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Multiple studies have demonstrated that cancer cells with microsatellite instability (MSI) are intolerant to loss of the Werner syndrome helicase (WRN), whereas microsatellite-stable (MSS) cancer cells are not. Therefore, WRN represents a promising new synthetic lethal target for developing drugs to treat cancers with MSI. Given the uncertainty of how effective inhibitors of WRN activity will prove in clinical trials, and the likelihood of tumours developing resistance to WRN inhibitors, alternative strategies for impeding WRN function are needed. Proteolysis-targeting chimeras (PROTACs) are heterobifunctional small molecules that target specific proteins for degradation. Here, we engineered the WRN locus so that the gene product is fused to a bromodomain (Bd)-tag, enabling conditional WRN degradation with the AGB-1 PROTAC specific for the Bd-tag. Our data revealed that WRN degradation is highly toxic in MSI but not MSS cell lines. In MSI cells, WRN degradation caused G 2 /M arrest, chromosome breakage and ATM kinase activation. We also describe a multi-colour cell-based platform for facile testing of selective toxicity in MSI versus MSS cell lines. Together, our data show that a degrader approach is a potentially powerful way of targeting WRN in MSI cancers and paves the way for the development of WRN-specific PROTAC compounds., (© 2024. The Author(s).)

Translationally controlled tumor protein (TCTP) is expressed in many tissues, particularly in human tumors. It plays a role in malignant transformation, apoptosis prevention, and DNA damage repair. The signaling mechanisms underlying TCTP regulation in cancer are only partially understood. Here, we investigated the role of mTORC1 in regulating TCTP protein levels, thereby modulating chemosensitivity, in human lung cancer cells and an A549 lung cancer xenograft model. The inhibition of mTORC1, but not mTORC2, induced ubiquitin/proteasome-dependent TCTP degradation without a decrease in the mRNA level. PLK1 activity was required for TCTP ubiquitination and degradation and for its phosphorylation at Ser 46 upon mTORC1 inhibition. Akt phosphorylation and activation was indispensable for rapamycin-induced TCTP degradation and PLK1 activation, and depended on S6K inhibition, but not mTORC2 activation. Furthermore, the minimal dose of rapamycin required to induce TCTP proteolysis enhanced the efficacy of DNA-damaging drugs, such as cisplatin and doxorubicin, through the induction of apoptotic cell death in vitro and in vivo. This synergistic cytotoxicity of these drugs was induced irrespective of the functional status of p53. These results demonstrate a new mechanism of TCTP regulation in which the mTORC1/S6K pathway inhibits a novel Akt/PLK1 signaling axis and thereby induces TCTP protein stabilization and confers resistance to DNA-damaging agents. The results of this study suggest a new therapeutic strategy for enhancing chemosensitivity in lung cancers regardless of the functional status of p53., (© 2021. The Author(s).)

To investigate the ability of theaflavins (TF) from black tea to protect dentin collagen against enzymatic degradation via cross-linking effect under clinically relevant conditions. 10-µm-thick dentin films were microtomed from dentin slabs of human molars. Following demineralization, films or slabs were treated with TF at two concentrations (0.4% and 2%) for 30 s. A well-known collagen cross-linker grape seed proanthocyanidins (PA) was used as control. Collagen cross-linking interactions and stabilization against enzymatic degradation were investigated by Fourier transform infrared spectroscopy, weight loss, hydroxyproline release, and scanning/transmission electron microscopy. Data were analyzed by ANOVA, Tukey's and Student's T test (α = 0.05%). The results showed collagen cross-linking and stabilization efficacy was dependent on TF/PA concentrations. At 2.0%, TF and PA offered nearly full protection to collagen; at 0.4%, TF exhibited a significantly better collagen stabilization effect than PA (P < 0.05), while untreated collagen was completely digested. It's concluded that TF cross-links dentin collagen within a clinically relevant time (30 s) and offers excellent collagen protection against enzymatic degradation, with efficacy comparable to or better than PA. The study supports the potential use of TF as a novel, promising collagen cross-linker for degradation resistant, long-lasting dentin bonding in composite restorations., (© 2021. The Author(s).)

Tendon extracellular matrix (ECM) mechanical unloading results in tissue degradation and breakdown, with niche-dependent cellular stress directing proteolytic degradation of tendon. Here, we show that the extracellular-signal regulated kinase (ERK) pathway is central in tendon degradation of load-deprived tissue explants. We show that ERK 1/2 are highly phosphorylated in mechanically unloaded tendon fascicles in a vascular niche-dependent manner. Pharmacological inhibition of ERK 1/2 abolishes the induction of ECM catabolic gene expression (MMPs) and fully prevents loss of mechanical properties. Moreover, ERK 1/2 inhibition in unloaded tendon fascicles suppresses features of pathological tissue remodeling such as collagen type 3 matrix switch and the induction of the pro-fibrotic cytokine interleukin 11. This work demonstrates ERK signaling as a central checkpoint to trigger tendon matrix degradation and remodeling using load-deprived tissue explants.

N-acetyl-para-amino phenol (APAP, usually named paracetamol), which is commonly used for its analgesic and antipyretic properties may lead to hepatotoxicity and acute liver damage in case of overdoses. Released cytokines and oxidative stress following acute liver damage may affect other organs' function notably the diaphragm, which is particularly sensitive to oxidative stress and circulating cytokines. We addressed this issue in a mouse model of acute liver injury induced by administration of APAP. C57BL/6J mice (each n = 8) were treated with N-acetyl-para-amino phenol (APAP) to induce acute drug caused liver injury and sacrificed 12 or 24 h afterwards. An untreated group served as controls. Key markers of inflammation, proteolysis, autophagy and oxidative stress were measured in diaphragm samples. In APAP treated animals, liver damage was proven by the enhanced serum levels of alanine aminotransferase and aspartate aminotransferase. In the diaphragm, besides a significant increase in IL 6 and lipid peroxidation, no changes were observed in key markers of the proteolytic, and autophagy signaling pathways, other inflammatory markers and fiber dimensions. The first 24 h of acute liver damage did not impair diaphragm atrophic pathways although it slightly enhanced IL-6 and lipid peroxidation. Whether longer exposure might affect the diaphragm needs to be addressed in future experiments.

Clinical reports have found that with the improvement of treatment, most septic patients are able to survive the severe systemic inflammatory response and to enter the immunoparalysis stage. Considering that immunoparalysis leads to numerous deaths of clinical sepsis patients, alleviation of the occurrence and development of immunoparalysis has become a top priority in the treatment of sepsis. In our study, we investigate the effects of oroxylin A on sepsis in cecal ligation and puncture (CLP) mice. We find that the 60 h + 84 h (30 mg/kg) injection scheme of oroxylin A induce the production of pro-inflammatory factors, and further significantly improves the survival of CLP mice during the middle or late stages of sepsis. Mechanistically, C/EBP-homologous protein (CHOP) is upregulated and plays anti-inflammatory roles to facilitate the development of immunoparalysis in CLP mice. Oroxylin A induces the transcription of E3 ligase F-box only protein 15 gene (fbxo15), and activated FBXO15 protein binds to CHOP and further mediates the degradation of CHOP through the proteasome pathway, which eventually relieves the immunoparalysis of CLP mice. Taken together, these findings suggest oroxylin A relieves the immunoparalysis of CLP mice by degrading CHOP through interacting with FBXO15.

Targeted protein degradation (TPD) has emerged as a powerful tool in drug discovery for the perturbation of protein levels using heterobifunctional small molecules. E3 ligase recruiters remain central to this process yet relatively few have been identified relative to the ~ 600 predicted human E3 ligases. While, initial recruiters have utilized non-covalent chemistry for protein binding, very recently covalent engagement to novel E3's has proven fruitful in TPD application. Herein we demonstrate efficient proteasome-mediated degradation of BRD4 by a bifunctional small molecule linking the KEAP1-Nrf2 activator bardoxolone to a BRD4 inhibitor JQ1.

