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.

WITHDRAWN: Mera: A scalable high throughput automated micro-physiological system.

The Publisher regrets that this article is an accidental duplication of an article previously published at http://dx.doi.org/10.

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

There is an urgent need for scalable Microphysiological Systems (MPS's) 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.

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.

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.

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).

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.

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.

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.

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.

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.

How do we fit ferroptosis in the family of regulated cell death?

In the last few years many new cell death modalities have been described. To classify different types of cell death, the term 'regulated cell death' was introduced to discriminate it from 'accidental cell death'. Regulated cell death involves the activation of genetically encoded molecular machinery that couples the presence of some signal to cell death.

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.

DNA-PK activity is associated with caspase-dependent myogenic differentiation.

Differentiation of myoblasts into myotubes is essential for skeletal muscle development and regeneration. Caspase-3 and caspase-9 are required for efficient myoblast differentiation. The caspase-activated endonuclease activity, CAD, and the DNA-damage repair protein XRCC1 have also been shown to be required to complete differentiation.

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).

New roles for old enzymes: killer caspases as the engine of cell behavior changes.

It has become increasingly clear that caspases, far from being merely cell death effectors, have a much wider range of functions within the cell. These functions are as diverse as signal transduction and cytoskeletal remodeling, and caspases are now known to have an essential role in cell proliferation, migration, and differentiation. There is also evidence that apoptotic cells themselves can direct the behavior of nearby cells through the caspase-dependent secretion of paracrine signaling factors.

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.

"Dead Cells Talking": The Silent Form of Cell Death Is Not so Quiet.

After more than twenty years of research, the molecular events of apoptotic cell death can be succinctly stated; different pathways, activated by diverse signals, increase the activity of proteases called caspases that rapidly and irreversibly dismantle condemned cell by cleaving specific substrates. In this time the ideas that apoptosis protects us from tumourigenesis and that cancer chemotherapy works by inducing apoptosis also emerged. Currently, apoptosis research is shifting away from the intracellular events within the dying cell to focus on the effect of apoptotic cells on surrounding tissues.

Activation of p73 and induction of Noxa by DNA damage requires NF-kappa B.

Although the transcription factor NF-kappaB is most clearly linked to the inhibition of extrinsic apoptotic signals such as TNFalpha by upregulating known anti-apoptotic genes, NF-kappaB has also been proposed to be required for p53-induced apoptosis in transformed cells. However, the involvement of NF-kappaB in this process is poorly understood. Here we investigate this mechanism and show that in transformed MEFs lacking NF-kappaB (p65-null cells) genotoxin-induced cytochrome c release is compromised.

p53-mediated induction of Noxa and p53AIP1 requires NFkappaB.

The intricate regulation of cell survival and cell death is critical for the existence of both normal and transformed cells. Two factors central to these processes are p53 and NFkappaB, with both factors having ascribed roles in both promoting and repressing cell death. Not surprisingly, a number of studies have previously reported interplay between p53 and NFkappaB.

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.

A non-apoptotic role for caspase-9 in muscle differentiation.

Caspases, a family of cysteine proteases most often investigated for their roles in apoptosis, have also been demonstrated to have functions that are vital for the efficient execution of cell differentiation. One such role that has been described is the requirement of caspase-3 for the differentiation of skeletal myoblasts into myotubes but, as yet, the mechanism leading to caspase-3 activation in this case remains elusive. Here, we demonstrate that caspase-9, an initiator caspase in the mitochondrial death pathway, is responsible for the activation of caspase-3 in differentiating C2C12 cells.

Identification of an inhibitor of caspase activation from heart extracts; ATP blocks apoptosome formation.

By revealing the biochemistry of apoptosis it is expected we will both improve our understanding of diseases where apoptosis plays an important role and aid the development of therapies for these disorders. Caspases are a family of proteases whose activity is required for apoptosis. In this study, a cell-free system was used to investigate the mechanism of caspase-9 activation in extracts from heart cells.

Intracellular nucleotides act as critical prosurvival factors by binding to cytochrome C and inhibiting apoptosome.

Cytochrome c (CC)-initiated Apaf-1 apoptosome formation represents a key initiating event in apoptosis. This process can be reconstituted in vitro with the addition of CC and ATP or dATP to cell lysates. How physiological levels of nucleotides, normally at high mM concentrations, affect apoptosome activation remains unclear.

Apo cytochrome c inhibits caspases by preventing apoptosome formation.

Caspases are cysteine proteases and potent inducers of apoptosis. Their activation and activity is therefore tightly regulated. There are several mechanisms by which caspases can be activated but one key pathway involves release of holo cytochrome c from mitochondria into the cytoplasm.