Cytoprotective Functions Research Articles

NRF2-Dependent Bioactivation of Mitomycin C as a Novel Strategy To Target KEAP1-NRF2 Pathway Activation in Human Cancer.

Activating mutations in the KEAP1-NRF2 pathway are found in approximately 25% of lung tumors, where the hijacking of NRF2's cytoprotective functions results in aggressive tumor growth, chemoresistance, and a poor prognosis for patients. There are currently no approved drugs which target aberrant NRF2 activation, which means that there is an urgent clinical need to target this orphan oncogenic pathway in human tumors. In this study, we used an isogenic pair of wild-type and Keap1 knockout cells to screen a range of chemotherapeutic and pathway-targeted anticancer drugs in order to identify compounds which display enhanced toxicity toward cells with high levels of Nrf2 activity. Through this approach, complemented by validation across a panel of eight human cancer cell lines from a range of different tissues, we identified the DNA-damaging agent mitomycin C to be significantly more toxic in cells with aberrant Nrf2 activation. Mechanistically, we found that the NRF2 target genes for cytochrome P450 reductase, NQO1, and enzymes in the pentose phosphate pathway are all responsible for the NRF2-dependent enhanced bioactivation of mitomycin C. As mitomycin C is already approved for clinical use, it represents as excellent drug repositioning candidate to target the currently untreatable NRF2 activation in human tumors.

Expression Profile of Mouse Gm20594, Nuclear-Encoded Humanin-Like Gene.

BackgroundMitochondrial-derived peptides (MDPs) such as MOTS-c and humanin have been studied for their cytoprotective functions. In mice, humanin-encoding Mtrnr2 is a mitochondrial pseudogene, and the humanin-like peptide is encoded by the nuclear Gm20594 gene. However, endogenous tissue-specific expression profiles of Gm20594 have not yet been identified.MethodsMtrnr1 and Gm20594 expression was profiled via reverse transcription using only oligo(dT) primers from tissues of C57BL6/J mice. To analyze altered expression upon mitochondrial biogenesis, C2C12 myocytes and brown adipocytes were differentiated. Mitochondrial DNA copy numbers were quantified for normalization.ResultsBoth Mtrnr1 and Gm20594 were highly expressed in brown adipose tissue. When normalized against mitochondrial content, Mtrnr1 was identified as being highly expressed in the duodenum, followed by the jejunum. In models of mitochondrial biogenesis, both Mtrnr1 and Gm20594 were upregulated during myocyte and brown adipocyte differentiation. Increased Mtrnr1 expression during brown adipocyte differentiation remained significant after normalization against mitochondrial DNA copy number, whereas myocyte differentiation exhibited biphasic upregulation and downregulation in early and late phases, respectively.ConclusionNuclear-encoded Gm20594 showed similar expression patterns of mitochondrial-encoded Mtrnr1. Brown adipose tissue presented the highest basal expression levels of Gm20594 and Mtrnr1. When normalized against mitochondrial DNA copy number, gut tissues exhibited the highest expression of Mtrnr1. Upregulation of Mtrnr1 during mitochondrial biogenesis is independent of mitochondrial content.

General Structural and Functional Features of Molecular Chaperones.

Molecular chaperones are a group of structurally diverse and highly conserved ubiquitous proteins. They play crucial roles in facilitating the correct folding of proteins in vivo by preventing protein aggregation or facilitating the appropriate folding and assembly of proteins. Heat shock proteins form the major class of molecular chaperones that are responsible for protein folding events in the cell. This is achieved by ATP-dependent (folding machines) or ATP-independent mechanisms (holders). Heat shock proteins are induced by a variety of stresses, besides heat shock. The large and varied heat shock protein class is categorised into several subfamilies based on their sizes in kDa namely, small Hsps (HSPB), J domain proteins (Hsp40/DNAJ), Hsp60 (HSPD/E; Chaperonins), Hsp70 (HSPA), Hsp90 (HSPC), and Hsp100. Heat shock proteins are localised to different compartments in the cell to carry out tasks specific to their environment. Most heat shock proteins form large oligomeric structures, and their functions are usually regulated by a variety of cochaperones and cofactors. Heat shock proteins do not function in isolation but are rather part of the chaperone network in the cell. The general structural and functional features of the major heat shock protein families are discussed, including their roles in human disease. Their function is particularly important in disease due to increased stress in the cell. Vector-borne parasites affecting human health encounter stress during transmission between invertebrate vectors and mammalian hosts. Members of the main classes of heat shock proteins are all represented in Plasmodium falciparum, the causative agent of cerebral malaria, and they play specific functions in differentiation, cytoprotection, signal transduction, and virulence.