Herpes simplex virus 1 (HSV-1) causes a number of clinical manifestations including cold sores, keratitis, meningitis and encephalitis. Although current drugs are available to treat HSV-1 infection, they can cause side effects such as nephrotoxicity. Moreover, owing to the emergence of drug-resistant HSV-1 strains, new anti-HSV-1 compounds are needed. Because many viruses exploit cellular host proteases and encode their own viral proteases for survival, we investigated the inhibitory effects of a panel of protease inhibitors (TLCK, TPCK, E64, bortezomib, or MG132) on HSV-1 replication and several host cell signaling pathways. We found that HSV-1 infection suppressed c-Raf-MEK1/2-ERK1/2-p90RSK signaling in host cells, which facilitated viral replication. The mechanism by which HSV-1 inhibited ERK signaling was mediated through the polyubiquitination and proteasomal degradation of Ras-guanine nucleotide-releasing factor 2 (Ras-GRF2). Importantly, the proteasome inhibitor MG132 inhibited HSV-1 replication by reversing ERK suppression in infected cells, inhibiting lytic genes (ICP5, ICP27 and UL42) expression, and overcoming the downregulation of Ras-GRF2. These results indicate that the suppression of ERK signaling via proteasomal degradation of Ras-GRF2 is necessary for HSV-1 infection and replication. Given that ERK activation by MG132 exhibits anti-HSV-1 activity, these results suggest that the proteasome inhibitor could serve as a novel therapeutic agent against HSV-1 infection.

The protozoan Giardia lamblia is the most common cause of parasitic gastrointestinal infection worldwide. The parasite developed sophisticated, yet not completely disclosed, mechanisms to escape immune system and growth in the intestine. To further understand the interaction of G. lamblia with host immune cells, we investigated the ability of parasites to modulate the canonical activation of mouse macrophages (Raw 264.7 cell line) and human monocyte-derived macrophages triggered by the TLR4 agonist, lipopolysaccharide (LPS). We observed that G. lamblia impairs LPS-evoked pro-inflammatory status in these macrophage-like cells through inhibition of cyclooxygenase-2 and inducible nitric oxide synthase expression and subsequent NO production. This effect was in part due to the activity of three G. lamblia proteases, a 135 kDa metalloprotease and two cysteine proteases with 75 and 63 kDa, that cleave the p65 RelA subunit of the nuclear factor-kappa B (NF-κB). Moreover, Tnf and Ccl4 transcription was increased in the presence of the parasite. Overall, our data indicates that G. lamblia modulates macrophages inflammatory response through impairment of the NF-κB, thus silencing a crucial signaling pathway of the host innate immune response.

Ibuprofen, an inhibitor of prostanoid biosynthesis, is a common pharmacological agent used for the management of pain, inflammation and fever. However, the chronic use of ibuprofen at high doses is associated with increased risk for cardiovascular, renal, gastrointestinal and liver injuries. The underlying mechanisms of ibuprofen-mediated effects on liver remain unclear. To determine the mechanisms and signaling pathways affected by ibuprofen (100 mg/kg/day for seven days), we performed proteomic profiling of male mice liver with quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) using ten-plex tandem mass tag (TMT) labeling. More than 300 proteins were significantly altered between the control and ibuprofen-treated groups. The data suggests that several major pathways including (1) energy metabolism, (2) protein degradation, (3) fatty acid metabolism and (4) antioxidant system are altered in livers from ibuprofen treated mice. Independent validation of protein changes in energy metabolism and the antioxidant system was carried out by Western blotting and showed sex-related differences. Proteasome and immunoproteasome activity/expression assays showed ibuprofen induced gender-specific proteasome and immunoproteasome dysfunction in liver. The study observed multifactorial gender-specific ibuprofen-mediated effects on mice liver and suggests that males and females are affected differently by ibuprofen.

Exposure to thalidomide during a critical window of development results in limb defects in humans and non-human primates while mice and rats are refractory to these effects. Thalidomide-induced teratogenicity is dependent on its binding to cereblon (CRBN), the substrate receptor of the Cul4A-DDB1-CRBN-RBX1 E3 ubiquitin ligase complex. Thalidomide binding to CRBN elicits subsequent ubiquitination and proteasomal degradation of CRBN neosubstrates including SALL4, a transcription factor of which polymorphisms phenocopy thalidomide-induced limb defects in humans. Herein, thalidomide-induced degradation of SALL4 was examined in human induced pluripotent stem cells (hiPSCs) that were differentiated either to lateral plate mesoderm (LPM)-like cells, the developmental ontology of the limb bud, or definitive endoderm. Thalidomide and its immunomodulatory drug (IMiD) analogs, lenalidomide, and pomalidomide, dose-dependently inhibited hiPSC mesendoderm differentiation. Thalidomide- and IMiD-induced SALL4 degradation can be abrogated by CRBN V388I mutation or SALL4 G416A mutation in hiPSCs. Genetically modified hiPSCs expressing CRBN E377V/V388I mutant or SALL4 G416A mutant were insensitive to the inhibitory effects of thalidomide, lenalidomide, and pomalidomide on LPM differentiation while retaining sensitivity to another known limb teratogen, all-trans retinoic acid (atRA). Finally, disruption of LPM differentiation by atRA or thalidomide perturbed subsequent chondrogenic differentiation in vitro. The data here show that thalidomide, lenalidomide, and pomalidomide affect stem cell mesendoderm differentiation through CRBN-mediated degradation of SALL4 and highlight the utility of the LPM differentiation model for studying the teratogenicity of new CRBN modulating agents.

A growing body of evidence implicates endoplasmic reticulum (ER) stress in the pathogenesis of chronic inflammatory and autoimmune disorders. Here, we demonstrate that the proinflammatory cytokine TNFα stimulates matrix metalloproteinase 9 (MMP9) at the ocular surface through a c-Fos-dependent mechanism of ER stress. We found positive reactivity of the molecular chaperone BiP/GRP78 in conjunctival epithelium of patients with ocular cicatricial pemphigoid and increased levels of BiP/GRP78, sXBP1 and GRP94 in human corneal epithelial cells treated with TNFα. Pharmacological blockade of ER stress in vitro using dexamethasone or the chemical chaperones TUDCA and 4PBA attenuated MMP9 expression and secretion in the presence of TNFα. Moreover, expression analysis of genes associated with inflammation and autoimmunity identified the c-Fos proto-oncogene as a mediator of ER stress responses in epithelial cells. Substantially less TNFα-induced MMP9 expression occurred when c-Fos signaling was suppressed with a function-blocking antibody. Taken together, these results indicate that activation of ER stress contributes to promote inflammation-mediated proteolytic activity and uncovers a target for restoring tissue homeostasis in ocular autoimmune disease.

Macrophages are an important component of the innate immune response. Priming and activation of macrophages is stimulated by cytokines (i.e IFNγ). However, growth hormone (GH) can also stimulate macrophage activation. Based on these observations, the goal of this work was to 1) to compare the transcriptome profile of macrophages activated in vitro with GH and IFNγ, and 2) to assess the impact of GH on key macrophage functional properties like reactive oxygen species (ROS) production and phagosomal proteolysis. To assess the global transcriptional and functional impact of GH on macrophage programming, bone marrow derived macrophages were treated with GH or IFNγ. Our data strongly support a potential link between GH, which wanes with age, and impaired macrophage function. The notable overlap of GH with IFNγ-induced pathways involved in innate immune sensing of pathogens and antimicrobial responses argue for an important role for GH in macrophage priming and maturation. By using functional assays that report on biochemical activities within the lumen of phagosomes, we have also shown that GH alters physiologically relevant processes such as ROS production and proteolysis. These changes could have far reaching impacts on antimicrobial capacity, signaling, and antigen presentation.

