Integrated Proteomics Characterization of NLRP3 Inflammasome Inhibitor MCC950 in Monocytic Cell Line Confirms Direct MCC950 Engagement with Endogenous NLRP3.

Inhibition of the NLRP3 inflammasome is a promising strategy for the development of new treatments for inflammatory diseases. MCC950 is a potent and selective small-molecule inhibitor of the NLRP3 pathway and has been validated in numerous species and disease models. Although the capacity of MCC950 to block NLRP3 signaling is well-established, it is still critical to identify the mechanism of action and molecular targets of MCC950 to inform and derisk drug development. Quantitative proteomics performed in disease-relevant systems provides a powerful method to study both direct and indirect pharmacological responses to small molecules to elucidate the mechanism of action and confirm target engagement. A comprehensive target deconvolution campaign requires the use of complementary chemical biology techniques. Here we applied two orthogonal chemical biology techniques: compressed Cellular Thermal Shift Assay (CETSA) and photoaffinity labeling chemoproteomics, performed under biologically relevant conditions with LPS-primed THP-1 cells, thereby deconvoluting, for the first time, the molecular targets of MCC950 using chemical biology techniques. In-cell chemoproteomics with inlysate CETSA confirmed the suspected mechanism as the disruption of inflammasome formation via NLRP3. Further cCETSA (c indicates compressed) in live cells mapped the stabilization of NLRP3 inflammasome pathway proteins, highlighting modulation of the targeted pathway. This is the first evidence of direct MCC950 engagement with endogenous NLRP3 in a human macrophage cellular system using discovery proteomics chemical biology techniques, providing critical information for inflammasome studies.

Tubulin CFEOM mutations both inhibit or activate kinesin motor activity.

Kinesin-mediated transport along microtubules is critical for axon development and health. Mutations in the kinesin Kif21a, or the microtubule subunit ß-tubulin, inhibit axon growth and/or maintenance resulting in the eye-movement disorder congenital fibrosis of the extraocular muscles (CFEOM). While most examined CFEOM-causing ß-tubulin mutations inhibit kinesin-microtubule interactions, Kif21a mutations activate the motor protein. These contrasting observations have led to opposed models of inhibited or hyperactive Kif21a in CFEOM. We show that, contrary to other CFEOM-causing ß-tubulin mutations, R380C enhances kinesin activity. Expression of ß-tubulin-R380C increases kinesin-mediated peroxisome transport in S2 cells. The binding frequency, percent motile engagements, run length and plus-end dwell time of Kif21a are also elevated on ß-tubulin-R380C compared with wildtype microtubules in vitro. This conserved effect persists across tubulins from multiple species and kinesins from different families. The enhanced activity is independent of tail-mediated kinesin autoinhibition and thus utilizes a mechanism distinct from CFEOM-causing Kif21a mutations. Using molecular dynamics, we visualize how ß-tubulin-R380C allosterically alters critical structural elements within the kinesin motor domain, suggesting a basis for the enhanced motility. These findings resolve the disparate models and confirm that inhibited or increased kinesin activity can both contribute to CFEOM. They also demonstrate the microtubule's role in regulating kinesins and highlight the importance of balanced transport for cellular and organismal health.

A cell-active cyclic peptide targeting the Nrf2/Keap1 protein-protein interaction.

The disruption of the protein-protein interaction (PPI) between Nrf2 and Keap1 is an attractive strategy to counteract the oxidative stress that characterises a variety of severe diseases. Peptides represent a complementary approach to small molecules for the inhibition of this therapeutically important PPI. However, due to their polar nature and the negative net charge required for binding to Keap1, the peptides reported to date exhibit either mid-micromolar activity or are inactive in cells. Herein, we present a two-component peptide stapling strategy to rapidly access a variety of constrained and functionalised peptides that target the Nrf2/Keap1 PPI. The most promising peptide, P8-H containing a fatty acid tag, binds to Keap1 with nanomolar affinity and is effective at inducing transcription of ARE genes in a human lung epithelial cell line at sub-micromolar concentration. Furthermore, crystallography of the peptide in complex with Keap1 yielded a high resolution X-ray structure, adding to the toolbox of structures available to develop cell-permeable peptidomimetic inhibitors.

