERTΔCT. Furthermore, flurbiprofen inhibited the formation of SERTΔCT aggregates. Studies using flurbiprofen enantiomers suggested that these effects of flurbiprofen on SERT were not mediated via cyclooxygenase inhibition. The levels of GRP78/BiP, an endoplasmic reticulum (ER) stress marker, were assessed to elucidate whether flurbiprofen can ameliorate SERTΔCT-induced ER stress. Interestingly, flurbiprofen induced GRP78/BiP expression only under ER stress conditions and not under steady-state conditions. In HRD1 E3 ubiquitin ligase knockdown cells, flurbiprofen affected the ER-associated degradation system. Collectively, the findings suggest that flurbiprofen may function as an inducer of molecular chaperones, in addition to functioning as a chemical chaperone.

Loss of proteostasis can occur due to mutations, the formation of aggregates, or general deficiency in the correct translation and folding of proteins. These phenomena are commonly observed in pathologies, but most significantly, loss of proteostasis characterizes aging. This loss leads to the chronic activation of stress responses and has a generally deleterious impact on the organism. While finding molecules that can alleviate these symptoms is an important step toward solutions for these conditions, some molecules might be mischaracterized on the way. 4‐phenylbutyric acid (4PBA) is known for its role as a chemical chaperone that helps alleviate endoplasmic reticulum (ER) stress, yet a scan of the literature reveals that no biochemical or molecular experiments have shown any protein refolding capacity. Here, we show that 4PBA is a conserved weak inhibitor of mRNA translation, both in vitro and in cellular systems, and furthermore—it does not promote protein folding nor prevents aggregation. 4PBA possibly alleviates proteostatic or ER stress by inhibiting protein synthesis, allowing the cells to cope with misfolded proteins by reducing the protein load. Better understanding of 4PBA biochemical mechanisms will improve its usage in basic science and as a drug in different pathologies, also opening new venues for the treatment of different diseases. [ABSTRACT FROM AUTHOR]

The aggregation of proteins into ordered fibrillar structures called amyloids, and their disintegration represent major unsolved problems that limit the therapeutic applications of several proteins. For example, insulin, commonly used for the treatment of diabetes, is susceptible to amyloid formation upon exposure to non-physiological conditions, resulting in a loss of its biological activity. Here, we report a novel amphiphilic molecule called PAD-S, which acts as a chemical chaperone and completely inhibits fibrillation of insulin and its biosimilars. Mechanistic investigations and molecular docking lead to the conclusion that PAD-S binds to key hydrophobic regions of native insulin, thereby preventing its self-assembly. PAD-S treated insulin was biologically active as indicated by its ability to phosphorylate Akt, a protein in the insulin signalling pathway. PAD-S is non-toxic and protects cells from insulin amyloid induced cytotoxicity. The high aqueous solubility and easy synthetic accessibility of PAD-S facilitates its potential use in commercial insulin formulations. Notably, PAD-S successfully disintegrated preformed insulin fibrils to non-toxic smaller fragments. Since the structural and mechanistic features of amyloids are common to several human pathologies, the understanding of the amyloid disaggregation activity of PAD-S will inform the development of small molecule disaggregators for other amyloids. [Display omitted] • PAD-S, a water-soluble small molecule, preferentially interacts with the hydrophobic regions of insulin. • The interaction of PAD-S with insulin leads to highly efficient amyloid inhibition and disintegration. • PAD-S treated insulin does not exhibit amyloid mediated cytotoxicity and is biologically active. [ABSTRACT FROM AUTHOR]

Background: Monocarboxylate transporter 8 (MCT8) deficiency is a rare genetic disease leading to a severe developmental delay due to a lack of thyroid hormones (THs) during critical stages of human brain development. Some MCT8-deficient patients are not as severely affected as others. Previously, we hypothesized that these patients' mutations do not affect the functionality but destabilize the MCT8 protein, leading to a diminished number of functional MCT8 molecules at the cell surface. Methods: We have already demonstrated that the chemical chaperone sodium phenylbutyrate (NaPB) rescues the function of these mutants by stabilizing their protein expression in an overexpressing cell system. Here, we expanded our previous work and used iPSC (induced pluripotent stem cell)-derived brain microvascular endothelial-like cells (iBMECs) as a physiologically relevant cell model of human origin to test for NaPB responsiveness. The effects on mutant MCT8 expression and function were tested by Western blotting and radioactive uptake assays. Results: We found that NaPB rescues decreased mutant MCT8 expression and restores transport function in iBMECs carrying patient's mutation MCT8-P321L. Further, we identified MCT10 as an alternative TH transporter in iBMECs that contributes to triiodothyronine uptake, the biological active TH. Our results indicate an upregulation of MCT10 after NaPB treatment. In addition, we detected an increase in thyroxine (T4) uptake after NaPB treatment that was not mediated by rescued MCT8 but an unidentified T4 transporter. Conclusions: We demonstrate that NaPB is suitable to stabilize a pathogenic missense mutation in a human-derived cell model. Further, it activates TH transport independent of MCT8. Both options fuel future studies to investigate repurposing the Food and Drug Administration-approved drug NaPB in selected cases of MCT8 deficiency. [ABSTRACT FROM AUTHOR]

