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Pharmacological characterization of synthetic serine palmitoyltransferase inhibitors by biochemical and cellular analyses
Highlights: • Tetrahydropyrazolopyridine derivatives were characterized as SPT inhibitors. • The SPT inhibitors exhibited dual inhibitory activities against SPT2 and SPT3. • A SPT inhibitor bound to the SPT2 or SPT3 subunit in SPT enzyme complexes. • One of the SPT inhibitors exhibited a significantly slow dissociation from SPT. • The SPT inhibitors suppressed ceramide content in a non-small-cell lung cancer cell line. Human serine palmitoyltransferase (SPT) is a PLP-dependent enzyme residing in the endoplasmic reticulum. It catalyzes the synthesis of 3-ketodihydrosphingosine (3-KDS) from the substrates palmitoyl-CoA and l-serine. It is a rate-limiting enzyme for sphingolipid synthesis in cells. In the present study, we characterized and pharmacologically profiled a series of tetrahydropyrazolopyridine derivatives that potently inhibit human SPT enzymatic activity, including two cell-active derivatives and one fluorescent-labelled derivative. These SPT inhibitors exhibited dual inhibitory activities against SPT2 and SPT3. We used a fluorescent-labelled probe to molecularly assess the inhibitory mechanism and revealed its binding to the SPT2 or SPT3 subunit in the small subunit (ss) SPTa/SPT1/SPT2/or ssSPTa/SPT1/SPT3 functional complexes. One of the SPT inhibitors exhibited a significantly slow dissociation from the SPT complex. We confirmed that our SPT inhibitors suppressed ceramide content in non-small-cell lung cancer cell line, HCC4006, by performing a target engagement analysis. The potency of ceramide reduction correlated to that observed in a recombinant SPT2 enzyme assay. We thus elucidated and provided a fundamental understanding of the molecular mode of action of SPT inhibitors and developed potent, cell-active SPT inhibitors that can be used to clarify the biological function of SPT.
Increased chemo-sensitivity by knockdown coilin expression involved acceleration of premature cellular senescence in HeLa cells
Coilin is a marker protein of the Cajal body (CB). Cajal bodies, functional nuclear structure, play important roles for the maturation of telomerase mRNAs. However, whether CB participates in the process of cell senescence is unknown. Cisplatin is a frequently used drug for the chemotherapy for various cancers, which was recently reported to be able to induce premature senescence of tumor cells. In this study, we found that when HeLa cells were treated with 2 μg/ml cisplatin for 4 days, stagnant cell growth, especially in cells stained positive of SA-β-gal, was accompanied with significant changes in CB morphologies. The removal of cisplatin allowed the recovery of normal CB appearance, but was not able to restore cells from senescent states. Knocking down coilin expression by siRNA attenuated the growth and reduced the viability of treated cells, and the decreased rate of CB formation correlated with increased staining of SA-β-gal. Interestingly, when coilin knocked-down cells exposed to cisplatin, the drug sensitivity as shown by the reduction of cell viability was significantly increased compared to the control siRNA transfection groups. Overexpression of coilin phosphomutants increased SA-β-gal fluorescence following treatments with cisplatin as compared to the wild type coilin transfection. Our results indicated that coilin was an important functional player that involved in cisplatin-induced premature cell senescence. It suggested that the modulation of coilin expression could be considered as a potential anti-tumor strategy to increase the sensitivity of chemotherapy through which drug-induced cell senescence was accelerated. - Highlights: • The morphology of Cajal body change in cisplatin inducing premature cells. • The release from cisplatin treatments resulted in the recovery of CB morphology. • Knocking down coilin expression accelerated cisplatin induced senescence. • The overexpression of coilin phosphomutants induced premature senescence.
MCPIP1 negatively regulate cellular antiviral innate immune responses through DUB and disruption of TRAF3-TBK1-IKKε complex
IFNβ innate immune plays an essential role in antiviral immune. Previous reports suggested that many important regulatory proteins in innate immune pathway may be modified by ubiquitin and that many de-ubiquitination (DUB) proteins may affect immunity. Monocyte chemotactic protein-inducing protein 1 (MCPIP1), one of the CCCH Zn finger-containing proteins, was reported to have DUB function, but its effect on IFNβ innate immune was not fully understood. In this study, we uncovered a novel mechanism that may explain how MCPIP1 efficiently inhibits IFNβ innate immune. It was found that MCPIP1 negatively regulates the IFNβ expression activated by RIG-I, STING, TBK1, IRF3. Furthermore, MCPIP1 inhibits the nuclear translocation of IRF3 upon stimulation with virus, which plays a key role in type I IFN expression. Additionally, MCPIP1 interacts with important modulators of IFNβ expression pathway including IPS1, TRAF3, TBK1 and IKKε. Meanwhile, the interaction between the components in TRAF3-TBK1-IKKε complex was disrupted by MCPIP1. These results collectively suggest MCPIP1 as an innate immune regulator encoded by the host and point to a new mechanism through which MCPIP1 negatively regulates IRF3 activation and type I IFNβ expression.
