Conserved and divergent chaperoning effects of Hsp60/10 chaperonins on protein folding landscapes

The GroEL/ES chaperonin cavity surface charge properties, especially the negative charges, play an important role in its capacity to assist intracavity protein folding. Remarkably, the larger fraction of GroEL/ES negative charges are not conserved among different bacterial species, resulting in a large variation in negative-charge density in the GroEL/ES cavity across prokaryotes. Intriguingly, eukaryotic GroEL/ES homologs have the lowest negative-charge density in the chaperonin cavity. This prompted us to investigate if GroEL’s chaperoning mechanism changed during evolution. Using a model in vivo GroEL/ES substrate, we show that the ability of GroEL/ES to buffer entropic traps in the folding pathway of its substrate was partially dependent upon the negative-charge density inside its cavity. While this activity of GroEL/ES was found to be essential for Escherichia coli, it has been perfected in some organisms and diminished in others. However, irrespective of their charges, all the tested homologs retained their ability to regulate polypeptide chain collapse and remove enthalpic traps from folding pathways. The ability of these GroEL/ES homologs to buffer mutational variations in a model substrate correlated with their negative-charge density. Thus, Hsp60/10 chaperonins in different organisms may have changed to accommodate a different spectrum of mutations on their substrates.

A multimeric, barrel-shaped complex of Hsp60 and Hsp10 proteins forms an essential chaperone (also known as chaperonin) system in most life forms (1). Representative members of this system are found in all prokaryotes—barring some tenericutes—and in the compartments of endosymbiotic origin in all eukaryotes. Escherichia coli Hsp60/Hsp10, also known as GroEL/GroES (referred to as GroEL/ES), is the best-studied member of the Hsp60/Hsp10 (referred to as Hsp60/10) family. Extensive studies on GroEL/ES as a canonical Hsp60/10 chaperone have revealed important mechanistic insights on the molecular mechanism of folding assistance by this group of chaperones but whether the chaperoning mechanisms of GroEL/ES persisted over evolution is still elusive (1, 2).Despite significant controversies regarding GroEL/ES’s chaperoning mechanism, it is well established that at least a subset of its substrates is assisted for its folding by encapsulation within the chaperonin cavity. Thus, the cavity charge property of the GroEL/ES and Hsp60/10s would be immensely important in determining their chaperoning mechanism. To address this, we computationally compared the GroEL/ES’s cavity charge property with several of its eubacterial, hyperthermophilic bacterial and some eukaryotic homologs. Interestingly, we found significant variation in cavity charge among the different Hsp60/10 homologs. While the net negative charge of the chaperonin cavity was mostly conserved in eubacterial GroEL/ES homologs, hyperthermophilic and eukaryotic homologs contain drastically decreased negatively charged cavities. To understand the changes in the chaperoning mechanism with altered cavity charges, we employed laboratory-evolved and endogenous in vivo substrates of GroEL/ES to compare the folding assistance offered by GroEL/ES and its representative homologs.We have shown that the negatively charged cavity wall of the GroEL/ES system acts like a high-density array of chemical chaperones to edit the protein-folding landscape. It reroutes folding to deviate from the path determined by the amino acid sequence of the substrate and take a route with a lower entropic barrier. Remarkably, the density of negative charges inside the cavity determined the capacity of this chaperonin to assist folding and buffer mutational variations of a model substrate in vivo. Since the density of this charge showed a large variation across different species, we posit that the evolvability of the chaperonin substrates varies between different organisms based on the cavity properties of the chaperonin. Interestingly, GroEL/ES also possesses a contrasting function of preventing nonnative contacts in its substrates. This function is shared between all the bacterial and eukaryotic Hsp60/10 chaperones tested. Thus, we show that this chaperonin system has the following two central mechanisms to assist folding: 1) a conserved mechanism to prevent the formation of nonnative contacts, and 2) a variable mechanism to aid folding with its negatively charged cavity by lowering entropic barriers.     