[Display omitted] The abnormal accumulation of fibrillar α-synuclein in the substantia nigra contributes to Parkinson's disease (PD). Chemical chaperones like 4-phenyl butyric acid (4PBA) show neuroprotective potential, but high doses are required. A derivative, 5-phenyl valeric acid (5PVA), has reported therapeutic potential for PD by reducing Pael-R expression. This study assessed 5PVA's efficacy in PD animals and its molecular mechanism. In vitro studies revealed 5PVA's anti-aggregation ability against alpha-synuclein and neuroprotective effects on SHSY5Y neuroblastoma cells exposed to rotenone. PD-like symptoms were induced in SD rats with rotenone, followed by 5PVA treatment at 100 mg/kg and 130 mg/kg. Behavioral analysis showed significant improvement in memory and motor activity with 5PVA administration. Histopathological studies demonstrated normal neuronal histoarchitecture in mid-brain tissue sections of 5PVA-treated animals compared to the PD group. mRNA studies revealed significant suppression in the expression of various protein folding and heat-shock protein markers in the 5PVA-treated group. In conclusion, 5PVA, with its anti-aggregation ability against alpha-synuclein, acts as a chemical chaperone, showing potential as a therapeutic candidate for PD treatment. [ABSTRACT FROM AUTHOR]

For hard tissue formation, cellular mechanisms, involved in protein folding, processing, and secretion play important roles in the endoplasmic reticulum (ER). In pathological and regeneration conditions, ER stress hinders proper formation and secretion of proteins, and tissue regeneration by unfolded protein synthesis. 4-Phenylbutyric acid (4PBA) is a chemical chaperone that alleviates ER stress through modulation in proteins folding and protein trafficking. However, previous studies about 4PBA only focused on the metabolic diseases rather than on hard tissue formation and regeneration. Herein, we evaluated the function of 4PBA in dentin regeneration using an exposed pulp animal model system via a local delivery method as a drug repositioning strategy. Our results showed altered morphological changes and cellular physiology with histology and immunohistochemistry. The 4PBA treatment modulated the inflammation reaction and resolved ER stress in the early stage of pulp exposure. In addition, 4PBA treatment activated blood vessel formation and TGF-β1 expression in the dentin-pulp complex. Micro-computed tomography and histological examinations confirmed the facilitated formation of the dentin bridge in the 4PBA-treated specimens. These results suggest that proper modulation of ER stress would be an important factor for secretion and patterned formation in dentin regeneration. [ABSTRACT FROM AUTHOR]

Cataract is one of the most prevalent protein aggregation disorders and still the most common cause of vision loss worldwide. The metabolically quiescent core region of the human lens lacks cellular or protein turnover; it has therefore evolved remarkable mechanisms to resist light-scattering protein aggregation for a lifetime. We now report that one such mechanism involves an unusually abundant lens metabolite, myo-inositol, suppressing aggregation of lens crystallins. We quantified aggregation suppression using our previously well-characterized in vitro aggregation assays of oxidation-mimicking human γD-crystallin variants and investigated myo-inositol’s molecular mechanism of action using solution NMR, negative-stain TEM, differential scanning fluorometry, thermal scanning Raman spectroscopy, turbidimetry in redox buffers, and free thiol quantitation. Unlike many known chemical chaperones, myo-inositol’s primary target was not the native, unfolded, or final aggregated states of the protein; rather, we propose that it was the rate-limiting bimolecular step on the aggregation pathway. Given recent metabolomic evidence that it is severely depleted in human cataractous lenses compared to age-matched controls, we suggest that maintaining or restoring healthy levels of myo-inositol in the lens may be a simple, safe, and globally accessible strategy to prevent or delay lens opacification due to age-onset cataract.

For hard tissue formation, cellular mechanisms, involved in protein folding, processing, and secretion play important roles in the endoplasmic reticulum (ER). In pathological and regeneration conditions, ER stress hinders proper formation and secretion of proteins, and tissue regeneration by unfolded protein synthesis. 4-Phenylbutyric acid (4PBA) is a chemical chaperone that alleviates ER stress through modulation in proteins folding and protein trafficking. However, previous studies about 4PBA only focused on the metabolic diseases rather than on hard tissue formation and regeneration. Herein, we evaluated the function of 4PBA in dentin regeneration using an exposed pulp animal model system via a local delivery method as a drug repositioning strategy. Our results showed altered morphological changes and cellular physiology with histology and immunohistochemistry. The 4PBA treatment modulated the inflammation reaction and resolved ER stress in the early stage of pulp exposure. In addition, 4PBA treatment activated blood vessel formation and TGF-β1 expression in the dentin-pulp complex. Micro-computed tomography and histological examinations confirmed the facilitated formation of the dentin bridge in the 4PBA-treated specimens. These results suggest that proper modulation of ER stress would be an important factor for secretion and patterned formation in dentin regeneration.