Inflammatory production of reactive oxygen species by Drosophila hemocytes activates cellular immune defenses
Highlights: • Biphasic hemocyte ROS response to infection in flies. • Early response high in non-phagocytic prohemocytes and crystal cells. • Delayed ROS in phagocytic cells. • ROS activate plasmatocyte spreading and adhesion. • ROS promote JNK-dependent crystal cell rupture. The production of reactive oxygen species (ROS) is a prominent response to infection among innate immune cells such as macrophages and neutrophils. To better understand the relationship between antimicrobial and regulatory functions of blood cell ROS, we have characterized the ROS response to infection in Drosophila hemocytes. Using fluorescent probes, we find a biphasic hemocyte ROS response to bacterial infection. In the first hour, virtually all hemocytes generate a transient ROS signal, with nonphagocytic cells including prohemocytes and crystal cells displaying exceptionally strong responses. A distinct, and more delayed ROS response starting at 90 min is primarily within cells that have engulfed bacteria, and is sustained for several hours. The early response has a clear regulatory function, as dampening or intensifying the intracellular ROS level has profound effects on plasmatocyte activation. In addition, ROS are necessary and sufficient to activate JNK signalling in crystal cells, and to promote JNK-dependent crystal cell rupture. These findings indicate that Drosophila will be a promising model in which to dissect the mechanisms of ROS stimulation of immune activation.
Design, synthesis, and structure-activity relationships of novel 4,7,12,12a-tetrahydro-5H-thieno[3′,2′:3,4]pyrido[1,2-b]isoquinoline and 5,8,12,12a-tetrahydro-6H-thieno[2′,3′:4,5]pyrido[2,1-a]isoquinoline derivatives as cellular activators of adenosine 5′-monophosphate-activated protein kinase (AMPK)
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Insight into molecular basis and dynamics of full-length CRaf kinase in cellular signaling mechanisms
Raf kinases play key roles in signal transduction in cells for regulating proliferation, differentiation, and survival. Despite decades of research into functions and dynamics of Raf kinases with respect to other cytosolic proteins, understanding Raf kinases is limited by the lack of their full-length structures at the atomic resolution. Here, we present the first model of the full-length CRaf kinase obtained from artificial intelligence/machine learning algorithms with a converging ensemble of structures simulated by large-scale temperature replica exchange simulations. Our model is validated by comparing simulated structures with the latest cryo-EM structure detailing close contacts among three key domains and regions of the CRaf. Our simulations identify potentially new epitopes of intramolecule interactions within the CRaf and reveal a dynamical nature of CRaf kinases, in which the three domains can move back and forth relative to each other for regulatory dynamics. The dynamic conformations are then used in a docking algorithm to shed insight into the paradoxical effect caused by vemurafenib in comparison with a paradox breaker PLX7904. In this study, we propose a model of Raf-heterodimer/KRas-dimer as a signalosome based on the dynamics of the full-length CRaf.
Atoms to Phenotypes: Molecular Design Principles of Cellular Energy Metabolism
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helixCAM: A platform for programmable cellular assembly in bacteria and human cells
Interactions between cells are indispensable for signaling and creating structure. The ability to direct precise cell-cell interactions would be powerful for engineering tissues, understanding signaling pathways, and directing immune cell targeting. In humans, intercellular interactions are mediated by cell adhesion molecules (CAMs). However, endogenous CAMs are natively expressed by many cells and tend to have cross-reactivity, making them unsuitable for programming specific interactions. Here, we showcase “helixCAM,” a platform for engineering synthetic CAMs by presenting coiled-coil peptides on the cell surface. helixCAMs were able to create specific cell-cell interactions and direct patterned aggregate formation in bacteria and human cells. Based on coiled-coil interaction principles, we built a set of rationally designed helixCAM libraries, which led to the discovery of additional high-performance helixCAM pairs. Finally, we applied this helixCAM toolkit for various multicellular engineering applications, such as spherical layering, adherent cell targeting, and surface patterning.
Tracer particle transport dynamics in the diffusive sandpile cellular automaton
The confinement properties of the diffusive running sandpile are characterized by tracking the motion of a population of marked grains of sand. It is found that, as the relative strength of the avalanching to the diffusive transport channel is varied, a point is reached at which the particle global confinement time and the probability density functions of the jump-sizes and waiting-times of the tracked grains experience a sudden change, thus revealing a dynamical transition, that is consistent with previous studies (Newman DE et al., Phys Rev Lett 2002;88(20):204304). Across this transition, the sandpile moves from a regime characterized by self-similarity and memory, where avalanches of all possible sizes dominate transport across the system, to another regime where transport is taken over by near system-size, quasi-periodic avalanches. Values for the fractional transport exponents that quantify effective transport across the sandpile prior to the transition are also obtained.
Principles Governing Establishment versus Collapse of HIV-1 Cellular Spread
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