Biosensors for inflammation as a strategy to engineer regulatory T cells for cell therapy

Engineered regulatory T cell (Treg cell) therapy is a promising strategy to treat patients suffering from inflammatory diseases, autoimmunity, and transplant rejection. However, in many cases, disease-related antigens that can be targeted by Treg cells are not available. In this study, we introduce a class of synthetic biosensors, named artificial immune receptors (AIRs), for murine and human Treg cells. AIRs consist of three domains: (a) extracellular binding domain of a tumor necrosis factor (TNF)-receptor superfamily member, (b) intracellular costimulatory signaling domain of CD28, and (c) T cell receptor signaling domain of CD3-ζ chain. These AIR receptors equip Treg cells with an inflammation-sensing machinery and translate this environmental information into a CD3-ζ chain–dependent TCR-activation program. Different AIRs were generated, recognizing the inflammatory ligands of the TNF-receptor superfamily, including LIGHT, TNFα, and TNF-like ligand 1A (TL1A), leading to activation, differentiation, and proliferation of AIR–Treg cells. In a graft-versus-host disease model, Treg cells expressing lymphotoxin β receptor–AIR, which can be activated by the ligand LIGHT, protect significantly better than control Treg cells. Expression and signaling of the corresponding human AIR in human Treg cells prove that this concept can be translated. Engineering Treg cells that target inflammatory ligands leading to TCR signaling and activation might be used as a Treg cell–based therapy approach for a broad range of inflammation-driven diseases.

Regulatory T cells (Treg cells) are a pivotal T cell population with various functions in the body. Treg cells foster tolerance against self-antigens, allergens, and commensals, thereby limiting self-reactivity of immune cells and excessive inflammation (1). In the last years, it became evident that Treg cells exert additional functions in safeguarding tissue homeostasis and tissue regeneration (2–5). Therefore, Treg cells are a promising candidate to be used as “living drugs” against autoimmune disorders and in transplantation, and first clinical trials have already proven the safety and efficacy of Treg-based cellular therapies (6–8). Concepts applying the chimeric antigen receptor (CAR) technology to Treg cells are being developed to enhance the potency of adoptive Treg cell therapy (9). Preclinical studies have demonstrated the superiority of engineered Treg cells with a CAR-guided antigen specificity over Treg cells with only a natural polyclonal T cell receptor (TCR) repertoire in reducing alloimmune reactions in graft-versus-host disease (GvHD) and graft rejection after transplantation (10–14). CAR–Treg cells were also effectively tested for the treatment of asthma, hemophilia A, experimental autoimmune encephalitis (EAE), and inflammatory bowel disease (IBD) in preclinical models (15–18).However, in human autoimmune diseases, the implicated autoantigens, which could serve as potential targets for CAR–Treg cells, are often unknown or vary strongly between individual patients. In addition, in many autoimmune and alloreactive diseases, multiple organs and tissues are affected without a uniform antigen that could be targeted by CAR–Treg cells. In contrast to this, the mediators of inflammation show a high redundancy as well as functional importance for the development of various inflammatory diseases, including autoimmunity and alloreactivity. Especially, cytokines of the tumor necrosis factor (TNF) superfamily are involved in many different inflammatory and autoimmune diseases. For example, therapeutic intervention with TNF receptor (TNFR) activation is an important treatment option for several inflammatory diseases (19). These considerations led us to develop a concept for engineered Treg cell therapy by generating artificial immune receptors (AIRs) that target these inflammatory mediators instead of tissue-specific antigens.We focused on targets of the TNF superfamily and chose receptors of the ligands LIGHT and LTα₁β₂, TNFα, and TNF-like ligand 1A (TL1A), as these cytokines have pleiotropic roles in numerous autoimmune diseases (19) and are expressed as membrane-bound inflammatory ligands. Preclinical disease models and patient data indicate that LIGHT and LTα₁β₂ signaling through their corresponding receptors lymphotoxin β receptor (LTBR) and herpesvirus entry mediator (HVEM/TNFRSF14) enhance pathology in IBD, autoimmune hepatitis, asthma, rheumatoid arthritis, multiple sclerosis, and GvHD (20). A role for TL1A and its receptor death receptor 3 (DR3) has been described in a variety of inflammatory diseases. Studies with DR3- or TL1A-blocking antibodies or TL1A- and DR3-deficient mice demonstrated a critical involvement for this receptor–ligand system in the induction and maintenance of chronic inflammation in IBD, arthritis, and EAE (21). Moreover, patient data and genome-wide association studies point to a fundamental impact of TL1A-DR3 in human disease (22). The most prominent member of the TNF family, TNFα itself, has been studied extensively since its discovery in 1984. Its proinflammatory function was revealed in multiple diseases, among them ankylosing spondylitis, IBD, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, and juvenile idiopathic arthritis (23).AIRs as a synthetic tool for cellular therapy allow Treg cells to sense and target these environmental inflammatory signals and translate them into a CD3-ζ chain–dependent TCR program, enabling Treg cells to fulfill their suppressive and tissue-protective functions, independent of a CAR- or TCR-specific antigen.     

