The Lumiptosome, an engineered luminescent form of the apoptosome can report cell death by using the same Apaf-1 dependent pathway.

Detection of the apoptosis signature becomes central in understanding cell death modes. We present here a whole-cell biosensor that detects Apaf-1 association and apoptosome formation using a split-luciferase complementary assay. Fusion of N-terminal (Nluc) and C-terminal (Cluc)-fragments of firefly luciferase to the N-terminus of human Apaf-1 was performed in HEK293 cells by using CRISPR-Cas9 technology. This resulted in a luminescent form of the apoptosome that we named 'Lumiptosome'. During Apaf-1 gene editing, a high number of knock-in events were observed without selection, suggesting that the Apaf-1 locus is important for the integration of exogenous transgenes. Since activation of caspase-9 is directly dependent on the apoptosome formation, measured reconstitution of luciferase activity should result from the cooperative association of Nluc-Apaf-1 and Cluc-Apaf-1. Time-response measurements also confirmed that formation of the apoptosome occurs prior to activation of caspase-3. Additionally, overexpression of the Bcl2 apoptosis regulator in transgenic and normal HEK293 cells confirmed that formation of the Lumiptosome depends on release of cytochrome c Thus, HEK293 cells that stably express the Lumiptosome can be utilized to screen pro- and anti-apoptotic drugs, and to examine Apaf-1-dependent cellular pathways.

Density Functional Theory (DFT)-Aided Structure Elucidation of Linear Diterpenes from the Irish Brown Seaweed Bifurcaria bifurcata.

Brown alga Bifurcaria bifurcata is an extraordinarily rich source of linear (acylic) diterpenes with enormous structural diversity. As part of our interest into secondary metabolites of the Irish seaweeds, here we report four new acyclic diterpenes (1-4) and seven known terpenoids (5-11) from the CHCl3 extract of B. bifurcata. The planar structures of the new metabolites were elucidated by means of 1D and 2D NMR, HRMS, and FT-IR spectroscopy. Since linear diterpenes are highly flexible compounds, the assignment of their stereochemistry by conventional methods, e.g., NOESY NMR, is difficult. Therefore, we employed extensive quantum-mechanical prediction of NMR chemical shifts and optical rotation analyses to identify the relative and absolute configurations of the new compounds 1-4. Several compounds moderately inhibited the human breast cancer cell line (MDA-MB-231) with IC50 values ranging from 10.0 to 33.5 µg/mL. This study not only demonstrates the vast capacity of the Irish B. bifurcata to produce highly oxygenated linear diterpenoids, but also highlights the potential of new methodologies for assignment of their stereogenic centers.

Oxygenated Acyclic Diterpenes with Anticancer Activity from the Irish Brown Seaweed Bifurcaria bifurcata.

Brown alga Bifurcaria bifurcata is a prolific source of bioactive acyclic (linear) diterpenes with high structural diversity. In the continuation of our investigations on Irish brown algae, we undertook an in-depth chemical study on the n-hexanes and chloroform subextracts of B. bifurcata that led to isolation of six new (1-6) and two known (7-8) acyclic diterpenes. Chemical structures of the compounds were elucidated by a combination of 1D and 2D NMR, HRMS, FT-IR, [α]D and vibrational circular dichroism (VCD) spectroscopy. Compounds 1-8, as well as three additional linear diterpenes (9-11), which we isolated from the same seaweed before, were tested against the human breast cancer cell line (MDA-MB-231). Several compounds moderately inhibited the growth of the MDA-MB-231 cell line with IC50 values ranging from 11.6 to 32.0 µg/mL. The present study carried out on the lipophilic extracts of the Irish B. bifurcata shows the enormous capacity of this seaweed to produce a large palette of acyclic diterpenes with diverse oxygenation and substitution patterns and promising bioactivities.

A Novel High-Throughput Screening Platform Identifies Itaconate Derivatives from Marine Penicillium antarcticum as Inhibitors of Mesenchymal Stem Cell Differentiation.