Farrerol Enhances Nrf2-Mediated Defense Mechanisms against Hydrogen Peroxide-Induced Oxidative Damage in Human Retinal Pigment Epithelial Cells by Activating Akt and MAPK.

Oxidative stress of the retinal pigment epithelium (RPE) is an essential element contributing to the progression of age-related macular degeneration (AMD). Notably, the activation of Nrf2 is regarded as an effective strategy for controlling oxidation. The novel 2,3-dihydroflavonoid compound farrerol, which is extracted from Rhododendron, possesses antioxidant properties. In this study, we investigated the mechanism by which farrerol protects against oxidative damage mediated by hydrogen peroxide (H2O2) in adult retinal pigment epithelial cell line 19 (ARPE-19) cells. Farrerol supplementation conspicuously reversed H2O2-related cell damage through declining the generation of intracellular reactive oxygen species (ROS) and MDA and increasing the concentrations of GSH and SOD. According to the results of the apoptosis assay, a farrerol pretreatment decreased the protein expression of the Bax/Bcl-2, cleaved caspase-3, PARP, caspase-8, and caspase-9 proteins. Furthermore, farrerol markedly activated Nrf2, thereby increasing the levels of antioxidant enzymes downstream of Nrf2, such as HO-1, NQO1, and GCLM. Knockdown of Nrf2 with a specific siRNA successfully suppressed farrerol-mediated HO-1 transcription and partially abolished the cytoprotective effect on ARPE-19 cells. Meanwhile, farrerol induced Akt and MAPK phosphorylation in a dose-related way. However, inhibiting Akt and MAPK substantially blocked the cytoprotective functions of farrerol. Therefore, farrerol enhanced Nrf2-mediated cytoprotection of oxidative damage caused by H2O2, which may be inseparable from the activation of Akt and MAPK.

Divergent erythroid megakaryocyte fates in Blvrb-deficient mice establish non-overlapping cytoprotective functions during stress hematopoiesis

Cytoprotective mechanisms of heme oxygenases function by derivatizing heme to generate carbon monoxide, ferrous iron, and isomeric biliverdins, followed by rapid NAD(P)H-dependent biliverdin reduction to the antioxidant bilirubin using two non-overlapping biliverdin reductases that display biliverdin isomer-restricted redox activity. Although cytoprotective functions of heme oxygenases are widely recognized, concomitant effects of downstream biliverdin reductases remain incomplete. A computational model predicated on murine hematopoietic single-cell transcriptomic data identified Blvrb as a biological driver linked to the tumor necrosis factor stress pathway as a predominant source of variation defining hematopoietic cell heterogeneity. In vivo studies using Blvrb-deficient mice established the dispensable role of Blvrb in steady-state hematopoiesis, although model validation using aged Blvrb-deficient mice established an important cytoprotective function in stress hematopoiesis with dichotomous megakaryocyte-biased hematopoietic recovery. Defective stress erythropoiesis was evident in Blvrb−/− spleens and in bone marrow erythroid development, occurring in conjunction with defective lipid peroxidation as a marker of oxidant mishandling. Cell autonomous effects on megakaryocyte lineage bias were documented using multipotential progenitor assays. These data provide the first physiological function of murine Blvrb in a non-redundant pathway of stress cytoprotection. Divergent effects on erythroid/megakaryocyte lineage speciation impute a novel redox-regulated mechanism for lineage partitioning.