Serine-threonine kinase 38 (STK38) is a member of the protein kinase A (PKA)/PKG/PKC-family implicated in the regulation of cell division and morphogenesis. However, the molecular mechanisms underlying STK38 stability remain largely unknown. Here, we show that treatment of cells with either heat or the calcium ionophore A23187 induced STK38 degradation. The calpain inhibitor calpeptin suppressed hyperthermia-induced degradation or the appearance of A23187-induced cleaved form of STK38. An in vitro cleavage assay was then used to demonstrate that calpain I directly cleaves STK38 at the proximal N-terminal region. Deletion of the N-terminal region of STK38 increased its stability against hyperthermia. We further demonstrated that the MAPKK kinase (MAP3K) MEKK2 prevented both heat- and calpain-induced cleavage of STK38. MEKK2 knockdown enhanced hyperthermia-induced degradation of STK38. We performed an in vitro MEKK2 assay and identified the key regulatory site in STK38 phosphorylated by MEKK2. Experiments with a phosphorylation-defective mutant demonstrated that phosphorylation of Ser 91 is important for STK38 stability, as the enzyme is susceptible to degradation by the calpain pathway unless this residue is phosphorylated. In summary, we demonstrated that STK38 is a calpain substrate and revealed a novel role of MEKK2 in the process of STK38 degradation by calpain.

Renal hypoxia and loss of proximal tubular cells (PTC) are relevant in diabetic nephropathy. Hypoxia inhibits hypoxia-inducible factor-1α (HIF-1α) degradation, which leads to cellular adaptive responses through HIF-1-dependent activation of gene hypoxia-responsive elements (HRE). However, the diabetic microenvironment represses the HIF-1/HRE response in PTC. Here we studied the mechanism and consequences of impaired HIF-1α regulation in human proximal tubular HK-2 cells incubated in hyperglycemia. Inhibition at different levels of the canonical pathway of HIF-1α degradation did not activate the HIF-1/HRE response under hyperglycemia, except when proteasome was inhibited. Further studies suggested that hyperglycemia disrupts the interaction of HIF-1α with Hsp90, a known cause of proteasomal degradation of HIF-1α. Impaired HIF-1α regulation in cells exposed to hyperglycemic, hypoxic diabetic-like milieu led to diminished production of vascular endothelial growth factor-A and inhibition of cell migration (responses respectively involved in tubular protection and repair). These effects, as well as impaired HIF-1α regulation, were reproduced in normoglycemia in HK-2 cells incubated with microparticles released by HK-2 cells exposed to diabetic-like milieu. In summary, these results highlight the role of proteasome-dependent mechanisms of HIF-1α degradation on diabetes-induced HK-2 cells dysfunction and suggest that cell-derived microparticles may mediate negative effects of the diabetic milieu on PTC.

Clp proteases play a central role in bacterial physiology and, for some bacterial species, are even essential for survival. Also due to their conservation among bacteria including important human pathogens, Clp proteases have recently attracted considerable attention as antibiotic targets. Here, we functionally reconstituted and characterized the ClpXP protease of Chlamydia trachomatis (ctClpXP), an obligate intracellular pathogen and the causative agent of widespread sexually transmitted diseases in humans. Our in vitro data show that ctClpXP is formed by a hetero-tetradecameric proteolytic core, composed of two distinct homologs of ClpP (ctClpP1 and ctClpP2), that associates with the unfoldase ctClpX via ctClpP2 for regulated protein degradation. Antibiotics of the ADEP class interfere with protease functions by both preventing the interaction of ctClpX with ctClpP1P2 and activating the otherwise dormant proteolytic core for unregulated proteolysis. Thus, our results reveal molecular insight into ctClpXP function, validating this protease as an antibacterial target.

Accumulating evidence indicates that inflammation participates in the pathophysiological progress from insulin resistance, obesity, metabolic abnormalities, and type 2 diabetes mellitus. Our previous study reveals that interleukin-4 (IL-4) inhibits adipogenesis and promotes lipolysis to decrease lipid deposits by enhancing the activity of hormone sensitive lipase (HSL). The present study further dissects and characterizes the molecular mechanism of IL-4 in regulating HSL expression and lipolytic activity in the terminal differentiated 3T3-L1 mature adipocytes. Our results showed that IL-4 increased cAMP which then enhanced PKA activity and subsequent phosphorylation of HSL and perilipin. The phosphorylated HSL (p-HSL) translocated from cytoplasm to the surface of lipid droplets and exhibited lipolytic function. After being phosphorylated, p-perilipin also facilitated lipolysis through interacting with p-HSL. The in vitro findings were further verified by in vivo study in which IL-4 exhibited pro-lipolytic activity and enhanced HSL activity. In summary, the net outcome of IL-4 treatment is to reduce lipid storage by promoting lipolysis through enhancing HSL activity via cAMP/PKA pathway, the major route leading to lipolysis.

The glycolytic rate in neurons is low in order to allow glucose to be metabolized through the pentose-phosphate pathway (PPP), which regenerates NADPH to preserve the glutathione redox status and survival. This is controlled by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), the pro-glycolytic enzyme that forms fructose-2,6-bisphosphate, a powerful allosteric activator of 6-phosphofructo-1-kinase. In neurons, PFKFB3 protein is physiologically inactive due to its proteasomal degradation. However, upon an excitotoxic stimuli, PFKFB3 becomes stabilized to activate glycolysis, thus hampering PPP mediated protection of redox status leading to neurodegeneration. Here, we show that selective inhibition of PFKFB3 activity by the small molecule AZ67 prevents the NADPH oxidation, redox stress and apoptotic cell death caused by the activation of glycolysis triggered upon excitotoxic and oxygen-glucose deprivation/reoxygenation models in mouse primary neurons. Furthermore, in vivo administration of AZ67 to mice significantly alleviated the motor discoordination and brain infarct injury in the middle carotid artery occlusion ischemia/reperfusion model. These results show that pharmacological inhibition of PFKFB3 is a suitable neuroprotective therapeutic strategy in excitotoxic-related disorders such as stroke.

The development of cardiovascular disease is intimately linked to elevated levels of low-density lipoprotein (LDL) cholesterol in the blood. Hepatic LDL receptor (LDLR) levels regulate the amount of plasma LDL. We identified the secreted zinc metalloproteinase, bone morphogenetic protein 1 (BMP1), as responsible for the cleavage of human LDLR within its extracellular ligand-binding repeats at Gly 171 ↓Asp 172 . The resulting 120 kDa membrane-bound C-terminal fragment (CTF) of LDLR had reduced capacity to bind LDL and when expressed in LDLR null cells had compromised LDL uptake as compared to the full length receptor. Pharmacological inhibition of BMP1 or siRNA-mediated knockdown prevented the generation of the 120 kDa CTF and resulted in an increase in LDL uptake into cells. The 120 kDa CTF was detected in the livers from humans and mice expressing human LDLR. Collectively, these results identify that BMP1 regulates cellular LDL uptake and may provide a target to modulate plasma LDL cholesterol.