Worldwide diffused diseases such as osteoarthritis, atherosclerosis or chronic kidney disease are associated with a tissue calcification process which may involve unexpected local stem cell differentiation. Current pharmacological treatments for such musculoskeletal conditions are weakly effective, sometimes extremely expensive and often absent. The potential to develop new therapies is represented by the discovery of small molecules modulating resident progenitor cell differentiation to prevent aberrant tissue calcification. The marine environment is a rich reserve of compounds with pharmaceutical potential and many novel molecules are isolated from macro and microorganisms annually. The potential of small molecules synthetized by marine filamentous fungi to influence the osteogenic and chondrogenic differentiation of human mesenchymal stem/stromal cells (hMSCs) was investigated using a novel, high-throughput automated screening platform. Metabolites synthetized by the marine-derived fungus Penicillium antarcticum were evaluated on the platform. Itaconic acid derivatives were identified as inhibitors of calcium elaboration into the matrix of osteogenically differentiated hMSCs and also inhibited hMSC chondrogenic differentiation, highlighting their capacity to impair ectopic calcification. Bioactive small molecule discovery is critical to address ectopic tissue calcification and the use of biologically relevant assays to identify naturally occurring metabolites from marine sources represents a strategy that can contribute to this effort.

Mesenchymal stem cells and a vitamin D receptor agonist additively suppress T helper 17 cells and the related inflammatory response in the kidney.

Mesenchymal stem cells (MSCs) suppress T helper (Th)17 cell differentiation and are being clinically pursued for conditions associated with aberrant Th17 responses. Whether such immunomodulatory effects are enhanced by coadministration of MSCs with other agents is not well known. In the present study, individual and combined effects of MSCs and the vitamin D receptor (VDR) agonist paricalcitol on Th17 induction were investigated in vitro and in a mouse model of sterile kidney inflammation (unilateral ureteral obstruction). In vitro, MSCs and paricalcitol additively suppressed Th17 differentiation, although only MSCs suppressed expression of Th17-associated transcriptions factors. Combined administration of MSCs and paricalcitol resulted in an early (day 3) reduction of intrarenal CD4(+) and CD8(+) T cells, CD11b(+)/lymphocyte antigen 6G(+) neutrophils, and inflammatory (lymphocyte antigen 6C(hi)) monocytes as well as reduced transcript for IL-17 compared with untreated animals. Later (day 8), obstructed kidneys of MSC/paricalcitol double-treated mice, but not mice treated with either intervention alone, had reduced tubular injury and interstitial fibrosis as well as lower numbers of neutrophils and inflammatory monocytes and an increase in the ratio between M2 (CD206(+)) and M1 (CD206(-)) macrophages compared with control mice. Adjunctive therapy with VDR agonists may enhance the immunosuppressive properties of MSCs in the setting of pathogenic Th17-type immune responses and related inflammatory responses.

Inhibition of protein synthesis and JNK activation are not required for cell death induced by anisomycin and anisomycin analogues.

Anisomycin was identified in a screen of clinical compounds as a drug that kills breast cancer cells (MDA16 cells, derived from the triple negative breast cancer cell line, MDA-MB-468) that express high levels of an efflux pump, ABCB1. We show the MDA16 cells died by a caspase-independent mechanism, while MDA-MB-468 cells died by apoptosis. There was no correlation between cell death and either protein synthesis or JNK activation, which had previously been implicated in anisomycin-induced cell death. In addition, anisomycin analogues that did not inhibit protein synthesis or activate JNK retained the ability to induce cell death. These data suggest that either a ribosome-ANS complex is a death signal in the absence of JNK activation or ANS kills cells by binding to an as yet unidentified target.

An atlas of caspase cleavage events in differentiating muscle cells.

Executioner caspases, such as caspase-3, are known to induce apoptosis, but in other contexts, they can control very different fates, including cell differentiation and neuronal plasticity. While hundreds of caspase substrates are known to be specifically targeted during cell death, we know very little about how caspase activity brings about non-apoptotic fates. Here, we report the first proteome identification of cleavage events in C2C12 cells undergoing myogenic differentiation and its comparison to undifferentiated or dying C2C12 cells. These data have identified new caspase substrates, including caspase substrates specifically associated with differentiation, and show that caspases are regulating proteins involved in myogenesis in myotubes, several days after caspase-3 initiated differentiation. Cytoskeletal proteins emerged as a major group of non-apoptotic caspase substrates. We also identified proteins with well-established roles in muscle differentiation as substrates cleaved in differentiating cells.

The Apaf-1*procaspase-9 apoptosome complex functions as a proteolytic-based molecular timer.