More than 100 different mutations in the RPE65 gene are associated with inherited retinal degeneration. Although some missense mutations have been shown to abolish isomerase activity of RPE65, the molecular bases leading to loss of function and retinal degeneration remain incompletely understood. Here we show that several missense mutations resulted in significant decrease in expression level of RPE65 in the human retinal pigment epithelium cells. The 26S proteasome non-ATPase regulatory subunit 13, a newly identified negative regulator of RPE65, mediated degradation of mutant RPE65s, which were misfolded and formed aggregates in the cells. Many mutations, including L22P, T101I, and L408P, were mapped on nonactive sites of RPE65. Enzyme activities of these mutant RPE65s were significantly rescued at low temperature, whereas mutant RPE65s with a distinct active site mutation could not be rescued under the same conditions. 4-phenylbutyrate (PBA) displayed a significant synergistic effect on the low temperature-mediated rescue of the mutant RPE65s. Our results suggest that a low temperature eye mask and PBA, a FDA-approved oral medicine, may provide a promising "protein repair therapy" that can enhance the efficacy of gene therapy for delaying retinal degeneration caused by RPE65 mutations.

Protein misfolding is involved in a large number of diseases, among which cystic fibrosis. Complex intra- and inter-domain folding defects associated with mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, among which p.Phe508del (F508del), have recently become a therapeutical target. Clinically approved correctors such as VX-809, VX-661, and VX-445, rescue mutant protein. However, their binding sites and mechanisms of action are still incompletely understood. Blind docking onto the 3D structures of both the first membrane-spanning domain (MSD1) and the first nucleotide-binding domain (NBD1), followed by molecular dynamics simulations, revealed the presence of two potential VX-809 corrector binding sites which, when mutated, abrogated rescue. Network of amino acids in the lasso helix 2 and the intracellular loops ICL1 and ICL4 allosterically coupled MSD1 and NBD1. Corrector VX-445 also occupied two potential binding sites on MSD1 and NBD1, the latter being shared with VX-809. Binding of both correctors on MSD1 enhanced the allostery between MSD1 and NBD1, hence the increased efficacy of the corrector combination. These correctors improve both intra-domain folding by stabilizing fragile protein–lipid interfaces and inter-domain assembly via distant allosteric couplings. These results provide novel mechanistic insights into the rescue of misfolded proteins by small molecules. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) is very sensitive to the nutritional and energy states of the cells. Disruption of ER homeostasis leads to the accumulation of unfolded/misfolded proteins in the ER lumen, which is defined as ER stress. ER stress triggers the unfolded protein response (UPR). It is suggested that chronic ER stress is associated with obesity and leptin resistance. We investigated the role of ER stress and the effect of the ER stress inhibitor phenylbutyric acid (PBA) of ER stress, in obesity, as well as their impact on leptin signaling. This study involved twenty-four lean and twenty-four leptin-deficient (ob/ob) mice divided into PBA- and vehicle-treated groups. Pancreatic islets were isolated, incubated with leptin for 48 h, and assayed for the expression of CHOP and XBP1s (UPR signaling indicators) and SOCS3 (regulator of leptin signaling) by RT-qPCR. The expression levels of XBP1s and CHOP were markedly increased in the ob/ob controls compared to other groups with and without leptin treatment. No significant differences in the XBP1s and CHOP expression levels were found between the PBA-treated ob/ob and lean mice. SOCS3 expression was significantly upregulated in the PBA-treated ob/ob mice compared to the ob/ob controls after leptin treatment; but no significant difference in the SOCS3 expression was found between the PBA-treated ob/ob and lean mice with and without leptin treatment. Our findings suggested that ER stress plays an important role in the pathology of obesity, while PBA reduces ER stress and may potentially ameliorate leptin signaling. [ABSTRACT FROM AUTHOR]

• At the intracellular level, 4-PBA prevents collagen accumulation and increases proteins secretion in OI osteoblasts, relieving stress and normalizing ER morphology. • At the extracellular level, 4-PBA increases collagen matrix incorporation in the OI murine models. • 4-PBA promotes OI osteoblasts mineralization by increasing alkaline phosphatase expression and activity. • Upon targeting osteoblast stress, an amelioration of extracellular matrix is achieved, making 4-PBA an appealing drug to complement current OI therapy. Most cases of dominantly inherited osteogenesis imperfecta (OI) are caused by glycine substitutions in the triple helical domain of type I collagen α chains, which delay collagen folding, and cause the synthesis of collagen triple helical molecules with abnormal structure and post-translational modification. A variable extent of mutant collagen ER retention and other secondary mutation effects perturb osteoblast homeostasis and impair bone matrix quality. Amelioration of OI osteoblast homeostasis could be beneficial both to osteoblast anabolic activity and to the content of the extracellular matrix they deposit. Therefore, the effect of the chemical chaperone 4-phenylbutyrate (4-PBA) on cell homeostasis, collagen trafficking, matrix production and mineralization was investigated in primary osteoblasts from two murine models of moderate OI, Col1a1+/G349C and Col1a2+/G610C. At the cellular level, 4-PBA prevented intracellular accumulation of collagen and increased protein secretion, reducing aggregates within the mutant cells and normalizing ER morphology. At the extracellular level, increased collagen incorporation into matrix, associated with more mature collagen fibrils, was observed in osteoblasts from both models. 4-PBA also promoted OI osteoblast mineral deposition by increasing alkaline phosphatase expression and activity. Targeting osteoblast stress with 4-PBA improved both cellular and matrix abnormalities in culture, supporting further in vivo studies of its effect on bone tissue composition, strength and mineralization as a potential treatment for classical OI. [ABSTRACT FROM AUTHOR]

