Accumulation of Glycogen and Upregulation of LEA-1 in C. elegans daf-2(e1370) Support Stress Resistance, Not Longevity

DAF-16-dependent activation of a dauer-associated genetic program in the C. elegans insulin/IGF-1 daf-2(e1370) mutant leads to accumulation of large amounts of glycogen with concomitant upregulation of glycogen synthase, GSY-1. Glycogen is a major storage sugar in C. elegans that can be used as a short-term energy source for survival, and possibly as a reservoir for synthesis of a chemical chaperone trehalose. Its role in mitigating anoxia, osmotic and oxidative stress has been demonstrated previously. Furthermore, daf-2 mutants show increased abundance of the group 3 late embryogenesis abundant protein LEA-1, which has been found to act in synergy with trehalose to exert its protective role against desiccation and heat stress in vitro, and to be essential for desiccation tolerance in C. elegans dauer larvae. Here we demonstrate that accumulated glycogen is not required for daf-2 longevity, but specifically protects against hyperosmotic stress, and serves as an important energy source during starvation. Similarly, lea-1 does not act to support daf-2 longevity. Instead, it contributes to increased resistance of daf-2 mutants to heat, osmotic, and UV stress. In summary, our experimental results suggest that longevity and stress resistance can be uncoupled in IIS longevity mutants.

A Chemical Chaperone That Prevents Insulin Fibrillation

Insulin, a peptide hormone, is susceptible to amyloid formation upon exposure to aberrant physiological conditions, result-ing in a loss of its bioactivity. For mitigating insulin aggregation, we report a molecule called PAD-S, which completely inhibit-ed insulin fibril formation, and preserved insulin in its soluble form. Circular Dichroism spectroscopy showed that PAD-S was able to maintain the native structure of insulin, thus acting as a chemical chaperone. Seeded aggregation kinetics suggest that PAD-S inhibited primary nucleation events during aggregation. This is consistent with molecular docking results which suggest that PAD-S binds strongly to native insulin monomers/dimers. Through a competitive binding experiment with ‘LVEALYL’ peptide, we conclude that PAD-S likely binds to the amyloid prone B11-B17 residues of insulin thereby prevent-ing its aggregation. PAD-S was also effective in disaggregating preformed insulin fibrils to non-toxic species. PAD-S treated insulin was functional as indicated by its ability to phosphorylate Akt. PAD-S was also highly effective in preventing the ag-gregation of insulin biosimilars. The low cellular cytotoxicity of PAD-S, and amelioration of aggregation-induced toxicity by PAD-S treated insulin further highlights its potential as an effective chemical chaperone.

Inhibition of histone deacetylation with vorinostat does not prevent tunicamycin-mediated acute kidney injury

Endoplasmic reticulum (ER) stress is associated with acute kidney injury (AKI) caused by various mechanisms, including antibiotics, non-steroidal anti-inflammatory drugs, cisplatin, and radiocontrast. Tunicamycin (TM) is a nucleoside antibiotic that induces ER stress and is a commonly used model of AKI. 4-phenylbutyrate (4-PBA) is a chemical chaperone and histone deacetylase (HDAC) inhibitor and has been shown to protect the kidney from ER stress, apoptosis, and structural damage in a tunicamycin model of AKI. The renal protection provided by 4-PBA is attributed to its ability to prevent misfolded protein aggregation and inhibit ER stress; however, the HDAC inhibitor effects of 4-PBA have not been examined in the TM-induced model of AKI. As such, the main objective of this study was to determine if histone hyperacetylation provides any protective effects against TM-mediated AKI. The FDA-approved HDAC inhibitor vorinostat was used, as it has no ER stress inhibitory effects and therefore the histone hyperacetylation properties alone could be investigated. In vitro work demonstrated that vorinostat inhibited histone deacetylation in cultured proximal tubular cells but did not prevent ER stress or protein aggregation induced by TM. Vorinostat induced a significant increase in cell death, and exacerbated TM-mediated total cell death and apoptotic cell death. Wild type male mice were treated with TM (0.5 mg/kg, intraperitoneal injection), with or without vorinostat (50 mg/kg/day) or 4-PBA (1 g/kg/day). Mice treated with 4-PBA or vorinostat exhibited similar levels of histone hyperacetylation. Expression of the pro-apoptotic protein CHOP was induced with TM, and not inhibited by vorinostat. Further, vorinostat did not prevent any renal damage or decline in renal function caused by tunicamycin. These data suggest that the protective mechanisms found by 4-PBA are primarily due to its molecular chaperone properties, and the HDAC inhibitors used did not provide any protection against renal injury caused by ER stress.

