Molecular and structural aspects of gasdermin family pores and insights into gasdermin-elicited programmed cell death.

Pyroptosis is a highly inflammatory and lytic type of programmed cell death (PCD) commenced by inflammasomes, which sense perturbations in the cytosolic environment. Recently, several ground-breaking studies have linked a family of pore-forming proteins known as gasdermins (GSDMs) to pyroptosis. The human genome encodes six GSDM proteins which have a characteristic feature of forming pores in the plasma membrane resulting in the disruption of cellular homeostasis and subsequent induction of cell death. GSDMs have an N-terminal cytotoxic domain and an auto-inhibitory C-terminal domain linked together through a flexible hinge region whose proteolytic cleavage by various enzymes releases the N-terminal fragment that can insert itself into the inner leaflet of the plasma membrane by binding to acidic lipids leading to pore formation. Emerging studies have disclosed the involvement of GSDMs in various modalities of PCD highlighting their role in diverse cellular and pathological processes. Recently, the cryo-EM structures of the GSDMA3 and GSDMD pores were resolved which have provided valuable insights into the pore formation process of GSDMs. Here, we discuss the current knowledge regarding the role of GSDMs in PCD, structural and molecular aspects of autoinhibition, and pore formation mechanism followed by a summary of functional consequences of gasdermin-induced membrane permeabilization.

Mechanisms and regulation underlying membraneless organelle plasticity control.

Evolution has enabled living cells to adopt their structural and functional complexity by organizing intricate cellular compartments, such as membrane-bound and membraneless organelles (MLOs), for spatiotemporal catalysis of physiochemical reactions essential for cell plasticity control. Emerging evidence and view support the notion that MLOs are built by multivalent interactions of biomolecules via phase separation and transition mechanisms. In healthy cells, dynamic chemical modifications regulate MLO plasticity, and reversible phase separation is essential for cell homeostasis. Emerging evidence revealed that aberrant phase separation results in numerous neurodegenerative disorders, cancer, and other diseases. In this review, we provide molecular underpinnings on (i) mechanistic understanding of phase separation, (ii) unifying structural and mechanistic principles that underlie this phenomenon, (iii) various mechanisms that are used by cells for the regulation of phase separation, and (iv) emerging therapeutic and other applications.

An overview of disease models for NLRP3 inflammasome over-activation.

Introduction: Inflammatory reactions, including those mediated by the NLRP3 inflammasome, maintain the body's homeostasis by removing pathogens, repairing damaged tissues, and adapting to stressed environments. However, uncontrolled activation of the NLRP3 inflammasome tends to cause various diseases using different mechanisms. Recently, many inhibitors of the NLRP3 inflammasome have been reported and many are being developed. In order to assess their efficacy, specificity, and mechanism of action, the screening process of inhibitors requires various types of cell and animal models of NLRP3-associated diseases.Areas covered: In the following review, the authors give an overview of the cell and animal models that have been used during the research and development of various inhibitors of the NLRP3 inflammasome.Expert opinion: There are many NLRP3 inflammasome inhibitors, but most of the inhibitors have poor specificity and often influence other inflammatory pathways. The potential risk for cross-reaction is high; therefore, the development of highly specific inhibitors is essential. The selection of appropriate cell and animal models, and combined use of different models for the evaluation of these inhibitors can help to clarify the target specificity and therapeutic effects, which is beneficial for the development and application of drugs targeting the NLRP3 inflammasome.

Molecular and Structural Basis of DNA Sensors in Antiviral Innate Immunity.