CROSSTALK BETWEEN AUTOPHAGY AND APOPTOSIS IN CD437-INDUCED А549 LUNG CARCINOMA CELL DEATH

Introduction. Synthetic retinoids CD437, an agonist of the gamma retinoic acid receptor (RARγ), not only induces growth arrest, but in contrast to retinoid acid, it also induces RARγ-independent apoptosis in many tumor cells through a unique mechanism that is independent of the retinoic acid receptor-mediated pathway. The aim of the study was to study the relationship between apoptosis and autophagy in CD437-induced cell death. Materials and methods. In this study we used 2D-culturing of lung carcinoma cells A549, immunocytochemistry, flow cytometry and fluorescence microscopy. Results. CD437 at concentrations between 0.2 and 5.0 μM increased the number of PI-positive cells in A549 lung cancer cells. The retinoid at concentrations close to IC 50 reduced the cell population in the G2/M-phase and arrest cell cycle in the S-phase. CD437 dose-dependent increased the number of apoptotic cells in the presence of non-cytotoxic concentrations of wortmanin, an irreversible inhibitor of anti-stress kinasе PI-3K, and LY 200192, a reversible inhibitor. CD437 also activated the biogenesis of autophagosomes, and there was a dose-dependent increase in the fluorescence intensity of monodansilkadaverine, a marker of autophagy. However, accumulation of LC-3B was not observed with an increase of CD437 concentration from 0.1 to 5.0 μM suggesting that the fusion between autophagic vacuoles and lysosomes was inhibited. zVAD-fmk, an irreversible caspase inhibitor, did not restore autophagy in A549 cells, and LC-3B levels did not change significantly with the increasing of CD437 concentration, indicating that CD437 was involved in autophagosome-lysosome fusion. When cells grew with non-cytotoxic concentrations of chlorokine, a late stage autophagy inhibitor, there were virtually no living cells at CD437 concentrations close to IC 50 . The additive effect of CD437 and chlorokine in inducing A549 cell death confirms that CD437 involved in fusion between autophagosomes and lysosomes required for final catabolism of autophagic material. Conclusions. The data obtained indicate that CD437 induced a failure of cytoprotective function of autophagy and apoptosis, that raise the question of combined therapy of CD437 with cytotoxic drugs in the treatment of lung carcinoma

Hsp70 in cancer: A double agent in the battle between survival and death.

The heat shock protein(Hsps) superfamily, also known as molecular chaperones, are highly conserved and present in all living organisms and play vital roles in protein fate. The HspA1A (Hsp70-1), calledHsp70 in this review, is expressed at low or undetectable levels in most unstressed normal cells, but numerous studies have shown that diverse types of tumor cells express Hsp70 at the plasma membrane that leads to resistance to programmed cell death and tumor progression. Hsp70 is released into the extracellular milieu in three forms including free soluble, complexed with cancer antigenic peptides, and exosome forms. Therefore, it seems to be a promising therapeutic target in human malignancies. However, a great number of studies have indicated that both intracellular and extracellular Hsp70 have a dual function. A line of evidence presented that intracellular Hsp70 has a cytoprotective function via suppression of apoptosis and lysosomal cell death (LCD) as well as that extracellular Hsp70 can promote tumorigenesis and angiogenesis. Other evidence showedintracellular Hsp70 can promote apoptosis and membrane-associated/extracellular Hsp70 can elicit antitumor innate and adaptive immune responses. Given the contradictory functions, as a "double agent,"could Hsp70 be a promising tool in the future of targeted cancer therapies? To answer this question, in this review, we will discuss the functions of Hsp70 in cancers besides inhibition and stimulation strategies fortargeting Hsp70 along with their challenges.

Network Pharmacology and bioinformatics analyses identify intersection genes of niacin and COVID-19 as potential therapeutic targets.

ObjectivesPatients with colorectal cancer (CRC) may be susceptible to the coronavirus disease-2019 (COVID-19). However, anti-CRC/COVID-19 treatment options are currently unavailable. Since niacin is a vitamin with cytoprotective and anti-inflammatory functions, this study aimed to evaluate the possible functional roles and underlying mechanisms of action of niacin as an anti-COVID-19 and -CRC therapy.InterventionsWe used a series of network pharmacology-based and computational analyses to understand and characterize the binding capacity, biological functions, pharmacological targets and therapeutic mechanisms of niacin in CRC/COVID-19.Measurements and main resultsWe revealed the clinical characteristics of CRC patients and COVID-19 patients, including predisposing genes, survival rate and prognosis. Moreover, the results of molecular docking analysis indicated that niacin exerted effective binding capacity in COVID-19. Further, we disclosed the targets, biological functions and signaling pathways of niacin in CRC/COVID-19. The analysis indicated that niacin could help in treating CRC/COVID-19 through cytoprotection, enhancement of immunologic functions, inhibition of inflammatory reactions and regulation of cellular microenvironment. Furthermore, five core pharmacological targets of niacin in CRC/COVID-19 were also identified, including BCL2L1, PTGS2, IL1B, IFNG and SERPINE1.ConclusionsThis study, for the first time, revealed the niacin-associated molecular functions and pharmacological targets for treating CRC/COVID-19, as COVID-19 remains a serious pandemic. But the findings were not validated in actual CRC patients infected with COVID-19, so further investigation is needed to confirm the potential use of niacin for treating CRC/COVID-19.