The infrapatellar fat pad (IFP) serves as a reservoir of Mesenchymal Stem Cells (MSC), and with adjacent synovium plays key roles in joint disease including the production of Substance P (SP) affecting local inflammatory responses and transmitting nociceptive signals. Here, we interrogate human IFP-derived MSC (IFP-MSC) reaction to inflammatory and pro-fibrotic environments (cell priming by TNFα/IFNγ and TNFα/IFNγ/CTGF exposure respectively), compared with bone marrow-derived MSC (BM-MSC). Naïve IFP-MSC exhibit increased clonogenicity and chondrogenic potential compared with BM-MSC. Primed cells experienced dramatic phenotypic changes, including a sharp increase in CD10, upregulation of key immunomodulatory transcripts, and secreted growth factors/cytokines affecting key pathways (IL-10, TNF-α, MAPK, Ras and PI3K-Akt). Naïve, and more so primed MSC (both) induced SP degradation in vitro, reproduced with their supernatants and abrogated with thiorphan, a CD10 inhibitor. These findings were reproduced in vivo in a rat model of acute synovitis, where transiently engrafted human IFP-MSC induced local SP reduction. Functionally, primed IFP-MSC demonstrated sustained antagonism of activated human peripheral blood mononuclear cells (PBMC) proliferation, significantly outperforming a declining dose-dependent effect with naïve cohorts. Collectively, our in vitro and in vivo data supports cell priming as a way to enhance the immunoregulatory properties of IFP-MSC, which selectively engraft in areas of active synovitis/IFP fibrosis inducing SP degradation, resulting in a cell-based product alternative to BM-MSC to potentially treat degenerative/inflammatory joint diseases.

Frailty is an age-associated condition, characterized by an inappropriate response to stress that results in a higher frequency of adverse outcomes (e.g., mortality, institutionalization and disability). Some light has been shed over its genetic background, but this is still a matter of debate. In the present study, we used network biology to analyze the interactome of frailty-related genes at different levels to relate them with pathways, clinical deficits and drugs with potential therapeutic implications. Significant pathways involved in frailty: apoptosis, proteolysis, muscle proliferation, and inflammation; genes as FN1, APP, CREBBP, EGFR playing a role as hubs and bottlenecks in the interactome network and epigenetic factors as HIST1H3 cluster and miR200 family were also involved. When connecting clinical deficits and genes, we identified five clusters that give insights into the biology of frailty: cancer, glucocorticoid receptor, TNF-α, myostatin, angiotensin converter enzyme, ApoE, interleukine-12 and -18. Finally, when performing network pharmacology analysis of the target nodes, some compounds were identified as potentially therapeutic (e.g., epigallocatechin gallate and antirheumatic agents); while some other substances appeared to be toxicants that may be involved in the development of this condition.

Functional imaging has become an important tool in oncology because it not only provides information about the size and localization of the tumour, but also about the pathophysiological features of the tumoural cells. One of the characteristic features of some tumour types is that their fast growth leads to deficient intratumoral vascularization, which results in low oxygen availability. To overcome this lack of oxygen, tumoural cells activate the neoangiogenic program by upregulating the transcription factor HIF-1α. Herein we report a non-invasive in vitro detection method of hypoxia using designed fluorescent peptide probes based on the oxygen-dependent degradation domain of HIF-1α. The fluorescent probe retains the oxygen-sensing capability of HIF-1α, so that it is stabilized under hypoxia and readily degraded by the proteasome under normoxia, thus providing direct information of the cellular oxygen availability.

c-Fms is the macrophage colony-stimulating factor (M-CSF) receptor, and intracellular signalling via the M-CSF/c-Fms axis mediates both innate immunity and bone remodelling. M-CSF-induced transient proteolytic degradation of c-Fms modulates various biological functions, and protein kinase C (PKC) signalling is activated during this proteolytic process via an unknown mechanism. Notably, the role of specific PKC isoforms involved in c-Fms degradation during osteoclast differentiation is not known. Here, we observed that inactivation of PKCδ by the biochemical inhibitor rottlerin, a cell permeable peptide inhibitor, and short hairpin (sh) RNA suppresses osteoclast differentiation triggered by treatment with M-CSF and receptor activator of NF-κB ligand. Interestingly, inhibition of PKCδ by either inhibitor or gene silencing of PKCδ accelerated M-CSF-induced proteolytic degradation of membrane-bound c-Fms via both the lysosomal pathway and regulated intramembrane proteolysis (RIPping), but did not affect c-fms expression at the mRNA level. Degradation of c-Fms induced by PKCδ inactivation subsequently inhibited M-CSF-induced osteoclastogenic signals, such as extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK), p38, and Akt. Furthermore, mice administered PKCδ inhibitors into the calvaria periosteum exhibited a decrease in both osteoclast formation on the calvarial bone surface and the calvarial bone marrow cavity, which reflects osteoclastic bone resorption activity. These data suggest that M-CSF-induced PKCδ activation maintains membrane-anchored c-Fms and allows the sequential cellular events of osteoclastogenic signalling, osteoclast formation, and osteoclastic bone resorption.

Limb-girdle muscular dystrophy type 2D (LGMD2D) is characterized by a progressive proximal muscle weakness. LGMD2D is caused by mutations in the gene encoding α-sarcoglycan (α-SG), a dystrophin-associated glycoprotein that plays a key role in the maintenance of sarcolemma integrity in striated muscles. We report here on the development of a new in vitro high-throughput screening assay that allows the monitoring of the proper localization of the most prevalent mutant form of α-SG (R77C substitution). Using this assay, we screened a library of 2560 FDA-approved drugs and bioactive compounds and identified thiostrepton, a cyclic antibiotic, as a potential drug to repurpose for LGMD2D treatment. Characterization of the thiostrepton effect revealed a positive impact on R77C-α-SG and other missense mutant protein localization (R34H, I124T, V247M) in fibroblasts overexpressing these proteins. Finally, further investigations of the molecular mechanisms of action of the compound revealed an inhibition of the chymotrypsin-like activity of the proteasome 24 h after thiostrepton treatment and a synergistic effect with bortezomib, an FDA-approved proteasome inhibitor. This study reports on the first in vitro model for LGMD2D that is compatible with high-throughput screening and proposes a new therapeutic option for LGMD2D caused by missense mutations of α-SG.

Liver receptor homologue-1 (LRH-1) plays a critical role in hepatic metabolism and disease. Here we show that LRH-1 protein stability is regulated by the cullin 4 (CUL4) E3 ubiquitin ligase complex. We found that DNA damage-binding protein 2 (DDB2) directly interacts with LRH-1 and functions as a substrate recognition component of CUL4-DDB1 to promote LRH-1 ubiquitination and proteasomal degradation. In human hepatoma (HepG2) cells, we observed that protein levels of endogenous LRH-1 are increased by insulin without a change in mRNA levels of LRH-1. However, overexpression of DDB2 impaired the insulin-stimulated increase in LRH-1 levels. In addition, DDB2 overexpression decreased LRH-1 transcriptional activation and expression of target genes, such as glucokinase, whereas knockdown of DDB2 increased the expression of glucokinase. Finally, we demonstrated that DDB2 knockdown increases glucose uptake and intracellular levels of glucose-6-phosphate in HepG2 cells. Our study reveals a novel regulatory mechanism of LRH-1 activity and suggests a role for DDB2 in hepatic glucose metabolism.

HSP90 is a ubiquitously expressed molecular chaperone implicated in the correct folding and maturation of a plethora of proteins including protein kinases and transcription factors. While disruption of chaperone activity was associated with augmented cancer cell death and decreased tumor growth both in vitro and in vivo, the regulation of HSP90 is not clearly understood. Here we report that treatment of cancer cells with cold physical plasma, an emerging and less aggressive tumor therapy, resulted in ROS generation which subsequently triggered the cleavage of HSP90. Notably, cleavage of HSP90 was followed by the degradation of PKD2, a crucial regulator of tumor growth and angiogenesis. Pre-sensitization of cancer cells with subliminal doses of PU-H71, an HSP90 inhibitor currently under clinical evaluation, followed by treatment with cold-plasma, synergistically and negatively impacted on the viability of cancer cells. Taken together, cold-plasma can be used in conjunction with pharmacologic treatment in order to target the expression and activity of HSP90 and the downstream client proteins implicated in various cancer cell capabilities.