Lafora disease (LD) is one of the progressive and fatal forms of a neurodegenerative disorder and is characterized by teenage-onset myoclonic seizures. Neuropathological changes in LD include the formation of abnormal glycogen as Lafora bodies, gliosis, and neuroinflammation. LD is caused by defects in the gene coding for phosphatase (laforin) or ubiquitin ligase (malin). Mouse models of LD, developed by targeted disruption of these two genes, develop most symptoms of LD and show increased susceptibility to induced seizures. Studies on mouse models also suggest that defective autophagy might contribute to LD etiology. In an attempt to understand the specific role of autophagy in LD pathogenesis, in this study, we fed LD animals with trehalose, an inducer of autophagy, for 3 months and looked at its effect on the neuropathology and seizure susceptibility. We demonstrate here that trehalose ameliorates gliosis, neuroinflammation, and endoplasmic reticulum stress and reduces susceptibility to induced seizures in LD animals. However, trehalose did not affect the formation of Lafora bodies, suggesting the epileptic phenotype in LD could be either secondary to or independent of Lafora bodies. Taken together, our results suggest that autophagy inducers can be considered as potential therapeutic molecules for Lafora disease. [ABSTRACT FROM AUTHOR]

Context: Monocarboxylate transporter 8 (MCT8) deficiency is a rare X-chromosomal inherited disease leading to severe cognitive impairment, muscular hypotonia and symptoms of peripheral thyrotoxicosis. Experimental approaches aiming to functionally rescue mutant MCT8 activity by the chemical chaperone phenylbutyrate (PB) demonstrated promising effects in vitro for several MCT8 missense mutations., Objective: The objective was to evaluate biochemical and clinical effects of PB in doses equivalent to those approved for the treatment of urea cycle disorders in a boy with MCT8 deficiency due to a novel MCT8 missense mutation c.703G > T (p.V235L)., Results: During a treatment period of 13 months, PB led to a significant decrease of elevated TSH and T3 serum concentrations, while fT4 increased. Weight z-score of the toddler remained remarkably stable during the treatment period. Neurodevelopmental assessments (BSID-III) revealed a slight increase of gross motor skills from developmental age 4 to 6 months. However, increasing liver enzyme serum activities and accumulation of phenylacetate (PAA) in urine led to treatment interruptions and dose alterations. In vitro analyses in MDCK1 cells confirmed the pathogenicity of MCT8 p.V235L. However, while PB increased expression of the mutant protein, it did not rescue T3 transport, suggesting a PB effect on thyroid function tests independent of restoring MCT8 activity., Conclusion: In a clinical attempt of PB treatment in MCT8 deficiency we observed a significant improvement of thyroid hormone function tests, tendencies towards body weight stabilization and slight neurodevelopmental improvement. Hepatotoxicity of PB may be a limiting factor in MCT8 deficiency and requires further investigation., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Reteplase is a non-glycosylated and recombinant form of tissue type plasminogen activator, which is produced in Escherichia coli. However, its overexpression usually leads to formation of inactive aggregates or inclusion bodies. In the present study, we report on the development of optimized processes for isolation, solubilization, and refolding of reteplase inclusion bodies to recover active protein. After protein overexpression in E. coli BL21 (DE3) inclusion bodies were isolated by cell disruption and repeated wash of pellet with buffer containing Triton X-100. To solubilize the inclusion bodies, different types, concentrations, pHs, and additives of denaturing agents were used. Rapid micro dilution method was applied for refolding of solubilized reteplase. Different chemical additives including sugars, alcohols, polymers, detergents, amino acids, kosmotropic, and chaotropic salts, reducing agents, and buffering agents were used in the refolding buffer. To evaluate the biological activity of refolded reteplase, an indirect chromogenic assay was performed. The best solubilizing agent for dissolving reteplase inclusion bodies was 6 M urea at pH 12. The optimized buffer for refolding of solubilized reteplase was found to be 1.15 M glucose, 9.16 mM imidazole, and 0.16 M sorbitol which resulted in high yield of biologically active protein. Our results indicate type, concentration, and pH of solvent and type, concentration, and combination of chemical additives can significantly influence the yield of inclusion bodies solubilization and refolding.

Catechin compounds have potential benefits for recombinant monoclonal antibody (Mab) production as chemical additives in cell culture media. In this study, four catechin compounds catechin (Cat), epicatechin (EC), gallocatechin‐gallate (GCG), and epigallocatechin‐gallate (EGCG) were added to cell culture media (at 50 μM) and their effects on the recombinant Chinese hamster ovary (CHO) cell culture, specific productivity, and Mab quality were assessed. The results indicate that the improvement of specific productivity was linked to cell growth inhibition. All catechins caused cell phase growth arrest by lowering the number of cells in the G1/G0 phase and increasing the cells in the S and G2/M phases. Late addition of the catechin resulted in a significantly higher final IgG concentration. Cat and EC caused an improvement in the final antibody titer of 1.5 ± 0.1 and 1.3 ± 0.1 fold, respectively. Catechins with a galloyl group (GCG and EGCG) arrested cell growth and reduced cell specific productivity at the concentrations tested. The Cat‐treated IgG was found to have reduced acidic species with a corresponding increase in the main peak. [ABSTRACT FROM AUTHOR]