Single-cell derived tumor organoids display diversity in HLA class I peptide presentation

Tumor heterogeneity is a major cause of therapeutic resistance. Immunotherapy may exploit alternative vulnerabilities of drug-resistant cells, where tumor-specific human leukocyte antigen (HLA) peptide ligands are promising leads to invoke targeted anti-tumor responses. Here, we investigate the variability in HLA class I peptide presentation between different clonal cells of the same colorectal cancer patient, using an organoid system. While clone-specific differences in HLA peptide presentation were observed, broad inter-clone variability was even more prevalent (15–25%). By coupling organoid proteomics and HLA peptide ligandomics, we also found that tumor-specific ligands from DNA damage control and tumor suppressor source proteins were prominently presented by tumor cells, coinciding likely with the silencing of such cytoprotective functions. Collectively, these data illustrate the heterogeneous HLA peptide presentation landscape even within one individual, and hint that a multi-peptide vaccination approach against highly conserved tumor suppressors may be a viable option in patients with low tumor-mutational burden.

HSPB5 (αB-crystallin) confers protection against paraquat-induced oxidative stress at the organismal level in a tissue-dependent manner

Oxidative stress is one of the major and continuous stresses, an organism encounters during its lifetime. Tissues such as the brain, liver and muscles are more prone to damage by oxidative stress due to their metabolic activity, differences in physiological and adaptive processes. One of the defence mechanisms against continuous oxidative stress is a set of small heat shock proteins. αB-Crystallin/HSPB5, a small heat shock protein, gets upregulated under stress and acts as a molecular chaperone. In addition to acting as a molecular chaperone, HSPB5 is shown to have a role in other cytoprotective functions such as inhibition of apoptosis, prevention of oxidative stress and stabilisation of cytoskeletal system. Such protection in vivo, at the organism level, particularly in a tissue-dependent manner, has not been investigated. We have expressed HSPB5 in fat body (liver), neurons and specifically in dopaminergic and motor neurons in Drosophila and investigated its protective effect against paraquat-induced oxidative stress. We observed that expression of HSPB5 in neurons and fat body confers protection against paraquat-induced oxidative stress. Expression in dopaminergic neurons showed a higher protective effect. Our results clearly establish the protective ability of HSPB5 in vivo; the extent of protection, however, varies depending on the tissue in which it is expressed. Interestingly, neuronal expression of HSPB5 resulted in an improvement in negative geotropic behaviour, whereas specific expression in muscle tissue did not show such a beneficial effect.

Sestrin1 exerts a cytoprotective role against oxygen-glucose deprivation/reoxygenation-induced neuronal injury by potentiating Nrf2 activation via the modulation of Keap1

Sestrin1 (Sesn1) acts as a stress-inducible protein that performs a remarkable cytoprotective function upon diverse cellular stresses. However, whether Sesn1 exerts a cytoprotective role in neurons following cerebral ischemia/reperfusion injury is unknown. The goal of this work was to evaluate the role of Sesn1 in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal injury in vitro. The induction of Sesn1 was found in neurons exposed to OGD/R treatment. The silencing of Sesn1 rendered neurons more vulnerable to OGD/R injury, while the up-regulation of Sesn1 ameliorated OGD/R-induced neuronal injury by reducing apoptosis and the generation of reactive oxygen species (ROS). Furthermore, the up-regulation of Sesn1 promoted the activity of the nuclear factor-erythroid 2-related factor 2 (Nrf2) by down-regulating the expression of the Kelchlike ECH-associated protein 1 (Keap1). The restoration of Keap1 or the suppression of Nrf2 remarkably abolished the Sesn1-induced neuroprotection effects in OGD/R-exposed neurons. In summary, our work indicates that Sesn1 is a remarkable neuroprotective protein that potentiates Nrf2 activation via Keap1 to ameliorate OGD/R-induced injury.