ERK5 Inhibition Induces Autophagy-Mediated Cancer Cell Death by Activating ER Stress

Autophagy is a highly conserved intracellular process that preserves cellular homeostasis by mediating the lysosomal degradation of virtually any component of the cytoplasm. Autophagy is a key instrument of cellular response to several stresses, including endoplasmic reticulum (ER) stress. Cancer cells have developed high dependency on autophagy to overcome the hostile tumor microenvironment. Thus, pharmacological activation or inhibition of autophagy is emerging as a novel antitumor strategy. ERK5 is a novel member of the MAP kinase family that is activated in response to growth factors and different forms of stress. Recent work has pointed ERK5 as a major player controlling cancer cell proliferation and survival. Therefore small-molecule inhibitors of ERK5 have shown promising therapeutic potential in different cancer models. Here, we report for the first time ERK5 as a negative regulator of autophagy. Thus, ERK5 inhibition or silencing induced autophagy in a panel of human cancer cell lines with different mutation patterns. As reported previously, ERK5 inhibitors (ERK5i) induced apoptotic cancer cell death. Importantly, we found that autophagy mediates the cytotoxic effect of ERK5i, since ATG5ˉ/ˉ autophagy-deficient cells viability was not affected by these compounds. Mechanistically, ERK5i stimulated autophagic flux independently of the canonical regulators AMPK or mTORC1. Moreover, ERK5 inhibition resulted in ER stress and activation of the Unfolded Protein Response (UPR) pathways. Specifically, ERK5i induced expression of the ER luminal chaperone BiP (a hallmark of ER stress), the UPR markers CHOP and ATF4, and the spliced form of XBP1. Pharmacological inhibition of UPR with chemical chaperone TUDC, or ATF4 silencing, resulted in impaired ERK5i-mediated UPR, autophagy and cytotoxicity. Overall, our results suggest that ERK5 inhibition induces autophagy-mediated cancer cell death by activating ER stress. Since ERK5 inhibition sensitizes cancer cells and tumors to chemotherapy, future work will determine the relevance of UPR and autophagy in the combined use of chemotherapy and ERK5i to tackle Cancer.

Sodium 4-Phenylbutyrate Reduces Ocular Hypertension by Degrading Extracellular Matrix Deposition via Activation of MMP9

Ocular hypertension (OHT) is a serious adverse effect of the widely prescribed glucocorticoid (GC) therapy and, if left undiagnosed, it can lead to glaucoma and complete blindness. Previously, we have shown that the small chemical chaperone, sodium-4-phenylbutyrate (PBA), rescues GC-induced OHT by reducing ocular endoplasmic reticulum (ER) stress. However, the exact mechanism of how PBA rescues GC-induced OHT is not completely understood. The trabecular meshwork (TM) is a filter-like specialized contractile tissue consisting of TM cells embedded within extracellular matrix (ECM) that controls intraocular pressure (IOP) by constantly regulating aqueous humor (AH) outflow. Induction of abnormal ECM deposition in TM is a hallmark of GC-induced OHT. Here, we investigated whether PBA reduces GC-induced OHT by degrading abnormal ECM deposition in TM using mouse model of GC-induced OHT, ex vivo cultured human TM tissues and primary human TM cells. We show that topical ocular eye drops of PBA (1%) significantly lowers elevated IOP in mouse model of GC-induced OHT. Importantly, PBA prevents synthesis and deposition of GC-induced ECM in TM. We report for the first time that PBA can degrade existing abnormal ECM in normal human TM cells/tissues by inducing matrix metalloproteinase (MMP)9 expression and activity. Furthermore, inhibition of MMPs activity by chemical-inhibitor (minocycline) abrogated PBA’s effect on ECM reduction and its associated ER stress. Our study indicates a non-chaperone activity of PBA via activation of MMP9 that degrades abnormal ECM accumulation in TM.

