Microtubules, composed of αβ-tubulin heterodimers, have remained popular anticancer targets for decades. Six known binding sites on tubulin dimers have been identified thus far, with five sites on β-tubulin and only one site on α-tubulin, hinting that compounds binding to α-tubulin are less well characterized. Cevipabulin, a microtubule-active antitumor clinical candidate, is widely accepted as a microtubule-stabilizing agent by binding to the vinblastine site. Our x-ray crystallography study reveals that, in addition to binding to the vinblastine site, cevipabulin also binds to a new site on α-tubulin. We find that cevipabulin at this site pushes the αT5 loop outward, making the nonexchangeable GTP exchangeable, which reduces the stability of tubulin, leading to its destabilization and degradation. Our results confirm the existence of a new agent binding site on α-tubulin and shed light on the development of tubulin degraders as a new generation of antimicrotubule drugs targeting this novel site.

Endothelial cells (ECs) form an active barrier between the circulation and the body. In addition to controlling transport of molecules between these 2 compartments, the endothelium is a major secretory organ, releasing proteins both into the circulation and into the vascular matrix. Although it is clearly important that proteins are correctly sorted into these 2 spaces, we currently know little of the polarization of this secretion or how it is controlled. Here, we present an optimized system for the analysis of polarized secretion and show that it allows the derivation of deep, robust proteomes from small numbers of primary ECs. We present the first endothelial apically and basolaterally secreted proteomes, demonstrating that ECs polarize the secretion of extracellular vesicle cargoes to the apical surface. Conversely, we find that protein secretion at the basolateral surface is focused on components of the extracellular matrix (ECM). Finally, we examine the role of liprin‐α1 in secretion toward the basolateral compartment and identify a subset of ECM components that share this route with fibronectin.—Wei, H., Sundararaman, A., Dickson, E., Rennie‐Campbell, L., Cross, E., Heesom, K. J., Mellor, H. Characterization of the polarized endothelial secretome. FASEB J. 33, 12277‐12287 (2019). http://www.fasebj.org

Liver fibrosis is an abnormal wound healing response and a common consequence of chronic liver diseases from infection or alcohol/xenobiotic exposure. At the cellular level, liver fibrosis is mediated by trans-differentiation of hepatic stellate cells (HSCs), which is driven by persistent hepatic and systemic inflammation. However, impaired enterohepatic circulation and gut dysbiosis may indirectly contribute to the liver fibrogenesis. The composition of the gut microbiota depends on diet composition and host factors. In this study, we examined chlorophyllin, derived from green pigment chlorophyll, on gut microbiota, the intestinal mucosal barrier, and liver fibrosis. BALB/c mice received carbon tetrachloride through intraperitoneal injection to induce liver fibrosis and chlorophyllin was administrated in drinking water. The effects of chlorophyllin on liver fibrosis were evaluated for (1) survival rate, (2) hepatic morphologic analysis, (3) inflammatory factors in both the small intestine and liver, and (4) gut microbiota. Our results indicate that oral administration of chlorophyllin could attenuate intestinal and hepatic inflammation and ameliorate liver fibrosis. Importantly, oral administration of chlorophyllin promptly rebalanced the gut microbiota, exhibiting down-regulation of the phylum Firmicutes and up-regulation of the phylum Bacteroidetes. In vitro experiments on intestinal epithelial cells showed that chlorophyllin exposure could inhibit NF-κB pathway via IKK-phosphorylation suppression. In conclusion, this study demonstrates potential application of chlorophyllin to regulate the intestinal microbiota and ameliorate hepatic fibrosis.

Here, we report a novel mechanism to selectively degrade target proteins. 3-(3-Phenoxybenzyl)amino-β-carboline (PAC), a tubulin inhibitor, promotes selective degradation of αβtubulin heterodimers. Biochemical studies have revealed that PAC specifically denatures tubulin, making it prone to aggregation that predisposes it to ubiquitinylation and then degradation. The degradation is mediated by a single hydrogen bond formed between the pyridine nitrogen of PAC and βGlu198, which is identified as a low-barrier hydrogen bond (LBHB). In contrast, another two tubulin inhibitors that only form normal hydrogen bonds with βGlu198 exhibit no degradation effect. Thus, the LBHB accounts for the degradation. We then screened for compounds capable of forming an LBHB with βGlu198 and demonstrated that BML284, a Wnt signaling activator, also promotes tubulin heterodimer degradation through the LBHB. Our study provided a unique example of LBHB function and identified a novel approach to obtain tubulin degraders.

Pelvic organ prolapse (POP) is a serious health issue that affects many adult women. Surgical treatments for POP patients comprise a common strategy in which scaffold materials are used to reconstruct the prolapsed pelvic. However, the existing materials for pelvic reconstruction cannot meet clinical requirements in terms of biocompatibility, mechanics and immunological rejection. To address these concerns, polypropylene (PP) mesh was selected because of its strong mechanical properties. Small intestinal submucosa (SIS) was used to modify the PP mesh via a mussel-inspired polydopamine coating to enhance its biocompatibility. The scanning electron microscopy (SEM) and atomic force microscopy (AFM) results demonstrated that SIS was successfully conjugated on the surface of the PP mesh. Moreover, the cytotoxicity results indicated that the PP mesh and SIS-modified PP mesh were safe to use. Furthermore, in vivo tests demonstrated that the fibroplasia around the implanted site in the SIS-modified PP mesh group was significantly less than the fibroplasia around the PP mesh group. In addition, the immunohistochemistry staining results indicated that the expression of pro-inflammatory macrophages (M1) was substantially lower and that the expression of pro-healing macrophages (M2) was higher in the SIS-modified PP mesh group. Furthermore, ELISA detection indicated that the expression of IL-1β and IL-6 in the SIS-modified PP mesh group was reduced compared with the PP mesh group. These findings suggest that a SIS-modified polypropylene hybrid mesh via a mussel-inspired polydopamine coating is a promising approach in pelvic reconstruction.