A study of auditory reaction time in different phases of the normal menstrual cycle

Reaction time is an indirect index of processing capabilities of the central nervous system. The present study was carried out to determine if there is any alteration of simple auditory reaction time across the normal menstrual cycle. In this study, reaction time of 100 female medical and paramedical students was recorded in different phases of their menstrual cycle namely premenstrual, menstrual, middle of proliferative, middle of secretory phase and on the expected day of ovulation. Results were expressed as mean, standard deviation and statistically analyzed using student's paired 't' test. On comparing each phase with the corresponding adjacent phases auditory reaction time was significantly increased (P<0.05) in premenstrual phase and on the expected day of ovulation. Thus fluctuating levels of sex steroids across normal menstrual cycle affect sensory motor association of an individual.     

Differential growth inhibitory effects of W. somnifera root and E. officinalis fruits on CHO cells

The Chinese Hamster ovary (CHO) cell line is widely used for measuring drug cytotoxicity and resistance. Therefore, the effects of two major Ayurvedic drugs (W. somnifera root and E. officinalis fruits) on the short and long-term growth of these cells were investigated. A standard 96-well plate assay was used to measure short-term growth. For assessment of long-term growth, the colony formation assay (CFA) was used, which measures clonogenic potential. This assay is the best measure of the cytotoxicity of anticancer drugs and the radio-sensitivity of tumor cells. As reported by others, the aqueous extracts of both herbal drugs were found to have short-term growth inhibitory effects on CHO cells when added to cells at the time of cell plating. However, this is the first report showing that these two herbal drugs have significantly different effects on the long-term growth of CHO cells. Thus, extracts of W. somnifera root, but not E. officinalis fruit, caused a reproducible, dose dependent, inhibition of colony formation of CHO cells.     

MTH1 expression is required for effective transformation by oncogenic HRAS

Due to sustaining elevated reactive oxygen species (ROS), oncogenic RAS-transformed cells upregulate redox-protective genes, among them the mammalian 8-oxodGTPase, MutT Homolog 1 (MTH1). We previously showed MTH1 abrogates RAS oncogene-induced senescence (OIS) in normal cells and that its inhibition compromises the tumorigenicity of established oncogenic RAS-harboring cancer cells. Here, we investigated how pre-transformation MTH1 levels in immortalized cells influence HRASV12-induced oncogenic transformation. We find MTH1 suppression prior to HRASV12 transduction into BEAS2B immortalized epithelial cells compromised maintenance of high RASV12- and oncogenic ROS-expressing cell populations. Furthermore, pre-transformation MTH1 levels modulated the efficiency of HRASV12-mediated soft agar colony formation. Downstream transformation-associated traits such as the epithelial-mesenchymal transition (EMT) were also compromised by MTH1 inhibition. These collective effects were observed to a greater degree in cells harboring high vs. low RASV12 levels, suggesting MTH1 is required for tumor cells to accumulate RAS oncoprotein. This is significant as, a priori, one cannot ascertain whether tumor-promoting adaptations wrought by introducing oncogenic RAS into an immortalized cell are capable of overcoming pre-transformation deficiencies. Our results suggest nucleotide pool sanitization comprises an important transformation-promoting requirement that, if compromised, cannot be adequately compensated post-transformation and thus is likely to affect optimal development and progression of RAS-driven tumors.     

Anticancer Potential of Myricanone,a Major Bioactive Component of Myrica cerifera: Novel Signaling Cascade for Accomplishing Apoptosis

Extract of Myrica cerifera bark has long been fruitfully used as a hepato-protective and anti-cancer drug in various complementary and alternative systems of medicine. Myricanone, its principal bioactive compound, had also been reported to have apoptosis-promoting ability. We evaluated its anti-cancer potential in vitro in HepG2 liver cancer cells and tried to understand the signal cascades involved in accomplishing apoptosis. Further, we ascertained by using a (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay (MTT) assay if it had cytotoxic effects on normal noncancerous liver cells (WRL-68). We deployed various tools and protocols, like phase contrast, scanning electron and fluorescence microscopies, performed an annexinV-FITC/PI assay and cell cycle analysis, and estimated the reactive oxygen species (ROS) generation and mitochondrial membrane depolarization through flow cytometry. Further, analyses of cytochrome-c translocation and of HSP70 and caspase expressions were also done by using immunoblota and Enzyme linked immunosorbent assay (ELISA). Results revealed that myricanone induced apoptosis in HepG2 cells through generation of ROS, depolarization of the mitochondrial membrane, early release of cytochrome-c, down-regulation of HSP70 and activation of a caspase cascade; it had no, or insignificant, cytotoxic effects in WRL-68 cells in vitro and in mice in vivo. Thus, myricanone has great potential for use in formulating an effective drug against both hepatotoxicity and hepatocellular cancer.