Activation of NRF2 by topical apocarotenoid treatment mitigates radiation-induced dermatitis

Radiation therapy is a frontline treatment option for cancer patients; however, the effects of radiotherapy on non-tumor tissue (e.g. radiation-induced dermatitis) often worsen patient quality of life. Previous studies have implicated the importance of redox balance in preventing dermatitis, specifically in reference to modulation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) signaling pathway. Due to the cytoprotective functions of transcriptional target genes of NRF2, we investigated how modulation of NRF2 expression could affect DNA damage, oxidative stress, and cell viability in response to radiotherapy. Specifically, it was noted that NRF2 knockdown sensitized human skin keratinocytes to ionizing radiation; likewise, genetic ablation of NRF2 in vivo increased radiosensitivity of murine epidermis. Oppositely, pharmacological induction of NRF2 via the apocarotenoid bixin lowered markers of DNA damage and oxidative stress, while preserving viability in irradiated keratinocytes. Mechanistic studies indicated that topical pretreatment using bixin as an NRF2 activator antagonized initial DNA damage by raising cellular glutathione levels. Additionally, topical application of bixin prevented radiation-induced dermatitis, epidermal thickening, and oxidative stress in the skin of SKH1 mice. Overall, these data indicate that NRF2 is critical for mitigating the harmful skin toxicities associated with ionizing radiation, and that topical upregulation of NRF2 via bixin could prevent radiation-induced dermatitis.

CpG Site-Specific Regulation of Metallothionein-1 Gene Expression.

Metal-binding inducible proteins called metallothioneins (MTs) protect cells from heavy-metal toxicity. Their transcription is regulated by metal response element (MRE)-binding transcription factor-1 (MTF1), which is strongly recruited to MREs in the MT promoters, in response to Zn and Cd. Mouse Mt1 gene promoter contains 5 MREs (a–e), and MTF1 has the highest affinity to MREd. Epigenetic changes like DNA methylation might affect transcription and, therefore, the cytoprotective function of MT genes. To reveal the CpG site(s) critical for Mt1 transcription, we analyzed the methylation status of CpG dinucleotides in the Mt1 gene promoter through bisulfite sequencing in P1798 mouse lymphosarcoma cells, with high or low MT expression. We found demethylated CpG sites near MREd and MREe, in cells with high expression. Next, we compared Mt1 gene-promoter-driven Lucia luciferase gene expression in unmethylated and methylated reporter vectors. To clarify the effect of complete and partial CpG methylation, we used M.SssI (CG→5mCG) and HhaI (GCGC→G5mCGC)-methylated reporter vectors. Point mutation analysis revealed that methylation of a CpG site near MREd and MREe strongly inhibited Mt1 gene expression. Our results suggest that the methylation status of this site is important for the regulation of Mt1 gene expression.

Clusterin secretion is attenuated by the proinflammatory cytokines interleukin-1β and tumor necrosis factor-α in models of cartilage degradation.

The protein clusterin has been implicated in the molecular alterations that occur in articular cartilage during osteoarthritis (OA). Clusterin exists in two isoforms with opposing functions, and their roles in cartilage have not been explored. The secreted form of clusterin (sCLU) is a cytoprotective extracellular chaperone that prevents protein aggregation, enhances cell proliferation and promotes viability, whereas nuclear clusterin acts as a pro-death signal. Therefore, these two clusterin isoforms may be putative molecular markers of repair and catabolic responses in cartilage and the ratio between them may be important. In this study, we focused on sCLU and used established, pathophysiologically relevant, in vitro models to understand its role in cytokine-stimulated cartilage degradation. The secretome of equine cartilage explants, osteochondral biopsies and isolated unpassaged chondrocytes was analyzed by western blotting for released sCLU, cartilage oligomeric protein (COMP) and matrix metalloproteinases (MMP) 3 and 13, following treatment with the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α. Release of sulfated glycosaminoglycans (sGAG) was determined using the dimethylmethylene blue assay. Clusterin messenger RNA (mRNA) expression was quantified by quantitative real-time polymerase chain reaction. MMP-3, MMP-13, COMP, and sGAG release from explants and osteochondral biopsies was elevated with cytokine treatment, confirming cartilage degradation in these models. sCLU release was attenuated with cytokine treatment in all models, potentially limiting its cytoprotective function. Clusterin mRNA expression was down-regulated 7-days post cytokine stimulation. These observations implicate sCLU in catabolic responses of chondrocytes, but further studies are required to evaluate its role in OA and its potential as an investigative biomarker.