Dynamic microtubules slow down during their shrinkage phase.

Microtubules are dynamic polymers that undergo stochastic transitions between growing and shrinking phases. The structural and chemical properties of these phases remain poorly understood. The transition from growth to shrinkage, termed catastrophe, is not a first-order reaction but rather a multistep process whose frequency increases with the growth time: the microtubule ages as the older microtubule tip becomes more unstable. Aging shows that the growing phase is not a single state but comprises several substates of increasing instability. To investigate whether the shrinking phase is also multistate, we characterized the kinetics of microtubule shrinkage following catastrophe using an in vitro reconstitution assay with purified tubulins. We found that the shrinkage speed is highly variable across microtubules and that the shrinkage speed of individual microtubules slows down over time by as much as several fold. The shrinkage slowdown was observed in both fluorescently labeled and unlabeled microtubules as well as in microtubules polymerized from tubulin purified from different species, suggesting that the shrinkage slowdown is a general property of microtubules. These results indicate that microtubule shrinkage, like catastrophe, is time dependent and that the shrinking microtubule tip passes through a succession of states of increasing stability. We hypothesize that the shrinkage slowdown is due to destabilizing events that took place during growth, which led to multistep catastrophe. This suggests that the aging associated with growth is also manifested during shrinkage, with the older, more unstable growing tip being associated with a faster depolymerizing shrinking tip.

Nicotinamide adenine dinucleotides and their precursor NMN have no direct effect on microtubule dynamics in purified brain tubulin.

Microtubules are dynamic cytoskeletal polymers that provide mechanical support for cellular structures, and play important roles in cell division, migration, and intracellular transport. Their intrinsic dynamic instability, primarily controlled by polymerization-dependent GTP hydrolysis, allows for rapid rearrangements of microtubule arrays in response to signaling cues. In neurons, increases in intracellular levels of nicotinamide adenine dinucleotide (NAD+) can protect against microtubule loss and axonal degeneration elicited by axonal transection. The protective effects of NAD+ on microtubule loss have been shown to be indirect in some systems, for example through the sirtuin-3 pathway. However, it is still possible that NAD+ and related metabolites have direct effects on microtubule dynamics to promote assembly or inhibit disassembly. To address this question, we reconstituted microtubule dynamics in an in vitro assay with purified bovine brain tubulin and examined the effects of NAD+, NADH, and NMN. We found that the compounds had only small effects on the dynamics at the plus and minus ends of the microtubules. Furthermore, these effects were not statistically significant. Consequently, our data support earlier findings that NADs and their precursors influence microtubule growth through indirect mechanisms.

Label-free high-speed wide-field imaging of single microtubules using interference reflection microscopy.

