Dynamic metabolic modeling of ATP allocation during viral infection.

Viral pathogens, like SARS-CoV-2, hijack the host's macromolecular production machinery, imposing an energetic burden that is distributed across cellular metabolism. To explore the dynamic metabolic tension between the host's survival and viral replication, we developed a computational framework that uses genome-scale models to perform dynamic Flux Balance Analysis of human cell metabolism during virus infections. Relative to previous models, our framework addresses the physiology of viral infections of non-proliferating host cells through two new features.

PARP7 negatively regulates the type I interferon response in cancer cells and its inhibition triggers antitumor immunity.

PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function. Here, we identify PARP7 as a negative regulator of nucleic acid sensing in tumor cells. Inhibition of PARP7 restores type I interferon (IFN) signaling responses to nucleic acids in tumor models.

A potent and selective PARP14 inhibitor decreases protumor macrophage gene expression and elicits inflammatory responses in tumor explants.

PARP14 has been implicated by genetic knockout studies to promote protumor macrophage polarization and suppress the antitumor inflammatory response due to its role in modulating interleukin-4 (IL-4) and interferon-γ signaling pathways. Here, we describe structure-based design efforts leading to the discovery of a potent and highly selective PARP14 chemical probe. RBN012759 inhibits PARP14 with a biochemical half-maximal inhibitory concentration of 0.

In Vitro and Cellular Probes to Study PARP Enzyme Target Engagement.

Poly(ADP-ribose) polymerase (PARP) enzymes use nicotinamide adenine dinucleotide (NAD) to modify up to seven different amino acids with a single mono(ADP-ribose) unit (MARylation deposited by PARP monoenzymes) or branched poly(ADP-ribose) polymers (PARylation deposited by PARP polyenzymes). To enable the development of tool compounds for PARP monoenzymes and polyenzymes, we have developed active site probes for use in in vitro and cellular biophysical assays to characterize active site-directed inhibitors that compete for NAD binding. These assays are agnostic of the protein substrate for each PARP, overcoming a general lack of knowledge around the substrates for these enzymes.

Enabling drug discovery for the PARP protein family through the detection of mono-ADP-ribosylation.

Poly-ADP-ribose polymerases (PARPs) are a family of enzymes responsible for transferring individual or chains of ADP-ribose subunits to substrate targets as a type of post-translational modification. PARPs regulate a wide variety of important cellular processes, ranging from DNA damage repair to antiviral response. However, most research to date has focused primarily on the polyPARPs, which catalyze the formation of ADP-ribose polymer chains, while the monoPARPs, which transfer individual ADP-ribose monomers, have not been studied as thoroughly.

Publisher Correction: SERPINB1-mediated checkpoint of inflammatory caspase activation.

In the version of this article initially published, the label (CASP4-CA-HA) above the second and fifth lanes in the right blot in Fig. 1e is incorrect; the correct label is CASP4-CA-HA. Also, the two labels at right above the plot in Fig.

SERPINB1-mediated checkpoint of inflammatory caspase activation.

Inflammatory caspases (caspase-1, caspase-4, caspase-5 and caspase-11 (caspase-1/-4/-5/-11)) mediate host defense against microbial infections, processing pro-inflammatory cytokines and triggering pyroptosis. However, precise checkpoints are required to prevent their unsolicited activation. Here we report that serpin family B member 1 (SERPINB1) limited the activity of those caspases by suppressing their caspase-recruitment domain (CARD) oligomerization and enzymatic activation.

Molecular mechanism for NLRP6 inflammasome assembly and activation.

Inflammasomes are large protein complexes that trigger host defense in cells by activating inflammatory caspases for cytokine maturation and pyroptosis. NLRP6 is a sensor protein in the nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)-containing (NLR) inflammasome family that has been shown to play multiple roles in regulating inflammation and host defenses. Despite the significance of the NLRP6 inflammasome, little is known about the molecular mechanism behind its assembly and activation.

Cryo-EM structures of ASC and NLRC4 CARD filaments reveal a unified mechanism of nucleation and activation of caspase-1.

Canonical inflammasomes are cytosolic supramolecular complexes that activate caspase-1 upon sensing extrinsic microbial invasions and intrinsic sterile stress signals. During inflammasome assembly, adaptor proteins ASC and NLRC4 recruit caspase-1 through homotypic caspase recruitment domain (CARD) interactions, leading to caspase-1 dimerization and activation. Activated caspase-1 processes proinflammatory cytokines and Gasdermin D to induce cytokine maturation and pyroptotic cell death.

Instant Hydrogelation Inspired by Inflammasomes.

Based on the recent near-atomic structures of the PYRIN domain of ASC in the protein filament of inflammasomes and the observation that the active form of vitamin B6 (pyridoxal phosphate, P5P) modulates the self-assembly of ASC, we rationally designed an N-terminal capped nonapeptide (Nap-FFKKFKLKL, 1) to form supramolecular nanofibers consisting of α-helix. The addition of P5P to the solution of 1 results in a hydrogel almost instantly (about 4 seconds). Several other endogenous small molecules (for example, pyridoxal, folinic acid, ATP, and AMP) also convert the solution of 1 into a hydrogel.

Cryo-EM Structure of Caspase-8 Tandem DED Filament Reveals Assembly and Regulation Mechanisms of the Death-Inducing Signaling Complex.

Caspase-8 activation can be triggered by death receptor-mediated formation of the death-inducing signaling complex (DISC) and by the inflammasome adaptor ASC. Caspase-8 assembles with FADD at the DISC and with ASC at the inflammasome through its tandem death effector domain (tDED), which is regulated by the tDED-containing cellular inhibitor cFLIP and the viral inhibitor MC159. Here we present the caspase-8 tDED filament structure determined by cryoelectron microscopy.