MicroRNAs as regulators of VEGFA and NFE2L2 in cancer.

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in post-transcriptional regulation of various genes, and their deregulation can lead to tumorigenesis. They may play the role of oncogenes or tumor suppressors by regulating different genes involved in cellular processes. One of the genes regulated by the miRNAs is the vascular endothelial growth factor A (VEGFA), which is responsible for angiogenesis. Angiogenesis is the process of formation of new blood vessels from pre-existing ones. This process plays an important role in tumor development, since it is responsible for the transport of nutrients required for tumor growth. Several studies have shown an increased expression of VEGFA in various cancers. Another gene regulated by miRNAs, the nuclear factor erythroid 2-like-2 (NFE2L2/NRF2), has a cytoprotective function and regulates cellular defense against oxidative stress. The NFE2L2 is the major regulator of cytoprotective agents and their oxidative damage to cells, which is down-regulated by Kelch-like ECH-associated protein 1 (KEAP1) at the post-transcriptional level. Regulation of the VEGFA and NFE2L2 by miRNAs has been observed in hepatocellular carcinoma and breast, lung, esophageal, endometrial, gastric, and ovarian cancer. This review highlights the role of miRNAs in the regulation of VEGFA and NFE2L2 and their relevance as therapeutic targets in various cancers.

Temporal changes guided by mesenchymal stem cells on a 3D microgel platform enhance angiogenesis in vivo at a low-cell dose

Therapeutic factors secreted by mesenchymal stem cells (MSCs) promote angiogenesis in vivo. However, delivery of MSCs in the absence of a cytoprotective environment offers limited efficacy due to low cell retention, poor graft survival, and the nonmaintenance of a physiologically relevant dose of growth factors at the injury site. The delivery of stem cells on an extracellular matrix (ECM)-based platform alters cell behavior, including migration, proliferation, and paracrine activity, which are essential for angiogenesis. We demonstrate the biophysical and biochemical effects of preconditioning human MSCs (hMSCs) for 96 h on a three-dimensional (3D) ECM-based microgel platform. By altering the macromolecular concentration surrounding cells in the microgels, the proangiogenic phenotype of hMSCs can be tuned in a controlled manner through cell-driven changes in extracellular stiffness and "outside-in" integrin signaling. The softest microgels were tested at a low cell dose (5 × 104 cells) in a preclinical hindlimb ischemia model showing accelerated formation of new blood vessels with a reduced inflammatory response impeding progression of tissue damage. Molecular analysis revealed that several key mediators of angiogenesis were up-regulated in the low-cell-dose microgel group, providing a mechanistic insight of pathways modulated in vivo. Our research adds to current knowledge in cell-encapsulation strategies by highlighting the importance of preconditioning or priming the capacity of biomaterials through cell-material interactions. Obtaining therapeutic efficacy at a low cell dose in the microgel platform is a promising clinical route that would aid faster tissue repair and reperfusion in "no-option" patients suffering from peripheral arterial diseases, such as critical limb ischemia (CLI).

N- and C-terminal regions of αB-crystallin and Hsp27 mediate inhibition of amyloid nucleation, fibril binding, and fibril disaggregation

Small heat-shock proteins (sHSPs) are ubiquitously expressed molecular chaperones that inhibit amyloid fibril formation; however, their mechanisms of action remain poorly understood. sHSPs comprise a conserved α-crystallin domain flanked by variable N- and C-terminal regions. To investigate the functional contributions of these three regions, we compared the chaperone activities of various constructs of human αB-crystallin (HSPB5) and heat-shock 27-kDa protein (Hsp27, HSPB1) during amyloid formation by α-synuclein and apolipoprotein C-II. Using an array of approaches, including thioflavin T fluorescence assays and sedimentation analysis, we found that the N-terminal region of Hsp27 and the terminal regions of αB-crystallin are important for delaying amyloid fibril nucleation and for disaggregating mature apolipoprotein C-II fibrils. We further show that the terminal regions are required for stable fibril binding by both sHSPs and for mediating lateral fibril-fibril association, which sequesters preformed fibrils into large aggregates and is believed to have a cytoprotective function. We conclude that although the isolated α-crystallin domain retains some chaperone activity against amyloid formation, the flanking domains contribute additional and important chaperone activities, both in delaying amyloid formation and in mediating interactions of sHSPs with amyloid aggregates. Both these chaperone activities have significant implications for the pathogenesis and progression of diseases associated with amyloid deposition, such as Parkinson's and Alzheimer's diseases.