Methamphetamine (Meth) exacerbates HIV-1 pathobiology by increasing virus transmission and replication and accelerating clinical progression to AIDS. Meth has been shown to alter the expression of HIV-1 co-receptors and impair intrinsic resistance mechanisms of immune cells. However, the exact molecular mechanisms involved in augmenting HIV-1 replication in T-cells are still not yet clear. Here, we demonstrate that pretreatment with Meth of CD4 + T-cells enhanced HIV-1 replication. We observed upregulation of CD4 + T-cell activation markers and enhanced expression of miR-34c-5p and miR-155 in these cells. Further, we noted activation of the sigma-1 receptor and enhanced intracellular Ca 2+ concentration and cAMP release in CD4 + T-cells upon Meth treatment, which resulted in increased phosphorylation and nuclear translocation of transcription factors NFκB, CREB, and NFAT1. Increased gene expression of IL-4 and IL-10 was also observed in Meth treated CD4 + T-cells. Moreover, proteasomal degradation of Ago1 occurred upon Meth treatment, further substantiating the drug as an activator of T-cells. Taken together, these findings show a previously unreported mechanism whereby Meth functions as a novel T-cell activator via the sigma-1 signaling pathway, enhancing replication of HIV-1 with expression of miR-34c-5p, and transcriptional activation of NFκB, CREB and NFAT1.

Albumin has a serum half-life of three weeks in humans and is utilized to extend the serum persistence of drugs that are genetically fused or conjugated directly to albumin or albumin-binding molecules. Responsible for the long half-life is FcRn that protects albumin from intracellular degradation. An in-depth understanding of how FcRn binds albumin across species is of importance for design and evaluation of albumin-based therapeutics. Albumin consists of three homologous domains where domain I and domain III of human albumin are crucial for binding to human FcRn. Here, we show that swapping of two loops in domain I or the whole domain with the corresponding sequence in mouse albumin results in reduced binding to human FcRn. In contrast, humanizing domain I of mouse albumin improves binding. We reveal that domain I of mouse albumin plays a minor role in the interaction with the mouse and human receptors, as domain III on its own binds with similar affinity as full-length mouse albumin. Further, we show that P573 in domain III of mouse albumin is required for strong receptor binding. Our study highlights distinct differences in structural requirements for the interactions between mouse and human albumin with their respective receptor, which should be taken into consideration in design of albumin-based drugs and evaluation in mouse models.

Chronic myelogenous leukemia (CML) is characterized by the oncogenic fusion protein, BCR-ABL protein kinase, against which clinically useful inhibitors have been developed. An alternative approach to treat CML is to degrade the BCR-ABL protein. Recently, potent degraders against BCR-ABL have been developed by conjugating dasatinib to ligands for E3 ubiquitin ligases. Since the degraders contain the dasatinib moiety, they also inhibit BCR-ABL kinase activity, which complicates our understanding of the impact of BCR-ABL degradation by degraders in CML growth inhibition. To address this issue, we chose DAS-IAP, as a potent BCR-ABL degrader, and developed a structurally related inactive degrader, DAS-meIAP, which inhibits kinase activity but does not degrade the BCR-ABL protein. DAS-IAP showed slightly weaker activity than DAS-meIAP in inhibiting cell growth when CML cells were treated for 48 h. However, DAS-IAP showed sustained growth inhibition even when the drug was removed after short-term treatment, whereas CML cell growth rapidly resumed following removal of DAS-meIAP and dasatinib. Consistently, suppression of BCR-ABL levels and downstream kinase signaling were maintained after DAS-IAP removal, whereas kinase signaling rapidly recovered following removal of DAS-meIAP and dasatinib. These results indicate that BCR-ABL degrader shows more sustained inhibition of CML cell growth than ABL kinase inhibitor.

Human T regulatory cells (T regs) express high levels of TNF receptor 2 (TNFR2). Ligation of TNFR2 with TNF, which can recognise both TNFR1 and TNFR2, or with a TNFR2-selective binding molecule, DARPin 18 (D18) activates canonical NF-κB signalling, assessed by IκBα degradation, and the magnitude of the response correlates with the level of TNFR2 expression. RNA-seq analysis of TNF- or D18-treated human T regs revealed that TNFR2 ligation induces transcription of NFKB2 and RELB, encoding proteins that form the non-canonical NF-κB transcription factor. In combination with IL2, D18 treatment is specific for T regs in (1) stabilising NF-κB-inducing kinase protein, the activator of non-canonical NF-κB signalling, (2) inducing translocation of RelB from cytosol to nucleus, (3) increasing cell cycle entry, and (4) increasing cell numbers. However, the regulatory function of the expanded T regs is unaltered. Inhibition of RelB nuclear translocation blocks the proliferative response. We conclude that ligation of TNFR2 by D18 enhances IL2-induced T regs proliferation and expansion in cell number through the non-canonical NF-κB pathway.

Fibronectin (FN) plays a major role in the stability and organization of the extracellular matrix (ECM). We have previously demonstrated that FN interacts directly with Hsp90, as well as showing that the Hsp90 inhibitor novobiocin results in FN turnover via a receptor mediated process. However, the receptor involved has not been previously identified. LRP1 is a ubiquitous receptor responsible for the internalisation of numerous ligands that binds both Hsp90 and FN, and therefore we investigated whether LRP1 was involved in novobiocin-mediated FN turnover. FN, LRP1 and Hsp90 could be isolated in a common complex, and inhibition of Hsp90 by novobiocin increased the colocalisation of FN and LRP1. Novobiocin induced an increase (at low concentrations) followed by a loss of FN that was primarily derived from extracellular matrix-associated FN and led to a concomitant increase in intracellular FN. The effect of novobiocin was specific to LRP1-expressing cells and could be recapitulated by an LRP1 blocking antibody and the allosteric C-terminal Hsp90 inhibitor SM253, but not the N-terminal inhibitor geldanamycin. Together these data suggest that LRP1 is required for FN turnover in response to Hsp90 inhibition by novobiocin, which may have unintended physiological consequences in contexts where C-terminal Hsp90 inhibition is to be used therapeutically.

High-risk human papillomaviruses (HR-HPVs) are the causative agents for the onset of several epithelial cancers in humans. The deregulated expression of the viral oncoproteins E6 and E7 is the driving force sustaining the progression of malignant transformation in pre-neoplastic lesions. Targeting the viral E6 oncoprotein through inhibitory compounds can counteract the survival of cancer cells due to the reactivation of p53-mediated pathways and represents an intriguing strategy to treat HPV-associated neoplasias. Here, we describe the development of a quantitative and easy-to-perform assay to monitor the E6-mediated degradation of p53 in living cells to be used for small-molecule testing. This assay allows to unbiasedly determine whether a compound can protect p53 from the E6-mediated degradation in cells, through a simple 3-step protocol. We validated the assay by testing two small molecules, SAHA and RITA, reported to impair the E6-mediated p53 degradation. Interestingly, we observed that only SAHA efficiently rescued p53, while RITA could not provide the same degree of protection. The possibility to specifically and quantitatively monitor the ability of a selected compound to rescue p53 in a cellular context through our LumiFluo assay could represent an important step towards the successful development of anti-HPV drugs.