When studying microtubules in vitro, label free imaging of single microtubules is necessary when the quantity of purified tubulin is too low for efficient fluorescent labelling or there is concern that labelling will disrupt function. Commonly used techniques for observing unlabelled microtubules, such as video enhanced differential interference contrast, dark-field and more recently laser-based interferometric scattering microscopy, suffer from a number of drawbacks. The contrast of differential interference contrast images depends on the orientation of the microtubules, dark-field is highly sensitive to impurities and optical misalignments. In addition, all of these techniques require costly optical components such as Nomarski prisms, dark-field condensers, lasers and laser scanners. Here we show that single microtubules can be imaged at high speed and with high contrast using interference reflection microscopy without the aforementioned drawbacks. Interference reflection microscopy is simple to implement, requiring only the incorporation of a 50/50 mirror instead of a dichroic in a fluorescence microscope, and with appropriate microscope settings has a similar signal-to-noise ratio to differential interference contrast and fluorescence. We demonstrated the utility of interference reflection microscopy by high-speed imaging and tracking of dynamic microtubules at 100 frames per second. In conclusion, the optical quality of interference reflection microscopy falls within the range of other microscope techniques, being inferior to some and superior to others, depending on the metric used and, with minimal microscope modification, can be used to study the dynamics of unlabelled microtubules. LAY DESCRIPTION: The cytoskeleton gives a cell its shape and plays a major role in its movement and division. It's also helps organise the content of cells and is the base for intracellular transport. Important components of the cytoskeleton are microtubules, which are hollow cylindrical beams (25 nm in diameter) that assemble from protein building blocks called tubulin. Deficiencies in microtubules are related to many diseases including cancer and Alzheimer. Given their important role, microtubules are heavily investigated in many laboratories. One way to study microtubules is to isolate them from cells and image them using light microscopy. Over the years a number of imaging techniques have been used. These techniques have a number of drawbacks which are addressed by ongoing efforts which this work is a part of. Here, we present a method based on light interference that produce high quality images of microtubules. The technique is cheap and easy to implement making it accessible to a wide base of researchers.

Mutations in TUBB8 and Human Oocyte Meiotic Arrest.

Background Human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. Methods We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other ß-tubulin isotypes was assessed in human oocytes, early embryos, sperm cells, and several somatic tissues by means of a quantitative reverse-transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one ß-tubulin polypeptide (α/ß-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. Results We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed ß-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/ß-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. Conclusions TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility. (Funded by the National Basic Research Program of China and others.).

Spatial control of microtubule length and lifetime by opposing stabilizing and destabilizing functions of Kinesin-8.

BACKGROUND: To function in diverse cellular processes, the dynamic behavior of microtubules (MTs) must be differentially regulated within the cell. In budding yeast, the spindle position checkpoint (SPOC) inhibits mitotic exit in response to mispositioned spindles. To maintain SPOC-mediated anaphase arrest, astral MTs must maintain persistent interactions with and/or extend through the bud neck. However, the molecular mechanisms that ensure the stability of these interactions are not known. RESULTS: The presence of an MT extending through and/or interacting with the bud neck is maintained by spatial control of catastrophe and rescue, which extends MT lifetime >25-fold and controls the length of dynamic MTs within the bud compartment. Moreover, the single kinesin-8 motor Kip3 alternately mediates both catastrophe and rescue of the bud MT. Kip3 accumulates in a length-dependent manner along the lattice of MTs within the bud, yet induces catastrophe spatially near the bud tip. Rather, this accumulation of Kip3 facilitates its association with depolymerizing MT plus ends, where Kip3 promotes rescue before MTs exit the bud. MT rescue within the bud requires the tail domain of Kip3, whereas the motor domain mediates catastrophe at the bud tip. In vitro, Kip3 exerts both stabilizing and destabilizing effects on reconstituted yeast MTs. CONCLUSIONS: The kinesin-8 Kip3 is a multifunctional regulator that differentially stabilizes and destabilizes specific MTs. Control over MT catastrophe and rescue by Kip3 defines the length and lifetime of MTs within the bud compartment of cells with mispositioned spindles. This subcellular regulation of MT dynamics is critical to maintaining mitotic arrest in response to mispositioned spindles.

Structure-function analysis of yeast tubulin.