The polyglutamine (polyQ) diseases are a group of inherited neurodegenerative diseases that include Huntington's disease, various spinocerebellar ataxias, spinal and bulbar muscular atrophy, and dentatorubral pallidoluysian atrophy. They are caused by the abnormal expansion of a CAG repeat coding for the polyQ stretch in the causative gene of each disease. The expanded polyQ stretches trigger abnormal β-sheet conformational transition and oligomerization followed by aggregation of the polyQ proteins in the affected neurons, leading to neuronal toxicity and neurodegeneration. Disease-modifying therapies that attenuate both symptoms and molecular pathogenesis of polyQ diseases remain an unmet clinical need. Here we identified arginine, a chemical chaperone that facilitates proper protein folding, as a novel compound that targets the upstream processes of polyQ protein aggregation by stabilizing the polyQ protein conformation. We first screened representative chemical chaperones using an in vitro polyQ aggregation assay, and identified arginine as a potent polyQ aggregation inhibitor. Our in vitro and cellular assays revealed that arginine exerts its anti-aggregation property by inhibiting the toxic β-sheet conformational transition and oligomerization of polyQ proteins before the formation of insoluble aggregates. Arginine exhibited therapeutic effects on neurological symptoms and protein aggregation pathology in Caenorhabditis elegans, Drosophila, and two different mouse models of polyQ diseases. Arginine was also effective in a polyQ mouse model when administered after symptom onset. As arginine has been safely used for urea cycle defects and for mitochondrial myopathy, encephalopathy, lactic acid and stroke syndrome patients, and efficiently crosses the blood-brain barrier, a drug-repositioning approach for arginine would enable prompt clinical application as a promising disease-modifier drug for the polyQ diseases. [ABSTRACT FROM AUTHOR]

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a human FAD‐dependent enzyme that plays a crucial role in the antioxidant defense system. A naturally occurring single‐nucleotide polymorphism (NQO1*2) in the NQO1 gene leads to an amino acid substitution (P187S), which severely compromises the activity and stability of the enzyme. The NQO1*2 genotype has been linked to a higher risk for several types of cancer and poor survival rate after anthracycline‐based chemotherapy. In this study, we show that a small molecular chaperone (N‐(2‐bromophenyl)pyrrolidine‐1‐sulfonamide) repopulates the native wild‐type conformation. As a consequence of the stabilizing effect, the enzymatic activity of the P187S variant protein is strongly improved in the presence of the molecular chaperone in vitro. [ABSTRACT FROM AUTHOR]

Background: Pilsicainide, classified as a relatively selective Na+ channel blocker, also has an inhibitory action on the rapidly-activating delayed-rectifier K+ current (IKr) through human ether-a-go-go-related gene (hERG) channels. We studied the effects of chronic exposure to pilsicainide on the expression of wild-type (WT) hERG proteins and WT-hERG channel currents, as well as on the expression of mutant hERG proteins, in a heterologous expression system. Methods: HEK293 cells stably expressing WT or mutant hERG proteins were subjected to Western blotting, immunofluorescence microscopy and patch-clamp experiments. Results: Acute exposure to pilsicainide at 0.03–10 μM influenced neither the expression of WT-hERG proteins nor WT-hERG channel currents. Chronic treatment with 0.03–10 μM pilsicainide for 48 h, however, increased the expression of WT-hERG proteins and channel currents in a concentration-dependent manner. Chronic treatment with 3 μM pilsicainide for 48 h delayed degradation of WT-hERG proteins and increased the channels expressed on the plasma membrane. A cell membrane-impermeant pilsicainide derivative did not influence the expression of WT-hERG, indicating that pilsicainide stabilized the protein inside the cell. Pilsicainide did not influence phosphorylation of Akt (protein kinase B) or expression of heat shock protein families such as HSF-1, hsp70 and hsp90. E4031, a chemical chaperone for hERG, abolished the pilsicainide effect on hERG. Chronic treatment with pilsicainide could also increase the protein expression of trafficking-defective mutant hERG, G601S and R752W. Conclusions: Pilsicainide penetrates the plasma membrane, stabilizes WT-hERG proteins by acting as a chemical chaperone, and enhances WT-hERG channel currents. This mechanism could also be applicable to modulations of certain mutant-hERG proteins.

The endoplasmic reticulum (ER) is responsible for folding secretory and membrane proteins, but disturbed ER proteostasis may lead to protein aggregation and subsequent cellular and clinical pathologies. Chemical chaperones have recently emerged as a potential therapeutic approach for ER stress-related diseases. Here, we identified 2-phenylimidazo[2,1-b]benzothiazole derivatives (IBTs) as chemical chaperones in a cell-based high-throughput screen. Biochemical and chemical biology approaches revealed that IBT21 directly binds to unfolded or misfolded proteins and inhibits protein aggregation. Finally, IBT21 prevented cell death caused by chemically induced ER stress and by a proteotoxin, an aggression-prone prion protein. Taken together, our data show the promise of IBTs as potent chemical chaperones that can ameliorate diseases resulting from protein aggregation under ER stress.