Muscle atrophy is one of the main characteristics of human ageing and physical inactivity, with resulting adverse health outcomes. To date, there are still no efficient therapeutic strategies for its prevention and/or treatment. However, during hibernation, bears exhibit a unique ability for preserving muscle in conditions where muscle atrophy would be expected in humans. Therefore, our objective was to determine whether there are components of bear serum which can control protein balance in human muscles. In this study, we exposed cultured human differentiated muscle cells to bear serum collected during winter and summer periods, and measured the impact on cell protein content and turnover. In addition, we explored the signalling pathways that control rates of protein synthesis and degradation. We show that the protein turnover of human myotubes is reduced when incubated with winter bear serum, with a dramatic inhibition of proteolysis involving both proteasomal and lysosomal systems, and resulting in an increase in muscle cell protein content. By modulating intracellular signalling pathways and inducing a protein sparing phenotype in human muscle cells, winter bear serum therefore holds potential for developing new tools to fight human muscle atrophy and related metabolic disorders.

Accumulating of evidence suggests that activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) exacerbates early brain injury (EBI) following subarachnoid hemorrhage (SAH) by provoking pro-inflammatory and pro-apoptotic signaling. Myeloid differentiation primary response protein 88 (MyD88) is an endogenous adaptor protein in the toll-like receptors (TLRs) and interleukin (IL) -1β family signaling pathways and acts as a bottle neck in the NF-κB and MAPK pathways. Here, we used ST2825, a selective inhibitor of MyD88, to clarify whether inhibiting MyD88 could provide neuroprotection in EBI following SAH. Our results showed that the expression of MyD88 was markedly increased at 24 h post SAH. Intracerebroventricular injection of ST2825 significantly reduced the expression of MyD88 at 24 h post SAH. Involvement of MAPKs and NF-κB signaling pathways was revealed that ST2825 inhibited SAH-induced phosphorylation of TAK1, p38 and JNK, the nuclear translocation of NF-κB p65, and degradation of IκBα. Further, ST2825 administration diminished the SAH-induced inflammatory response and apoptosis. As a result, SAH-induced EBI was alleviated and neurological deficits caused by SAH were reversed. Our findings suggest that MyD88 inhibition confers marked neuroprotection against EBI following SAH. Therefore, MyD88 might be a promising new molecular target for the treatment of SAH.

Exopolysaccharides (EPS) from lactic acid bacteria (LAB) have been reported to play vital parts in the modulation of cell-cycle and apoptosis in cancer cells. However, the mechanisms by which EPS regulate the proliferation and apoptosis of cancer cells remain incompletely understood. We thus used different cancer cells to evaluate the anticancer ability and to investigate the underlying molecular mechanism of EPS from Lactobacillus plantarum NCU116 (EPS116). Our studies showed that EPS116 inhibited the proliferation of cancer cells in a cell type manner, and remarkably repressed the growth and survival of CT26 through induction of apoptosis. Moreover, EPS116 increased the expression of pro-apoptotic genes, including Fas, Fasl and c-Jun, induced the phosphorylation of c-Jun in CT26 cells. Furthermore, TLR2 (Toll like receptor 2) was upregulated by EPS116, and the CT26 cells with TLR2 knockdown were found to be insensitive to EPS116, suggesting that the anti-cancer activity of EPS116 may be TLR2-dependent. Taken together, the suppressive efficacy of EPS116 on the proliferation of CT26 cells may be mediated via TLR2 and the activation of c-Jun dependent Fas/Fasl-mediated apoptotic pathway. Our study has, for the first time, shown that EPS from LAB induced c-Jun dependent Fas/Fasl-mediated apoptosis via TLR2 in CT26 cells.

Acute myeloid leukemia (AML) is a malignancy of myeloid progenitor cells that are blocked in differentiation. Acute promyelocytic leukemia (APL) is a rare form of AML, which generally presents with a t(15;17) translocation causing expression of the fusion protein PML-RARA. Pharmacological doses of all-trans retinoic acid (ATRA) induce granulocytic differentiation of APL cells leading to cure rates of >80% if combined with conventional chemotherapy. Autophagy is a lysosomal degradation pathway for the removal of cytoplasmic content and recycling of macromolecules. ATRA induces autophagy in ATRA-sensitive AML and APL cells and autophagy inhibition attenuates ATRA-triggered differentiation. In this study, we aimed at identifying if the autophagy-linked FYVE-domain containing protein (ALFY/WDFY3) is involved in autophagic degradation of protein aggregates contributes to ATRA therapy-induced autophagy. We found that ALFY mRNA levels increase significantly during the course of ATRA-induced differentiation of APL and AML cell lines. Importantly ALFY depletion impairs ATRA-triggered granulocytic differentiation of these cells. In agreement with its function in aggrephagy, knockdown of ALFY results in reduced ATRA-induced proteolysis. Our data further suggest that PML-RARα is an autophagy substrate degraded with the help of ALFY. In summary, we present a crucial role for ALFY in retinoid triggered maturation of AML cells.

Colorectal cancer is the fourth leading cause of cancer-related deaths worldwide. Here, we investigated the anticancer effect of Rhus coriaria extract (RCE) on HT-29 and Caco-2 human colorectal cancer cells. We found that RCE significantly inhibited the viability and colony growth of colon cancer cells. Moreover, RCE induced Beclin-1-independent autophagy and subsequent caspase-7-dependent apoptosis. Blocking of autophagy by chloroquine significantly reduced RCE-induced cell death, while blocking of apoptosis had no effect on RCE-induced cell death. Mechanistically, RCE inactivated the AKT/mTOR pathway by promoting the proteasome-dependent degradation of both proteins. Strikingly, we also found that RCE targeted Beclin-1, p53 and procaspase-3 to degradation. Proteasome inhibition by MG-132 not only restored these proteins to level comparable to control cells, but also reduced RCE-induced cell death and blocked the activation of autophagy and apoptosis. The proteasomal degradation of mTOR, which occurred only 3 hours post-RCE treatment was concomitant with an overall increase in the level of ubiquitinated proteins and translated stimulation of proteolysis by the proteasome. Our findings demonstrate that Rhus coriaria possesses strong anti-colon cancer activity through stimulation of proteolysis as well as induction of autophagic and apoptotic cell death, making it a potential and valuable source of novel therapeutic cancer drug.

Muscle atrophy is one of the serious complications of chronic kidney disease (CKD). Dysregulation of mitochondrial quality control (MQC) process, including decrease mitochondrial biogenesis, impair mitochondrial dynamics and induce activation of mitophagy, play an important role in mediating muscle wasting. This study aimed to observe effects of Jian-Pi-Yi-Shen (JPYS) decoction on muscle atrophy in CKD rats and explore its possible mechanism on regulation of MQC processes. The 5/6 nephrectomised rats were randomly allocated into 2 groups: CKD group and JPYS group. Besides, a sham-operated rats as sham group. All rats were treated for 6 weeks. Results showed that administration of JPYS decoction prevented body weight loss, muscle loss, muscle fiber size decrease, muscle protein degradation, and increased muscle protein systhesis. In addition, JPYS decoction increased the mitochondrial content and biogenesis proteins, and down-regulated the autophagy and mitophagy proteins. Furthermore, JPYS decoction increased mitochondrial fusion proteins, while decreased mitochondrial fission proteins. In conclusion, JPYS decoction increased mitochondrial content and biogenesis, restore the balance between fission and fusion, and inhibited autophagy-lysosome pathway (mitophagy). Collectively, our data showed that JPYS decoction to be beneficial to muscle atrophy in CKD, which might be associated with the modulation of MQC process.