Microtubules play essential roles in a wide variety of cellular processes including cell division, motility, and vesicular transport. Microtubule function depends on the polymerization dynamics of tubulin and specific interactions between tubulin and diverse microtubule-associated proteins. To date, investigation of the structural and functional properties of tubulin and tubulin mutants has been limited by the inability to obtain functional protein from overexpression systems, and by the heterogeneous mixture of tubulin isotypes typically isolated from higher eukaryotes. The budding yeast, Saccharomyces cerevisiae, has emerged as a leading system for tubulin structure-function analysis. Yeast cells encode a single beta-tubulin gene and can be engineered to express just one of two alpha isotypes. Moreover, yeast allows site-directed modification of tubulin genes at the endogenous loci expressed under the native promoter and regulatory elements. These advantageous features provide a homogeneous and controlled environment for analysis of the functional consequences of specific mutations. Here, we present the techniques to generate site-specific tubulin mutations in diploid and haploid cells, assess the ability of the mutated protein to support cell viability, measure overall microtubule stability, and define changes in the specific parameters of microtubule dynamic instability. We also outline strategies to determine whether mutations disrupt interactions with microtubule-associated proteins. Microtubule-based functions in yeast are well defined, which allows the observed changes in microtubule properties to be related to the role of microtubules in specific cellular processes.

Synthesis and Biological Evaluation of a Biotinylated Paclitaxel With an Extra-Long Chain Spacer Arm.

A biotinylated paclitaxel derivative with an extra-long-chain (LC-LC-Biotin) spacer arm was synthesized using an improved synthetic reaction sequence. The biotinylated paclitaxel analogue retained excellent microtubule stabilizing activity in vitro. Furthermore, it was shown that this analogue can simultaneously engage streptavidin and the binding site on microtubules, making it suitable for localization studies or for the attachment of paclitaxel to solid substrates via a streptavidin linkage.

An inherited TUBB2B mutation alters a kinesin-binding site and causes polymicrogyria, CFEOM and axon dysinnervation.

Microtubules are essential components of axon guidance machinery. Among ß-tubulin mutations, only those in TUBB3 have been shown to cause primary errors in axon guidance. All identified mutations in TUBB2B result in polymicrogyria, but it remains unclear whether TUBB2B mutations can cause axon dysinnervation as a primary phenotype. We have identified a novel inherited heterozygous missense mutation in TUBB2B that results in an E421K amino acid substitution in a family who segregates congenital fibrosis of the extraocular muscles (CFEOM) with polymicrogyria. Diffusion tensor imaging of brains of affected family members reveals aberrations in the trajectories of commissural projection neurons, implying a paucity of homotopic connections. These observations led us to ask whether axon dysinnervation is a primary phenotype, and why the E421K, but not other, TUBB2B substitutions cause CFEOM. Expression of exogenous Tubb2b-E421K in developing callosal projection neurons is sufficient to perturb homotopic connectivity, without affecting neuronal production or migration. Using in vitro biochemical assays and yeast genetics, we find that TUBB2B-E421K αß-heterodimers are incorporated into the microtubule network where they alter microtubule dynamics and can reduce kinesin localization. These data provide evidence that TUBB2B mutations can cause primary axon dysinnervation. Interestingly, by incorporating into microtubules and altering their dynamic properties, the E421K substitution behaves differently than previously identified TUBB2B substitutions, providing mechanistic insight into the divergence between resulting phenotypes. Together with previous studies, these findings highlight that ß-tubulin isotypes function in both conserved and divergent ways to support proper human nervous system development.

An engineered substance P variant for receptor-mediated delivery of synthetic antibodies into tumor cells.

We have developed and tested a robust delivery method for the transport of proteins to the cytoplasm of mammalian cells without compromising the integrity of the cell membrane. This receptor-mediated delivery (RMD) technology utilizes a variant of substance P (SP), a neuropeptide that is rapidly internalized upon interaction with the neurokinin-1 receptor (NK1R). Cargos in the form of synthetic antibody fragments (sABs) were conjugated to the engineered SP variant (SPv) and efficiently internalized by NK1R-expressing cells. The sABs used here were generated to bind specific conformational forms of actin. The internalized proteins appear to escape the endosome and retain their binding activity within the cells as demonstrated by co-localization with the actin cytoskeleton. Further, since the NK1R is over-expressed in many cancers, SPv-mediated delivery provides a highly specific method for therapeutic utilization of affinity reagents targeting intracellular processes in diseased tissue.