During stress-induced apoptosis, the initiator caspase-9 is activated by the Apaf-1 apoptosome and must remain bound to retain significant catalytic activity. Nevertheless, in apoptotic cells the vast majority of processed caspase-9 is paradoxically observed outside the complex. We show herein that apoptosome-mediated cleavage of procaspase-9 occurs exclusively through a CARD-displacement mechanism, so that unlike the effector procaspase-3, procaspase-9 cannot be processed by the apoptosome as a typical substrate. Indeed, procaspase-9 possessed higher affinity for the apoptosome and could displace the processed caspase-9 from the complex, thereby facilitating a continuous cycle of procaspase-9 recruitment/activation, processing, and release from the complex. Owing to its rapid autocatalytic cleavage, however, procaspase-9 per se contributed little to the activation of procaspase-3. Thus, the Apaf-1 apoptosome functions as a proteolytic-based 'molecular timer', wherein the intracellular concentration of procaspase-9 sets the overall duration of the timer, procaspase-9 autoprocessing activates the timer, and the rate at which the processed caspase-9 dissociates from the complex (and thus loses its capacity to activate procaspase-3) dictates how fast the timer 'ticks' over.

Mera: A scalable high throughput automated micro-physiological system.

There is an urgent need for scalable Microphysiological Systems (MPS's)1 that can better predict drug efficacy and toxicity at the preclinical screening stage. Here we present Mera, an automated, modular and scalable system for culturing and assaying microtissues with interconnected fluidics, inbuilt environmental control and automated image capture. The system presented has multiple possible fluidics modes. Of these the primary mode is designed so that cells may be matured into a desired microtissue type and in the secondary mode the fluid flow can be re-orientated to create a recirculating circuit composed of inter-connected channels to allow drugging or staining. We present data demonstrating the prototype system Mera using an Acetaminophen/HepG2 liver microtissue toxicity assay with Calcein AM and Ethidium Homodimer (EtHD1) viability assays. We demonstrate the functionality of the automated image capture system. The prototype microtissue culture plate wells are laid out in a 3 × 3 or 4 × 10 grid format with viability and toxicity assays demonstrated in both formats. In this paper we set the groundwork for the Mera system as a viable option for scalable microtissue culture and assay development.

Autophagosomal IkappaB alpha degradation plays a role in the long term control of tumor necrosis factor-alpha-induced nuclear factor-kappaB (NF-kappaB) activity.

Transcription factor NF-κB is persistently activated in many chronic inflammatory diseases and cancers. The short term regulation of NF-κB is well understood, but little is known about the mechanisms of its long term activation. We studied the effect of a single application of TNF-α on NF-κB activity for up to 48 h in intestinal epithelial cells. Results show that NF-κB remained persistently activated up to 48 h after TNF-α and that the long term activation of NF-κB was accompanied by a biphasic degradation of IκBα. The first phase of IκBα degradation was proteasome-dependent, but the second was not. Further investigation showed that TNF-α stimulated formation of autophagosomes in intestinal epithelial cells and that IκBα co-localized with autophagosomal vesicles. Pharmacological or genetic blockade of autophagosome formation or the inhibition of lysosomal proteases decreased TNF-α-induced degradation of IκBα and lowered NF-κB target gene expression. Together, these findings indicate a role of autophagy in the control of long term NF-κB activity. Because abnormalities in autophagy have been linked to ineffective innate immunity, we propose that alterations in NF-κB may mediate this effect.

Non-Canonical Roles of Apoptotic Caspases in the Nervous System.

Caspases are a family of cysteine proteases that predominantly cleave their substrates after aspartic acid residues. Much of what we know of caspases emerged from investigation a highly conserved form of programmed cell death called apoptosis. This form of cell death is regulated by several caspases, including caspase-2, caspase-3, caspase-7, caspase-8 and caspase-9. However, these "killer" apoptotic caspases have emerged as versatile enzymes that play key roles in a wide range of non-apoptotic processes. Much of what we understand about these non-apoptotic roles is built on work investigating how "killer" caspases control a range of neuronal cell behaviors. This review will attempt to provide an up to date synopsis of these roles.

Apoptosome Formation through Disruption of the K192-D616 Salt Bridge in the Apaf-1 Closed Form.