Poly(ADP-ribose) polymerase (PARP) inhibitors represent a promising strategy toward the treatment of triple-negative breast cancer (TNBC), which is often associated to genomic instability and/or BRCA mutations. However, clinical outcome is controversial and no benefits have been demonstrated in wild type BRCA cancers, possibly due to poor drug bioavailability and low nuclear delivery. In the attempt to overcome these limitations, we have developed H-Ferritin nanoformulated olaparib (HOla) and assessed its anticancer efficacy on both BRCA-mutated and non-mutated TNBC cells. We exploited the natural tumor targeting of H-Ferritin, which is mediated by the transferrin receptor-1 (TfR1), and its physiological tropism toward cell nucleus. TNBC cell lines over-expressing TfR-1 were successfully recognized by H-Ferritin, displaying a fast internalization into the cells. HOla induced remarkable cytotoxic effect in cancer cells, exhibiting 1000-fold higher anticancer activity compared to free olaparib (Ola). Accordingly, HOla treatment enhanced PARP-1 cleavage, DNA double strand breaks and Ola delivery into the nuclear compartment. Our findings suggest that H-Ferritin nanoformulation strongly enhances cytotoxic efficacy of Ola as a stand-alone therapy in both BRCA-mutated and wild type TNBC cells, by promoting targeted nuclear delivery.

In this study, the authors investigated the anti-melanogenic effects of 3,8-dihydroxyquinoline (jineol) isolated from Scolopendra subspinipes mutilans, the mechanisms responsible for its inhibition of melanogenesis in melan-a cells, and its antioxidant efficacy. Mushroom tyrosinase activities and melanin contents were determined in melan-a cells, and the protein and mRNA levels of MITF, tyrosinase, TYRP-1, and TYRP-2 were assessed. Jineol exhibited significant, concentration-dependent antioxidant effects as determined by DPPH, ABTS, CUPRAC, and FRAP assays. Jineol significantly inhibited mushroom tyrosinase activity by functioning as an uncompetitive inhibitor, and markedly inhibited melanin production and intracellular tyrosinase activity in melan-a cells. In addition, jineol abolished the expressions of tyrosinase, TYRP-1, TYRP-2, and MITF, thereby blocking melanin production and interfering with the phosphorylations of ERK1/2 and p38. Furthermore, specific inhibitors of ERK1/2 and p38 prevented melanogenesis inhibition by jineol, and the proteasome inhibitor (MG-132) prevented jineol-induced reductions in cellular tyrosinase levels. Taken together, jineol was found to stimulate MAP-kinase (ERK1/2 and p38) phosphorylation and the proteolytic degradation pathway, which led to the degradations of MITF and tyrosinase, and to suppress the productions of melanin.

Colon cancer is one of the most common cancers. In this study, we isolated a lignan [(-)-(2R,3R)-1,4-O-diferuloylsecoisolariciresinol, DFS] from Alnus japonica (Betulaceae) and investigated its biological activity and mechanism of action on colon cancer. DFS reduced the viability of colon cancer cells and induced cell cycle arrest. DFS also suppressed β-catenin nuclear translocation and β-catenin target gene expression through a reduction in FoxM1 protein. To assess the mechanism of the action of DFS, we investigated the effect of DFS on endogenous and exogenous FoxM1 protein degradation in colon cancer cells. DFS-induced FoxM1 protein degradation was suppressed by lysosomal inhibitors, chloroquine and bafilomycin A1, but not by knock-down of proteasomal proteins. The mechanism of DFS for FoxM1 degradation is lysosomal dependent, which was not reported before. Furthermore, we found that FoxM1 degradation was partially lysosomal-dependent under normal conditions. These observations indicate that DFS from A. japonica suppresses colon cancer cell proliferation by reducing β-catenin nuclear translocation. DFS induces lysosomal-dependent FoxM1 protein degradation. This is the first report on the lysosomal degradation of FoxM1 by a small molecule. DFS may be useful in treating cancers that feature the elevated expression of FoxM1.

Hepatocellular carcinoma (HCC) is one of the most prevalent and malignant cancers with high inter- and intra-tumor heterogeneity. A central common signaling mechanism in cancer is proline-directed phosphorylation, which is further regulated by the unique proline isomerase Pin1. Pin1 is prevalently overexpressed in human cancers including ~70% of HCC, and promotes tumorigenesis by activating multiple cancer-driving pathways. However, it was challenging to evaluate the significance of targeting Pin1 in cancer treatment until the recent identification of all-trans retinoic acid (ATRA) as a Pin1 inhibitor. Here we systematically investigate functions of Pin1 and its inhibitor ATRA in the development and treatment of HCC. Pin1 knockdown potently inhibited HCC cell proliferation and tumor growth in mice. ATRA-induced Pin1 degradation inhibited the growth of HCC cells, although at a higher IC50 as compared with breast cancer cells, likely due to more active ATRA metabolism in liver cells. Indeed, inhibition of ATRA metabolism enhanced the sensitivity of HCC cells to ATRA. Moreover, slow-releasing ATRA potently and dose-dependently inhibited HCC growth in mice. Finally, chemical or genetic Pin1 ablation blocked multiple cancer-driving pathways simultaneously in HCC cells. Thus, targeting Pin1 offers a promising therapeutic approach to simultaneously stop multiple cancer-driving pathways in HCC.

HIV maturation inhibitors are an emerging class of anti-retroviral compounds that inhibit the viral protease-mediated cleavage of the Gag, CA-SP1 (capsid-spacer peptide 1) peptide to mature CA. The first-in-class maturation inhibitor bevirimat (BVM) displayed potent activity against HIV-1 clade B but was ineffective against other HIV-1 clades including clade C. Another pyridone-based maturation inhibitor, PF-46396 displayed potent activity against HIV-1 clade B. In this study, we aimed at determining the activity of PF-46396 against HIV-1 clade C. We employed various biochemical and virological assays to demonstrate that PF-46396 is effective against HIV-1 clade C. We observed a dose dependent accumulation of CA-SP1 intermediate in presence of the compound. We carried out mutagenesis in the CA- SP1 region of HIV-1 clade C Gag and observed that the mutations conferred resistance against the compound. Many mutations inhibited Gag processing thereby reducing virus release in the absence of the compound. However, presence of PF-46396 rescued these defects and enhanced virus release, replication capacity and infectivity of HIV-1 clade C. These results put together identify PF-46396 as a broadly active maturation inhibitor against HIV-1 clade B and C and help in rational designing of novel analogs with reduced toxicity and increased efficacy for its potential use in clinics.

This work aimed to establish an effective approach to evaluate the quality of frozen fish, focusing on changes in fish tissue structure and chemical composition during storage. Fresh tilapia samples were treated by coating with tangerine peel (TP) extract and then stored at -4, -8 and -18 °C, respectively, for 40 days. The frozen fish tissues were analyzed for structural and chemical changes. Fractal dimension, which quantifies the porous structure formed in the tissue samples, texture properties including hardness and springiness, and moisture content and water activity all decreased during the storage, while the extents of lipid oxidation, measured as peroxide value and thiobarbituric acid concentration, and protein degradation, monitored with total volatile basic nitrogen and trichloroacetic acid soluble peptides, increased. The change rates of these parameters decreased with decreasing the storage temperature and by applying TP extract. A model was developed for predicting fractal dimension, which indicated the quality of preserved tilapia and thus can be used to predict the shelf life under different storage temperatures. The results demonstrated that TP extract could extend the shelf life of frozen tilapia by 35-45% by inhibiting changes in tissue structure, moisture loss, lipid oxidation and protein degradation during frozen storage., Competing Interests: The authors declare no competing financial interests.