Osteogenesis imperfecta (OI) types VII, VIII and IX, caused by recessive mutations in cartilage-associated protein (CRTAP), prolyl-3-hydroxylase 1 (P3H1) and cyclophilin B (PPIB), respectively, are characterized by the synthesis of overmodified collagen. The genes encode for the components of the endoplasmic reticulum (ER) complex responsible for the 3-hydroxylation of specific proline residues in type I collagen. Our study dissects the effects of mutations in the proteins of the complex on cellular homeostasis, using primary fibroblasts from seven recessive OI patients. In all cell lines, the intracellular retention of overmodified type I collagen molecules causes ER enlargement associated with the presence of protein aggregates, activation of the PERK branch of the unfolded protein response and apoptotic death. The administration of 4-phenylbutyrate (4-PBA) alleviates cellular stress by restoring ER cisternae size, and normalizing the phosphorylated PERK (p-PERK):PERK ratio and the expression of apoptotic marker. The drug also has a stimulatory effect on autophagy. We proved that the rescue of cellular homeostasis following 4-PBA treatment is associated with its chaperone activity, since it increases protein secretion, restoring ER proteostasis and reducing PERK activation and cell survival also in the presence of pharmacological inhibition of autophagy. Our results provide a novel insight into the mechanism of 4-PBA action and demonstrate that intracellular stress in recessive OI can be alleviated by 4-PBA therapy, similarly to what we recently reported for dominant OI, thus allowing a common target for OI forms characterized by overmodified collagen. This article has an associated First Person interview with the first author of the paper.

The cellular response to endoplasmic reticulum (ER) stress accompanies plasma cell maturation and is one of triggers and cofactors of the local inflammatory response. Chemical chaperones, low-molecular substances that eliminate pathological ER stress, are proposed as means of treating pathologies associated with ER stress. The aim of this study was to evaluate the effect and mechanisms of influence of chemical chaperones on the humoral response in a low-dose model of allergy. The allergic immune response was induced in BALB/c mice by repeated administration of ovalbumin at a dose of 100 ng for 6 weeks. Some animals were injected with both the antigen and the chemical chaperones, TUDCA (tauroursodeoxycholic acid) or 4-PBA (4-phenylbutyrate). Administration of TUDCA, but not 4-PBA, suppressed production of allergen-specific IgE (a 2.5-fold decrease in titer). None of the chemical chaperones affected the production of specific IgG1. The effect of TUDCA was associated with suppression of the switch to IgE synthesis in regional lymph nodes. This phenomenon was associated with suppressed expression of genes encoding cytokines involved in type 2 immune response, especially Il4 and Il9, which in turn could be caused by suppression of IL-33 release. In addition, TUDCA significantly suppressed expression of the cytokine APRIL, and to a lesser extent, BAFF. Thus, TUDCA inhibition of the allergy-specific IgE production is due to suppression of the release of IL-33 and a decrease in the production of type 2 immune response cytokines, as well as suppression of the expression of the cytokines APRIL and BAFF.

Misfolded or unfolded proteins are accumulated in lumen of endoplasmic reticulum (ER) in ER stress condition. It has been implicated in many pathological conditions such as Alzheimer's disease, diabetic retinopathy, atherosclerosis, β-cell apoptosis and lung inflammation. We found a series of N-substituted-2-arylcarbonylhydrazinecarbothioamides to potently decrease ER stress signal, showing up to almost 300-fold better activity than 1-hydroxynaphthoic acid and tauro-ursodesoxycholic acid, positive controls, respectively. Structure−activity relationship (SAR) study showed that 2-arylcarbonyl moiety is critical for the activity of the hydrazinecarbothioamide analogues and side chains tethering on thioamide moiety were relatively insensitive to the activity. Some analogues were found to consistently exert the potency under more physiologically relevant condition where ER stress was induced by palmitic acid. ER stress markers such as CHOP and phosphorylated eIF2α and PERK were accordingly decreased in western blotting upon treatment of compound 4h. Potential ER stress inhibitory activity and novel structures could provide a novel platform for new chemical chaperone and therapy for protein misfolding diseases. [ABSTRACT FROM AUTHOR]

In the past two decades, significant advances have been made in the etiology of lipid disorders. Concomitantly, the discovery of liporegulatory functions of certain short-chain fatty acids has generated interest in their clinical applications. In particular, butyric acid (BA) and its derivative, 4-phenylbutyric acid (PBA), which afford health benefits against lipid disorders while being generally well tolerated by animals and humans have been assessed clinically. This review examines the evidence from cell, animal and human studies pertaining to the lipid-regulating effects of BA and PBA, their molecular mechanisms and therapeutic potential. Collectively, the evidence supports the view that intakes of BA and PBA benefit lipid homeostasis across biological systems. We reviewed the evidence that BA and PBA downregulate de novo lipogenesis, ameliorate lipotoxicity, slow down atherosclerosis progression, and stimulate fatty acid β-oxidation. Central to their mode of action, BA appears to function as a histone deacetylase (HDAC) inhibitor while PBA acts as a chemical chaperone and/or a HDAC inhibitor. Areas of further inquiry include the effects of BA and PBA on adipogenesis, lipolysis and apolipoprotein metabolism. [ABSTRACT FROM AUTHOR]