DNA viruses are a source of great morbidity and mortality throughout the world by causing many diseases; thus, we need substantial knowledge regarding viral pathogenesis and the host's antiviral immune responses to devise better preventive and therapeutic strategies. The innate immune system utilizes numerous germ-line encoded receptors called pattern-recognition receptors (PRRs) to detect various pathogen-associated molecular patterns (PAMPs) such as viral nucleic acids, ultimately resulting in antiviral immune responses in the form of proinflammatory cytokines and type I interferons. The immune-stimulatory role of DNA is known for a long time; however, DNA sensing ability of the innate immune system was unraveled only recently. At present, multiple DNA sensors have been proposed, and most of them use STING as a key adaptor protein to exert antiviral immune responses. In this review, we aim to provide molecular and structural underpinnings on endosomal DNA sensor Toll-like receptor 9 (TLR9) and multiple cytosolic DNA sensors including cyclic GMP-AMP synthase (cGAS), interferon-gamma inducible 16 (IFI16), absent in melanoma 2 (AIM2), and DNA-dependent activator of IRFs (DAI) to provide new insights on their signaling mechanisms and physiological relevance. We have also addressed less well-understood DNA sensors such as DEAD-box helicase DDX41, RNA polymerase III (RNA pol III), DNA-dependent protein kinase (DNA-PK), and meiotic recombination 11 homolog A (MRE11). By comprehensive understanding of molecular and structural aspects of DNA-sensing antiviral innate immune signaling pathways, potential new targets for viral and autoimmune diseases can be identified.

Mitotic motor CENP-E cooperates with PRC1 in temporal control of central spindle assembly.

Error-free cell division depends on the accurate assembly of the spindle midzone from dynamic spindle microtubules to ensure chromatid segregation during metaphase-anaphase transition. However, the mechanism underlying the key transition from the mitotic spindle to central spindle before anaphase onset remains elusive. Given the prevalence of chromosome instability phenotype in gastric tumorigenesis, we developed a strategy to model context-dependent cell division using a combination of light sheet microscope and 3D gastric organoids. Light sheet microscopic image analyses of 3D organoids showed that CENP-E inhibited cells undergoing aberrant metaphase-anaphase transition and exhibiting chromosome segregation errors during mitosis. High-resolution real-time imaging analyses of 2D cell culture revealed that CENP-E inhibited cells undergoing central spindle splitting and chromosome instability phenotype. Using biotinylated syntelin as an affinity matrix, we found that CENP-E forms a complex with PRC1 in mitotic cells. Chemical inhibition of CENP-E in metaphase by syntelin prevented accurate central spindle assembly by perturbing temporal assembly of PRC1 to the midzone. Thus, CENP-E-mediated PRC1 assembly to the central spindle constitutes a temporal switch to organize dynamic kinetochore microtubules into stable midzone arrays. These findings reveal a previously uncharacterized role of CENP-E in temporal control of central spindle assembly. Since CENP-E is absent from yeast, we reasoned that metazoans evolved an elaborate central spindle organization machinery to ensure accurate sister chromatid segregation during anaphase and cytokinesis.

Methylation of PLK1 by SET7/9 ensures accurate kinetochore-microtubule dynamics.

Faithful segregation of mitotic chromosomes requires bi-orientation of sister chromatids, which relies on the sensing of correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying PLK1 activation have been extensively studied, the regulatory mechanisms that couple PLK1 activity to accurate chromosome segregation are not well understood. In particular, PLK1 is implicated in stabilizing kinetochore-microtubule attachments, but how kinetochore PLK1 activity is regulated to avoid hyperstabilized kinetochore-microtubules in mitosis remains elusive. Here, we show that kinetochore PLK1 kinase activity is modulated by SET7/9 via lysine methylation during early mitosis. The SET7/9-elicited dimethylation occurs at the Lys191 of PLK1, which tunes down its activity by limiting ATP utilization. Overexpression of the non-methylatable PLK1 mutant or chemical inhibition of SET7/9 methyltransferase activity resulted in mitotic arrest due to destabilized kinetochore-microtubule attachments. These data suggest that kinetochore PLK1 is essential for stable kinetochore-microtubule attachments and methylation by SET7/9 promotes dynamic kinetochore-microtubule attachments for accurate error correction. Our findings define a novel homeostatic regulation at the kinetochore that integrates protein phosphorylation and methylation with accurate chromosome segregation for maintenance of genomic stability.