Dysfunction of energy metabolism is involved in inflammatory bowel disease (IBD). This study was designed to investigate the potential of astragaloside IV (ASIV), an active ingredient of Radix Astragalus, to ameliorate colonic mucosal injury, with focusing on the implication of energy restoration in the underlying mechanism. Experimental colitis model was established in rats by injecting 2,4,6-trinitrobenzene sulfonic acid (TNBS) through anus. After 24 hours, ASIV was administrated once daily by gavage for 6 days. On day 1 and day 7, colon tissue was collected for macroscopic and histological examination, ELISA, Western blot and immunohistochemical analysis. TNBS impaired colonic mucosa with an injured epithelial architecture, increased inflammatory cell infiltration, and decreased colonic blood flow. Lgr5 positive cell number in crypt and β-catenin nuclear translocation were down-regulated by TNBS treatment. TNBS induced epithelial F-actin disruption and junctional protein degradation. Furthermore, adenosine triphosphate (ATP) content and ATP synthase subunit β expression in the colon tissue were significantly decreased after TNBS stimulation. All of the aforementioned alterations were relieved by ASIV post-treatment. The present study revealed that ASIV promoted mucosal healing process in TNBS-induced colitis, which was most likely attributed to regulating energy metabolism., Competing Interests: The authors declare no competing financial interests.

Inducing degradation of oncoproteins by small molecule compounds has the potential to avoid drug resistance and therefore deserves to be exploited for new therapies. Oridonin is a natural compound with promising antitumor efficacy that can trigger the degradation of oncoproteins; however, the direct cellular targets and underlying mechanisms remain unclear. Here we report that oridonin depletes BCR-ABL through chaperon-mediated proteasomal degradation in leukemia. Mechanistically, oridonin poses oxidative stress in cancer cells and directly binds to cysteines of HSF1, leading to the activation of this master regulator of the chaperone system. The resulting induction of HSP70 and ubiquitin proteins and the enhanced binding to CHIP E3 ligase hence target BCR-ABL for ubiquitin-proteasome degradation. Both wild-type and mutant forms of BCR-ABL can be efficiently degraded by oridonin, supporting its efficacy observed in cultured cells as well as mouse tumor xenograft assays with either imatinib-sensitive or -resistant cells. Collectively, our results identify a novel mechanism by which oridonin induces rapid degradation of BCR-ABL as well as a novel pharmaceutical activator of HSF1 that represents a promising treatment for leukemia., Competing Interests: The authors declare no competing financial interests.

Mammalian Eag1 (Kv10.1) potassium (K + ) channels are widely expressed in the brain. Several mutations in the gene encoding human Eag1 K + channel have been associated with congenital neurodevelopmental anomalies. Currently very little is known about the molecules mediating protein synthesis and degradation of Eag1 channels. Herein we aim to ascertain the protein degradation mechanism of rat Eag1 (rEag1). We identified cullin 7 (Cul7), a member of the cullin-based E3 ubiquitin ligase family, as a novel rEag1 binding partner. Immunoprecipitation analyses confirmed the interaction between Cul7 and rEag1 in heterologous cells and neuronal tissues. Cul7 and rEag1 also exhibited significant co-localization at synaptic regions in neurons. Over-expression of Cul7 led to reduced protein level, enhanced ubiquitination, accelerated protein turn-over, and decreased current density of rEag1 channels. We provided further biochemical and morphological evidence suggesting that Cul7 targeted endoplasmic reticulum (ER)- and plasma membrane-localized rEag1 to the proteasome and the lysosome, respectively, for protein degradation. Cul7 also contributed to protein degradation of a disease-associated rEag1 mutant. Together, these results indicate that Cul7 mediates both proteasomal and lysosomal degradations of rEag1. Our findings provide a novel insight to the mechanisms underlying ER and peripheral protein quality controls of Eag1 channels.

Chronic obstructive pulmonary disease (COPD) is characterized by reduced lung function associated with increased local and systemic inflammatory markers, such as TNFα and IL-1β. Glucocorticoids are used to treat this chronic disease, however their efficacy is low and new drugs are very much required. 17-oxo-DHA is a cyclooxygenase-2-dependent, electrophilic, α,β-unsaturated keto-derivative of docosahexaenoic acid with anti-inflammatory properties. We evaluated the action of 17-oxo-DHA alone or in combination with the steroid fluticasone propionate (FP) in peripheral blood mononuclear cells (PBMCs) from COPD patients and healthy individuals exposed to lipopolysaccharide. We show that PBMCs from COPD patients released higher levels of TNFα and IL-1β compared to controls. 17-oxo-DHA displayed strong anti-inflammatory effects. The addition of 17-oxo-DHA in combination with FP showed enhanced anti-inflammatory effects through the modulation of transcriptional and post-transcriptional mechanisms. 17-oxo-DHA, but not FP, was able to suppress the release of mature IL-1β through inhibition of the NLRP3 inflammasome. Furthermore, 17-oxo-DHA inhibited inflammasome-dependent degradation of the glucocorticoid receptor (GR). Our findings suggest that 17-oxo-DHA in combination with FP or other steroids might achieve higher therapeutic efficacy than steroids alone. Combined treatment might be particularly relevant in those conditions where increased inflammasome activation may lead to GR degradation and steroid-unresponsive inflammation.

We have previously shown that ES-62, a phosphorylcholine (PC)-containing glycoprotein secreted by the parasitic filarial nematode Acanthocheilonema viteae targets dendritic cell (DC) responses, specifically by suppressing TLR4 signalling to inhibit Th1/Th17-driven inflammation. We have now investigated the molecular mechanisms underpinning such immunomodulation and show here that ES-62-mediated downregulation of protein kinase C-δ (PKC-δ), a TLR4-associated signalling mediator required for full activation of LPS-driven pro-inflammatory responses, is associated with induction of a low level of autophagic flux, as evidenced by upregulation and trafficking of p62 and LC3 and their consequent autophagolysosomal degradation. By contrast, the classical TLR4 ligand LPS, strongly upregulates p62 and LC3 expression but under such canonical TLR4 signalling this upregulation appears to reflect a block in autophagic flux, with these elements predominantly degraded in a proteasomal manner. These data are consistent with autophagic flux acting to homeostatically suppress proinflammatory DC responses and indeed, blocking of PKC-δ degradation by the autophagolysosomal inhibitors, E64d plus pepstatin A, results in abrogation of the ES-62-mediated suppression of LPS-driven release of IL-6, IL-12p70 and TNF-α by DCs. Thus, by harnessing this homeostatic regulatory mechanism, ES-62 can protect against aberrant inflammation, either to promote parasite survival or serendipitously, exhibit therapeutic potential in inflammatory disease.

Ganoderma lucidum exerts antitumor activity, but the mechanism of G. lucidum polysaccharides on cancer is unclear. Here, we demonstrated that a fucose-containing fraction of Ling-Zhi (FFLZ) reduced tumor size and suppressed metastasis in vivo. Furthermore, FFLZ inhibited breast cancer cell migration and altered the epithelial-to-mesenchymal transition (EMT) phenotype. Transforming growth factor-β receptor (TGFR) pathways act as key mediators to promote tumor progression and metastasis. We found that FFLZ down-regulated TGFR and downstream signaling pathways, including the phosphorylation of Smad2/3 and the expression of Smad4. In an investigation of the underlying mechanisms, we found that FFLZ enhanced the Smurf2-dependent ubiquitination of TGFR by disrupting the balance of the lipid rafts, promoted the "re-localization" of the TGFR to the caveolae, and facilitated the degradation of TGFR. Together, our data indicated that FFLZ is associated with the inhibition of EMT and the prevention of metastasis by promoting ubiquitination-dependent TGFR degradation and abolishing TGFR signaling pathways. Moreover, the combination of FFLZ and trastuzumab synergistically inhibited the viability of certain trastuzumab-resistant human breast cancer cells. In summary, our current findings indicate that FFLZ is a potential therapeutic or dietary supplemental agent for cancer patients and that it functions via the caveolin-1/Smad7/Smurf2-dependent ubiquitin-mediated degradation of TGFR.