Excessive stimulation of ionotropic glutamate receptor is associated with glutamate-mediated excitotoxicity, thereby causing oxidative imbalance and mitochondrial dysfunction, resulting in the excitotoxic death of neurons. Eminent role of endoplasmic reticulum under glutamate-induced excitotoxicity has been highlighted in numerous literatures which have been observed to trigger endoplasmic reticulum stress (ER stress) as well as regulating oxidative stress. However, combating ER stress in excitotoxic neurons can provide a novel approach to alleviate the mitochondrial dysfunctioning and ROS generation. Therefore, we propose to investigate the cross-communication of α-amino-3-hydroxy-5-methyl-4-isoxzole-propionate (AMPA) excitotoxicity-induced oxidative injury with ER stress by employing ER stress inhibitor—4-phenlybutyric acid (4-PBA). Male SD rats were divided into four groups viz sham group (group 1), AMPA (10 mM)-induced excitotoxic group (group 2), curative group of AMPA-induced excitotoxic animals given 4-PBA at a dose of 100 mg/kg body weight (group 3), and alone 4-PBA treatment group (100 mg/kg body weight) (group 4). Animals were sacrificed after 15 days of treatment, and hippocampi were analyzed for histopathological examination, ROS, inflammatory markers, mitochondrial dysfunction, and ER stress markers. AMPA-induced excitotoxicity exhibited a significant increase in the levels of ROS, upregulated ER stress markers, inflammation markers, and compromised mitochondrial functioning in the hippocampus. However, 4-PBA administration significantly curtailed the AMPA-induced excitotoxic insult. This study suggests that targeting ER stress with a chemical chaperone can provide a better therapeutic intervention for neurological disorders involving excitotoxicity, and thus, it opens a new avenue to screen chemical chaperones for the therapeutic modalities. [ABSTRACT FROM AUTHOR]

Autosomal recessive bestrophinopathy (ARB) is a retinopathy caused by mutations in the bestrophin-1 protein, which is thought to function as a Ca2+-gated Cl− channel in the basolateral surface of the retinal pigment epithelium (RPE). Using a stably transfected polarised epithelial cell model, we show that four ARB mutant bestrophin-1 proteins were mislocalised and subjected to proteasomal degradation. In contrast to the wild-type bestrophin-1, each of the four mutant proteins also failed to conduct Cl− ions in transiently transfected cells as determined by whole-cell patch clamp. We demonstrate that a combination of two clinically approved drugs, bortezomib and 4-phenylbutyrate (4PBA), successfully restored the expression and localisation of all four ARB mutant bestrophin-1 proteins. Importantly, the Cl− conductance function of each of the mutant bestrophin-1 proteins was fully restored to that of wild-type bestrophin-1 by treatment of cells with 4PBA alone. The functional rescue achieved with 4PBA is significant because it suggests that this drug, which is already approved for long-term use in infants and adults, might represent a promising therapy for the treatment of ARB and other bestrophinopathies resulting from missense mutations in BEST1.

An elevated level of endoplasmic reticulum (ER) stress is considered an aggravating factor for inflammatory bowel disease (IBD). To develop an ER-stress attenuator that is effective against colitis, 4-phenylbutyric acid (4-PBA), a chemical chaperone that alleviates ER stress, was conjugated with acidic amino acids to yield 4-PBA-glutamic acid (PBA-GA) and 4-PBA-aspartic acid (PBA-AA) conjugates. The PBA derivatives were converted to 4-PBA in the cecal contents, and the conversion was greater with PBA-GA than that with PBA-AA. After oral administration of PBA-GA (oral PBA-GA), up to 2.7 mM PBA was detected in the cecum, whereas 4-PBA was not detected in the blood, indicating that PBA-GA predominantly targeted the large intestine. In 2,4-dinitrobenzenesulfonic acid-induced colitis in rats, oral PBA-GA alleviated the damage and inflammation in the colon and substantially reduced the elevated levels of ER stress marker proteins in the inflamed colon. Moreover, PBA-GA was found to be as effective as the currently used anti-IBD drug, sulfasalazine. In conclusion, PBA-GA is a colon-targeted prodrug of 4-PBA and is effective against rat colitis probably via the attenuation of ER stress in the inflamed colon.

Alzheimer's disease (AD) is an extremely devastating neurodegenerative disease, and there is no cure for it. AD is specified as the misfolding and aggregation of amyloid-β protein (Aβ) and abnormalities in hyperphosphorylated tau protein. Current approaches to treat Alzheimer's disease have had some success in slowing down the disease's progression. However, attempts to find a cure have been largely unsuccessful, most likely due to the complexity associated with AD pathogenesis. Hence, a shift in focus to better understand the molecular mechanism of Aβ processing and to consider alternative options such as chaperone proteins seems promising. Chaperone proteins act as molecular caretakers to facilitate cellular homeostasis under standard conditions. Chaperone proteins like heat shock proteins (Hsps) serve a pivotal role in correctly folding amyloid peptides, inhibiting mitochondrial dysfunction, and peptide aggregation. For instance, Hsp90 plays a significant role in maintaining cellular homeostasis through its protein folding mechanisms. In this review, we analyze the most recent studies from 2020 to 2023 and provide updates on Aβ regulation by Hsp90, BRICHOS domain chaperone, and distinctive newly reported chaperones.