A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly.

Myeloid cells assemble inflammasomes in response to infection or cell damage; cytosolic sensors activate pro-caspase-1, indirectly for the most part, via the adaptors ASC and NLRC4. This leads to secretion of proinflammatory cytokines and pyroptosis. To explore complex formation under physiological conditions, we generated an alpaca single domain antibody, VHHASC, which specifically recognizes the CARD of human ASC via its type II interface.

Molecular basis of caspase-1 polymerization and its inhibition by a new capping mechanism.

Inflammasomes are cytosolic caspase-1-activation complexes that sense intrinsic and extrinsic danger signals, and trigger inflammatory responses and pyroptotic cell death. Homotypic interactions among Pyrin domains and caspase recruitment domains (CARDs) in inflammasome-complex components mediate oligomerization into filamentous assemblies. Several cytosolic proteins consisting of only interaction domains exert inhibitory effects on inflammasome assembly.

Plasticity in PYD assembly revealed by cryo-EM structure of the PYD filament of AIM2.

Absent in melanoma 2 (AIM2) is an essential cytosolic double-stranded DNA receptor that assembles with the adaptor, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1 to form the AIM2 inflammasome, which leads to proteolytic maturation of cytokines and pyroptotic cell death. AIM2 contains an N-terminal Pyrin domain (PYD) that interacts with ASC through PYD/PYD interactions and nucleates ASC filament formation. To elucidate the molecular basis of AIM2-induced ASC polymerization, we generated AIM2 filaments fused to green fluorescent protein (GFP) and determined its cryo-electron microscopic (cryo-EM) structure.

The Inflammasome Adaptor ASC Induces Procaspase-8 Death Effector Domain Filaments.

Inflammasomes mediate inflammatory and cell death responses to pathogens and cellular stress signals via activation of procaspases-1 and -8. During inflammasome assembly, activated receptors of the NLR or PYHIN family recruit the adaptor protein ASC and initiate polymerization of its pyrin domain (PYD) into filaments. We show that ASC filaments in turn nucleate procaspase-8 death effector domain (DED) filaments in vitro and in vivo.

Cryo-EM structure of the activated NAIP2-NLRC4 inflammasome reveals nucleated polymerization.

The NLR family apoptosis inhibitory proteins (NAIPs) bind conserved bacterial ligands, such as the bacterial rod protein PrgJ, and recruit NLR family CARD-containing protein 4 (NLRC4) as the inflammasome adapter to activate innate immunity. We found that the PrgJ-NAIP2-NLRC4 inflammasome is assembled into multisubunit disk-like structures through a unidirectional adenosine triphosphatase polymerization, primed with a single PrgJ-activated NAIP2 per disk. Cryo-electron microscopy (cryo-EM) reconstruction at subnanometer resolution revealed a ~90° hinge rotation accompanying NLRC4 activation.

Drosophila muller f elements maintain a distinct set of genomic properties over 40 million years of evolution.

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D.

Structural mechanisms of inflammasome assembly.

Inflammasomes are supramolecular signaling complexes that activate a subset of caspases known as the inflammatory caspases, an example of which is caspase 1. Upon stimulation by microbial and damage-associated signals, inflammasomes assemble to elicit the first line of host defense via the proteolytic maturation of cytokines interleukin-1β and interleukin-18, and by induction of pyroptotic cell death. Inflammasome assembly requires activation of an upstream sensor, a downstream effector and, in most cases, an adaptor molecule such as apoptosis-associate speck-like protein containing a caspase recruitment domain (ASC).

Unified polymerization mechanism for the assembly of ASC-dependent inflammasomes.

Inflammasomes elicit host defense inside cells by activating caspase-1 for cytokine maturation and cell death. AIM2 and NLRP3 are representative sensor proteins in two major families of inflammasomes. The adaptor protein ASC bridges the sensor proteins and caspase-1 to form ternary inflammasome complexes, achieved through pyrin domain (PYD) interactions between sensors and ASC and through caspase activation and recruitment domain (CARD) interactions between ASC and caspase-1.

Heat shock protein 70 inhibitors. 2. 2,5'-thiodipyrimidines, 5-(phenylthio)pyrimidines, 2-(pyridin-3-ylthio)pyrimidines, and 3-(phenylthio)pyridines as reversible binders to an allosteric site on heat shock protein 70.

The discovery and development of heat shock protein 70 (Hsp70) inhibitors is currently a hot topic in cancer. In the preceding paper in this issue ( 10.1021/jm401551n ), we have described structure-activity relationship studies in the first Hsp70 inhibitor class rationally designed to bind to a novel allosteric pocket located in the N-terminal domain of the protein.

Crystal structure of the F27G AIM2 PYD mutant and similarities of its self-association to DED/DED interactions.

Absent in melanoma 2 (AIM2) is a cytoplasmic double-stranded DNA sensor involved in innate immunity. It uses its C-terminal HIN domain for recognizing double-stranded DNA and its N-terminal pyrin domain (PYD) for eliciting downstream effects through recruitment and activation of apoptosis-associated Speck-like protein containing CARD (ASC). ASC in turn recruits caspase-1 and/or caspase-11 to form the AIM2 inflammasome.

Molecular mechanism for p202-mediated specific inhibition of AIM2 inflammasome activation.

Mouse p202 containing two hemopoietic expression, interferon inducibility, nuclear localization (HIN) domains antagonizes AIM2 inflammasome signaling and potentially modifies lupus susceptibility. We found that only HIN1 of p202 binds double-stranded DNA (dsDNA), while HIN2 forms a homotetramer. Crystal structures of HIN1 revealed that dsDNA is bound on face opposite the site used in AIM2 and IFI16.