The molecular mechanism of apoptosome activation through conformational changes of Apaf-1 auto-inhibited form remains largely enigmatic. The crystal structure of Apaf-1 suggests that some ionic bonds, including the bond between K192 and D616, are critical for the preservation of the inactive "closed" form of Apaf-1. Here, a split luciferase complementation assay was used to monitor the effect of disrupting this ionic bond on apoptosome activation and caspase-3 activity in cells. The K192E mutation, predicted to disrupt the ionic interaction with D616, increased apoptosome formation and caspase activity, suggesting that this mutation favors the "open"/active form of Apaf-1. However, mutation of D616 to alanine or lysine had different effects. While both mutants favored apoptosome formation such as K192E, D616K cannot activate caspases and D616A activates caspases poorly, and not as well as wild-type Apaf-1. Thus, our data show that the ionic bond between K192 and D616 is critical for maintaining the closed form of Apaf-1 and that disrupting the interaction enhances apoptosome formation. However, our data also reveal that after apoptosome formation, D616 and K192 play a previously unsuspected role in caspase activation. The molecular explanation for this observation is yet to be elucidated.

Non-lethal message from the Holy Land: The first international conference on nonapoptotic roles of apoptotic proteins.

Apoptosis is a major form of programmed cell death (PCD) that eliminates unnecessary and potentially dangerous cells in all metazoan organisms, thus ensuring tissue homeostasis and many developmental processes. Accordingly, defects in the activation of the apoptotic pathway often pave the way to disease. After several decades of intensive research, the molecular details controlling the apoptosis program have largely been unraveled, as well as the regulatory mechanisms of caspase activation during apoptosis. Nevertheless, an ever-growing list of studies is suggesting the essential role of caspases and other apoptotic proteins in ensuring nonlethal cellular functions during normal development, tissue repair, and regeneration. Moreover, if deregulated, these novel nonapoptotic functions can also instigate diseases. The difficulty of identifying and manipulating the caspase-dependent nonlethal cellular processes (CDPs), as well as the nonlethal functions of other cell death proteins (NLF-CDPs), meant that CDPs and NLF-CDPs have been only curiosities within the apoptotic field; however, the recent technical advancements and the latest biological findings are assigning an unanticipated biological significance to these nonapoptotic functions. Here, we summarize the various talks presented in the first international conference fully dedicated to discuss CDPs and NFL-CDPs and named 'The Batsheva de Rothschild Seminar on Non-Apoptotic Roles of Apoptotic Proteins'. The conference was organized between September 22, 2019, and 25, 2019, by Eli Arama (Weizmann Institute of Science), Luis Alberto Baena-Lopez (University of Oxford), and Howard O. Fearnhead (NUI Galway) at the Weizmann Institute of Science in Israel, and hosted a large international group of researchers.

A robust platform for high-throughput screening of therapeutic strategies for acute and chronic spinal cord injury.

Astrocytes and microglia are critical regulators of inflammatory cascade after spinal cord injury (SCI). Existing glial in vitro studies do not replicate inflammatory phases associated with SCI. Here, we report an in vitro model of mixed glial culture where inflammation is induced by the administration of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-1ß, and interleukin-6) to promote pathologically relevant "acute" and "chronic" inflammatory phases. We observed SCI relevant differential modulation of inflammatory pathways, cytokines, chemokines, and growth factors over 21 days. Mitochondrial dysfunction was associated with a cytokine combination treatment. Highly expressed cytokine induced neutrophil chemoattractant (CINC-3) chemokine was used as a biomarker to establish an enzyme-linked immunosorbent assay-based high-throughput screening (HTS) platform. We screened a 786-compound drug library to demonstrate the efficacy of the HTS platform. The developed model is robust and will facilitate in vitro screening of anti-reactive glial therapeutics for the treatment of SCI.

Loss of WD2 subdomain of Apaf-1 forms an apoptosome structure which blocks activation of caspase-3 and caspase-9.

Split luciferase complementary assay has been used to investigate the effect of WD domain deletion on Apaf-1 oligomerization. Apaf-1 is an adaptor molecule in formation of apoptosome that activates caspase-9, an activation that is a key event in the mitochondrial cell death pathway. Structural studies suggest that normally Apaf-1 is held in an inactive conformation by intramolecular interactions between Apaf-1's nucleotide binding domain and one of its WD40 domains (WD1). In the prevailing model of Apaf-1 activation, cytochrome c binds to sites in WD1 and in Apaf-1's second WD40 domain (WD2), moving WD1 and WD2 closer together and rotating WD1 away from the nucleotide binding domain. This allows Apaf-1 to bind dATP or ATP and to form the apoptosome, which activates caspase-9. This model predicts that cytochrome c binding to both WD domains is necessary for apoptosome formation and that an Apaf-1 with only WD1 will be locked in an inactive conformation that cannot be activated by cytochrome c. Here we investigated the effect of removing one WD domain (Apaf-1 1-921) on Apaf-1 interactions and caspase activation. Apaf-1 1-921 could not activate caspase-9, even in the presence of cytochrome c. These data show that a single WD domain is sufficient to lock Apaf-1 in an inactive state and this state cannot be altered by cytochrome c.