Graphical abstract Highlights • Conjugation of tauroursodeoxycholic acid (TUDCA) to Lys-27 of exendin-4 (Ex4). • TUDCA-Lys27-Ex4 (TUM1-Ex4) showed strong activity as a GLP-1 receptor agonist. • TUM1-Ex4 reduced the expression of GRP78 and levels of phosphorylated eIF2α. Abstract Endoplasmic reticulum stress has been considered a major cause of pancreatic β-cell dysfunction and apoptosis leading to diabetes. Glucagon-like peptide-1 receptor activation and chemical chaperones have been known to reduce endoplasmic reticulum stress and improve β-cell function and survival. The purpose of this study was to prepare and evaluate the chemical chaperone tauroursodeoxycholic acid-conjugated exendin-4 as a protective agent for pancreatic β-cells. Mono-tauroursodeoxycholic acid-Lys27-exendin-4 conjugate (TUM1-Ex4) showed better receptor binding affinity than other conjugates with strong in vitro insulinotropic activity in rat pancreatic β-cells and in vivo hypoglycemic activity in type 2 diabetic db/db mice. In INS-1 cells under endoplasmic reticulum stress induced by thapsigargin, TUM1-Ex4 promoted cell survival in a dose-dependent manner. In western blot analysis, TUM1-Ex4 reduced the expression of the endoplasmic reticulum stress marker GRP78 and phosphorylation of the translation initiation factor eIF2α. These results reveal that TUM1-Ex4 accelerates translational recovery and contributes to β-cell protection and survival. The present study indicates that the chemical chaperone-coupled glucagon-like peptide-1 receptor agonist is a feasible therapeutic strategy to enhance β-cell function and survival. [ABSTRACT FROM AUTHOR]

Mutations in α-actinin-4 (actinin-4) result in hereditary focal segmental glomerulosclerosis (FSGS) in humans. Actinin-4 mutants induce podocyte injury because of dysregulation of the cytoskeleton and proteotoxicity. Injury may be associated with endoplasmic reticulum (ER) stress and polyubiquitination of proteins. We assessed if the chemical chaperone 4-phenylbutyrate (4-PBA) can ameliorate the proteotoxicity of an actinin-4 mutant. Actinin-4 K255E, which causes FSGS in humans (K256E in the mouse), showed enhanced ubiquitination, accelerated degradation, aggregate formation, and enhanced association with filamentous (F)-actin in glomerular epithelial cells (GECs). The mutant disrupted ER function and stimulated autophagy. 4-PBA reduced actinin-4 K256E aggregation and its tight association with F-actin. Transgenic mice that express actinin-4 K256E in podocytes develop podocyte injury, proteinuria, and FSGS in association with glomerular ER stress. Treatment of these mice with 4-PBA in the drinking water over a 10-wk period significantly reduced albuminuria and ER stress. Another drug, celastrol, which enhanced expression of ER and cytosolic chaperones in GECs, tended to reduce actinin-4 aggregation but did not decrease the tight association of actinin-4 K256E with F-actin and did not reduce albuminuria in actinin-4 K256E transgenic mice. Thus, chemical chaperones, such as 4-PBA, may represent a novel therapeutic approach to certain hereditary glomerular diseases. [ABSTRACT FROM AUTHOR]

The clinical phenotype in osteogenesis imperfecta (OI) is attributed to the dominant negative function of mutant type I collagen molecules in the extracellular matrix, by altering its structure and function. Intracellular retention of mutant collagen has also been reported, but its effect on cellular homeostasis is less characterized. Using OI patient fibroblasts carrying mutations in the α1(I) and α2(I) chains we demonstrate that retained collagen molecules are responsible for endoplasmic reticulum (ER) enlargement and activation of the unfolded protein response (UPR) mainly through the eukaryotic translation initiation factor 2 alpha kinase 3 (PERK) branch. Cells carrying α1(I) mutations upregulate autophagy, while cells with α2(I) mutations only occasionally activate the autodegradative response. Despite the autophagy activation to face stress conditions, apoptosis occurs in all mutant fibroblasts. To reduce cellular stress, mutant fibroblasts were treated with the FDA-approved chemical chaperone 4-phenylbutyric acid . The drug rescues cell death by modulating UPR activation thanks to both its chaperone and histone deacetylase inhibitor abilities. As chaperone it increases general cellular protein secretion in all patients' cells as well as collagen secretion in cells with the most C-terminal mutation. As histone deacetylase inhibitor it enhances the expression of the autophagic gene Atg5 with a consequent stimulation of autophagy. These results demonstrate that the cellular response to ER stress can be a relevant target to ameliorate OI cell homeostasis. [ABSTRACT FROM AUTHOR]

Abstract: It is known that endoplasmic reticulum (ER) stress in cells and extracellular vesicles (EVs) plays a significant role in cancer cells, therefore the evaluation of compounds that can regulate ER stress and EV secretion would be a suitable system for further screening and development of new drugs. In this study, we evaluated chemical chaperones derived from natural products based on monitoring Bip/GRP78 promoter activity during cancer cell growth, at the level of the single cell, by a bioluminescence microscopy system that had several advantages compared with fluorescence imaging. It was found that several chemical chaperones, such as ferulic acid (FA), silybin, and rutin, affected the activity. We visualized EVs from cancer cells using bioluminescence imaging and showed that several EVs could be observed when using CD63 fused with NanoLuc luciferase, which has a much smaller molecular weight and higher intensity than conventional firefly luciferase. We then examined the effects of the chemical chaperones on EVs from cancer cells by bioluminescence imaging and quantified the expression of CD63 in these EVs. It was found that the chemical chaperones examined in this study affected CD63 levels in EVs. These results showed that imaging at the level of the single cell using bioluminescence is a powerful tool and could be used to evaluate chemical chaperones and EVs from cancer cells. This approach may produce new information in this field when taken together with conventional and classical methods. [ABSTRACT FROM AUTHOR]