Targeted inhibition of activated protein C by a non-active-site inhibitory antibody to treat hemophilia

Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Based on the hypothesis that specific inhibition of APC’s anticoagulant but not its cytoprotective activity can be beneficial for hemophilia therapy, 2 types of inhibitory monoclonal antibodies (mAbs) are tested: A type I active-site binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as shown by X-ray crystallography. Both mAbs increase thrombin generation and promote plasma clotting. Type I blocks all APC activities, whereas type II preserves APC’s cytoprotective function. In normal monkeys, type I causes many adverse effects including animal death. In contrast, type II is well-tolerated in normal monkeys and shows both acute and prophylactic dose-dependent efficacy in hemophilic monkeys. Our data show that the type II mAb can specifically inhibit APC’s anticoagulant function without compromising its cytoprotective function and offers superior therapeutic opportunities for hemophilia.

Albumin Protects Lung Cells against Acrolein Cytotoxicity and Acrolein-Adducted Albumin Increases Heme Oxygenase 1 Transcripts.

Albumin is an abundant protein in the lung lining fluid that forms an interface between lung epithelial cells and the external environment. In the lung, albumin can be targeted for adduction by inhaled acrolein. Acrolein, an α,β-unsaturated aldehyde, reacts with biomolecules via Michael addition at the β-carbon or Schiff base formation at the carbonyl carbon. To gain insight into acrolein's mode of action, we investigated in vitro albumin-acrolein reactivity and the consequence of albumin adduction by acrolein on cytotoxicity and transcript changes in NCI-H441 and human airway epithelial cells (HAEC). Albumin protected NCI-H441 cells from acrolein toxicity. In addition, albumin inhibited acrolein-induced increase of transcripts associated with cellular stress response, activating transcription factor 3 (ATF3), and antioxidant response, heme oxygenase 1 (HMOX1) in HAEC cells. Acrolein-adducted albumin itself increased HMOX1 transcripts but not ATF3 transcripts. The HMOX1 transcript increase was inhibited by hydralazine, a carbonyl scavenger, suggesting that the carbonyl group of acrolein-adducted albumin mediated HMOX1 transcript increase. In acutely exposed C57BL/6J mice, bronchoalveolar lavage protein carbonylation increased. Acrolein-adducted albumin Cys34 was identified by nLC-MS/MS. These findings indicate that adduction of albumin by acrolein confers a cytoprotective function by scavenging free acrolein, decreasing a cellular stress response, and inducing an antioxidant gene response. Further, these results suggest that β-carbon reactivity may be required for acrolein's cytotoxicity and ATF3 transcript increase, and the carbonyl group of acrolein-adducted albumin can induce HMOX1 transcript increase.

Selective Modulation of the Protease Activated Protein C Using Exosite Inhibiting Aptamers.

Activated protein C (APC) is a serine protease with anticoagulant and cytoprotective activities. Nonanticoagulant APC mutants show beneficial effects as cytoprotective agents. To study, if such biased APC signaling can be achieved by APC-binding ligands, the aptamer technology has been used. A G-quadruplex-containing aptamer, G-NB3, has been selected that binds to the basic exosite of APC with a KD of 0.2 nM and shows no binding to APC-related serine proteases or the zymogen protein C. G-NB3 inhibits the inactivation of activated cofactors V and VIII with IC50 values of 11.6 and 13.1 nM, respectively, without inhibiting the cytoprotective and anti-inflammatory functions of APC as tested using a staurosporine-induced apoptosis assay and a vascular barrier protection assay. In addition, G-NB3 prolongs the plasma half-life of APC through inhibition of APC-serine protease inhibitor complex formation. These physicochemical and functional characteristics qualify G-NB3 as a promising therapeutic agent usable to enhance the cytoprotective functions of APC without increasing the risk of APC-related hemorrhage.