The Multifaceted Roles of Proline in Cell Behavior

Herein, we review the multifaceted roles of proline in cell biology. This peculiar cyclic imino acid is: (i) A main precursor of extracellular collagens (the most abundant human proteins), antimicrobial peptides (involved in innate immunity), salivary proteins (astringency, teeth health) and cornifins (skin permeability); (ii) an energy source for pathogenic bacteria, protozoan parasites, and metastatic cancer cells, which engage in extracellular-protein degradation to invade their host; (iii) an antistress molecule (an osmolyte and chemical chaperone) helpful against various potential harms (UV radiation, drought/salinity, heavy metals, reactive oxygen species); (iv) a neural metabotoxin associated with schizophrenia; (v) a modulator of cell signaling pathways such as the amino acid stress response and extracellular signal-related kinase pathway; (vi) an epigenetic modifier able to promote DNA and histone hypermethylation; (vii) an inducer of proliferation of stem and tumor cells; and (viii) a modulator of cell morphology and migration/invasiveness. We highlight how proline metabolism impacts beneficial tissue regeneration, but also contributes to the progression of devastating pathologies such as fibrosis and metastatic cancer.

Hypercholesterolemia Impairs Clearance of Neutrophil Extracellular Traps and Promotes Inflammation and Atherosclerotic Plaque Progression

Objective: Hypercholesterolemia-induced NETosis and accumulation of neutrophil extracellular traps (NETs) in the atherosclerotic lesion exacerbates inflammation and is causally implicated in plaque progression. We investigated whether hypercholesterolemia additionally impairs the clearance of NETs mediated by endonucleases such as DNase1 and DNase1L3 and its implication in advanced atherosclerotic plaque progression. Approach and Results: Using a mouse model, we demonstrate that an experimental increase in the systemic level of NETs leads to a rapid increase in serum DNase activity, which is critical for the prompt clearance of NETs and achieving inflammation resolution. Importantly, hypercholesterolemic mice demonstrate an impairment in this critical NET-induced DNase response with consequent delay in the clearance of NETs and defective inflammation resolution. Administration of TUDCA, a chemical chaperone that relieves endoplasmic reticulum stress, rescued the hypercholesterolemia-induced impairment in the NET-induced DNase response suggesting a causal role for endoplasmic reticulum stress in this phenomenon. Correction of the defective DNase response with exogenous supplementation of DNase1 in Apoe −/− mice with advanced atherosclerosis resulted in a decrease in plaque NET content and significant plaque remodeling with decreased area of plaque necrosis and increased collagen content. From a translational standpoint, we demonstrate that humans with hypercholesterolemia have elevated systemic extracellular DNA levels and decreased plasma DNase activity. Conclusions: These data suggest that hypercholesterolemia impairs the NET-induced DNase response resulting in defective clearance and accumulation of NETs in the atherosclerotic plaque. Therefore, strategies aimed at rescuing this defect could be of potential therapeutic benefit in promoting inflammation resolution and atherosclerotic plaque stabilization.

4-Phenylbutyric acid improves free fatty acid-induced hepatic insulin resistance in vivo

Plasma free fatty acids (FFAs) are elevated in obesity and can induce insulin resistance via endoplasmic reticulum (ER) stress. However, it is unknown whether hepatic insulin resistance caused by the elevation of plasma FFAs is alleviated by chemical chaperones. Rats received one of the following i.v. treatments for 48 h: saline, intralipid plus heparin (IH), IH plus the chemical chaperone 4-phenylbutyric acid (PBA), or PBA alone and a hyperinsulinemic-euglycemic clamp was performed during the last 2 h. PBA co-infusion normalized IH-induced peripheral insulin resistance, similar to our previous findings with an antioxidant and an IκBα kinase β (IKKβ) inhibitor. Different from our previous results with the antioxidant and IKKβ inhibitor, PBA also improved IH-induced hepatic insulin resistance in parallel with activation of Akt. Unexpectedly, IH did not induce markers of ER stress in the liver, but PBA prevented IH-induced elevation of phosphorylated eukaryotic initiation factor-2α protein in adipose tissue. PBA tended to decrease circulating fetuin-A and significantly increased circulating fibroblast growth factor 21 (FGF21) without affecting markers of activation of hepatic protein kinase C-δ or p38 mitogen-activated protein kinase that we have previously involved in hepatic insulin resistance in this model. In conclusion: (i) PBA prevented hepatic insulin resistance caused by prolonged plasma FFA elevation without affecting hepatic ER stress markers; (ii) the PBA effect is likely due to increased FGF21 and/or decreased fetuin-A, which directly signal to upregulate Akt activation.