Apoptosome-dependent myotube formation involves activation of caspase-3 in differentiating myoblasts.

Caspase-2, -9, and -3 are reported to control myoblast differentiation into myotubes. This had been previously explained by phosphatidylserine exposure on apoptotic myoblasts inducing differentiation in neighboring cells. Here we show for the first time that caspase-3 is activated in the myoblasts undergoing differentiation. Using RNAi, we also demonstrate that differentiation requires both cytochrome c and Apaf-1, and by using a new pharmacological approach, we show that apoptosome formation is required. We also show that Bid, whose cleavage links caspase-2 to the mitochondrial death pathway, was required for differentiation, and that the caspase cleavage product, tBid, was generated during differentiation. Taken together, these data suggest that myoblast differentiation requires caspase-2 activation of the mitochondrial death pathway, and that this occurs in the cells that differentiate. Our data also reveal a hierarchy of caspases in differentiation with caspase-2 upstream of apoptosome activation, and exerting a more profound control of differentiation, while caspases downstream of the apoptosome primarily control cell fusion.

Droplet Combinations: A Scalable Microfluidic Platform for Biochemical Assays.

Droplet-based microfluidics holds enormous potential for transforming high-throughput drug screening. Miniaturization through droplets in combination with automation contributes to reduce reagent use and analysis time as well as minimizing or eliminating labor-intensive steps leading to associated reductions in cost. In this paper, we demonstrate the potential of automated and cost-effective microfluidic droplet-generating technology in the context of an enzymatic activity assay for screening collagenase inhibitors. Experimental results show reproducible and accurate creation and mixing of droplet combinations resulting in biochemical data comparable to data produced by an industry standard instrument. This microfluidic platform that can generate and combine multiple droplets represents a promising tool for high-throughput drug screening.

A new split-luciferase complementation assay identifies pentachlorophenol as an inhibitor of apoptosome formation.

The expense and time required for in vivo reproductive and developmental toxicity studies have driven the development of in vitro alternatives. Here, we used a new in vitro split luciferase-based assay to screen a library of 177 toxicants for inhibitors of apoptosome formation. The apoptosome contains seven Apoptotic Protease-Activating Factor-1 (Apaf-1) molecules and induces cell death by activating caspase-9. Apaf-1-dependent caspase activation also plays an important role in CNS development and spermatogenesis. In the in vitro assay, Apaf-1 fused to an N-terminal fragment of luciferase binds to Apaf-1 fused to a C-terminal fragment of luciferase and reconstitutes luciferase activity. Our assay indicated that pentachlorophenol (PCP) inhibits apoptosome formation, and further investigation revealed that PCP binds to cytochrome c. PCP is a wood preservative that reduces male fertility by ill-defined mechanisms. Although the data show that PCP inhibited apoptosome formation, the concentration required suggests that other mechanisms may be more important for PCP's effects on spermatogenesis. Nonetheless, the data demonstrate the utility of the new assay in identifying apoptosome inhibitors, and we suggest that the assay may be useful in screening for reproductive and developmental toxicants.

Viral hijacking of host caspases: an emerging category of pathogen-host interactions.

Viruses co-evolve with their hosts, and many viruses have developed mechanisms to suppress or modify the host cell apoptotic response for their own benefit. Recently, evidence has emerged for the opposite strategy. Some viruses have developed the ability to co-opt apoptotic caspase activity to facilitate their own proliferation. In these strategies, viral proteins are cleaved by host caspases to create cleavage products with novel activities which facilitate viral replication. This represents a novel and interesting class of viral-host interactions, and also represents a new group of non-apoptotic roles for caspases. Here we review the evidence for such strategies, and discuss their origins and their implications for our understanding of the relationship between viral pathogenesis and programmed cell death.